Process for preparing maytansinoid antibody conjugates

TECHNICAL MEMORY

PROCESS FOR PREPARING ANTIBODY AND MAITANSINOID CONJUGATES

FIELD OF THE INVENTION

This invention pertains to a process for preparing conjugates of substantially high purity and stability, wherein the conjugates comprise a drug-chemically coupled cell-binding agent.

BACKGROUND OF THE INVENTION

Cancer treatment has progressed significantly with the development of pharmaceuticals that more efficiently target and kill cancer cells. To this end, researchers have taken advantage of 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, radionucleotides, and certain chemotherapeutic drugs have been chemically linked to monoclonal antibodies that bind to tumor-associated or tumor-specific cell surface antigens (see, for example, US Patent Applications International WO 00/02587, WO 02/060955, and WO 02/092127, US Patent Nos. 5,475,092, 6,340,701, and 6,171,586, US Patent Application Publication No. 2003/0004210 A1, and Ghetie et al., J. Immunol. Methods, 112:267-277 (1988). Such compounds are typically referred to as toxin, radionucleotide, and drug "conjugates", 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 release and/or activation of the drug's cytotoxic activity. The selectivity produced by drug conjugates minimizes toxicity towards normal cells, thus improving drug tolerance in the patient.

Processes for conjugating antibodies to sulfhydryl-containing cytotoxic agents such as maytansinoids have previously been described (see, for example, US Patent Nos. 5,208,020, 5,416,064, and 6,441,163). For example, US Patent Nos. 5,208,020 and 5,416,064 describe a process for making maytansinoid-antibody conjugates in which the antibody is first modified with a heterobifunctional reagent as described in US Patent Nos. 4,149,003, 4,563,304 and 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 (SEO) has also been described (see, for example, Liu et al., Procc. Nati. Acad. Sci. (USA), 93: 8618-8623 (1996 ), and Chari et al., Cancer Research, 52: 127-131 (1992)).

The previously described processes for the manufacture of antibody-drug conjugates are complex because they are hampered with steps that are cumbersome, to make or produce immunoconjugates that are less pure or less stable than optimally desirable. For example, conjugation at a pH between 6.0 and 6.5 is not optimal for producing stable, pure conjugates. Furthermore, the conjugation reaction under these conditions is generally slow and inefficient, leading to excessive time requirement and material consumption.

It would be desirable to modify or eliminate one or more manufacturing steps without compromising product quality, such as purity and/or stability. It would be further desirable to have additional purification options than have been described thus far in that some options will be more efficient with certain combinations of cell-binding agents, linkers, and drugs than 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. The invention provides such a method. These and other advantages of the invention, as well as additional aspects of the invention, will become apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a process for preparing a conjugate of substantially high purity and stability comprising a cell-binding agent chemically coupled to a drug. The process comprises (a) contacting a cell-binding agent with a bifunctional cross-linking reagent to covalently affix a bond 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 tangential flow filtration, absorption chromatography, adsorption filtration, selective precipitation, or combinations thereof, and thus prepare a first purified mixture of cell-binding agents having linkers bound to them, (c) conjugating a drug to the cell binding agents having linkers attached thereto in the first mixture purified by reacting the cell binding agents having linkers attached thereto with a drug in a solution having a pH of about 4 to about 9 to prepare a second mixture comprising (i) cell binding agent chemically coupled through drug binding, (ii) reaction by-products, and (d) subjecting the second mixture to tangential flow filtration, adsorption chromatography, adsorption filtration, selective precipitation, or combinations thereof to purify the chemically coupled cell-binding agents through drug-bindings from other components of the mixture. second mix and thus prepare a second purified mix.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a process for preparing drug-cell binding agent conjugates of substantially high purity and stability. Said compositions can be used to treat diseases because of 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) greater than 90%, preferably greater than 95%, of conjugated species are monomeric, and/or (b) the level of free drug in the conjugate preparation is less than of 2% (in relation to the total drug).

In this regard, the process of the invention comprises (a) modifying the cell binding agent with a bifunctional crosslinking reagent to covalently attach a linker to the cell binding agent and thus prepare a first mixture comprising cell binding agents having linked linkers thereto, (b) subjecting the first mixture to tangential flow filtration, adsorption chromatography, adsorption filtration, selective precipitation, or combinations thereof, to purify the cell-binding agents having linkers bound thereto from other components of the first mixture and thus prepare a first purified mixture of cell-binding agents having linkers bound thereto, (c) conjugating a drug to 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 of about 4 to about 9 to prepare a second mixture comprising (i) cell binding agent chemically coupled through the linker to the drug, (ii) free drug, and (iii) reaction by-products, and (d) subjecting the second mixture to tangential flow filtration, adsorption chromatography, adsorption filtration, selective precipitation, or a combination thereof, to remove unconjugated drugs, reactants, and by-products, as well as to obtain conjugates. of substantially purified drug-cellular binding agent.

Preferably, tangential flow filtration (TFF, also known as cross-flow filtration, ultrafiltration, and diafiltration), and/or adsorption chromatography resins are used in the purification steps. However, when TFF was used in the first purification step (step b) in (step c), a conjugation at pH of 6.0 - 6.5 was used, and a resin for adsorption chromatography was used in the second purification step ( step d), it was preferred that the resin for adsorption chromatography is not an ion exchange resin. In other preferred embodiments, TFF is used in both purification steps, adsorption chromatography resins were used in both purification steps. Alternatively, a resin for adsorption chromatography was used in the first purification step, and TFF was used in the second purification step. A combination of TFF and an adsorption chromatography resin can be used in the first and/or second purification step as well.

Any of the suitable TFF systems can be used, including a Pellicon type system (Millipore, Billerica, MA), a Sartocon Cartridge system (Sartorius AG, Edgewood, NY), and a Centrasette type system (Pall Corp., East Hills, NY).

Any adsorption chromatography resin can be used. Preferred adsorption chromatography resins include hydroxyapatite chromatography resins, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixing mode ion exchange chromatography, immobilized metal (IMAC), stained ligand chromatography, affinity chromatography, reverse phase chromatography, and combinations thereof. Examples of suitable hydroxyapatite resins include ceramic hydroxyapatite (CHT Type I and Type II, BioRad Laboratories, Hercules, CA), HA Ultragel hydroxyapatite (Pall Corp, East Hills, NY), and ceramic fluoroapatite (Type I and Type II CFT). 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 Macro-Prep Methyl and Macro-Prep t-Butyl resins. (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 stained ligand resins include Blue Sepharose resin (GE HealthCare, Piscataway, NJ) and Affi-fel 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, lectin affinity resins, eg, Lentil Lectin resin. Sepharose (GE Health Care, Piscataway, NJ), where the cell binding agent possesses appropriate lectin binding sites. Alternatively an antibody specific to the cell binding agent can be used. Such an antibody can be immobilized on, for example, Sepharose 4 Fast Flow resin (GE HealthCare, Piscataway, NJ). Examples of suitable reverse phase resins include C4, C6, and C18 resins (Grace Vydac, Hesperia, CA).

In accordance with the method of the invention, a first mixture is produced comprising a cell binding agent having linkers attached to it, 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 process. In this regard, the first mixture can be purified using tangential flow filtration (TFF), for example membrane-based tangential flow filtration process, adsorption chromatography, adsorption filtration, or selective precipitation, or any other suitable purification process. , as well as combinations thereof. This first purification step provides a first purified mixture, i.e. an increasing concentration of the cell binding agent having linkers bound thereto and a decreasing amount of the unbound bifunctional crosslinker reagent, compared to the first mixture before purification according to the invention.

After purification of the first mixture to obtain a first purified mixture of cell binding agents having linkers bound thereto, a drug is conjugated to the cell binding agents having linkers bound thereto in the first purified mixture by reaction of the cell-binding agents having linkers attached thereto with a drug in a solution having a pH of from about 4 to about 9, whereupon a second mixture is produced comprising (i) the cell binding agent chemically coupled through the linker to the drug, (ii) free drug, and (iii) reaction by-products. While the conjugation reaction is carried out at a pH of about 4 to about 9, the reaction is preferably carried out at a pH of about 6 or less or at a pH of about 6.5 or higher, more preferably at a pH of about 4 to about 6 or at a pH from about 6.5 to about 9, and especially at a pH from 4 to less than 6 or at a pH greater than 6.5 to 9. When the conjugation step is carried out at a pH of about 6.5 or higher, some sulfhydryl-containing drugs may have a tendency to dimerize through disulfide bond formation. Removal of trace metals and/or oxygen from the reaction mixture may be required, as well as optional addition of antioxidants or use of linkers with more reactive displacers, or addition of drug in more than one aliquot. to allow efficient reaction in such a situation.

Optionally, purification of the modified cell binding agent can be omitted. In such a situation, the drug may be added simultaneously with the crosslinking reagent or at the same time, eg 1, 2, 3, or more hours after addition of the crosslinking reagent to the cell binding agent.

The method of the invention may optionally include the addition of sucrose in the conjugation step, used in the method of the invention to increase the solubility and recovery of drug-cell binding agent conjugates. Desirably, the sucrose is added at a concentration of from about 0.1% (w/v) to about 20% (w/v) (for example, about 0.1% (w/v), about 5% (w/v) about 10 % (w/v) about 15% (w/v), or about 20% (w/v)). Preferably, the sucrose is added at a concentration of from about 1% (w/v) to about 10% (w/v) (for example, about 2% (w/v), about 4% (w/v), about 6% (w/v) or about 8% (w/v) In addition the conjugation reaction may also comprise the addition of a regulating agent Any suitable regulating agent known in the art may 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 can be purified using tangential flow filtration (TFF), for example a membrane-based tangential flow filtration process, adsorption chromatography, adsorption filtration, selective precipitation, or any other suitable purification process. , as well as combinations thereof, which are set forth herein. This second purification step provides a second purified mixture, i.e., an increasing concentration of the cell-binding agent chemically coupled through the linkers to the drug and a decreasing amount of one or more other components of the second mixture, compared to the second mixture. second mixture before purification according to the invention. The cell-binding agent can be any suitable agent that binds to a cell, typically and preferably an animal cell (eg, a human cell). The cell binding agent is preferably a peptide or polypeptide. Suitable cell-binding agents include, for example, antibodies (for example, monoclonal antibodies and fragments thereof), lymphokines, hormones, growth factors, nutrient transport molecules (for example, transferrin), and any other agent or molecule that specifically binds to a target molecule on the surface of a cell.

The term "antibody", as used herein, refers to any immunoglobulin, any immunoglobulin fragment, such as Fab, F(ab')2, dsFv, sFv, diabodies, and triabodies, or chimeric immunoglobulins, which may bind to an antigen on the surface of a cell (eg, containing a complementarity determining region (CDR)). Any suitable antibody can be used as the cell binding agent. One skilled in the art will appreciate that the selection of a suitable antibody will depend on the cell population to be considered. 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. of the invention. Cell surface expression profiles are known for a wide variety of cell types, including tumor cell types, or, if not known, can be determined using routine molecular biology and histochemical techniques.

The antibody can be polyclonal or monoclonal, but is more preferably a monoclonal antibody. As used herein, "polyclonal" antibodies refer to heterogeneous populations of antibody molecules, typically contained in the serum 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 a variety of techniques known to those skilled in the art, including standard hybridoma technology (see, for example, Khóler & Milstein, Eur J. Immunol., 5: 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CHS Press (1988), and CA Janeway et al. (eds.), Immunobiology, 5-. Ed., Garland Publishing, New York, NY (2001)). In summary, the hybridoma's method of producing monoclonal antibodies typically involves injecting any suitable animal, typically and preferably a mouse, with an antigen (ie, immunogen). The animal is subsequently sacrificed, and B cells isolated from its spleen are fused with human myeloma cells. A hybrid cell (ie, a "hybridoma") is produced, which proliferates indefinitely and consequently secretes high titers of an antibody with the desired specificity in vitro. Any suitable 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 secreting a single species of antibody to the antigen. Because each hybridoma is a clone derived from fusion with a single B cell, all antibody molecules produced are identical in structure, including their antigenic binding site and isotype. Monoclonal antibodies can also be generated using other suitable techniques including EBV-hybridoma technology (see, for example, 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, for example, Huse et al., Science , 246:1275-81 (1989)), or phage display libraries comprising antibody fragments, such as Fab and scFv (single chain variable region) (see, for example, US Patents 5,885,793 and 5,969,108, and US Pat. International Patent Application Nos. 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 known to those skilled in the art and are described herein. With respect to human antibodies, one skilled in the art will appreciate that polyclonal antibodies can be isolated from the serum of human subjects vaccinated or immunized with an appropriate antigen. Alternatively, human antibodies can be generated by adapting known techniques for producing human antibodies in non-human animals such as mice (see, for example, US Patents 5,545,806, 5,569,825, and 5,714,352, and US Patent Application Publication No. 2002/0197266 A1).

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

To this end, phage display can be used to generate the antibody. In this regard, phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard recombinant DNA and molecular biology techniques (see, for example, 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 hybridoma production, so that human antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, for example). example, Janeway et al., supra, Huse et al., supra, and US Patent No. 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 Patent Nos. 5,545,806 and 5,569,825 and Janeway et al., supra.

More preferably, the antibody is a humanized antibody. As used herein, a "humanized" antibody is one in which the complementarity regions (CDRs) of a mouse monoclonal antibody, which form the antigen-binding circuitry of the antibody, are grafted onto the backbone of a molecule. of human antibody. Due to the similarity of the major structures of human and mouse antibodies, it is generally accepted in the art that this procedure produces a monoclonal antibody that is antigenically identical to a human antibody but binds to the same antigen as the mouse monoclonal antibody from the which 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., supra, 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 resurfacing technology described in US Patent 5,639,641 and Pedersen et al., J. Mol. BioL, 235: 959-973 (1994). While the antibody employed in the conjugate of the composition of the invention is most preferably a humanized monoclonal antibody, a human monoclonal antibody and a mouse monoclonal antibody as described above are within the scope of the invention. Antibody fragments that have at least one antigenic binding site, and therefore recognize and bind to at least one antigen or receptor present on the surface of a target cell, are also within the scope of the invention. In this regard, proteolytic fragmentation of an intact antibody molecule can produce a variety of 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 referred to as Fab fragments, when they retain the antigenic binding activity of the parent antibody molecule. Fragmentation of an antibody molecule with the enzymatic pepsin normally produces two antibody fragments, one of which retains both antigen-binding arms of the antibody molecule, and is therefore referred to as the F(ab') fragment.₂. Reduction of an F(ab') 2 fragment with dithiothreitol or mercaptoethylamine produces a fragment referred to as a Fab' fragment. A single chain variable region (sFv) antibody fragment, consisting of a truncated Fab fragment comprising the variable (V) domain of an antibody heavy chain linked to a V domain of an antibody light chain via a synthetic peptide , can be generated using routine recombinant DNA technology techniques (see, eg, Janeway et al., supra). Similarly, disulfide stabilized variable region (dsFv) fragments can be prepared by recombinant DNA technology (see, eg, Protein Engineering, 7: 697-704 (1994)). Antibody fragments in the context of the invention, however, are not limited to these exemplary types of antibody fragments. Any antibody fragment that recognizes and binds to a desired cell surface receptor or antigen can be used. Additionally, antibody fragments are described in, for example, Parsham, 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). Antigen-antibody binding can be assayed using any method known in the art available, such as, for example, radioimmunoassay (RIA), ELISA, Western staining, immunoprecipitation, and comparative inhibition assays (see, for example, Janeway et al. , supra, and US Patent Application Publication No. 2002/0197266 A1).

Furthermore, the antibody can be a chimeric antibody or an antigenic binding fragment thereof. By "chimeric" it is meant that the antibody comprises at least two immunoglobulins, or fragments thereof, obtained or derived from at least two different species (for example, two different immunoglobulins, such as the human immunoglobulin constant region combined with a variable region of rodent immunoglobulin). The antibody can also be a domain antibody (dAb) or an antigenic binding fragment thereof, such as, for example, a camelid antibody (see, for example, Desmyter et al., Nature, Struct. Biol. 3: 752 , (1996)), or a shark antibody, such as, for example, a novel receptor antigen (IgNAR) (see, for example, 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 invention. For example, the monoclonal antibody J5 is a rodent IgG2a antibody that is specific for Communal Acute Lymphoblastic Leukemia Antigen (CALLA) (Ritz et al., Nature, 283: 583-585 (1980), and can be used in target cells that express CALLA (eg, acute lymphoblastic leukemia cells). The MY9 monoclonal antibody is a rodent IgGI antibody that specifically binds to the CD33 antigen (Griffin et al., Leukemia Res., 8: 521 (1984), and can be used to target CD33-expressing cells, eg, myelogenous cells. (AML)).

Similarly, the anti-B4 monoclonal antibody (also referred to as B4) is a rodent IgG1 antibody that binds to the CD19 antigen on B cells (Nadler et al., J. Immunol, 131: 244-250 (1983)). , and can be used on target B cells or diseased cells that express CD19 (eg, non-Hodgkin's lymphoma cells and chronic lymphoblastic leukemia cells). N901 is a rodent monoclonal antibody that binds to the CD56 (neural cell adhesion molecule) antigen found on cells of neuroendocrine origin, including small cell lung tumor, which can be used in the conjugate to target cells of neuroendocrine origin . The J5, MY9 and B4 antibodies are preferably resurfaced or humanized prior to use as part of the conjugate. The resurfacing or humanization of antibodies is described in, for example, Roguska et al., Proc. nati. Acad. Sci. USA, 91: 969.73(1994).

In addition, the C242 monoclonal antibody binds to the CanAg antigen (see, for example, US Patent 5,552,293), and can be used to target CanAg conjugates that are expressed in tumors, such as colon rectal, pancreatic, cell lung cancers. not small, and pancreatic. HuC242 is a humanized form of the C242 monoclonal antibody (see, for example, US Patent No. 5,552,293). Whose hybridoma HuC242 is deposited under the ECACC Identification Number 900112601. HuC242 can be prepared using CDR grafting methodology (see, for example, US Patent Nos. 5,585,089, 5,693,761, and 5,693,762) or resurfacing technology (see, for example, US Patent No. 5,639,641). HuC242 can be used to conjugate tumor cells expressing the CanAg antigen, such as, for example, colonectal, pancreatic, non-small cell lung, and gastric cancer cells.

To terminate in ovarian cancer and prostate cancer cells, an anti-MUC1 antibody can be used as the cell-binding agent in the conjugate. Anti-MUC1 antibodies include, for example, anti-HMFG-2 (see, for example, Taylor-Papadimitriou et al., Int. J. Cancer, 28: 17-21 (1981)), hCTM01 (see, for example, van Hof et al., Cancer Res., 56: 5179-5185)), and D56. Prostate cancer cells can also be targeted with the conjugate using a prostate-specific anti-membrane antigen (PSMA) as the cell-binding agent, such as J591 (see, for example, Liu et al., Cancer Res. 57: 3629 -3634 81997)). In addition, cancer cells that express the Her2 antigen, such as breast, prostate and ovarian cancer cells, can be attacked using the antibody trastuzumab. Anti-IGF-IR antibodies that bind to 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 rituzumab (see, for example, Patents 5,639,641 and 5,665,357, US Provisional Patent Application No. 60 /242,332 (which is related to Patent Application Publication No. 2005/0118183 A1), International Patent Application WO 02/16401, Pedersen et al., supra, Roguska et al., supra, Liu et al., supra, Nadler et al., supra, Colomer et al., Cancer Invest., 19:49 -56 (2001), Heider et al., Eur. J. Cancer, 31-:2385-2391 (1995), Welt et al., J. Clin. OncoL, 12:1193-1203 (1994), and Maloney et al., Blood, 90:2188-2195 (1997)). More preferably, the antibody is the humanized monoclonal antibody huN901 or the humanized monoclonal antibody huMy9-6. Other preferred antibodies include CNTO95, huDS6, huB4, and huC242. Other humanized monoclonal antibodies are known in the art and may be used in connection with the invention.

While the cell binding agent is preferably an antibody, the cell binding agent can also be a different antibody molecule. Suitable non-antibody molecules include, for example, interferons (for example, interferon alpha, beta, or gamma), lymphokines (for example, interleukin 2 (IL-2), IL-3, IL-4, or IL-6) , hormones (for example, insulin), growth factors (for example, EGF, TGF-alpha, FGF, and VEGF), colony-stimulating factors (for example, G-CSF, M-CSF, and GM-CSF (see, eg, Burgess, Immunology Today, 5:155-158 (1984)) somatostatin, and transferrin (see, eg, O'Keefe et al., J. Biol. Chem., 260: 932-937 (1985)). For example, GM-CSF, which binds to myeloid cells, can be used as a cell-binding agent to target acute myelogenous leukemia cells. In addition, IL-2, which binds to cells

Activated T cells can be used for prevention of transplant graft rejection, for therapy and prevention of graft-versus-host diseases, and for treatment of acute T-cell leukemia. Epidermal growth factor (EGF) can be used for squamous cell cancers such as lung cancer and head and neck cancer. Somatostatin can be used to target neuroblastoma cells and other types of tumor cells.

The conjugate may comprise any suitable drug, typically a cytotoxic agent. A "cytotoxic agent," as used herein, refers to any compound that results in the death of a cell, induces cell death, or decreases cell viability. Suitable cytotoxic agents include, for example, maytansinoids and maytansinoid analogs, taxoids, CC-1065 and CC-1065 analogs, and dolastatin and dolastatin analogs. In a preferred embodiment of the invention, the cytotoxic agent is a maytansinoid, including maytansinol and maytansinol analogues. Maytansinols are compounds that inhibit microtubule formation and are highly toxic to mammalian cells. Examples of suitable maytansinol analogues include those that have a modified aromatic ring and those that have modifications at other positions. Said maytansinoids are described in, for example, US Pat. 4,256,746, 4,294,757, 4,307,016, 4313,946, 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, y 6,333,410.

Examples of analogues of maytansinoids having a modified aromatic ring include: (1) C19 dechloro (US Patent No. 4,256,746) (prepared by LAH reduction of ansamitosin P2), (C-20-hydroxy (or C- 20-desmethyl) +/- C-19-dechloro (US Patent Nos. 4,361,650 and 4,307, Streptomyces or Actinomyces 016) (prepared by demethylation using or dechlorination using LAH), and (3) C-20-desmethoxy¡, C- 20-acyloxy (-OCOR), +/- dechloro (US Patent No. 4,294,757) (prepared by acylation using acyl chlorides).

Examples of maytansinol analogues having modifications at positions other than an aromatic ring include: (1) C-9-SH (US Patent No. 4,424,219) (prepared by the reaction of maytansinol with H₂S or P2S5), (2) C-14-alkoxymethyl (desmethoxy/CH2OR) (US Patent No. 4,331,598), (3) C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAr) (US Patent No. 4,450,254) (prepared from Nocardiá), (4) C-15-hydroxy/acyloxy (US Patent No. 4,364,866) (prepared by the conversion of maytansinol by Streptomyces), ( 5) C-15-methoxy¡ (US Patent No. 4,313,946) (isolated from Trevia mediflora), (6) C-18-N-desmethyl (US Patent Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces), and (7) 4,5-deoxy (US Pat. 4,371,533) (prepared by reduction of maytansinol with titanium trichloride/LAH).

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

<img file="ECSP088212A_D0001.tif" />

C1 α>

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

<img file="ECSP088212A_D0002.tif" />

Other maytansines may be used in the context of the invention, including, for example, thiol- and dffllide-containing maytansinoids that possess a mono- or di-alkyl 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 a group acyl possessing an interrupted sulfhydryl group, wherein the carbon atom of the acyl group possessing thiol functionality has one or two substituents, said substituents being CH₃, C₂h₅, linear or branched alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or aromatic heterocyclic or heterocycloalkyl radical, and wherein additionally one of substituents may be H, and wherein the acyl group has a straight chain extension of at least three carbon atoms between the carbonite functionality and the sulfur atom.

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

<img file="ECSP088212A_D0003.tif" />

where Y' represents (CR7R8)i(CRg=CRio)pC=CqA_r(CR5Rg)_mD._LI(CR₁₁=CR₁2)r(C=C)_yesB._he(CR3R4)_laughed-CR₁R2SZ, where Rj and R₂ are each independently CH₃, C₂h₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or aromatic heterocyclic radical or heterocycloalkyl, and where R₂ it can also be H, where A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simple or substituted aryl, or aromatic heterocyclic or heterocycloalkyl radical, where R₃,R₄,R₅,R₆,R₇,R₈,R_g,R_{1 B} and R₁₂ are each independently H, CH₃, C₂h₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or aromatic heterocyclic radical, or heterocycloalkyl, where I, m, n , o, p, q, r, s, and t are each independently zero or an integer from 1 to 5, provided that at least two of I, m, n, o, p, q, r, s, and t are not simultaneously zero, and where Z is H, SR, or COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or linear alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or aromatic heterocyclic or heterocycloalkyl radical.

Preferred embodiments of Formula (III) include compounds of Formula (III) wherein (a) Ri is H, R₂ is methyl and Z is H, (b) Ri and R₂ are methyl and Z is H, (c) R₁ is H,R₂ is methyl, and Z is -SCH₃, and (d) R! and R₂ are methyl, and Z is -SCH₃.

Said additional maytansines also include compounds represented by Formulas (IV-L), (IV-D), or (IV-D,L)

<img file="ECSP088212A_D0004.tif" />

EITHER

<img file="ECSP088212A_D0005.tif" />

<img file="ECSP088212A_D0006.tif" />

EITHER

<img file="ECSP088212A_D0007.tif" />

or where Y represents (CRyRgMCRsReMCRsRACR^SZ, where Ri and R₂ are each independently CH₃, C₂h₅, linear alkyl, or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or aromatic heterocyclic or heterocycloalkyl radical, and where R₂ can also be H, where R₃,R₄,R₅,R₆,R₇ and R₈ are each independently H, CH₃, C₂h₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or aromatic heterocyclic or heterocycloalkyl radical, where I, m, yn are each independently an integer from to 1 5, and further n can be zero, where Z is H, SR, or COR where R is linear or branched alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or aromatic heterocyclic radical or heterocycloalkyl, and where May represents a maytansinoid having the side chain at C-3, C-14 hydroxymethyl, C -15 hydroxy, or C20 demethyl.

Preferred embodiments of Formulas (IV-L), (IV-D) and (IV-DL) include compounds of (IV-L), (IV-D) and (IV-D,L) wherein (a) R1 is H, R2 is methyl, R5, R6, R7, and R8 are each Η, I and m are each 1, n is 0, and Z is H, (b) R1 and R2 are methyl, R5, R6, R7 , and R8 are each Η, I and m are 1, n is 0, and Z is H, (c) R1 is H, R2 is methyl, R5, R6, R7, and R8 are each Η, I and m are 1 , n is 0, and Z is -SCH3, or (d) R1 and R2 are methyl, R5, R6, R7, and R8 are each Η, I and m are 1, n is 0, and Z is -SCH3.

Preferably, the cytotoxic agent is represented by Formula (IV-L).

The additional maytansines

Formula (V):

Preferred compounds also include compounds represented by the where Y represents (CRyRgMCRsRgMCR^nCR-^SZ, where Ri and R₂ are each independently CH₃, C₂h₅, linear alkyl, or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or aromatic heterocyclic or heterocycloalkyl radical, and where R₂ can also be H, where R₃,R₄,R₅,R₆,R₇ and R₈ are each independently H, CH₃, C₂h₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or aromatic heterocyclic or heterocycloalkyl radical, where I, m, yn are each independently an integer from a 1 to 5, and further n can be zero, where Z is H, SR, or COR where R is linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or aromatic heterocyclic or heterocycloalkyl radical.

Preferred embodiments of Formula (V) include compounds of Formula (V) wherein (a) Ri is H, R₂ is methyl, R₅,R₆,R₇, and Rs are each Η, I and m are each 1, n is 0, and Z is H, (b) Ri and R₂are methyl, R5, R6, R7, and R8 are each Η, I and m are 1, n is 0, and Z is H, (c) R1 is H, R2 is methyl, R5, R6, R7, and R8 are Η, I and m are each 1, n is 0, and Z is -SCH3, or (d) R1 and R2 are methyl, R5, R6, R7, R8 are each Η, I and m are 1, n is 0, and Z is -SCH3.

Still further preferred maytansines include compounds represented by Formulas (Vl-L), (Vl-D), or (VI-D,L):

<img file="ECSP088212A_D0008.tif" />

(VI-L) (VLD)

<img file="ECSP088212A_D0009.tif" />

where and₂ represents (CR₇R.₈)i(CR₅R.₆)_m(CR₃R.₄)_noCR₁R.₂SZ, where Rt and R₂ are each independently CH₃, C₂h₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or aromatic heterocyclic or heterocycloalkyl radical, and wherein R₂ can also be H, where R₃,R₄,R₅,R₆,R₇ and R₈ are each independently H, CH₃, C₂h₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or aromatic heterocyclic or heterocycloalkyl radical, where I, m, yn are each independently an integer from a 1 to 5, and also n can be zero, where Z₂ is SR, or COR where R is linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or aromatic heterocyclic or heterocycloalkyl radical , and where May is a maytansinoid.

Additional preferred maytansines include compounds represented by Formula (Vil)

<img file="ECSP088212A_D0010.tif" />

(VII), where Y2' represents (CR7R8)i(CR₉=CR₁o)p(C=C)qA_r(CR5R6)mD_L.J.(CRi₁=CR₁₂)_r(C=C)_yesBt(CR3R)₄)n-CR₁R.₂SZ, where Ri and R₂ are each independently CH₃, C₂h₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or aromatic heterocyclic radical or heterocycloalkyl, and where R2 can also be H, in where A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simple or substituted aryl, or aromatic heterocyclic or heterocycloalkyl radical, where R₃,R₄,R₅,R₆,R₇,R₈,R₉, R n , and R_i2 are each independently H, CH₃, C₂h₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic or branched alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or aromatic heterocyclic radical, or heterocycloalkyl, where I, m, n , o, p, q, r, s, and t are each independently zero or an integer from 1 to 5, provided that at least two of I, m, n, o, p, q, r, s, and t are not zero at the same time, and where Z2 is H,

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

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

In addition to maytansinoids, the cytotoxic agent used in the conjugate can be a taxane or a derivative thereof. Taxanes are a family of compounds that includes paclitaxel (Taxol®, a natural cytotoxic product, and docetaxel (Taxotere®), a semi-synthetic derivative, both of which are widely used in the treatment of cancer. Taxanes are mitotic needle-like poisons that inhibit tubulin depolymerization, resulting in cell death. While docetaxel and paclitaxel are useful agents in the treatment of cancer, their anti-tumor activity is limited because of their non-specific toxicity towards normal cells. Additionally, compounds such as paclitaxel and docetaxel by themselves are not potent enough to be used in cell-binding agent conjugates.

A preferred taxane for use in preparing a cytotoxic conjugate is the taxane of Formula (VIII):

<img file="ECSP088212A_D0011.tif" />

(HIV)

Methods for synthesizing taxanes that can be used in the context of the invention, along with methods for conjugating taxanes to cell-binding agents such as antibodies, are described in detail in US Patent Nos. 5,416,064, 5,475,092, 6,340,701, 6,372,738, 6,436,931, 6,596,757, 6,706,708, and 6,716,821, and US Patent Application Publication No. 2004/0024049 A1.

The cytotoxic can also be CC-1065 or a derivative thereof. CC-1065 is a potent anti-tumor antibiotic isolated from the culture broth of Streptomyces zelensis. CC-1065 is approximately 1000 times more potent in vitro than commonly used anti-cancer drugs, such as doxorubicin, methotrexate, and vincristine (Bhuyan et al., Cancer Res. 42: 35323537 (1982)). CC-1065 and its analogs are described in US Patent Nos. 5,585,499, 5,8846,545, 6,340,701, and 6,372,738. The cytotoxic potency of CC-1065 has been correlated with its alkylating activity and its DNA-binding or DNA-intercalating activity. These two activities reside in separate parts of the molecule. In this regard, the alkylating activity is contained in the cyclopropapyrroloindole (CPI) subunit and the binding activity resides in the two pyrroloindole subunits of CC1065.

Various analogs of CC-1065 are known in the art and can also be used as the cytotoxic agent in the conjugate (see, for example, Wapehoski et al., J. Med. Chem. 31: 590-603 (1988)). A series of analogs of CC-1065 have been developed, in which the CPI portion is replaced by a cyclopropabencindole (CBI) portion (Boger et al., J. Org. Chem. 55: 5823-5833 (1990), and Boger et al. collaborators, Bioorg. Med. Lett., 1: 115-120 (1991)). These CC-1065 analogs maintain the high in vitro potency of the parent drug, without causing delayed toxicity in mice. Like CC-1065, these compounds are alkylating agents to covalently bind to the DNA minor stretch to cause cell death.

The therapeutic efficiency of CC-1065 analogues can be greatly improved by changing the in vivo distribution through targeted delivery to a tumor site, resulting in lower non-target tissue toxicity, and thus, lower systemic toxicity. To this end, conjugates of CC-1065 analogs and derivatives have been generated with cell-binding agents that specifically target tumor cells (see, for example, US Pat. 5,475,092, 5,585,499, and 5,846,545).

These conjugates typically exhibit highly targeted, specific cytotoxicity in vitro, and anti-tumor activity in human tumor xenograft models (see eg, Chari et al., Cancer Res., 55: 4079-4084 (1995)).

Methods for synthesizing CC-1065 analogs are described in detail in US Patent Nos. 5,475,092, 5,585,499, 5,846,545, 6,534,660, 6,585,618, and 6,756,397 and Patent Application Publication No. 2003/0195365 A1.

Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin, tubulisin and tubulisin analogues, duocarmycin and duocarmycin analogues, dolostatin and dolostatin analogues may also be used in the context of the invention . Doxarubicin and daunorubicin compounds (see, for example, US Patent No. 6,630,579) can also be used as the drug.

Conjugated drugs can be prepared by in vitro methods. In order to link a drug or prodrug to the antibody, a linker group is used. Suitable linking groups are well known in the art and include disulfide groups, labile acid groups, photolabile groups, labile peptidase groups, and labile esterase 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. Drug molecules can also be linked to a cell binding agent through an intermediate carrier molecule such as serum albumin.

In accordance with the invention, the cell binding agent is modified by reacting a bifunctional crosslinking reagent with the cell binding agent, thus resulting in the covalent attachment of a linker molecule to the cell binding agent. 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 portion of the linker portion is provided by the drug. In this regard, the drug comprises a linker portion that is part of a larger linker molecule that is used to join the cell-binding agent to the drug. For example, to form the maytansinoid DM1, the side chain on the hydroxyl group at C-3 of maytansine is modified to have a free sulfhydryl (SH) group. This thiolate form of maytansine can react with a modified cell-binding agent to form a conjugate. Consequently, the final linkage is assembled from two components, one of which is provided by the crosslinking reagent, while the other is provided by the DM1 side chain.

Any suitable bifunctional cross-linking reagent may be used in connection with the invention, provided that the linker reagent is provided for retention of therapeutic characteristics, eg cytotoxicity, and objectivity of the drug and cell-binding agent, respectively.

Preferably, the linker molecule links the drug to the cell-binding agent via chemical linkers (as described above), such that the drug and the cell-binding agent are chemically coupled (eg, covalently linked) to each other.

Preferably, the linker reagent is a fragmentable linker. More preferably, the linker is cleaved under mild conditions, ie conditions in a cell under which drug activity is not affected. Examples of suitable cleavable linkers include disulfide bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds. Disulfide-containing bonds are breakable bonds through disulfide exchange, which can take place under physiological conditions. Acid labile bonds are breakable bonds 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 bonds. Photolabile bonds are useful on the body surface and in many body cavities that are accessible to light. In addition, infrared rays can penetrate tissue. Leaving peptidase bonds can be used to cleave certain peptides in inner and outer cells (see for example, Trouet et al., Proc. Nati. Acad. Sci. USA, 79: 626-629 81982), and Umemoto et al., Int. J Cancer, 43: 677-684 (1989)).

Preferably, the drug is linked to a cell binding agent through a disulfide bond. The linker molecule comprises a reactive chemical group that can react with the cell-binding agent. Preferred reactive chemical groups for reaction with the cell binding agent are N-succimidyl esters and N-sulfosuccinimidyl esters. Additionally the linker molecule comprises a reactive chemical group, preferably a dithiopyridyl group, which can react with the drug to form a disulfide bond. Particularly preferred binding molecules include, for example, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (see, for example, Carlsson et al., Biochem. J., 173: 723-737 (1978)), 4 - N-succinimidyl (2-pyridyldithio)butanoate (SPDB) (see, for example, US Pat. 4,563,304), N-succinimidyl 4-(2-pyridyldithiojpentanoate (SPP) (see, for example, CAS Registry Number 341498-08-6), and other reactive crosslinkers described in US Patent No. 6,913,748, which is incorporated herein in its entirety by reference.

Although fragmentable linkers are preferably used in the method of the invention, a non-fragmentable linker can also be used to generate the above-described conjugate. A non-fragmentable linker is any chemical moiety that is capable of linking a drug, such as a maytansinoid, a taxane, or an analog of CC-1065, to a cell-binding agent in a stable covalent manner. Consequently, non-fragmentable linkers are substantially resistant to acid-induced fragmentation. Light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions under which the drug or cell-binding agent remains active.

Suitable cross-linking reagents that form non-fragmentable linkers between a drug and a fractible linker agent are well known in the art. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or succinimidyl ester moiety for reaction with the cell binding agent, as well as a maleimido- or haloacetyl-based moiety for drug reaction. Crosslinking reagents comprising a maleimido-based moiety include N-succinimidyl 4-(maleimidomethyl-cyclohexanecarboxylate (SMCC), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxy-1-carboxy-(6-amidocaproate) , which is a “long chain” analogue of SMCC (LC-SMCC), kmaleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N. (a-male¡m¡midoacetox¡)-succin¡m¡de ester (AMAS), 6-(βmaleimidopropionamidojhexanoate succinimidyl (SMPH), 4-(p- N-succinimidyl-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl) isocyanate (PMPI).Crosslinking reagents comprising a haloacetyl-based moiety include N-succinimidyl 4-(iodoacetyl)-amlnobenzoate (SIAB), N-iodoacetate - succinimidyl (SIA), N-succinimidyl bromoacetate (SBA), and

N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

Other crosslinking reagents lacking a sulfur atom that forms non-fragmentable linkers may also be used in the method of the invention. Said linkers can be derived from dlcarboxylic acid based moieties. Suitable dicarboxylic acid-based moieties include, but are not limited to, α,ω-dicarboxylic acids of General Formula (IX):

HCOOC-X_rAND_no-Z_m-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 having 3 to 10 carbon atoms, Z is an aromatic group substituted or unsubstituted having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group wherein the heteroatom is selected from N, O, or S, and wherein I, m, and n are each 0 or 1, to condition that I, m, n are not all zero at the same time.

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

Alternatively, as described in US Patent No. 6,441,163, B1, the drug may first be modified to introduce a suitable reactive ester to react with a cell-binding agent. Reaction of these maytansinoids containing a suitable binding moiety with a cell binding agent provides another method of producing a cleavable or non-cleavable maytansinoid-cell binding agent conjugate.

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

The following examples further illustrate the invention but are of course not construed as limiting its scope in any way.

EXAMPLE 1

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

The monoclonal antibody huN901 (8 mg/mL final concentration) was incubated with N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP, 5.6-fold molar excess) for approximately 180 min at 20 °C in 50 mM buffer. potassium phosphate (pH 7.5) containing 50 mM NaCI, 2 mM EDTA, and 5% ethanol. In a first set, 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 NaCI and 2 mM EDTA. In a second set, 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 NaCI (pH 6.5 ), and 2 mM EDTA using a 10,000 molecular weight cutoff membrane (UltracelTM 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 steps. Antibody to linker ratios were also determined by treatment with dithiothreitol to release pyridine2-thione, which had an extinction coefficient of 8.080 ΜΊ cm'.¹ at 343nM. Drug/antibody ratios were determined spectrophotometrically (280 nm and 252 nm wavelengths) for the conjugation step. In addition, the removal of small molecular species related to SPP was measured by HPLC Hisep.

The resulting data is shown in Table 1.

Table 1. Purification Methods for Modified huN901 Using G-25F versus TFF

<td colspan="3">Resin</td><td>you</td>

<td></td><td></td><td>Sephadex^you</td><td>F</td>

<td></td><td></td><td>^m G25F</td><td>F</td>

<td>Stage of</td><td>Stage performance (%)</td><td> 94</td><td> 98</td>

<td>Modification</td><td></td><td></td><td></td>

<td></td><td>Connect/Ratio</td><td> 4.9</td><td> 4.</td>

<td></td><td>Antibody</td><td></td><td> 9</td>

<td></td><td>small molecules</td><td> 0.2</td><td> 0.</td>

<td></td><td>related to SPP(%)</td><td></td><td> 2</td>

<td>Stage of</td><td>Drug Ratio/</td><td> 3.7</td><td> 3.</td>

<td>Conjugation</td><td>Antibody</td><td></td><td> 7</td>

As shown in Table 1, the use of TFF produced the drug conjugate product of at least equivalent quality to the non-adsorbent chromatography (G25) process, while being more convenient and transportable to scale.

EXAMPLE 2

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

The huB4 antibody was modified with N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB, 5.4-fold molar excess) for 120 min at room temperature in 50 mM potassium phosphate buffer (pH. 6.5) containing 50 mM NaCI, 2 mM EDTA, and 5% ethanol. In a first group, the reaction mixture was purified using Sephadex™ G25F resin as described in Example 1. In a second group, the reaction mixture was loaded onto a ceramic hydroxyapatite (CHT) column, Bio-Rad Laboratories, Hercules, CA), which was equilibrated in 12.5 mM potassium phosphate buffer (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 yield of 91% and a linker/antibody ratio of 4.2. The second group had a yield of 89% and a linker/antibody ratio of 4.2.

The CNTO95 antibody (final concentration of 10 mg/mL) was modified with 4-(N-succinimidyl 2-pyridylthiojbutanoate (SPDB, 4.5-fold molar excess) for 120 min at 20 °C in 10 mM sodium phosphate buffer (pH of 7.5) containing 2.5% sucrose and 5% ethanol.In a first group the reaction mixture was purified using SephadexTM G25F resin in 12.5 mM sodium phosphate buffer (pH 6.5) containing 12.5 mM NaCl and 0.5mM EDTA. In a second set, 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 50 mM potassium phosphate buffer (pH 7.5), containing 50 mM NaCI.

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

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

EXAMPLE 3

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

In a first experiment, the CNTO95 antibody was modified and purified as described in Example 2. The modified antibody was then divided into two groups. In the first group, conjugation was performed in 12.5 mM pH 6.5 potassium phosphate buffer containing 12.5 mM NaCI, 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 is shown in Table 2.

Table 2. Drug/Antibody Ratio in Conjugation Reaction pH 6.5 versus 7.5.

<td>Time</td><td>Proportion of</td><td>Proportion of</td>

<td>of</td><td>drug/antibody at pH</td><td>drug/antibody at pH</td>

<td>reaction</td><td>conjugation of 6.5</td><td>conjugation of 7.5</td>

<td>(hours)</td><td></td><td></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 shown by the data set forth in Table 2, conjugation proceeds 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 min 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 equivalently purified except that the chromatography buffer was adjusted to pH 7.5. Both samples were conjugated with DM4 (1.7-fold molar excess over the bound linker) for 18 hours at room temperature in a final dimethylacetamide (DMA) concentration of 3%.

Therefore, sample (a) was conjugated at pH 6.5, and sample (b) was conjugated at pH 7.5. 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 free drug release analysis at intervals. The resulting data is shown in Table 3.

Table 3: Free Drug Release over Time of Conjugated Samples at pH of

6.5 and 7.5.

<td>Time</td><td>conjugation pH of 6.5</td><td>conjugation pH of 7.5</td>

<td>(months)</td><td></td><td></td>

<td> 0.5</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 shown by the data set forth in Table 3, the release of free drug is substantially slower than sample (b) that had been conjugated at pH 7.5 relative to sample (a) that had been conjugated at pH 7.5. 6.5. Accordingly, the drug conjugate product prepared at pH. Of 7.5 was shown to be stable with respect to free drug release over time as compared to the conjugated drug product prepared at pH 6.5. Conjugation at pH 7.5 also showed better drug incorporation than pH 6.5 thus requiring the use of less drug.

EXAMPLE 4

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

The huN901 monoclonal antibody (8 mg/mL final concentration) was incubated with N-succinimidyl 4-(2-pyridyldithiojpentanoate (SPP, 5.6-fold molar excess) for approximately 180 minutes at 20 °C in 50 mM potassium phosphate buffer ( pH of 7.5) containing 50 mM of

NaCI, 2 mM EDTA, and 5% ethanol. In a first set, the reaction mixture was purified using a Sephadex™ G25F resin column equilibrated and eluted in 50 mM sodium citrate buffer (pH 5.0) containing 50 mM NaCI and 2 mM EDTA. In a second set 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. Both samples were conjugated with DM4 (1.7-fold molar excess over the linked linker) for 3, 19, 25, 48, and 120 hours at room temperature in a final dimethylacetamide (DMA) concentration of 3%.

Therefore, the first set of samples was conjugated in 50 mM sodium citrate buffer (pH 5.0) containing 50 mM NaCI and 2 mM EDTA, and the second set of samples was conjugated in 50 mM sodium citrate buffer (pH 5.0). sodium phosphate (pH 6.5), containing 50 mM NaCI and 2 mM EDTA. Samples were then purified using a Sephadex™ G25F resin column equilibrated and 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 had an extinction coefficient of 8.00 M'¹ cm'¹ at 343nM. Drug/antibody ratios (280 nm and 252 nm inda lengths) for the conjugation step were determined spectrophotometrically.

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 in huN901 modified with SPP as a

Conjugation pH Function

<td>Reaction time</td><td>Drug/Antibody Molar Ratio</td>

<td>(hours)</td><td>conjugation pH conjugation pH 5.0 6.5</td>

<td> 3</td><td> 2.43 2.97</td>

<td> 19</td><td> 3.38 3.28</td>

<td> 25</td><td>3.41 NOT</td>

Table 4 (Continued)

<td>Reaction time</td><td>Drug/Antibody Molar Ratio</td>

<td>(hours)</td><td>conjugation pH conjugation pH 5.0 6.5</td>

<td> 48</td><td> 3.46 3.17</td>

<td> 120</td><td> 3.44 2.85</td>

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

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

Table 5. Effect of Conjugation pH on Conjugated Monomer Level During Conjugation of SPP-modified huN901 with DM1.

Time of

Conjugated monomer (%) reaction conjugation pH conjugation pH (hours)

<img file="ECSP088212A_D0012.tif" />

6.5

98.0

98.2

NT

98.3

97.8

As is obvious from the data set forth in Table 5, the conjugate that is made by conjugating the drug-modified antibody at pH 5.0 had a higher level of conjugated monomer than the conjugate made at a conjugation pH of 6.5.

EXAMPLE 5

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

The BIWA 4 antibody was modified with SPP (molar excess of SPP as shown in Table 6) for 120-140 min at room temperature in 50 mM potassium phosphate buffer (pH 6.5), 50 mM NaCI, 2 mM EDTA, and 5% ethanol. Aliquots of the modified antibody were purified on NAP25 separation columns equilibrated in buffers having various pH values ​​(pH 4.6 to 6.5). pH buffers from 4.6 to 5.9 consisted of 35 mM sodium citrate, 150 mM sodium chloride, and 2 mM EDTA. The pH buffer of 6.5 was PBS with 2 mM EDTA.

The modified antibody of each pH was conjugated to DM1 (1.7-fold molar excess over linker) in dimethylacetamide (DMA, 3% final concentration). After incubation for 17-18 hours at room temperature, samples of the conjugated antibody were purified by chromatography on NAP 25 columns equilibrated in PBS (pH 6.5). Linker/antibody ratios (L/A in Table 6) were determined by treatment with dithiothreitol to release pyridin-2-thione, which had a standard extinction coefficient of 8,000 M'.¹ cm'¹ at 343nM. Drug/antibody ratios were determined spectrophotometrically (280 nm and 252 nm wavelengths) for the conjugation step. Conjugated monomer, high molecular weight species, and low molecular weight species were determined by SEC _ HPLC using a TSKG3000WXL column equilibrated and grown in 0.2 M potassium phosphate buffer (pH. 7) containing 0.2 M potassium chloride. and 20% isopropanol.

The results of these analyzes are shown in Table 6.

Table 6. Characteristics of the Drug Conjugate Product in Relation to pH.

<td>TO</td><td>B.</td><td>C.</td><td>D.</td><td>AND</td><td>F</td><td>G.</td><td>h</td>

<td> 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> 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> 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> 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> 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>

A = pH of the regulator

B = Molar excess of SPP

C = L/A, ratio of connector/antibody

D = D/A, drug/antibody ratio

E = Monomer (%)

F = High molecular weight species (%)

G = Low molecular weight species (%)

H = Conjugation step yield (%)

The data set forth in Table 6 demonstrate that conjugation of SPP-modified BIWA to DM1 was efficient at pH less than 6.0, compared to pH 4.5. 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, levels of conjugated monomer, high molecular weight species, and low molecular weight species were more optimal, and yields were improved, at lower pH.

EXAMPLE 6

This example demonstrates that the step of purifying the modified antibody can optionally be eliminated. The drug can be added simultaneously with the bifunctional modifying reagent or some time later.

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 4.6-fold molar excess of SPDB over antibody for 120 minutes at 20 °C The modifying 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 SephadexTM G25F resin (standard four step process), equilibrated and eluted in 12.5 mM potassium phosphate buffer (pH 7.5) containing 12.5 mM NaCl, and was subsequently conjugated to DM4 (1.7-fold molar excess of drug over the ligated linker) at a final modified antibody concentration of 10 mg/ml in 12.5 mM potassium phosphate buffer (pH 7.5) containing 12.5 mM of NaCI 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 process), without being further purified.

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

Linker/antibody (L/A) ratios were determined by treatment with dithiothreitol to release pindin-2-thione, which had a standard extinction coefficient of 8.080 M'.¹ crn^-1at 343nM. Drug/antibody (D/A) ratios and yields were determined spectrophotometrically (wavelengths 280 nm and 252 nm) for the conjugation step. The monomer percentages were assayed by SEC-HPLC. Free drug percentages were assayed by HPLC on a Hisep column. The results of these analyzes are set forth in Table 7.

Table 7. Optimal Elimination of Purification Step for the Modified Antibody

<td>parameters</td><td>4 stage process</td><td>Process of stages</td>

<td>initial SPBD</td><td>4.6x</td><td>4.6x</td>

<td>THE</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>monomer (%)</td><td> 95.8</td><td> 96.1</td>

<td>Free drug (%)</td><td> 2.4</td><td> 1.1</td>

L/A, ratio of connector/antibody

D/A, drug/antibody ratio

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

EXAMPLE 7

This example demonstrates an improved means of purifying antibody that has been modified with a bifunctional modifying reagent and then conjugated to a maitansionide. The antibody huN901 modified with SPP (7-fold molar excess) and purified on SephadexTM G25F resin, as described in Example 1, was conjugated with the maytansinoid DM1 (1.7-fold molar excess on the linker, dissolved in dimethylacetamide (DMA). , final concentration of 3%).

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

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

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

Conjugated monomer was determined by SEC-HPCL using a TSKG3000SW resin column._XL equilibrated and grown in 0.2 M potassium phosphate buffer at pH 7.0, containing 0.2 M 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 289 nm).

The results of these analyzes are set forth in Table 8.

Table 8: Comparison of Conjugation Purification Stage

<td>sample of</td><td>Stage of</td><td>monomer</td><td>Performance</td>

<td>conjugate</td><td>purification</td><td>conjugate (%)</td><td>of the stage</td>

<td></td><td>conjugation</td><td></td><td> (%)</td>

<td>1 (checking)</td><td>resin G25F</td><td> 93.2</td><td> 85</td>

<td>2 (invention)</td><td>T.F.F.</td><td> 92.8</td><td> 85</td>

<td>3 (invention)</td><td>MEP resin</td><td> 94.5</td><td> 74</td>

<td></td><td>hypercell</td><td></td><td></td>

<td>4 (invention)</td><td>Resin</td><td> 96.3</td><td> 81</td>

<td></td><td>UNosphere</td><td></td><td></td>

<td>5 (invention)</td><td>CHT resin</td><td> 97.9</td><td> 72</td>

<td>6 (invention)</td><td>SP resin</td><td> 95.1</td><td> 81</td>

<td></td><td>sepharose</td><td></td><td></td>

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

In addition to CHT (hydroxyapatite ceramic), CFT (fluoroapatite ceramic) can also be used under similar chromatographic conditions. Alternatively both the CHT and CFT resins can be used in n-adsorbent mode so that the desired monomeric substantially conjugated product is not retained by the resins, while high molecular weight species are retained and thus separated from the desired product.

Although a standard buffer/solvent 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 any of the chromatographic steps described herein and provide other benefits over the standard process. For example, the 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 linker incorporated into the huB4 antibody was approximately 10% higher than for standard conditions. Furthermore, these conditions are more compatible with loading onto resins such as cation exchange resins and CHT.

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

The use of “un (before consonant) and “un” (before vowel) and “el, la” and similar referents in the context of describing the invention (especially in the context of the following claims) are construed to cover both the singular as well as the plural, unless otherwise indicated herein or clearly contraindicated by the context. The terms “comprising”, “having”, “including” and “containing” were construed as open-ended terms (i.e. meaning “including but not limited to”) unless otherwise indicated. manner. The citation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value that falls in the range, unless otherwise noted herein, and each separate value is incorporated into the specification as if individually cited herein. All of the methods described herein may be performed in any order unless otherwise stated herein or clearly contraindicated by the context. The use of any and all examples, or exemplary language (for example, "such as") provided herein, is intended merely to better illuminate the invention and does not present a limitation on the scope of the invention unless otherwise noted. claim differently. No language in the specification shall be construed as indicating any element not claimed as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations from the preferred embodiments may become obvious to those skilled in the art from reading the above description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend that the invention be practiced other than as specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is embraced by the invention unless otherwise indicated herein or clearly contraindicated by the context.

NOVELTY OF THE INVENTION

Having described the present invention, what is contained in the following is considered novel, and therefore is claimed as property: