NZ734179A - Calicheamicin derivative-carrier conjugates - Google Patents

Abstract

Disclosed are methods for preparing monomeric cytotoxic drug/carrier conjugates with a drug loading significantly higher than in previously reported procedures and with decreased aggregation and low conjugate fraction (LCF) are described. Cytotoxic drug derivative/antibody conjugates, compositions comprising the conjugates and uses of the conjugates are also described. Monomeric calicheamicin derivative/anti-CD22 antibody conjugates, compositions comprising the conjugates and uses of the conjugates are also described.

Description

CALICHEAMICIN DERIVATIVE-CARRIER CONJUGATES This is a divisional application of New Zealand patent application 716039, itself divided out of New Zealand patent application 626698, itself divided out of New Zealand patent application 611951, itself divided out of 597162, divided out of NZ application 586071, itself divided out of NZ application 573563, itself divided out of NZ application 555781, itself divided out of NZ application 536928, the contents of which are incorporated herein by reference in their entirety.

Field of the Invention The present invention relates to methods for the production of compositions comprising monomeric cytotoxic drug/carrier conjugates (the “conjugates”) with higher drug loading and substantially reduced low conjugate fraction (LCF). The invention also relates to compositions comprising anti-CD22 antibody-monomeric calichemicin conjugates. Also bed are conjugates, method(s) of purification of the conjugates, pharmaceutical compositions comprising the ates, and uses of the conjugates.

Background of the Invention Drug conjugates developed for systemic pharmacotherapy are target-specific cytotoxic agents. The concept involves coupling a therapeutic agent to a r molecule with specificity for a defined target cell population. Antibodies with high affinity for antigens are a natural choice as targeting moieties. With the availability of high affinity monoclonal antibodies, the prospects of dy-targeting therapeutics have become promising. Toxic nces that have been ated to onal dies include toxins, lecular-weight cytotoxic drugs, ical response modifiers, and radionuclides. Antibody-toxin conjugates are frequently termed immunotoxins, s immunoconjugates consisting of antibodies and low-molecular-weight drugs such as methothrexate and Adriamycin are called chemoimmunoconjugates. Immunomodulators contain biological se modifiers that are known to have regulatory functions such as lymphokines, growth factors, and ment-activating cobra venom factor (CVF).

Radioimmunoconjugates consist of radioactive es, which may be used as therapeutics to kill cells by their ion or used for imaging. Antibody-mediated specific delivery of cytotoxic drugs to tumor cells is expected to not only augment their anti-tumor cy, but also prevent nontargeted uptake by normal tissues, thus increasing their therapeutic indices Described are immunoconjugates comprising an antibody as a targeting vehicle and having specificity for antigenic determinants on the surface of malignant cells conjugated to a cytotoxic drug. Also described are cytotoxic drug-antibody conjugates, wherein the antibody has specificity for antigenic determinants on B-malignancies, lymphoproliferative disorders and chronic inflammatory diseases. Also bed are methods for producing conjugates and to their therapeutic use(s).

A number of antibody-based therapeutics for treating a variety of diseases including cancer and rheumatoid arthritis have been approved for clinical use or are in clinical trials for y of malignancies including B-cell malignancies such as Non-Hodgkin’s lymphoma. One such antibody based therapeutic is rituximab (Rituxan™), an unlabelled chimeric human g1 (+mg1V-region) antibody, which is ic for cell surface antigen CD20, which is expressed on B-cells. These dy based eutics rely either on complement-mediated cytotxicity (CDCC) or anibody-dependent cellular cytotoxicity (ADCC) against B cells, or on the use of radionuclides, such as 131I or 90Y, which have associated preparation and use ms for clinicians and patients. Consequently, there is a need for the generation of immunoconjugates which can overcome the omings of current antibody based therapeutics to treat a variety of malignancies including hematopoietic malignancies like non-Hodgkin’s lymphoma (NHL), which can be produced easily and efficiently, and which can be used repeatedly without inducing an immune response.

Immunoconjugates comprising a member of the potent family of antibacterial and antitumor agents, known tively as the calicheamicins or the LL-E33288 complex, (see U.S. Patent No. 4,970,198 (1990)), was ped for use in the treatment of myelomas. The most potent of the calicheamicins is designated γ1, which is herein referenced simply as gamma. These compounds contain a methyltrisulfide that can be reacted with appropriate thiols to form disulfides, at the same time introducing a onal group such as a hydrazide or other functional group that is useful in attaching a calicheamicin derivative to a carrier. (See U.S. Patent No. 5,053,394). The use of the monomeric calicheamicin derivative/carrier conjugates in developing therapies for a wide variety of cancers has been limited both by the availability of specific targeting agents (carriers) as well as the conjugation methodologies which result in the formation of protein aggregates when the amount of the eamicin tive that is conjugated to the carrier (i.e., the drug loading) is increased. Since higher drug loading increases the nt potency of the ate, it is desirable to have as much drug loaded on the carrier as is consistent with retaining the affinity of the carrier protein. The presence of aggregated protein, which may be nonspecifically toxic and immunogenic, and therefore must be removed for therapeutic applications, makes the scale-up process for the production of these conjugates more difficult and decreases the yield of the products.

The amount of calicheamicin loaded on the carrier protein (the drug loading), the amount of aggregate that is formed in the conjugation reaction, and the yield of final purified monomeric conjugate that can be obtained are all related. A compromise must therefore be made between higher drug g and the yield of the final monomer by adjusting the amount of the reactive calicheamicin derivative that is added to the ation reaction.

The tendency for cytotoxic drug conjugates, especially calicheamicin conjugates to aggregate is especially problematic when the conjugation reactions are performed with the s described in U.S. Patent No. 296 and U.S. Patent No. 5,773,001, which are incorporated herein in their entirety. In this case, a large percentage of the conjugates produced are in an aggregated form, and it is quite difficult to purify conjugates made by these original processes (CMA-676 process) for therapeutic use. For some carrier ns, conjugates with even modest loadings are virtually impossible to make except on a small scale. Consequently, there is a critical need to improve methods for conjugating xic drugs, such as the calicheamicins, to carriers which minimize the amount of aggregation and thereby allow for a higher drug loading as possible with a reasonable yield of product.

Previously, conjugation methods for preparing monomeric calicheamicin derivative/carrier with higher drug loading/yield and decreased aggregation were disclosed (see U.S. Patent No. 5,714,586 and U.S. Patent No. 5,712,374, incorporated herein in their entirety). gh these processes resulted in conjugate preparations with substantially reduced ate content, it was discovered later that it produced conjugates containing undesirably high levels (45-65% HPLC Area %) of a low conjugated fraction (LCF), a fraction consisting mostly of unconjugated antibody. The presence of the LCF in the product is an inefficient use of the antibody as it does not contain the cytotoxic drug. It may also compete with the calicheamicin-carrier conjugate for the target and ially reduce the tagetability of the latter resulting in reduced efficacy of the xic drug. Therefore, an improved conjugation process that would result in significantly lower levels of the LCF and have acceptable levels of aggregation, without significantly altering the physical properties of the molecule, is desirable.

Summary of the Invention In one aspect, the ion provides a method for the preparation of a stable lyophilized composition comprising monomeric eamicin tive/anti-CD22 antibody conjugates having the a, Pr(-X-W)m, wherein, Pr is an anti-CD22 antibody comprising SEQ ID NO: 1 for CDR-H1, residues 50-66 of SEQ ID NO: 27 for , SEQ ID NO: 3 for CDR-H3, SEQ ID NO: 4 for CDR-L1, SEQ ID NO: 5 for CDR-L2 and SEQ ID NO: 6 for CDR-L3; X is a hydrolyzable linker that that is capable of releasing the calicheamicin from the conjugates after binding and entry into target cells; W is a calicheamicin; m is the average g for a purified conjugation product such that the calicheamicin constitutes 4-10% of the conjugate by weight; and (-X-W)m is a calicheamicin derivative, the method comprising: (a) dissolving the monomeric calicheamicin derivative/anti-CD22 antibody conjugates to a final concentration 0.25 mg/mL in a solution comprising a cryoprotectant at a concentration of % by weight, electrolytes at a concentration of 0.01 M to 0.1 M, a solubility facilitating agent at a concentration of 0.005-0.05% by weight, buffering agent at a concentration of 5-50 mM such that the final pH of the solution is 7.8-8.2, and water; (b) dispensing the solution into vials; (c) freezing the solution at a ng temperature of -35ºC to -50ºC; (d) subjecting the solution to an initial freeze drying step at a primary drying pressure of 20 to 80 microns at a shelf-temperature at -10ºC to -40ºC for 24 to 78 hours, forming a freeze-dried product y; and (e) subjecting the freeze-dried product of step (d) to a secondary drying step at a drying pressure of 20 to 80 microns at a shelf temperature of +10ºC to +35ºC for at least 8 hours.

In another aspect, the ion provides a stable lyophilized formulation prepared by the method of the invention.

In the description in this specification reference may be made to subject matter which is not within the scope of the appended claims. That subject matter should be readily identifiable by a person skilled in the art and may assist in g into practice the invention as defined in the appended claims.

Also described are methods for the production of monomeric cytotoxic drug derivative/carrier conjugates (the “conjugates”) with higher g and substantially d low conjugate fraction (LCF). Also described are the production of monomeric calichemicin tive-carrier ates, conjugates produced by the method described, itions comprising the conjugates produced by the method described, method(s) of purification of the conjugates, and to use of the conjugates described. Particularly described is a method(s) for ing monomeric calichemicin derivative-anti-CD22 antibody conjugate (CMC-544).

In one embodiment, disclosed is an improved conjugation process for the production of monomeric cytotoxic drug derivative/carrier conjugates (the “conjugates”) that s in significantly lower levels of the LCF (below 10 percent) without any icant alteration of the physical or chemical properties of the molecule. Also disclosed is a further improvement to the conjugation process which s in not only a significant reduction in the levels of the LCF, but also results in a significant reduction in aggregation from previously disclosed processes, and produces substantially increased drug loading. The monomeric drug tive/carrier ates (the “conjugates”) described herein have the formula: Pr(-X-W)m wherein: Pr is a proteinaceous carrier, X is a linker that ses a product of any ve group that can react with a proteinaceous carrier, W is a cytotoxic drug; m is the average loading for a purified conjugation product such that the cytotoxic drug constitutes 7 - 9% of the conjugate by weight; and (-X-W)m is a cytotoxic drug derivative.

The monomeric drug derivative/carrier conjugates (the “conjugates”), in one embodiment, are generated by a method comprising the steps of: (1) adding the cytotoxic drug derivative to the proteinaceous carrier wherein the cytotoxic drug derivative is 4.5 - 11% by weight of the proteinaceous carrier; (2) incubating the xic drug derivative and a naceous carrier in a non-nucleophilic, protein-compatible, buffered solution having a pH in the range from about 7 to 9 to produce a monomeric cytotoxic drug/carrier conjugate, wherein solution further comprises (a) a suitable organic cosolvent, and (b) an additive comprising at least one C6-C18 carboxylic acid or its salt, and n the incubation is conducted at a temperature ranging from about 30ºC to about 35ºC for a period of time ranging from about 15 minutes to 24 hours; and(3) subjecting the conjugate produced in step (2) to a chromatographic tion s to separate monomeric cytotoxic drug derivative/ proteinaceous carrier conjugates with a loading in the range of 4 - 10 % by weight cytotoxic drug and with low ated fraction (LCF) below 10 percent from unconjugated proteinaceous carrier, cytotoxic drug derivative, and aggregated conjugates.

In one embodiment, the proteinaceous carrier of the conjugate is ed from a group consisting of hormones, growth factors, antibodies, antibody fragments, antibody mimics, and their cally or enzymatically engineered counterparts.

In a one embodiment, the proteinaceous carrier is an antibody. In a preferred embodiment, the antibody is selected from a group consisting of a monoclonal antibody, a chimeric dy, a human antibody, a zed antibody, a single chain antibody, a Fab fragment and a F(ab)2 fragment.

In another embodiment, the humanized antibody is directed against the cell surface antigen CD22.

In a preferred embodiment, the humanized anti-CD22 antibody is a CDR-grafted antibody, and comprises a light chain variable region 5/44-gL1 (SEQ ID NO:19), and a heavy chain variable region 5/44-gH7 (SEQ ID NO:27).

In another preferred ment, the humanized anti-CD22 antibody is a CDR-grafted antibody comprising a light chain having a ce set forth in SEQ ID NO: 28.

In yet another preferred embodiment, the humanized anti-CD22 antibody is a CDR- grafted antibody comprising a heavy chain having a sequence set forth in SEQ ID NO:30.

In another preferred embodiment, the humanized anti-CD22 antibody is a CDR-grafted antibody comprising a light chain having a sequence set forth in SEQ ID NO: 28 and a heavy chain having a sequence set forth in SEQ ID NO: 30.

In another embodiment, the humanized anti-CD22 antibody is a CDR-grafted antibody that is a variant antibody obtained by an affinity maturation protocol and has increased specificity for human CD22.

In another embodiment, the cytotoxic drug used to generate the monomeric cytotoxic drug/carrier conjugate is either an inhibitor of n polymerization, an alkylating agent that binds to and disrupts DNA, an inhibitor protein synthesis, an inhibitor of tyrosine In one embodiment, cytotoxic drug is selected from pa, s, vincristine, ubicin, doxorubicin, epirubicin, esperamicins, actinomycin, mycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins/auristatins, hemiasterlins and maytansinoids.

In a red embodiment, wherein the cytotoxic drug is calicheamicin, and is selected from gamma calicheamicin or N-acetyl gamma calicheamicin derivative.

In yet another embodiment, the cytotoxic drug is onalized with 3-mercapto methyl butanoyl hydrazide and conjugated to a proteinaceous carrier via a hydrolyzable linker that is e of releasing the cytotoxic drug from the conjugate after binding and entry into target cells.

In a preferred embodiment, the hydrolyzable linker is 4-(4-acetylphenoxy) butanoic acid (AcBut).

In yet another embodiment, octanoic acid or its salt, or decanoic acid ot its salt is used as an additive during the ation process to decrease aggregation and increase drug loading.

In yet another embodiment, the conjugates are purified by chromatographic separation process of step.

In one embodiment, the tographic tion process used to separate the monomeric drug derivative-carrier conjugate is size exclusion chromarography (SEC).

In another embodiment, the chromatographic separation process used to separate the ric drug derivative-carrier conjugate is HPLC, FPLC or Sephacryl S-200 chromatography.

In a preferred embodiment, the chromatographic separation process used to separate the monomeric drug tive-carrier conjugate is hydrophobic interaction chromatography (HIC). In a particularly preferred embodiment, HIC is carried out using Phenyl Sepharose 6 Fast Flow chromatographic medium, Butyl Sepharose 4 Fast Flow chromatographic , Octyl Sepharose 4 Fast Flow tographic medium, Toyopearl Ether-650M chromatographic medium, Macro-Prep methyl HIC medium or Macro-Prep t-Butyl HIC medium.

In a more particularly preferred embodiment, HIC is carried out using Butyl Sepharose 4 Fast Flow chromatographic medium.

Also bed is a monomeric cytotoxic drug derivative/carrier ate produced by the method described herein. In a red embodiment, the cytotoxic drug used is calicheamicin and the carrier used is an antibody.

In another preferred embodiment, the antibody is selected from a group consisting of a monoclonal dy, a chimeric antibody, a human antibody, a humanized antibody, a single chain antibody, a Fab fragment and a F(ab)2 fragment. In a more particularly preferred embodiment, a humanized antibody directed against the cell surface antigen, CD22 is used.

In one embodiment, the humanized anti-CD22 antibody is a CDR-grafted antibody, and comprises a light chain variable region 5/44-gL1 (SEQ ID NO:19), and a heavy chain variable region 5/44-gH7 (SEQ ID NO:27).

In another embodiment, the humanized anti-CD22 antibody is a CDR-grafted antibody comprising a light chain having a sequence set forth in SEQ ID NO: 28.

In a preferred embodiment, the humanized anti-CD22 antibody is a CDR-grafted comprising a heavy chain having a sequence set forth in SEQ ID NO:30.

In another preferred embodiment, the humanized anti-CD22 antibody is a afted antibody comprising a light chain having a sequence set forth in SEQ ID NO: 28 and a heavy chain having a sequence set forth in SEQ ID NO: 30.

In still another embodiment, the humanized anti-CD22 antibody is a CDR-grafted antibody that is a t antibody obtained by an affinity maturation protocol has increased specificity for human CD22.

In a red embodiment the cytotoxic drug derivative is gamma calicheamicin or an N- acetyl gamma calicheamicin.

In one embodiment, the calicheamicin tive is functionalized with 3-mercapto methyl butanoyl hydrazide.

In another embodiment, the linker used to ate the drug to the carrier is a hydrolyzable linker that is capable of releasing the xic drug from the conjugate after binding and entry into target cells. In a preferred embodiment, the hydrolyzable linker is cetylphenoxy) butanoic acid (AcBut).

Another embodiment is directed to a monomeric calicheamicin derivative/anti-CD22 antibody conjugate having the formula, Pr(-X-S-S-W)m wherein: Pr is an anti-CD22 antibody; X is a hydrolyzable linker that comprises a product of any reactive group that can react with an antibody; W is a calicheamicin l; m is the average loading for a purified ation product such that the calicheamicin constitutes 4 - 10% of the conjugate by weight; and (-X-S-S-W)m is a calicheamicin derivative, and generated by the process described herein.

In one embodiment, the dy is selected from a group consisting of a monoclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single chain antibody, a Fab nt and a F(ab)2 fragment. In a preferred ment, the antibody is an anti-CD22 antibody that has specificity for human CD22, and comprises a heavy chain wherein the le domain comprises a CDR having at least one of the sequences given as H1 in Figure 1 (SEQ ID NO:1) for CDR-H1, as H2 in Figure 1 (SEQ ID NO:2) or H2’ (SEQ ID NO:13) or H2’’ (SEQ ID NO:15) or H2’’’ (SEQ ID NO:16) for CDR-H2 or as H3 in Figure 1 (SEQ ID NO:3) for CDR-H3, and comprises a light chain wherein the variable domain ses a CDR having at least one of the sequences given as L1 in Figure 1 (SEQ ID NO:4) for CDR-L1, as L2 in Figure 1 (SEQ ID NO:5) for CDR-L2 or as L3 in Figure 1 (SEQ ID NO:6) for CDR-L3.

In another preferred embodiment, the anti-CD22 antibody comprises a heavy chain wherein the variable domain comprises a CDR having at least one of the sequences given in SEQ ID NO:1 for CDR-H1, SEQ ID NO:2 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:16 for CDR-H2 or SEQ ID NO:3 for CDR-H3, and a light chain wherein the variable domain ses a CDR having at least one of the sequences given in SEQ ID NO:4 for CDR-L1, SEQ ID NO:5 for CDR-L2 or SEQ ID NO:6 for CDR-L3.

In yet another preferred embodiment, the anti-CD22 antibody comprises SEQ ID NO:1 for , SEQ ID NO: 2 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:16 for CDR-H2, SEQ ID NO:3 for CDR-H3, SEQ ID NO:4 for CDR-L1, SEQ ID NO:5 for CDR-L2 and SEQ ID NO:6 for CDR-L3.

In another embodiment, the humanized anti-CD22 antibody is a CDR-grafted anti-CD22 antibody and comprises a variable domain comprising human acceptor framework s and non-human donor CDRs.

In another embodiment, the humanized D22 antibod has a human acceptor framework wherein regions of the variable domain of the heavy chain of the antibody are based on a human sub-group I consensus sequence and comprise non-human donor residues at positions 1, 28, 48, 71 and 93. In another ment, the humanized dy further ses non-human donor residues at positions 67 and 69.

In one preferred embodiment, the CDR-grafted humanized antibody comprises a variable domain of the light chain comprising a human acceptor framework region based on a human sub-group I consensus sequence and further comprising non-human donor residues at positions 2, 4, 37, 38, 45 and 60. In another embodiment, the afted antibody further comprises a non-human donor residue at position 3.

In yet another embodiment, the CDR-grafted antibody comprises a light chain variable region 5/44-gL1 (SEQ ID NO:19) and a heavy chain le region 5/44-gH7 (SEQ ID NO:27).

In another ment, the CDR-grafted antibody comprises a light chain having the sequence as set forth in SEQ ID NO: 28 and a heavy chain having the sequence as set forth in SEQ ID NO:30.

In yet another embodiment, the CDR-grafted dy comprises a light chain having the sequence as set forth in SEQ ID NO: 28 and a heavy chain having the sequence as set forth in SEQ ID NO: 30.

In one embodiment, the anti-CD22 CDR-grafted antibody is a variant antibody obtained by an affinity maturation protocol and has increased specificity for human CD22.

In another embodiment, the anti-CD22 antibody is a chimeric antibody comprising the sequences of the light and heavy chain variable domains of the monoclonal dy set forth in SEQ ID NO:7 and SEQ ID NO:8 respectively.

In yet another embodiment, the anti-CD22 antibody comprises a hybrid CDR with a truncated donor CDR sequence wherein the g portion of the donor CDR is replaced by a ent sequence and forms a functional CDR.

In a particularly preferred embodiment, the cytotoxic drug derivative is either gamma calicheamicin or N-acetyl gamma calicheamicin derivative.

In another aspect, the invention is ed to a method for the preparation of a stable lyophilized composition of a ric cytotoxic drug derivative/carrier conjugate. In one embodiment, the stable lyophilized composition of the monomeric cytotoxic drug derivative/carrier conjugate is prepared by (a) dissolving the monomeric cytotoxic drug derivative/carrier conjugate to a final concentration of 0.5 to 2 mg/mL in a solution comprising a cryoprotectant at a concentration of % by weight, a polymeric bulking agent at a concentration of 0.5-1.5% by weight, electrolytes at a concentration 0.01M to 0.1 M, a solubility facilitating agent at a concentration of 0.005-.05% by weight, buffering agent at a tration of 5-50 mM such that the final pH of the on is 7.8-8.2, and water; (b) dispensing the above solution into vials at a temperature of +5 °C to +10 °C; (c) freezing the solution at a freezing temperature of -35 °C to –50 °C; (d) subjecting the frozen solution to a initial freeze drying step at a y drying pressure of 20 to 80 microns at a shelf temperature at –10 °C to –40 °C for 24 to 78 hours; and (e) subjecting the freeze-dried product of step (d) to a ary drying step at a drying pressure of 20 to 80 microns at a shelf ature of +10°C to + 35°C for 15 to 30 hours.

In one embodiment, the cryoprotectant used in the lization of the cytotoxic drug/carrier conjugate is ed from alditol, mannitol, sorbitol, inositol, polyethylene glycol, aldonic acid, uronic acid, aldaric acid, aldoses, ketoses, amino sugars, alditols, inositols, glyceraldehydes, arabinose, lyxose, pentose, ribose, xylose, galactose, glucose, hexose, idose, e, talose, heptose, glucose, fructose, gluconic acid, sorbitol, lactose, ol, methyl α-glucopyranoside, maltose, isoascorbic acid, ascorbic acid, lactone, sorbose, glucaric acid, erythrose, threose, arabinose, allose, altrose, gulose, idose, talose, erythrulose, ribulose, se, psicose, tagatose, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, sucrose, trehalose, inic acid, arabinans, fructans, fucans, galactans, uronans, s, mannans, xylans, levan, fucoidan, carrageenan, galactocarolose, pectins, pectic acids, amylose, pullulan, en, ectin, cellulose, dextran, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthan gum, starch, sucrose, glucose, lactose, ose, ethylene glycol, hylene glycol, polypropylene glycol, glycerol and pentaerythritol.

In a preferred embodiment, the cryoprotectant is sucrose, which is present at a concentration of 1.5% by weight.

In one embodiment, the polymeric bulking agent used during the lyophilization process is selected from Dextran 40 or hydroxyethyl starch 40, and is at a concentration of 0.9% by weight.

In another embodiment, the electrolyte used in the lyophilization solution is sodium chloride, which is present at a concentration of 0.05 M.

In a preferred embodiment, a solubility facilitating agent is used during the lyophilization process. Usually this solubility facilitating agent is a surfactant. In a particularly preferred embodiment, the surfactant is Polysorbate 80, which is present at a concentration of 0.01% by .

In one embodiment, the ing agent used is tromethamine, which is present at a concentration of 0.02 M. it is desirable for the pH of the solution to be 8.0 at the start of the lyophilization process. The solution containing the cytotoxic drug/carrier conjugate is dispensed into vials at a temperature of +5 °C prior to the start of the s.

In a preferred embodiment, the solution in the vials is frozen at a temperature of -45 °C; the frozen solution is subjected to an initial freeze drying step at a primary drying pressure of 60 microns and at a shelf temperature of –30 °C for 60 hours; the freeze-dried product is subjected to a secondary drying step at a drying pressure 60 microns at a shelf temperature of +25°C for 24 hours.

Also described is a composition comprising a therapeutically effective dose of a monomeric cytotoxic drug derivative/carrier conjugate ed by the method bed herein.

In one ment, the carrier in the monomeric cytotoxic drug derivative/carrier conjugate is a naceous carrier selected from hormones, growth factors, antibodies and dy mimics.

In a preferred embodiment, the proteinaceous carrier is a human onal antibody, a chimeric antibody. a human antibody or a a humanized antibody.

In a preferred embodiment, the humanized antibody is directed against the cell surface antigen CD22.

In a particularly preferred embodiment, the anti-CD22 antibody has specificity for human CD22, and comprises a heavy chain wherein the variable domain comprises a CDR having at least one of the sequences given as H1 in Figure 1 (SEQ ID NO:1) for CDRH1 , as H2 in Figure 1 (SEQ ID NO:2) or H2’ (SEQ ID NO:13) or H2’’ (SEQ ID NO:15) or H2’’’ (SEQ ID NO:16) for CDR-H2 or as H3 in Figure 1 (SEQ ID NO:3) for CDR- H3, and comprises a light chain wherein the variable domain comprises a CDR having at least one of the sequences given as L1 in Figure 1 (SEQ ID NO:4) for CDR-L1, as L2 in Figure 1 (SEQ ID NO:5) for CDR-L2 or as L3 in Figure 1 (SEQ ID NO:6) for CDR-L3.

In another preferred embodiment, anti-CD22 antibody has a heavy chain n the variable domain comprises a CDR having at least one of the sequences given in SEQ ID NO:1 for CDR-H1, SEQ ID NO:2 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:16 for CDR-H2 or SEQ ID NO:3 for CDR-H3, and a light chain wherein the variable domain comprises a CDR having at least one of the sequences given in SEQ ID NO:4 for CDR-L1, SEQ ID NO:5 for CDR-L2 or SEQ ID NO:6 for CDR-L3.

In yet another preferred embodiment, the antibody molecule comprises SEQ ID NO:1 for CDR-H1, SEQ ID NO: 2 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:16 for CDR-H2, SEQ ID NO:3 for CDR-H3, SEQ ID NO:4 for CDR-L1, SEQ ID NO:5 for CDR-L2 and SEQ ID NO:6 for CDR-L3.

In a particularly preferred embodiment, the humanized anti-CD22 antibody is a CDR- grafted humanized anti-CD22 antibody and comprises a light chain variable region 5/44- gL1 (SEQ ID NO:19), and a heavy chain variable region 5/44-gH7 (SEQ ID NO:27).

In another particularly red embodiment, the humanized anti-CD22 antibody is a CDR-grafted antibody having specificity for human CD22 and comprises a light chain having a ce set forth in SEQ ID NO: 28 and a heavy chain having a sequence set forth in SEQ ID NO:30.

In r preferred embodiment, the zed anti-CD22 antibody is a CDR-grafted antibody having icity for human CD22 and ses a light chain having a sequence set forth in SEQ ID NO: 28 and a heavy chain having a sequence set forth in SEQ ID NO: 30.

In one embodiment, the CDR-grafted antibody is a variant dy, which hasincreased specificity for human CD22, and dy is obtained by an affinity maturation protocol.

In one embodiment, the monomeric cytotoxic drug is calicheamicin and is selected from gamma calicheamicin or yl eamicin.

In one embodiment, the composition may optionally n additional bioactive agent.

Such a bioactive agent may be a cytotoxic drug, a growth factor or a hormone.

Also described is a method of treating a subject with a erative disorder by stering to the subject a therapeutically effective dose of the composition as described. The ition may be administered subcutaneously, intraperitoneally, intravenously, intraarterially, intramedullarly, intrathecally, transdermally, transcutaneously, intranasaly, topically, entereally, intravaginally, gually or rectally. In a preferred embodiment, the composition is administered intravenously.

In one embodiment, the composition is administered to a human subject suffering from a proliferative disorder such as cancer. In a preferred embodiment, the cancer is a B-cell malignancy. The B-cell malignancy may be a leukemia or ma which express cell surface antigen CD22.

In yet another embodiment, the cancer is a carcinoma or a a.

Also described is a method of treating a B-cell malignancy by stering to a patient with such malignancy a therapeutically effective composition comprising a cytotoxic drug-anti-CD22-antibody conjugate as described herein. In a preferred embodiment, the B-cell malignancy is a lymphoma, particularly Non-Hodgkin’s lymphoma.

In one embodiment, the cytotoxic drug used to prepare the conjugates as described is selected from the group consisting of a calicheamicins, pa, taxanes, stine, daunorubicin, doxorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins/auristatins, hemiasterlins, sinoids and esperamicins.

In a preferred embodiment, the cytotoxic drug is is gamma calicheamicin or N-acetyl calicheamicin.

In another embodiment, the treatment comprises administering the cytotoxic drug conjugate with one or more bioactive agents ed from antibodies, growth factors, hormones, cytokines, anti-hormones, xanthines, interleukins, interferons and cytotoxic drugs.

In a preferred embodiment, the bioactive agent is an antibody, and is directed against a cell surface antigen expressed on B-cell malignancies. In a preferred embodiment, the antibody directed against cell surface antigens sed on B-cell malignancies is selected from a group consisting of anti-CD19, D20 and anti-CD33 antibodies.

Such antibodies include the D20 dy, mab (Rituxan™).

In r embodiment, the ive agents are cytokines or growth s and include, but are not limited to, interleukin 2 (IL-2), TNF, CSF, GM-CSF and G-CSF.

In r embodiment, bioactive agents are hormones and include estrogens (diethylstilbestrol, estradiol), androgens (testosterone, Halotestin), progestins (Megace, Provera), and corticosteroids (prednisone, dexamethasone, hydrocortisone).

In yet anothet embodiment, the bioactive agent is a cytotoxic drug selected from doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, ntrone, epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, abine, trifluridine, ancitabine, abine, azacitidine, doxifluridine, pentostatin, broxuridine, capecitabine, cladribine, decitabine, floxuridine, fludarabine, gougerotin, puromycin, tegafur, tiazofurin, adriamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine rexate, flurouracils, etoposide, taxol, taxol analogs and mitomycin.

In a preferred embodiment, the therapeutically effective composition of the cytotoxic drug-anti-CD22-antibody conjugate is administered er with one or more combinations of cytotoxic agents as a part of a treatment regimen, wherein the ation of cytotoxic agents is selected from:CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone and procarbazine); CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone); COP phosphamide, vincristine, prednisone); CAP-BOP (cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine and prednisone); m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, and leucovorin; ProMACE-MOPP (prednisone, methotrexate, doxorubicin, hosphamide, etoposide, leukovorin, mechloethamine, vincristine, prednisone and procarbazine); ProMACE-CytaBOM (prednisone, methotrexate, doxorubicin, hosphamide, etoposide, leukovorin, cytarabine, bleomycin and vincristine); MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leukovorin); MOPP (mechloethamine, vincristine, prednisone and procarbazine); ABVD (adriamycin/doxorubicin, bleomycin, stine and dacarbazine); MOPP alternating with ABV (adriamycin/doxorubicin, cin, vinblastine); MOPP alternating with ABVD, ChlVPP (chlorambucil, vinblastine, procarbazine, prednisone); IMVP-16 (ifosfamide, rexate, etoposide); MIME (methyl-gag, ifosfamide, methotrexate, etoposide); DHAP (dexamethasone, high-dose cytaribine and cisplatin); ESHAP (etoposide, methylpredisolone, HD cytarabine, and cisplatin); CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin); CAMP (lomustine, mitoxantrone, cytarabine and prednisone); CVP-1 (cyclophosphamide, vincristine and prednisone); and DHAP (cisplatin, ose cytarabine and dexamethasone).

In a preferred embodiment, the therapeutically ive ition of the xic drug-anti-CD22-antibody conjugate is administered prior to the administration of one or more of the above combinations of cytotoxic drugs. In another preferred embodiment, the therapeutically ive composition of the cytotoxic drug-anti-CD22-antibody conjugate is administered subsequent to the administration of one or more of the above combinations of cytotoxic drugs as a part of a treatment regimen.

Also described is a method of treating aggressive lymphomas comprising administering to a patient in need of said ent a therapeutically effective ition of a monomeric calicheamicin derivative-anti-CD22-antibody conjugate together with one or more bioactive agents.

Yet another embodiment is directed to the use of the composition in ng a subject with a proliferative disordersuch as cancer. In particular the cancer is a B-cell malignancy, which express CD22 antigen on the cell surface. In particular, the B-cell malignancy is either a leukemia or a lymphoma. In one embodiment, the cancer is a carcinoma or a leukemia.

In one embodiment, a therapeutically effective dose of the composition is administered subcutaneously, intraperitoneally, intravenously, intraarterially, intramedullarly, hecally, transdermally, transcutaneously, intranasaly, topically, entereally, intravaginally, sublingually or rectally.

In a preferred embodiment, the therapeutically effective dose of the pharmaceutical composition is administered intravenously.

Also described is the use of a monomeric calicheamicin derivative/anti-CD22 antibody ate as described for use in the treatment of a t with a B-cell malignancy such as Non-Hodgkin’s lymphoma. In one embodiment, the monomeric calicheamicin derivative/anti-CD22 antibody conjugate is administered with one or more bioactive agents.

In one ment, the bioactive agents are selected from a group consisting of antibodies, growth factors, hormones, cytokines, anti-hormones, xanthines, interleukins, interferons and cytotoxic drugs.

In a preferred embodiment, the bioactive agent is an antibody ed against a cell surface antigen expressed on B-cell malignancies, such as anti-CD19, anti-CD20 and anti-CD33 dies. In a preferred embodiment, the anti-CD20 antibody is rituximab (Rituxan™).

In another embodiment, bioactive agents include nes or growth factors such as interleukin 2 , TNF, CSF, GM-CSF and G-CSF or es, which include estrogens (diethylstilbestrol, estradiol), androgens (testosterone, Halotestin), tins (Megace, Provera), and corticosteroids (prednisone, dexamethasone, hydrocortisone).

In another embodiment, bioactive agent is a cytotoxic drug selected from doxorubicin, daunorubicin, idarubicin, aclarubicin, cin, mitoxantrone, epirubicin, carubicin, mycin, menogaril, bicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, broxuridine, capecitabine, cladribine, decitabine, floxuridine, fludarabine, gougerotin, puromycin, tegafur, tiazofurin, adriamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, stine, stine, ntrone, bleomycin, mechlorethamine, prednisone, procarbazine methotrexate, racils, etoposide, taxol, taxol analogs, and mitomycin.

In a preferred embodiment, the therapeutically effective dose of the monomeric calicheamicin derivative/anti-CD22 antibody conjugate is administered together with one or more combinations of cytotoxic agents as a part of a treatment regimen, wherein the combination of xic agents is selected from: CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone and procarbazine); CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone); COP (cyclophosphamide, vincristine, prednisone); CAP-BOP (cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine and prednisone); m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, and leocovorin); ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leukovorin, mechloethamine, vincristine, prednisone and bazine); ProMACE-CytaBOM (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leukovorin, cytarabine, bleomycin and vincristine); MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leukovorin); MOPP oethamine, vincristine, prednisone and bazine); ABVD (adriamycin/doxorubicin, bleomycin, vinblastine and dacarbazine); MOPP (mechloethamine, vincristine, prednisone and procarbazine) alternating with ABV (adriamycin/doxorubicin, bleomycin, vinblastine); MOPP (mechloethamine, vincristine, prednisone and procarbazine) alternating with ABVD (adriamycin/doxorubicin, bleomycin, vinblastine and dacarbazine); ChlVPP (chlorambucil, vinblastine, procarbazine, prednisone); IMVP-16 (ifosfamide, rexate, ide); MIME (methyl-gag, ifosfamide, methotrexate, etoposide); DHAP (dexamethasone, high-dose cytaribine and cicplatin); ESHAP (etoposide, methylpredisolone, HD cytarabine, and cisplatin); ) (cyclophosphamide, etoposide, procarbazine, prednisone and cin); CAMP (lomustine, mitoxantrone, cytarabine and prednisone); CVP-1 (cyclophosphamide, vincristine and prednisone); and DHAP atin, high-dose cytarabine and dexamethasone).

In one preferred embodiment, the monomeric calicheamicin derivative/anti-CD22 antibody conjugate is administered prior to the administration of one or more ations of cytotoxic agents as a part of a treatment regimen.

In another preferred embodiment, the therapeutically effective dose of the monomeric calicheamicin derivative/anti-CD22 antibody conjugate is administered subsequent to the administration of one or more combinations of cytotoxic agents as a part of a treatment regimen.

In yet another preferred embodiment, the therapeutically effective dose of the monomeric calicheamicin derivative/anti-CD22 antibody conjugate is administered together with an dy directed against a cell surface antigen on B-cell malignancies, and optionally comprising one or more combinations of cytotoxic agents as a part of a treatment regimen.

Also described is the use of the ric calicheamicin derivative/anti-CD22 antibody ate as described in the manufacture of a medicament for the treatment of a proliferative disorder. Such a medicament can be used to treat B-cell proliferative ers either alone or in combination with other ive agents.

Brief Description of the Drawings Figure 1 shows the amino acid ce of the CDRs of mouse monoclonal antibody /44 (SEQ ID NOS:1 to 6); Figure 2 shows the complete sequence of the light chain variable domain of mouse monoclonal antibody 5/44; Figure 3 shows the te sequence of the heavy chain variable domain of mouse onal antibody 5/44; Figure 4 shows the strategy for l of the glycosylation site and reactive lysine in CDR-H2; Figure 5 shows the graft design for the 5/44 light chain sequence; Figure 6 shows the graft design for the 5/44 heavy chain sequence; Figure 7 shows the vectors pMRR14 and pMRR10.1; Figure 8 shows the Biacore assay results of the chimeric 5/44 mutants; Figure 9 shows the oligonucleotides for 5/44 gH1 and gL1 gene assemblies; Figure 10 shows the intermediate vectors pCR2.1(544gH1) and pCR2.1(544gL1); Figure 11 shows the oligonucleotide cassettes used to make further grafts; Figure 12 shows the competition assay between fluorescently labelled mouse 5/44 antibody and grafted variants; Figure 13 shows the full DNA and protein sequence of the grafted heavy and light chains; Figure 14 is a schematic representation of an antibody-NAc-gamma calicheamicin DMH conjugate; Figure 15 shows the effect of CMC-544 on growth of RAMOS B-cell lymphoma. CMC- 544 made by the CMC conjugation ure was evaluated in B-cell lymphoma afts in nude mice. Animals with tumor xenografts were injected intraperitoneally (ip) with varying doses of CMC-544 or its murine antibody counterpart made by the CMC conjugation procedure on days 1, 5 and 9. In this study, shown in Figure 5, CMC- 544 and its murine antibody counterpart were shown to be efficacious in inhibiting, in a dose-dependent manner, the growth of RAMOS B-cell lymphoma; Figure 16 shows the effect of CMC-544 on large B-cell lymphomas in an in vivo xenograft model in nude mice. As shown in Figure 3, administration of CMC-544 (160 μg/Kg) to large RAMOS lymphoma-bearing mice on days 1, 5, and 9 caused gradual regression of the pre-existing ma mass and by day 20, 3 out of 4 tumor-bearing mice were tumor-free. Monitoring these tumor-free mice up to day 50 did not indicate any re-growth of regressed RAMOS lymphoma. In contrast, an isotype matched control, 6, had no effect on the tumor growth. Four out of five CMA-676 treated large tumor-bearing mice had to be sacrificed before day 15 because their tumor burden reached close to 15% of their body weight; Figure 17 compares the effects of CMC-544 made with the CMA and the CMC ation conjugation procedures on the growth of RL lymphoma. Figure 4 shows the results of a representative experiment in which staged RL lymphoma-bearing mice received two different doses (80 and 320 μg/Kg of conjugated calicheamicin) of CMC- 544 made using the CMA conjugation ure (labeled "OP") and the CMC conjugation procedure (labeled "NP") using the standard dosing schedule. Figure 17 shows that the observed umor efficacy was dose-dependent as expected and there was no difference in the efficacies of either of the two CMC-544 preparations. In contrast, ugated NAc-gamma calicheamicin DMH administered intraperitoneal at 160 μg/Kg was inactive; and Figure 18 shows that Rituxan™-treated large RL lymphoma is susceptible to CMC-544.

To determine whether B-cell lymphomas grown after the discontinuation of the commercially available, anti-CD20 Rituxan™ treatment were still responsive to the CMC-544 treatment, RL lymphomas were treated with Rituxan™ for three weeks. Upon cessation of Rituxan™ therapy, RL lymphomas grew rapidly to the size of ~1 g mass at which time they were treated with CMC-544 at the intraperitoneal dose of 160 μg/Kg on days 1, 5, and 9. Figure 8 shows that these RL lymphomas were still responsive to CMC- 544 with 80% of mice becoming tumor-free by day 60.

Detailed Description of the Invention The conjugates described herein comprise a cytotoxic drug tized with a linker that includes any ve group that reacts with a proteinaceous carrier to form a cytotoxic drug derivative-proteinaceous carrier conjugate. Specifically, the conjugates comprise a cytotoxic drug derivatized with a linker that includes any reactive group which reacts with an dy used as a proteinaceous carrier to form a cytotoxic drug derivativeantibody conjugate. Specifically, the antibody reacts against a cell surface antigen on B- cell ancies. Described below is an improved s for making and purifying such conjugates. The use of particular cosolvents, additives, and specific on conditions er with the separation process s in the formation of a monomeric cytotoxic drug derivative anti-CD22 antibody conjugate with a significant reduction in the LCF. The monomeric form as opposed to the ated form has significant therapeutic value, and minimizing the LCF and substabtially ng aggregation results in the utilization of the antibody starting material in a therapeutically meaningful manner by ting the LCF from ing with the more highly conjugated fraction (HCF).

I. CARRIERS The carriers/targeting agents described herein are preferably proteinaceous carriers/targeting agents. Included as carrier/targeting agents are es, growth factors, antibodies, antibody fragments, antibody , and their genetically or enzymatically engineered counterparts, hereinafter referred to singularly or as a group as ers”. The essential property of a carrier is its ability to recognize and bind to an antigen or receptor associated with undesired cells and be subsequently internalized.

Examples of carriers that are able are disclosed in U.S. Patent No. 5,053,394, which is incorporated herein in its entirety. Preferred carriers for use as described herein are antibodies and antibody mimics.

A number of non-immunoglobulin protein scaffolds have been used for generating antibody mimics that bind to antigenic epitopes with the specificity of an antibody (PCT publication No. WO 00/34784). For example, a “minibody” ld, which is related to the immunoglobulin fold, has been designed by deleting three beta strands from a heavy chain variable domain of a monoclonal antibody (Tramontano et al., J. Mol. Recognit. 7:9, 1994). This n includes 61 residues and can be used to present two hypervariable loops. These two loops have been randomized and products selected for antigen binding, but thus far the framework appears to have somewhat limited utility due to solubility problems. Another framework used to display loops is tendamistat, a n which specifically inhibits mammalian alpha-amylases and is a 74 residue, six-strand beta-sheet sandwich held together by two disulfide bonds, (McConnell and Hoess, J.

Mol. Biol. 250:460, 1995). This scaffold includes three loops, but, to date, only two of these loops have been examined for randomization ial.

Other ns have been tested as frameworks and have been used to display randomized residues on alpha helical surfaces (Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Protein Eng. 8:601, 1995), loops between alpha helices in alpha helix bundles (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by ide bridges, such as those of the small protease inhibitors (Markland et al., Biochemistry :8045, 1996; Markland et al., Biochemistry 35:8058, 1996; Rottgen and Collins, Gene 164;243, 1995; Wang et al., J. Biol. Chem. 270:12250, 1995).

Examples of antibody rs that may be used as described herein e monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies and biologically active fragments thereof. ably such antibodies are directed against cell e antigens sed on target cells and/or tissues in proliferative disorders such as cancer. Examples of specific antibodies directed against cell surface antigens on target cells include without tion, antibodies against CD22 antigen which is xpressed on most B-cell lymphomas; G5/44, a humanized form of a murine anti-CD22 monoclonal antibody; antibodies against cell surface antigen CD33, which is prevalent on certain human myeloid tumors especially acute myeloid leukemia; hP67.6, a humanized form of the anti-CD33 murine antibody (see U.S. Patent No. 5,773,001); an antibody against the PEM antigen found on many tumors of lial origin designated mP67.6 (see I.D.

Bernstein et al., J. Clin. Invest. 79:1153 (1987) and I.D. Bernstein et al., J. Immunol. 128:867-881 (1992)); and humanized antibody against the Lewis Y carbohydrate antigen over expressed on many solid tumors ated hu3S193, (see U.S. Patent No 6,310,185 B1). In addition, there are several commercially available dies such as rituximab (RituxanTM) and trastuzumab (HerceptinTM), which may also be used as carriers/targeting agents. Rituximab (RituxanTM) is a chimeric anti-CD20 antibody used to treat various B- call lymphomas and trastuzumab (HerceptinTM) is a humanized anti-Her2 antibody used to treat breast cancer. ified herein for use as a carrier is a CDR-grafted humanized antibody molecule directed against cell surface antigen CD22, designated G5/44. This antibody is a humanized form of a murine anti-CD22 onal antibody that is directed against the cell surface n CD22, which is prevalent on certain human mas. The term “a CDR-grafted antibody molecule” as used herein refers to an antibody molecule wherein the heavy and/or light chain contains one or more complementarity determining region (CDRs) (hereinafter CDR) (including, if d, a modified CDR) from a donor antibody (e.g., a murine onal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g., a human antibody). ably, such a CDR-grafted antibody has a variable domain sing human acceptor framework regions as well as one or more of the donor CDRs referred to above.

When the CDRs are grafted, any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.

Examples of human frameworks, which can be useful in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al. Seq. of Proteins of l.

Interest, 1:310-334 (1994)). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain and EU, LAY and POM can be used for both the heavy chain and the light chain.

In a CDR-grafted antibody, it is preferred to use as the or antibody one having chains which are homologous to the chains of the donor antibody. The acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.

Also, in a CDR-grafted antibody, the framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently occurring residues for that acceptor chain class or type.

Alternatively, selected residues in the acceptor framework regions may be d so that they pond to the e found at the same position in the donor dy.

Such s should be kept to the minimum necessary to recover the affinity of the donor dy. A protocol for selecting residues in the acceptor framework regions, which may need to be changed, is set forth in PCT Publication No.: WO 91/09967, which is incorporated herein in its entirety.

Donor residues are residues from the donor antibody, i.e., the antibody from which the CDRs were originally derived.

The antibody may comprise a heavy chain wherein the variable domain ses as CDR-H2 (as defined by Kabat et al., (supra)) an H2’ in which a ial glycosylation site sequence has been removed in order to increase the affinity of the antibody for the antigen.

Alternatively or additionally, the antibody may comprise a heavy chain wherein the variable domain ses as CDR-H2 (as defined by Kabat et al., (supra)) an H2’’ in which a lysine residue is at position 60. This lysine residue, which is located at an exposed position within CDR-H2, and is considered to have the potential to react with ation agents resulting in a reduction of antigen binding affinity, is substituted with an alternative amino acid.

Additionally, the antibody may comprise a heavy chain wherein the variable domain comprises as CDR-H2 (as d by Kabat et al., )) an H2’’’ in which both the potential glycosylation site sequence and the lysine residue at position 60, are substituted with alternative amino acids.

The antibody molecule as described herein may comprise: a complete antibody molecule, having full length heavy and light chains; a biologically active nt thereof, such as a Fab, modified Fab, Fab’, F(ab’)2 or Fv fragment; a light chain or heavy chain monomer or dimer; a single chain antibody, e.g., a single chain Fv in which the heavy and light chain variable s are joined by a peptide linker. Similarly, the heavy and light chain variable regions may be combined with other antibody domains as appropriate.

The antibody molecule as bed herein may also include a modified Fab fragment wherein the modification is the addition of one or more amino acids to allow for the attachment of an effector or er molecule to the inal end of its heavy chain.

Preferably, the additional amino acids form a modified hinge region containing one or two cysteine residues to which the effector or reporter molecule may be attached.

The constant region s of the antibody le, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may or may not be required. For e, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used, especially of the IgG1 and IgG3 isotypes when the antibody molecule is intended for therapeutic uses and antibody effector functions are required.

Alternatively, IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and dy effector functions are not required or desired.

The antibody molecule has a binding affinity of at least 5x10-8 M, preferably at least 1x10-9 M, more preferably at least 0.75x10-10 M, and most preferably at least 0.5x10-10 M.

In one ment, described are immunotoxin conjugates and methods for making these conjugates using antibody variants or antibody mimics. In a preferred embodiment, ts of the dy molecule are directed against CD22 and display improved affinity for CD22. Such variants can be ed by a number of affinity maturation protocols including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol.

Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998).

Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the r molecule(s) including antibodies as described herein. Bacterial, for example E. coli, and other microbial systems may be used, in part, for expression of antibody fragments such as Fab and F(ab’)2 fragments, and especially Fv fragments and single chain antibody nts, for example, single chain Fvs. Eukaryotic, e.g. mammalian, host cell expression systems may be used for production of larger dy molecules, including complete antibody molecules. Suitable mammalian host cells include CHO, myeloma, yeast cells, insect cells, hybridoma cells, NSO, VERO or PER C6 cells. Suitable expression systems also include enic animals and plants.

II. Therapeutic Agents The eutic agents suitable for use as described herein are cytotoxic drugs that inhibit or disrupt tubulin polymerization, alkylating agents that bind to and disrupt DNA, and agents which inhibit protein synthesis or essential cellular proteins such as protein kinases, enzymes and cyclins. es of such cytotoxic drugs e, but are not limited to thiotepa, taxanes, vincristine, ubicin, doxorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, icin, dolastatins/auristatins, hemiasterlins, esperamicins and maytansinoids. Preferred cytotoxic drugs are the calicheamicins, which are an example of the methyl trisulfide antitumor antibiotics. Examples of calicheamicins suitable for use in the present invention are sed, for example, in U.S. Patent No. 4,671,958; U.S. Patent No. 4,970,198, U.S. Patent No. 5,053,394, U.S. Patent No. 5,037,651; and U.S. Patent No. ,079,233, which are incorporated herein in their entirety. Preferred calicheamicins are the gamma-calicheamicin derivatives or the N-acetyl gamma-calicheamicin derivatives.

III. CYTOTOXIC DRUG DERIVATIVE/CARRIER CONJUGATES The conjugates described herein have the formula Pr(-X-S-S-W)m Pr is a proteinaceous carrier, X is a linker that comprises a t of any reactive group that can react with a proteinaceous r, W is the cytotoxic drug; m is the average loading for a purified conjugation product such that the calicheamicin constitutes 4 - 10% of the conjugate by weight; and (-X-W)m I is a xic drug derivative Preferably, X has the formula (CO - Alk1 - Sp1 - Ar - Sp2 - Alk2 - C(Z1) = Q - Sp) Alkl and Alk2 are independently a bond or branched or unbranched (Cl-C10) alkylene chain; Sp1 is a bond, -S-, -O-, -CONH-, -NHCO-, -NR'-, -N(CH2CH2)2N-, or -X-Ar'-Y-(CH2)n-Z wherein X, Y, and Z are independently a bond, -NR'-, -S-, or -O-, with the proviso that when n = 0, then at least one of Y and Z must be a bond and Ar' is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C1-C5) alkyl, (C1-C4) alkoxy, (C1-C4) thioalkoxy, halogen, nitro, -COOR', -CONHR', -(CH2)nCOOR', - S(CH2)nCOOR', -O(CH2)nCONHR', or -S(CH2)nCONHR', with the proviso that when Alk1 is a bond, Sp1 is a bond; n is an integer from 0 to 5; R' is a branched or unbranched (C1-C5) chain ally substituted by one or two groups of -OH, ) alkoxy, (C1-C4) thioalkoxy, halogen, nitro, (C1-C3) lamino, or ) trialkylammonium -A- where A- is a pharmaceutically acceptable anion completing a salt; Ar is 1,2-, 1,3-, or enylene optionally substituted with one, two, or three groups of (C1-C6) alkyl, (C1-C5) alkoxy, (C1-C4) koxy, halogen, nitro, -COOR', -CONHR', - O(CH2)nCOOR', -S(CH2)nCOOR', -O(CH2)nCONHR', or -S(CH2)nCONHR' wherein n and R' are as hereinbefore defined or a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene or with each naphthylidene or phenothiazine optionally substituted with one, two, three, or four groups of (C1-C6) alkyl, (C1-C5) alkoxy, (C1-C4) koxy, halogen, nitro, -COOR', -CONHR', -O(CH2)nCOOR', -S(CH2)nCOOR', or -S(CH2)nCONHR' wherein n and R' are as defined above, with the proviso that when Ar is phenothiazine, Sp1 is a bond only connected to nitrogen; Sp2 is a bond, -S-, or -O-, with the proviso that when Alk2 is a bond, Sp2 is a bond; Z1 is H, (C1-C5) alkyl, or phenyl ally substituted with one, two, or three groups of (C1-C5) alkyl, (C1-C5) alkoxy, (C1-C4) koxy, halogen, nitro, -COOR', -ONHR', - O(CH2)nCOOR', )nCOOR', -O(CH2)nCONHR', or -S(CH2)nCONHR' wherein n and R' are as defined above; Sp is a straight or branched-chain divalent or trivalent (C1-C18) radical, divalent or ent aryl or heteroaryl radical, divalent or trivalent (C3-C18) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-aryl (C1-C18) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C1-C18) radical or nt or trivalent (C2-C18) unsaturated alkyl radical, wherein heteroaryl is preferably furyl, thienyl, N-methylpyrrolyl, pyridinyl, N-methylimidazolyl, oxazolyl, pyrimidinyl, quinolyl, isoquinolyl, N-methylcarbazoyl, aminocourmarinyl, or phenazinyl and wherein if Sp is a trivalent radical, Sp can be additionally substituted by lower (C1-C5) dialkylamino, lower (C1-C5) alkoxy, hydroxy, or lower ) alkylthio groups; and Q is =NHNCO-, =NHNCS-, NH-, =NHNCSNH-, or =NHO-.

Preferably, Alk1 is a branched or unbranched (C1-C10) alkylene chain; Sp' is a bond, -S-, - O-, -CONH-, -NHCO-, or -NR' wherein R' is as hereinbefore defined, with the proviso that when Alk1 is a bond, Sp1 is a bond; Ar is 1,2-, 1,3-, or enylene optionally substituted with one, two, or three groups of (C1-C6) alkyl, (C1-C5) alkoxy, (C1-C4) thioalkoxy, halogen, nitro, -COOR', -CONHR', - O(CH2)nCOOR', -S(CH2)nCOOR', -O(CH2)nCONHR', or -S(CH2)nCONHR' wherein n and R' are as hereinbefore defined, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7- ylidene each optionally substituted with one, two, three, or four groups of (C1-C6) alkyl, (C1-C5) alkoxy, ) thioalkoxy, halogen, nitro, , - CONHR', -O(CH2)nCOOR', -S(CH2)nCOOR', -O(CH2)nCONHR', or -S(CH2)nCONHR'.

Z1 is ) alkyl, or phenyl ally substituted with one, two, or three groups of (C1- C5) alkyl, (C1-C4) alkoxy, (C1-C4) thioalkoxy, halogen, nitro, , -CONHR', - O(CH2)nCOOR', -S(CH2)nCOOR', -O(CH2)nCONHR', or -S(CH2)nCONHR'; Alk2 and Sp2 are together a bond; and Sp and Q are as immediately d above.

U.S. Patent No. 5,773,001, incorporated herein in its entirety, discloses linkers that can be used with nucleophilic derivatives, particularly hydrazides and related nucleophiles, prepared from the calicheamicins. These linkers are especially useful in those cases, where better activity is obtained when the linkage formed between the drug and the linker is hydrolyzable. These linkers contain two functional groups. One group typically is a carboxylic acid that is utilized to react with the carrier. The acid functional group, when properly activated, can form an amide linkage with a free amine group of the carrier, such as, for example, the amine in the side chain of a lysine of an antibody or other proteinaceous carrier. The other functional group commonly is a carbonyl group, i.e., an aldehyde or a ketone, which will react with the riately modified therapeutic agent.

The carbonyl groups can react with a hydrazide group on the drug to form a hydrazone linkage. This linkage is yzable, allowing for release of the therapeutic agent from the conjugate after binding to the target cells.

A most preferred bifunctional linker useful in the present invention is 4-(4- acetylphenoxy) butanoic acid ), which s in a red product wherein the conjugate consists of β-calicheamicin, cheamicin or N-acetyl γ-calicheamicin functionalized by reacting with 3-mercaptomethyl butanoyl hydrazide, the AcBut linker, and a human or humanized IgG antibody targeting carrier.

IV. MONOMERIC CONJUGATION The natural hydrophobic nature of many cytotoxic drugs including the calicheamicins creates difficulties in the preparation of monomeric drug conjugates with good drug loadings and reasonable yields which are necessary for therapeutic ations. The increased hydrophobicity of the linkage provided by linkers, such as the AcBut linker, disclosed in U.S. Patent No. 5,773,001, as well as the increased covalent distance separating the therapeutic agent from the carrier (antibody), exacerbate this problem. ation of cytotoxic drug tive/carrier conjugates with higher drug loadings occurs due to the hydrophobic nature of the drugs. The drug loading often has to be limited to obtain reasonable quantities of monomeric product. In some cases, such as with the conjugates in U.S. Patent No. 5,877,296, it is often difficult to make conjugates in useful yields with useful loadings for therapeutic applications using the on conditions disclosed in U.S. Patent No. 5,053,394 due to excessive aggregation. These reaction conditions utilized DMF as the co-solvent in the conjugation reaction. Methods which allow for higher drug loadings/yield without aggregation and the inherent loss of material are therefore needed. ements to reduce aggregation are described in U.S. Patent Nos. 5,712,374 and ,714,586, which are incorporated herein in their entirety. Disclosed in the patents are proteinaceous carriers including, but not limited to, proteins such as human or humanized dies that are used to target the cytotoxic therapeutic agents herein, such as, for example, hP67.6 and the other humanized antibodies sed herein, the use of a nonnucleophilic , protein-compatible, buffered solution containing (i) propylene glycol as a cosolvent and (ii) an additive comprising at least one C6-C18 ylic acid was found to generally produce monomeric cytotoxic drug derivative derivative/carrier conjugates with higher drug loading/yield and decreased aggregation having excellent activity. Preferred acids described therein are C7 to C12 acids, and the most preferred acid is octanoic acid (such as ic acid) or its salts. Preferred buffered solutions for conjugates made from N-hydroxysuccinimide (Osu) esters or other comparably activated esters are atebuffered saline (PBS) or Nhydroxyethyl zine-N'ethanesulfonic acid (HEPES buffer). The buffered solution used in such conjugation reactions cannot n free amines or nucleophiles. Those who are skilled in the art can readily determine acceptable s for other types of conjugates. Alternatively, the use of a non-nucleophilic, protein-compatible, buffered solution ning t-butanol without the additional additive was also found to produce monomeric calicheamicin derivative/carrier conjugates with higher drug loading/yield and decreased aggregation.

The amount of cosolvent needed to form a monomeric conjugate varies somewhat from protein to protein and can be determined by those of ry skill in the art without undue experimentation. The amount of additive ary to effectively form a monomeric conjugate also varies from antibody to antibody. This amount can also be determined by one of ordinary skill in the art without undue experimentation. Additions of propylene glycol in amounts ranging from 10% to 60%, preferably 10% to 40%, and most preferably about 30% by volume of the total solution, and an additive comprising at least one C6-C18 carboxylic acid or its salt, preferably caprylic acid or its salt, in amounts ranging from 20 mM to 100 mM, preferably from 40 mM to 90 mM, and most preferably about 60 mM to 90 mM are added to conjugation reactions to produce monomeric cytotoxic drug derivative/carrier conjugates with higher drug g/yield and decreased aggregation. Other protein-compatible organic cosolvents other than propylene glycol, such as ethylene , ethanol, DMF, DMSO, etc., can also be used. Some or all of the organic cosolvent is used to transfer the drug into the conjugation mixture.

Alternatively, the concentration of the C6-C18 carboxylic acid or its salt can be increased to 150-300 mM and the ent dropped to 1-10%. In one embodiment, the carboxylic acid is octanoic acid or its salt. In a preferred ment, the carboxylic acid is decanoic acid or its salt. In a preferred embodiment, the carboxylic acid is caprylic acid or its salt, which is present at a concentration of 200 mM caprylic acid together with 5% propylene glycol or l.

In another alternative embodiment, nol at concentrations ranging from 10% to 25%, preferably 15%, by volume of the total solution may be added to the conjugation reaction to produce monomeric cytotoxic drug derivative/carrier conjugates with higher drug loading/yield and decreased aggregation.

These established conjugation conditions were applied to the formation of 6 (Gemtuzumab Ozogamicin), which is now commercially sold as MylotargTM. Since introduction of this treatment for acute myeloid leukemia (AML), it has been learned through the use of ion-exchange tography that the calicheamicin is not distributed on the dy in a uniform manner. Most of the calicheamicin is on approximately half of the antibody, while the other half exists in a LCF that contains only small amounts of eamicin. uently, there is a critical need to improve the methods for conjugating cytotoxic drugs such as calicheamicins to carriers, which minimize the amount of aggregation and allow for a higher uniform drug loading with a significantly improved yield of the conjugate product.

A specific example is that of the G5/44-NAc-gamma-calicheamicin DMH AcBut conjugate, which is referred to as CMC-544 and is generically shown in Figure 14. The reduction of the amount of the LCF to <10% of the total antibody was desired for development of CMC-544, and various options for ion of the levels of the low conjugated fraction were ered. Other attributes of the conjugate, such as antigen binding and xicity, must not be affected by the ultimate solution. The options considered ed genetic or physical modification of the antibody molecule, chromatographic tion techniques, or modification of the reaction conditions.

Reaction of the G5/44 dy with NAc-gamma-calicheamicin DMH AcBut OSu using the old reaction conditions (CMA-676 Process Conditions) resulted in a product with similar physical properties (drug loading, low conjugated fraction (LCF), and aggregation) as CMA-676. However, the high level (50-60%) of LCF present after conjugation was deemed rable. Optimal reaction conditions were determined through statistical experimental design methodology in which key reaction variables such as temperature, pH, calicheamicin derivative input, and additive concentration, were evaluated. Analysis of these experiments demonstrated that calicheamicin input and additive concentration had the most significant effects on the level of the low conjugated on LCF and aggregate formation, while temperature and pH exerted smaller influences. In additional experiments, it was also shown that the concentrations of protein carrier (antibody) and cosolvent (ethanol) were similarly of lesser importance red to calicheamicin input and additive concentration) in controlling LCF and aggregate levels. In order to reduce the LCF to <10%, the calicheamicin tive input was increased from 3% to 8.5% (w/w) relative to the amount of dy in the reaction. The additive was changed from octanoic acid or its salt at a concentration of 200 mM (CMA process) to decanoic acid or its salt at a tration of 37.5 mM. The conjugation reaction proceeded better at slightly elevated temperature (30-35oC) and pH (8.2-8.7).

The reaction conditions incorporating these changes reduced the low conjugated fraction (LCF) to below 10 percent while increasing calicheamicin loading, and is hereinafter referred to as CMC-544 Process Condition or “new” process condition. A comparison of the results obtained with the 6 and CMC-544 Process Conditions is shown in Table 1.

Table 1: ison of the CMA-676 (Gemtuzumab Ozogamicin) and CMC-544 Process Conditions Conditions/Results CMA-676 Process 4 Process Conditions Conditions 2. Calicheamicin Input 3.0% (w/w powder 8.5% (w/w) weight basis) 3. Additive Identity and Octanoic 4. Decanoic acid/Sodium tration acid/Sodium ate; 200 mM decanoate; 37.5 Temperature 5. 26°C 31-35°C PH 7.8 8.2-8.7 Calicheamicin g 2.4-3.5 7.0-9.0 nt by weight; by UV assay) Low Conjugated Fraction 45-65 HPLC Area % <10% (LCF) (before purification) Aggregation (before ~5% <5% purification) Aggregation (after ≤2% <2% purification) The increase in calicheamicin input increased the drug loading from 2.5-3.0 weight percent to 7.0-9.0 (most typically 7.5-8.5) weight percent, and resulted in no increase in protein aggregation in the reaction. Due to reduction of aggregate and LCF, the CMC- 544 Process conditions ed in a more homogeneous t. CMC-544 has been reproducibly prepared by this new conjugation procedure at the multi-gram antibody scale.

In the foregoing reactions, the tration of antibody can range from 1 to 15 mg/ml and the concentration of the calicheamicin derivative, e.g., N-Acetyl gammacalicheamicin DMH AcBut OSu ester (used to make the conjugates shown in Figure 14), ranges from about % by weight of the antibody. The cosolvent was ethanol, for which good results have been demonstrated at concentrations ranging from 6 to 11.4% e basis). The reactions are performed in PBS, HEPES, N-(2- Hydroxyethyl)piperazine-N’-(4-butanesulfonic acid) (HEPBS), or other compatible buffer at a pH of 8 to 9, at a ature ranging from 30º C to about 35º C, and for times ranging from 15 minutes to 24 hours. Those who are skilled in the art can readily determine acceptable pH ranges for other types of conjugates. For various antibodies the use of slight variations in the combinations of the aforementioned additives have been found to improve drug loading and monomeric conjugate yield, and it is understood that any ular protein carrier may require some minor alterations in the exact ions or choice of additives to achieve the optimum results.

V. CONJUGATE PURIFICATION AND SEPARATION ing conjugation, the monomeric conjugates may be separated from unconjugated reactants (such as proteinaceous carrier molecules/antibodies and free cytotoxic drug/calicheamicin) and/or aggregated form of the conjugates by conventional s, for example size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), or chromatofocusing (CF).

The purified conjugates are monomeric, and usually contain from 4 to 10% by weight cytotoxic drug/calicheamicin. In a preferred embodiment, the conjugates are purified using hydrophobic interaction chromatography (HIC) In the processes previously used for production-scale manufacturing of cytotoxic drug/calicheamicin-antibody conjugates (CMA-676 process), the sole post-conjugation separation step employed was size exclusion chromatography (SEC). While this step is quite effective at both removing aggregated conjugate and in accomplishing buffer ge for formulation, it is ctive at reducing the LCF content. Consequently, the SEC-based process relies entirely on the chemistry of the conjugation on to control the LCF content of the final product. Another antage of SEC is the limitation of the volume of conjugate reaction e applied to the column (typically not exceeding 5 t of the process column bed volume). This severely limits the batch size (and therefore production capacity) that can be supported in a given production space. Finally, SEC purification process also results in significant dilution of the conjugate solution, which places aints on the protein concentration that can be dependably achieved in formulation.

When a cytotoxic drug has a highly hydrophobic , such as a calicheamicin derivative, and is used in a conjugate, hydrophobic ction chromatography (HIC) is a preferred candidate to provide effective separation of conjugated and unconjugated antibody. HIC presents three key advantages over SEC: (1) it has the capability to efficiently reduce the LCF content as well as aggregate; (2) the column load capacity for HIC is much higher; and (3) HIC avoids excessive dilution of the product.

A number of high-capacity HIC media suitable for production scale use such as Butyl, Phenyl and Octyl Sepharose 4 Fast Flow ham Biosciences, Piscataway, NJ) could ively separate unconjugated and aggregates of the conjugate from monomeric conjugated components following conjugation s.

VI. COMPOSITIONS AND FORMULATIONS Also described is a process for the preparation of a therapeutic or diagnostic composition/formulation comprising admixing the monomeric cytotoxic drug derivative/carrier conjugate as described together with a pharmaceutically acceptable excipient, diluent or carrier.

The monomeric cytotoxic drug derivative/carrier conjugate may be the sole active ingredient in the therapeutic or stic ition/formulation or may be accompanied by other active ingredients ing other antibody ingredients, for example anti-CD19, anti-CD20, anti-CD33, anti-T cell, anti-IFNγ or anti-LPS antibodies, or non-antibody ingredients such as cytokines, growth factors, hormones, anti-hormones, cytotoxic drugs and xanthines.

Cytokines and growth factors that may be used to treat proliferative disorders such as cancer, and which may be used together with the cytotoxic drug derivative/ carrier conjugates described include interferons, interleukins such as interleukin 2 (IL-2), TNF, CSF, GM-CSF and G-CSF.

Hormones commonly used to treat proliferative disorders such as cancer and which may be used together with the cytotoxic drug derivative/ carrier conjugate described include estrogens ylstilbestrol, estradiol), androgens (testosterone, Halotestin), progestins e, Provera), and osteroids isone, dexamethasone, ortisone).

Antihormones such as antiestrogens (tamoxifen), antiandrogens (flutamide) and antiadrenal agents are commonly used to treat proliferative disorders such as , and may be used together with the cytotoxic drug derivative/ carrier conjugate described. herapeutic/antineoplastic agents ly used to treat proliferative disorders such as cancer, and which may be used together with the cytotoxic drug derivative/ carrier conjugate described include, but are not limited to Adriamycin, cisplatin, carboplatin, vinblastine, stine, cin, methotrexate, doxorubicin, flurouracils, etoposide, taxol and its various analogs, and mitomycin.

The pharmaceutical compositions/formulations should preferably comprise a therapeutically effective amount of the conjugate. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or tative effect. For any conjugate, the eutically effective dose can be estimated initially either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The precise effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the t, diet, time and frequency of administration, drug combination(s), on sensitivities and nce/response to therapy. This amount can be ined by routine experimentation and is within the judgment of the clinician. Generally, an effective dose will be from 0.01 mg/m2 to 50 mg/m2, preferably 0.1 mg/m2 to 20 mg/m2, more preferably about 15 mg/m2, which dose is calculated on the basis of the proteinaceous carrier. itions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones. The dose at which the monomeric xic drug derivative/ antibody conjugate is administered depends on the nature of the condition to be treated, the grade of the malignant lymphoma or leukemia and on whether the conjugate is being used prophylactically or to treat an existing condition.

The ncy of dose will depend on the ife of the conjugate and the duration of its effect. If the ate has a short half-life (e.g., 2 to 10 hours) it may be necessary to give one or more doses per day. Alternatively, if the conjugate le has a long halflife (e.g., 2 to 15 days) it may only be necessary to give a dosage once per day, once per week or even once every 1 or 2 .

A composition may also contain a pharmaceutically acceptable carrier for administration of the antibody conjugate. The carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic.

Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulfates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.

Pharmaceutically acceptable carriers in eutic compositions/formulations may additionally contain liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such itions. Such carriers enable the compositions to be formulated as tablets, pills, s, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.

Preferred forms for administration include forms suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion.

Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preserving, izing and/or sing agents.

Although the stability of the buffered conjugate solutions is adequate for a short time, long-term stability is poor. To enhance stability of the ate and to increase its shelf life, the antibody-drug conjugate may be lyophilized to a dry form, for titution before use with an appropriate sterile liquid. The problems associated with lyophilization of a protein solution are well documented. Loss of secondary, tertiary and quaternary structure can occur during freezing and drying processes. Consequently, cryoprotectants may have to be included to act as an amorphous izer of the conjugate and to maintain the structural integrity of the protein during the lization process. In one embodiment, the cryoprotectant useful in the present invention is a sugar alcohol, such as alditol, mannitol, sorbitol, inositol, hylene glycol and combinations thereof. In another embodiment, the cryoprotectant is a sugar acid, including an aldonic acid, an uronic acid, an aldaric acid, and combinations thereof.

The cryoprotectant useful in this invention may also be a carbohydrate. Suitable carbohydrates are aldehyde or ketone compounds containing two or more hydroxyl groups. The carbohydrates may be cyclic or linear and include, for example, aldoses, s, amino sugars, alditols, inositols, aldonic acids, uronic acids, or aldaric acids, or combinations thereof. The carbohydrate may also be a mono-, a di-, or poly-, carbohydrate, such as for example, a disaccharide or polysaccharide. Suitable carbohydrates e for e, glyceraldehydes, arabinose, lyxose, pentose, ribose, xylose, galactose, glucose, hexose, idose, mannose, talose, heptose, glucose, fructose, gluconic acid, sorbitol, lactose, mannitol, methyl α-glucopyranoside, maltose, isoascorbic acid, ascorbic acid, lactone, sorbose, ic acid, erythrose, threose, arabinose, allose, altrose, gulose, idose, talose, erythrulose, ribulose, xylulose, psicose, se, glucuronic acid, ic acid, glucaric acid, galacturonic acid, onic acid, glucosamine, galactosamine, sucrose, trehalose or inic acid, or derivatives thereof. Suitable polycarbohydrates include for example arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galactocarolose, pectins, pectic acids, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, or starch. Among particularly useful carbohydrates are sucrose, e, e, trehalose, and combinations thereof. Sucrose is a particularly useful cryoprotectant.

Preferably, the cryoprotectant useful in the t invention is a carbohydrate or “sugar” alcohol, which may be a polyhydric alcohol. dric compounds are compounds that n more than one hydroxyl group. Preferably, the polyhydric compounds are linear.

Suitable polyhydric compounds include, for e, glycols such as ethylene glycol, polyethylene glycol, and polypropylene glycol, glycerol, or pentaerythritol; or combinations thereof.

In some preferred embodiments, the cryoprotectant agent is sucrose, trehalose, mannitol, or sorbitol.

Once formulated, the itions of the ion can be stered directly to the t. The subjects to be d can be animals. However, it is preferred that the itions are adapted for administration to human subjects.

The compositions of the present invention may be administered by any number of routes including, but not d to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (see PCT Publication No.: WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. Hyposprays may also be used to ster the compositions of the invention. Typically, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to ion may also be prepared.

Direct ry of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion.

Dosage treatment may be a single dose schedule or a multiple dose schedule.

It will be appreciated that the active ingredient in the composition will be a cytotoxic drug/proteinaceous carrier conjugate. As such, it will be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition will need to contain agents which protect the proteinaceous carrier from degradation but which release the conjugate once it has been absorbed from the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991).

Also described is a monomeric calicheamicin derivative/ zed D22 antibody (G5/44), CMC-544, for use in treating proliferative disorders characterized by cells expressing CD22 antigen on their surface.

Also described is the use of CMC-544 in the manufacture of a composition or medicament for the treatment of a proliferative disorder terized by cells expressing CD22.

CMC-544 may also be ed in any therapy where it is desired to reduce the level of cells expressing CD22 that are present in the t being treated with the composition or medicament disclosed herein. ically, the composition or medicament is used to treat humans or animals with proliferative disorders namely lymphomas and ias, which express CD22 antigen on the cell surface. These CD22-expressing cells may be circulating in the body or be present in an undesirably large number localized at a particular site in the body.

CMC-544 may also be preferably used for treatment of malignancies of B-lymphocyte lineage including lymphomas and leukemias, most preferably Non-Hodgkin’s Lymphoma (NHL), acute lymphocytic leukemia (ALL), multiple myeloma, acute lymphocyte leukemia (ALL) and chronic cytic leukemia (CLL). CMC-544 can be used alone or in combination with other bioactive agents to treat subjects suffering from B-cell malignancies.

Bioactive agents commonly used include growth factors, cytokines, and cytotoxic drugs.

Cytotoxic drugs commonly used to treat proliferative disorders such as cancer, and which may be used together with CMC-544 include, anthracycline such as bicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, and valrubicin for up to three days; a pyrimidine or purine side such as cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, broxuridine, capecitabine, cladribine, decitabine, floxuridine, fludarabine, gougerotin, puromycin, tegafur, and tiazofurin. Other chemotherapeutic/antineoplastic agents that may be stered in combination with CMC-544 include adriamycin, cisplatin, carboplatin, hosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine rexate, flurouracils, ide, taxol and its various analogs, and mitomycin. CMC-544 may be administered concurrently with one or more of these therapeutic agents. Alternatively, CMC-544 may be administered sequentially with one or more of these therapeutic agents.

CMC-544 may also be administered alone, concurrently, or sequentially with a combination of other bioactive agents such as growth factors, cytokines, steroids, antibodies such as anti-CD20 dy, mab an™), and chemotherapeutic agents as a part of a treatment regimen. Established treatment regimens for the treatment of malignant proliferative disorders include CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone and procarbazine), CHOP phosphamide, doxorubicin, vincristine, and prednisone), COP phosphamide, vincristine, prednisone), CAP-BOP (cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine and prednisone), m-BACOD (methotrexate, bleomycin, bicin, cyclophosphamide, vinvristine, dexamethasone, and leocovorin, ProMACE-MOPP (prednisone, rexate, doxorubicin, cyclophosphamide, etoposide, leukovorin, mechloethamine, vincristine, prednisone and procarbazine), ProMACE-CytaBOM (prednisone, methotrexate, bicin, cyclophosphamide, etoposide, leukovorin, cytarabine, bleomycin and vincristine), MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, fixed dose prednisone, bleomycin and leukovorin), MOPP (mechloethamine, stine, prednisone and procarbazine), ABVD (adriamycin/doxorubicin, bleomycin, vinblastine and dacarbazine), MOPP alternating with ABV (adriamycin/doxorubicin, bleomycin, vinblastine), and MOPP alternating with ABVD and ChlVPP (chlorambucil, vinblastine, procarbazine, prednisone). Therapy may comprise an induction therapy phase, a idation therapy phase and a maintenance therapy phase. CMC-544 may also be administered alone, concurrently, or sequentially with any of the above identified therapy regimens as a part of induction therapy phase, a consolidation therapy phase and a maintenance therapy phase.

The conjugates described herein may also be administered together with other bioactive and chemotherapeutic agents as a part of combination chemotherapy regimen for the treatment of relapsed aggressive mas. Such a treatment n includes IMVP- 16 amide, rexate, etoposide), MIME (methyl-gag, ifosfamide, rexate, ide), DHAP (dexamethasone, high-dose cytaribine and cicplatin), ESHAP (etoposide, methylpredisolone, high-dose cytarabine, and cisplatin), CEPP(B) phosphamide, etoposide, procarbazine, prednisone and bleomycin), CAMP (lomustine, ntrone, cytarabine and prednisone), CVP-1 (cyclophosphamide, vincristine and prednisone),and DHAP (cisplatin, cytarabine and dexamethasone).

Additional treatment regimens for aggressive lymphomas may e in phase 1 a first line of treatment with CHOP(cyclophosphamide, doxorubicin, vincristine, and prednisone)-rituximab (Rituxan™)-CMC-544, followed in phase 2 and phase 3 with CHOP-rituximab (Rituxan™), CHOP-CMC 544 or CHOP-rituximab (Rituxan™)-CMC- 544. Alternatively, phase 1 may have a first line of treatment with COP (cyclophosphamide, vincristine, and prednisone)-rituximab (Rituxan™)-CMC-544, followed in phase 2 and phase 3 with COP-rituximab (Rituxan™), COP-CMC-544 or COP-rituximab an™)-CMC-544. In a further embodiment, treatment of ive lymphomas may include a first or second line of treatment with the antibody drug conjugate CMC-544 in phase 1, followed in phase 2 and 3 with CMC-544 and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), CMC-544 and COP (cyclophosphamide, vincristine, and prednisone), CMC-544 with rituximab (Rituxan™) or rituximab (Rituxan™) alone. In yet another embodiment, the treatment of aggresive lymphomas may include a first or line line of treatment with the antibody drug conjugate CMC-544 followed in phase 2 and 3 with 4 alone or in combination with other treatment regimens including, but not limited to, ESHOP (etoposide, predisolone, high-dose (HD) cytarabine, vincristine and cisplatin), EPOCH, 6 (ifosfamide, methotrexate, ide), ASHAP, MIME (methyl-gag, ifosfamide, methotrexate, 40 etoposide) and ICE (ifosfamide, cyclophosphamide, and etoposide).

Also described is a method of treating human or animal ts suffering from or at risk of a proliferative disorder characterized by cells sing CD22, the method sing administering to the subject an effective amount of CMC-544 as bed.

The present invention is further bed below in specific working examples, which are intended to further describe the invention without limiting its scope.

Example 1 Generation of Candidate Antibodies A panel of antibodies against CD22 were selected from hybridomas using the following selection criteria: binding to Daudi cells, internalization on Daudi cells, binding to peripheral blood mononuclear cells (PBMC), internalization on PBMC, affinity (greater than 10-9M), mouse g1 and production rate. 5/44 was selected as the preferred antibody.

I. Gene Cloning and Expression of a Chimeric 5/44 Antibody le a) ation Of 5/44 Hybridoma Cells And RNA Preparation Therefrom Hybridoma 5/44 was generated by conventional hybridoma technology following immunization of mice with human CD22 protein. RNA was prepared from 5/44 hybridoma cells using a RNEasy kit (Qiagen, Crawley, UK; Catalogue No. 74106). The RNA obtained was reverse transcribed to cDNA, as described below. b) Distribution of CD22 on NHL Tumors An immunohistochemistry study was undertaken to examine the incidence and distribution of staining using the 5/44 anti-CD22 monoclonal antibodies. l anti- CD20 and anti-CD79a antibodies were included in the study to m B cell areas of A total of 50 tumours were studied and these were rized as follows by using the Working Formulation and REAL classification systems: • 7 B lymphoblastic leukemia/lymphoma (High/l) • 4 B-CLL/small lymphocytic lymphoma (Low/A) • 3 lymphoplasmacytoid/Immunocytoma (Low/A) • 1 Mantle cell (Int/F) • 14 Follicle center ma (Low to Int/D) • 13 Diffuse large cell lymphoma (Int to High/G,H) • 6 Unclassifiable (K) • 2 T cell lymphomas Forty B cell lymphomas were positive for CD22 antigen with the 5/44 antibody at 0.1 mg/ml and a further six became positive when the concentration was sed to 0.5 mg/ml. For the remaining two B cell tumors that were negative at 0.1 mg/ml, there was insufficient tissue remaining to test at the higher concentration. However, parallel testing with another D22 dy designated 6/13, which gave stronger staining than 5/44, resulted in all 48 B cell lymphomas staining positive for CD22.

Thus, it is possible to conclude that the CD22 antigen is widely expressed on B cell lymphomas and thus provides a suitable target for immunotherapy in NHL. c) PCR Cloning of 5/44 VH and VL cDNA sequences coding for the variable domains of 5/44 heavy and light chains were sized using reverse transcriptase to produce single stranded cDNA copies of the mRNA present in the total RNA. This was then used as the template for amplification of the murine V-region sequences using specific oligonucleotide primers by the Polymerase Chain Reaction (PCR). i) cDNA Synthesis cDNA was synthesized in a 20 μl reaction volume containing the following reagents: 50mM Tris-HCl pH 8.3, 75 mM KC1, 10 mM dithiothreitol, 3 mM MgC12, 0.5 mM each deoxyribonucleoside triphosphate, 20 units RNAsin, 75 ng random hexanucleotide primer, 2 μg 5/44 RNA and 200 units y Murine Leukemia Virus reverse riptase. After incubation at 42oC for 60 minutes, the on was terminated by heating at 95oC for 5 s. ii) PCR Aliquots of the cDNA were subjected to PCR using combinations of primers specific for the heavy and light . Degenerate primer pools designed to anneal with the conserved sequences of the signal peptide were used as forward primers. These sequences all contain, in order, a restriction site (VL SfuI; VH HindIII) starting 7 nucleotides from their 5’ ends, the sequence GCCGCCACC (SEQ ID , to allow optimal translation of the resulting mRNAs, an initiation codon and 20-30 nucleotides