Status - withdrawn from the market in 2010

Organizations involved:

Wyeth – Manuf.; R&D; Tech.; World mark.

Pfizer, Inc. – Parent

Celltech Biologics plc – R&D; Tech.; Former

Fred Hutchinson Cancer Research Center – R&D; Tech.

Protein Design Labs, Inc. – Tech.

Lonza Biologics plc – Tech.

Alusuise-Lonza Group – Parent

Genentech, Inc. – Tech.

University of Glasgow – Tech.

Lederle Laboratories – Former

Columbia University – Tech.; Patent dispute

UCB Group S.A. – Patent dispute

Cross ref.: See Monoclonal Antibodies (entry #300).

Description: Mylotarg is a lyophilized (freeze-dried) formulation of an immunotoxin, gemtuzumab ozogamicin, formed from the chemical conjugation of a recombinant humanized monoclonal antibody (hP67.6) expressed by murine (mouse) NS0 cells and having specificity for CD33 receptors (on leukemic cells) with calicheamicin, a potent bacterial toxin (also referred to as a cytotoxin or antibiotic). The conjugate is formed by covalent linkage (condensation) of a bifunctional linker, 4-acetylphenylacetic acid (AcBut linker), with calicheamicin N-acetyl gamma dimethyl hydrazide (NAc-gamma calicheamicin DMH) and the hP67.6 monoclonal antibody (Mab). The recombinant hP67.6 portion is manufactured by suspension culture of a transformed murine NS0 cell line. Three purification steps substantively remove and/or inactivate retroviruses – low pH (acid) treatment, DAEA-Sepharose chromatography, and viral (nano)filtration. Calicheamicin is obtained from fermentation of Micromonaspora echinospora ssp. calichensis bacteria.

The recombinant hP67.6 Mab is chemically linked to N-acetyl-gamma calicheamicin by a bifunctional linker group. Two (to three) calicheamicin molecules are chemically conjugated with the CD33 monoclonal antibody. Mylotarg has an average loading of calicheamicin on the antibody of 2-3 moles/mole (2-3 calicheamicin attached to each Mab via the AcBut linker). Mylotarg has a molecular weight of 151-153 kDa (reported by FDA as 154 kDa).

Mylotarg was the first immunotoxin (“antibody-targeted chemotherapy”), formed from linkage of an antibody with a (cyto)toxin, to be approved by FDA. Mylotarg was also the first drug specifically approved to treat relapsed acute myelogenic leukemia (AML), with all other therapeutics used for AML unapproved and used off-label for this indication.

The CD33 monoclonal antibody portion of gemtuzumab ozogamicin binds to acute myelogenous leukemia (CD33+) cells, the molecule enters the cells, and calicheamicin is released and kills the cells. CD33 is a sialic acid-dependent adhesion glycoprotein expressed by leukemic blast cells and immature cells of myelomonocytic origin. CD33 antigen is also found on other bone marrow hematopoietic cells, but not on normal pluripotent progenitor (stem) cells.

Calicheamicin is an very potent antitumor agent, over 1,000 times more potent than adriamycin. Once released inside the cell, calicheamicin binds in a sequence-specific manner to the minor groove of DNA, undergoes rearrangement, and exposes free radicals, leading to breakage of double-stranded DNA, and resulting in cell apoptosis (programmed cell death). Calicheamicin is a member of the eneiynes class of antibiotics/antitumor agents having an enediyne group – a large carbon ring containing triple bonds separated by a double bond. These structures readily cyclize (Bergman cycloaromatization reaction), forming benzene, via a highly reactive 1,4-benzenoid diradical intermediate. This diradical can oxidatively cleave DNA. Calicheamicin and related compounds contain a methyltrisulfide group that can be reacted with appropriate thiols to form disulfides, allowing linkage with antibodies and other proteins.

Mylotarg is packaged in 20 mL vials each containing 5 mg of drug conjugate (protein equivalent). Inactive excipients are dextran 40, sucrose, sodium chloride, and monobasic and dibasic sodium phosphate (amounts not reported). The product contains no preservatives.

Nomenclature: CD33 immunotoxin, rDNA [BIO]; Mylotarg [TR]; gemtuzumab ozogamicin [FDA USAN]; Recombinant Humanized Anti-CD33 Monoclonal Antibody (HP67.6 Antibody)-Calicheamicin Drug Conjugate [FDA]; immunoglobulin G4 (anti-(human CD33 (antigen)) (human-mouse monoclonal hP67.6 gamma4-chain)), disulfide with human- mouse monoclonal hP67 6kappa-chain, dimer, methyl ((1R,4Z,8S, 13E)-8-((2-O-(4-(acetylethylamino)-2,4-dideoxy-3-O-methyl-alpha-L-threo-pentopyranosyl)-4,6-dideoxy-4-(((2,6-dideoxy-4-S-(4-((6-deoxy-3-O-methyl-alpha-L-mannopyranosyl)oxy)-3-iodo-5,6-dimethoxy-2-methylbenzoyl)-4-thio-beta-D-ribo-hexopyranosyl)oxy)amino)-beta-D-glucopyranosyl)oxy)-13-(2-((3-((1-(4-(4-amino-4- oxobutoxy)phenyl)ethylidene)hydrazino)-1,1-dimethyl-3-oxopropyl)dithio)ethylidene)-1-hydroxy-11- oxobicyclo(7.3.1)trideca-4,9-diene-2,6-diyn-10-yl)carbamate conjugate [CAS]; 220578-59-6 [CAS RN]; CMA-676 [SY]; hP67.6 monoclonal antibody/N-acetyl-gamma calicheamicin conjugate [SY]; N-acetyl-gamma calicheamicin/hP67.6 monoclonal antibody conjugate [SY]; calicheamicin-CD33 monoclonal antibody conjugate [SY]; CD33 monoclonal antibody/N-acetyl-gamma calicheamicin conjugate [SY]; humanized mP67.6 Mab/N-acetyl-gamma calicheamicin conjugate [SY]; NDC 0008-4510-01 [NDC]

Biological.: Gemtuzumab ozogamicin has three components: a humanized recombinant antibody, hP67.6, targeted against the CD33 antigen, a derivative of the potent cytotoxic agent calicheamicin, and a linker molecule connecting the antibody to the calicheamicin. Such monoclonal antibodies are ideally suited for the treatment of acute myelogenous leukemia (AML) because of the accessibility of leukemic cells in the blood, bone marrow, spleen, and lymph nodes. The antibody portion of gemtuzumab binds specifically to the CD33 antigen on the surface of myeloid leukemia cells. Gemtuzumab ozogamicin is internalized within cells, and calicheamicin is cleaved from the molecule. The hydrazone linkage of the conjugate is presumably hydrolyzed in the acidic environment of the endosome/lysosomes through which the conjugate is routed after cell internalization. This results in release of the cytotoxic agent, NAC-gamma calicheamicin DMH, some of which binds to DNA, causing apoptosis and cell death. Studies in animals have shown that unconjugated calicheamicin derivatives represent < 4% of total derivatives in plasma, indicating that calicheamicin remains bound within gemtuzumab ozogamicin in the bloodstream.

A CD33 murine IgG1 monoclonal antibody, p67.6, originally developed by Fred Hutchinson Cancer Research Center (Seattle, WA), was licensed by Wyeth-Ayerst, now Wyeth. This monoclonal antibody component of the molecule was humanized by Celltech Group Ltd., using its own and technology licensed from Protein Design Labs. Inc. (PDL). Humanization of this Mab involved insertion of a CD33 antigen-binding portion of murine p67.6 into a human IgG4 monoclonal antibody framework, yielding hP67.6. About 98.3% of the amino acids sequence of the hP67.6 monoclonal antibody (constant and framework regions) is of human origin; with the remainder, the complementarity-determining regions (CDR) or epitope-binding regions, being of murine (mouse) origin (from p67.6).

The CD33 antigen is a cellular 67 kDa glycoprotein that functions as a sialic acid-dependent adhesion protein. CD33 is expressed on leukemic myeloid colony-forming cells and on about 90% of acute myelogenous leukemia (AML) myeloblasts, including leukemic clonogenic precursors. CD33 is not expressed on pluripotent hematopoietic stem cells and is not present in other tissues. In vitro studies with CD33 antibodies show rapid internalization by target cells. This allows CD33 antibodies to specifically deliver cytotoxic agents into leukemia cells. Gemtuzumab ozogamicin has an in vitro IC50 ≤0.005 ng/mL against target cells and a specificity index of 4,700 (indicating high ratio of specific activity/toxicity against target cells and lack of toxicity to non-target cells).

The calicheamicins are small molecule (~1.5 kDa) potent antitumor antibiotics that were initially identified by their ability to damage DNA in screening tests. They bind DNA in the minor groove and cause site-specific double strand DNA breakage. Calicheamicins contain two “domains.” The enediyne portion on reductive activation is responsible for DNA cleavage, and the aryltetrasaccharide tail anchors the whole molecule to the DNA minor groove. The aryltetrasaccharide moiety can efficiently inhibit the binding of transcription factors to a target DNA containing TCCT, generally considered to be the preferred canonical calicheamicin binding sequence. In this way, the drug can specifically interfere with DNA-related biological processes, e.g., transcription, and those that depend on protein-DNA interactions. Calicheamicin can interfere with biological processes not simply by cleaving free DNA, but also by displacing a DNA-binding protein through competition or modulation of DNA structure.

Companies.: Mylotarg was jointly developed by Lederle Labs., now Wyeth, and Celltech Group plc. Lederle Labs. began research with calicheamicins in the 1980s. Mylotarg is marketed in the U.S. and internationally by Wyeth and affiliates. Celltech receives unspecified royalties on sales of Mylotarg, and reported receiving £1.5 million in 2003 and 2002 royalties.

In March 2004, Wyeth researchers received the Discoverers Award from the Pharmaceutical Research and manufacturers of America (PhRMA) for development of Mylotarg.

Wyeth is now merged into Pfizer, Inc..

Manufacture: Calicheamicin is manufactured by Wyeth (Pearl River, NY; formerly Lederle Labs.), by conventional fermentation of Micromonaspora echinospora bacteria. The monoclonal antibody is manufactured by Wyeth using Celltech’s proprietary NS0 murine host cell culture technology, and the glutamine synthetase (GS) selection/expression system (originally developed by Celltech, now commercially licensed by Lonza Biologics plc). Bulk conjugate is shipped to Wyeth facilities in Carolina, Puerto Rico (PR), for finishing and packaging. See the Tech. transfer section for further manufacturing information.

FDA class: Drug NDA

Approvals: Date = 20000517; first approval, NDA 21174; accelerated approval; orphan drug designation (granted 11/1999; expires 5/2007)

indications: [full text of "INDICATIONS AND USAGE” section of product insert/labeling, 1/29/2006]:

Mylotarg is indicated for the treatment of patients with CD33 positive acute myeloid leukemia in first relapse who are 60 years of age or older and who are not considered candidates for other cytotoxic chemotherapy. The safety and efficacy of Mylotarg in patients with poor performance status and organ dysfunction has not been established.

The effectiveness of Mylotarg is based on OR rates (see CLINICAL STUDIES section). There are no controlled trials demonstrating a clinical benefit, such as improvement in disease-related symptoms or increased survival, compared to any other treatment. [Note, see the Trials section below regarding OR (objective response) rates].

Status: The initial IND was filed in Nov. 1994. An NDA for accelerated approval was submitted by Wyeth/American Home Products Corp. on October 29, 1999, and received priority review status. The FDA’s Oncologic Drugs Advisory Committee on March 17, 2000 voted that there was sufficient evidence of improved safety and acceptable efficacy to support accelerated approval for Mylotarg for the treatment of patients aged 60 or older with CD33+ relapsed AML. Mylotarg was approved under accelerated approval provisions on May 19, 2000 (approval time of about 7.5 months, ~.62 years). Mylotarg was designated an orphan drug in Nov. 1999. Mylotarg was launched on June 1, 2000. Approval was granted under Subpart H [21 CFR 314.510 (Subpart H)], i.e., based on a surrogate endpoint(s) or on an effect on a clinical endpoint other than survival or irreversible morbidity. Mylotarg has received multiple supplemental NDA approvals since its original May 2000 NDA approval, with most of these for labeling revisions and control supplements.

Mylotarg has not received European Union (EU) approval. In Sept. 2007, the CHMP, EMEA/EU, issued a negative opinion for Mylotarg to treat some patients with acute myeloid leukaemia. The CHMPdecided not to recommend approval, because only a small number of the 277 patients enrolled in the three main trials achieved complete remission, and also because there were no data comparing efficacy to other products. Wyeth planned to request a re-examination of the negative opinion, and continue to work towards EU approval.

On June 22, 2010, Pfizer (which had acquired Wyeth) announced withdrawal of Mylotarg's FDA approval, with the U.S. being the product's major market, i.e., the product was discontinued. Pfizer voluntarily withdraw Mylotarg after a post-approval study (SWOG S0106) raised concerns about safety and clinical benefits. Mylotarg is the first drug approved under the accelerated review program to be withdrawn from the market.

Tech. transfer: U.S. 4,970,198 assigned to American Cyanamid Co. (Lederle Labs.; now Wyeth) describes calicheamicins (LL-E33288 complex) with antibacterial and antitumor activities. Calicheamicin derivatives are also described in 5,037,651 and 5,079,233. All of these compounds contain a methyltrisulfide that can be reacted with appropriate thiols to form disulfides allowing linkage with antibodies. Compounds useful for the synthesis of conjugates with carrier molecules are described in 5,053,394. Conjugates are described in 5,773,001, assigned to American Cyanamid. See also EP 689845.

The FDA Orange Book had reported that 4,970,198 expired in Nov. 2007. The Orange Book now has no entry for the product (1/2013).

The source murine CD33 antibody, m-P67.6, disclosed in J. Clin. Invest., 79, 1153, 1987, was licensed to Lederle/Wyeth by the Fred Hutchinson Cancer Research Center.

U.S. patent 5,877,296, Hamann, et al., “Process for preparing conjugates of methyltrithio antitumor agents,” March 2, 1999, and related patents assigned to American Cyanamid Co. (Lederle Labs.; now Wyeth), describe methods for forming antibody (and other molecule) conjugates with disulfide calicheamicin analogs and their derivatives. The linker molecules, e.g., 4-acetylphenylacetic acid, used to conjugate the cytotoxin at one end and the recombinant monoclonal antibody at the other end requires a carbonyl group on one end for formation of a Schiff’s base, particularly a hydrazone, and a carboxylic acid on the other end. Gemtuzumab ozogamicin is the conjugate of 4-acetylphenylacetic acid condensed with calicheamicin N-acetyl gamma dimethyl hydrazide and CD33 monoclonal antibody. The linker molecule is condensed with calicheamicin N-acetyl gamma dimethyl hydrazide, and the exposed linker’s carboxylic acid group is activated and linked with lysine residues of the antibody. These constructs can be fine-tuned by varying the structure of the linker to “design in” the optimum amount of hydrolytic stability/instability for maximum toxicity to the target cells with minimal toxicity to the non-target cells. Conjugates of methyltrisulfide antibiotics were found to be unexpectedly stable, relative to carbohydrate based conjugates, without loss of activity. In some cases, the conjugates are 100 times more potent than the corresponding conjugates made by the carbohydrate-based linker method, and show reduced cytotoxicity against non-target cell lines. This results in conjugates with up to 10,000-fold selectivity between target and non-target cell lines. This patent covering Mylotarg (and related patents) includes descriptions of the humanization of the murine m-P67.6 Mab to the humanized h-P67.6 Mab and its expression in mammalian NS0 cells using vectors using the GS amplification system.

Wyeth has obtained a nonexclusive license for use of monoclonal antibody humanization design technology/patents from Protein Design Labs. (PDL). Based on a Jan. 2003 CIBC World Markets Report on PDL, the company receives royalties of 3.25% on sales of Mylotarg. The basic PDL technology for antibody humanization is disclosed by Winter in U.S. Pat. No. 5,225,539 and by Adair in WO 91/09967. In Dec. 2010, the U.S. Patent and Trademark Office (the PTO) terminated its interference proceeding between certain PDL's Queen et al., U.S. Patent No. 5,585,089, (the '089 Patent) and certain pending claims of Adair et al., U.S. Application No. 08/846,658, (the '658 Application), assinged to UCB Pharma S.A., in favor of PDL '089 Patent. The PTO held that the involved claims in the '658 Application, were not patentable.

The situation concerning recombinant monoclonal antibody patents, licensing, cross-licensing and disputes is very complex. See the “Tech. transfer (rDNA)” section of the Monoclonal Antibodies entry (#300) further information. Currently, Genentech holds a very strong position in U.S recombinant chimeric and humanized monoclonal antibody design/construction and expression patents with its “New Cabilly” patent (6,113,415), which includes claims from a revoked patent (“Boss” patent) assigned to Celltech Group plc, and a cross-licensing agreement between Genentech and Celltech. With Celltech having assisted in the design and development of Mylotarg, particularly antibody design/construction and expression, and with Genentech’s “New Cabilly” patent including the claims of Celltech’s revoked “Boss” patent and Celltech having (cross)licensed its patents to Genentech for licensing, it seems likely either that Wyeth has taken a license from Genentech or that Genentech will pressure (or file suit against) Wyeth to license its patents.

The glutamine synthetase (GS) gene amplification/expression system used to select and amplify transformed NS0 cells for expression is based patents issued to Celltech Biologics plc, now assigned to Lonza Group plc, subsidiary of Alusuise-Lonza Group. Related patents include U.S. 5,770,359 and 5,747,308 (coassigned to the University of Glasgow). These patents describe dominant selectable markers for use in co-amplification of non-selected genes and transformation of host cell lines to glutamine independence. The GS gene is used in recombinant vectors as a selectable marker along with (an)other gene(s) for expression in cells deficient in GS, e.g., NS0 cells. Only successfully transformed cells are capable of producing their own GS enzyme and surviving in glutamine-deficient culture media, with this allowing easy, reliable selection and amplification of transformed cells. Over 40 companies have licensed GS System technology for various uses. Lonza recently reported that continued development work has enabled yields to be increased from well under 1 gram per liter to 1 to 3 grams per liter.

Wyeth (originally Genetics Inst.) was a licensee of Columbia University’s patents concerning cotransformation, a broadly-useful genetic engineering method allowing selection and isolation of transformed cells. The original patents and license expired in 2000, but Columbia received another patent in 2002 and was again seeking royalties, which Wyeth and other companies challenged in court. Recently, the University decided not to continue to press infringement suits and seek royalties, but the patent office is reexaming the relevant patent, and the university could against pursue infringement and royalties at a later date. See the “Tech. transfer” section of the Recombinant DNA Products entry (#100) for further information.

Trials: The effectiveness of Mylotarg is based on objective response (OR) rates, a surrogate marker for clinical efficacy. There are no controlled trials demonstrating clinical efficacy, such as improvement in disease-related symptoms or survival, compared to any other treatment. In three multi-national Phase II trials, involving 142 patients, Mylotarg monotherapy produced a 26% overall remission rate in patients aged 60 and older with CD33-positive AML in first relapse (with poor prognosis). Mylotarg achieved this acceptable rate of remission, similar to combination chemotherapy, with an excellent safety profile (compared to chemotherapy). Median duration of overall survival for the 142 patients was 5.9 months. The most common side-effects were transient fever, chills and rigors, and infrequent hypotension and shortness of breath. Almost all patients experienced grade 4 neutropenia (low platelet counts) and thrombocytopenia lasting 2-5 weeks after Mylotarg administration. Hair loss, a common side effect of cancer chemotherapy, was not associated with Mylotarg. Mylotarg’s safety profile enabled patients to be treated in an outpatient setting.

Medical: The recommended dosage for treatment of acute myeloid leukemia (AML) is 9 mg/m2 of body surface, administered as a 2-hour intravenous infusion. The recommended regimen is two doses, 14 days apart. Mylotarg can be administered in outpatient settings.

Mylotarg exerts its effects on both cancerous and normal myeloid cells, resulting in severe myelosuppression or immunosuppression (e.g., Grade 3 or 4 neutropenia). Careful hematologic monitoring is required. Full recovery from the hematologic toxicities is not required for the second dose. Since hematopoietic stem cells (myeloid precursors) are spared, immune suppression is reversible and temporary (~6 months). Mylotarg can produce a postinfusion symptom complex of fever and chills and less commonly hypotension and dyspnea during the first 24 hours after administration.

Disease: AML is characterized by a rapid accumulation of abnormal white blood cells in the blood and bone marrow, resulting in severe anemia, infection, and hemorrhage during the course of the disease. If untreated, AML is a rapidly fatal disease. Even with treatment, only about 20% of AML patients survive five or more years. Most treated AML patients have leukemic relapses. Relapsed AML patients typically require prolonged hospitalization, and their prognosis is poor.

AML is the most common type of acute leukemia in adults. The American Cancer Society has estimated that 9,700 new cases of AML occurred in the U.S. in 2000. More than three-fourths of patients with AML are over the age of sixty. Approximately 75% of patients with AML ultimately relapse (poor prognosis), and few patients achieve complete remission.

Market: Upon withdrawal from the U.S market in 2010, an estimated <2,500 patients were receiving Mylotarg treatment annually.

Total sales were reported to be ~$20 million in 2004 and 2003. By now, 2007 sales could be much higher, e.g., doubled to about $40 million, but this is miniscule for a company such as Wyeth.

Mylotarg sales are low, not unexpected for a product with orphan designation and narrow indications:. Mylotarg is approved only for second-line use in patients over age 60 having failed traditional chemotherapy or where chemotherapy is contraindicated. Mylotarg is an important treatment option for older patients with relapsed CD33-positive AML who frequently cannot tolerate conventional combination chemotherapy. Its use may be expected to increase as the U.S. population ages.

The 2007 Average Wholesale Price (AWP) is $2,756.64, and the Direct Price (Manufacturer’s discount price) is $2,297.20. For comparison, the 2005 Average Wholesale Price (AWP) was $2,590.80 ($2,546.28 in 2004), and the Direct Cost (Manufacturer’s discount price) was $2,164.00 ($2,121.90 in 2004)).