Hematology Meeting Reports 2008;5(6):74-77 SESSION V J.F. DiJoseph1,3 CMC-544 (inotuzumab ozogamicin): K. Khandke1 M.M. Dougher1 A CD22-targeted immunoconjugate of D.Y. Evans1 D.C. Armellino1 calicheamicin P.R. Hamann2 N.K. Damle1 1Oncology Discovery CMC-544 (Inotuzumab ozogam- calichDMH inside the cells. Research, 2Chemical and icin) is a CD22-targeted immuno- CalichDMH is a derivative of a Screening Sciences Wyeth Research, Pearl River, NY; conjugate of calicheamicin cur- potent cytotoxic agent gamma 3Wyeth Research, Pearl River, rently being evaluated in phase III calicheamicin, a natural product NY, USA clinical trials in patients with non- synthesized by a strain of Hodgkin’s B-cell lymphoma. Micromonospora echinospora. CMC-544 is the product of a col- Upon internalization, the conju- laboration between Wyeth and gate is taken up by the lysosomes, UCB-Celltech. CMC-544 has where, under the acidic conditions demonstrated significant clinical in the lysosomes, the acid-labile activity in both follicular and dif- hydrazone functional group with- fuse large B-cell lymphoma in the AcBut linker is hydrolyzed patients who had failed multiple allowing for the release of therapies.1,2 CMC-544 (Figure 1) calichDMH. CalichDMH diffuses is a humanized IgG4 anti-CD22 into the nucleus where it binds antibody (G5/44) covalently DNA in the minor grove and linked to N-acetyl gamma undergoes thiol-dependent struc- calicheamicin dimethyl hydrazide tural changes in its enediyne moi- (CalichDMH) via an acid-labile ety (war-head) to generate a di- 4-(4-acetylphenoxy)butanoic acid radical that abstracts hydrogens (AcBut) linker.3,4 CD22 is a B- from the phosphodiester back- lymphoid lineage-specific differ- bone of DNA producing double- entiation antigen expressed on the strand DNA breaks leading to cell surface of both normal and malig- death.5-7 nant B cells. CD22 is not For calicheamicin conjugates, expressed on hematopoietic pro- the linker stability was optimized genitors of the B-lymphoid line- with the selection of the spacer age. CD22 efficiently internalizes and the attached groups for plas- upon binding to an antibody mak- ma stability versus the ability to ing it an ideal candidate for target- release calichDMH in the cellular ed drug delivery. Targeting malig- lysosomal compartment.8 The nant B-cells through CD22 by the AcBut linker with the hydrazone CD22 specific antibody G5/44 functional group produced the could, therefore, be an efficient best balance between stability in way to deliver calicheamicin to pH 7.4 plasma and hydrolysis in destroy malignant B-cells. the acidic (pH 4.5) lysosomal CMC-544 binds CD22 with compartment. The stability of subnanomolar affinity, and upon CMC-544 was confirmed in both binding to CD22, is rapidly inter- human plasma and serum by mon- nalized delivering conjugated itoring the generation of | 74 | New Drugs in Hematology Figure 1. CMC-544: inotuzumab ozogamicin. CD22-mediated cellular internalization of the conjugate. In various in vivo models of human B-cell lymphomas, CMC-544 caused dose- dependent regression of both small and large established B-lymphoma xenografts.3 It was active over the dose range of 10 µg of conju- gated calichDMH/kg (minimum efficacious dose) to 160 µg of conjugated calichDMH/kg (dose producing long term xenograft cures, for these studies CMC-544 was administered 3 times, Q4Dx3). CMC-544 caused long-term survival of SCID mice with systemically dis- seminated human B-lymphoma.10 In this dis- Figure 2. Stability of CMC-544 in human blood. seminated human B-lymphoma model, when suboptimal doses of CMC-544 were combined with suboptimal doses of rituximab, superior extractable unconjugated calichDMH from the anti-tumor activity was derived by the combi- antibody over a 4 day period using plasmon nation of these agents over either drug admin- resonance analysis.9 In either human plasma or istered alone.11 The targeting antibody G5/44 serum, CMC-544 remained relatively stable, had no effect on the growth of B-cell lym- hydrolyzing at a rate of 1.5-2% per day. phomas xenografts in nude mice over a wide (Figure 2). range of dosages, consistent with its effector CMC-544 has been studied extensively in function-deficient IgG4 isotype. CMC-544 preclinical models of human B-cell lym- therefore, is regarded as an antibody-targeted phoma. CMC-544 binds CD22 with a high chemotherapy agent rather than an immuno- affinity (KD=235 pM). It inhibited the in vitro therapeutic agent such as rituximab. Largely + growth of a number of CD22 cell lines (IC50s due to its tumor-targeted drug-delivery capa- 6-300 pM) much more potently than unconju- bility, it is anticipated that CMC-544 will have gated calichDMH, consistent with the active a better therapeutic index than that of conven- Hematology Meeting Reports 2008;2(5) | 75 | J.F. DiHoseph et al. (AUC0-∞) of CMC-544 in tumor-bearing mice than that in non-tumor-bearing mice is consis- tent with the tumor uptake of CMC-544. Acute lymphocytic leukemia (ALL) is pri- marily a B-cell or pre-B-cell malignancy. ALL blasts differentially express a number of B- lymphoid specific antigens including CD19, CD20 and CD22. The CD22 expressed on these blasts may allow preferential targeting by CMC-544. In order to investigate the effect of CMC-544 in ALL, mice were injected with Reh cells (a CD22+ ALL derived cell line) in the lateral tail vein and monitored for disease symptoms.13 Mice treated with vehicle (PBS) Figure 3. Effect of CMC-544 on human CD45+ cells engrafted in developed hind-limb paralysis. All of the vehi- bone marrow of scid mice with disseminated ALL. Mice were injected intravenously with Reh ALL cells. At the time of the cle-treated mice succumbed to the disseminat- appearance of hind limb paralysis in the vehicle-treated mice, ed disease by day 77. The average survival bone marrow cells were collected and analyzed by FACs for the time for the group was 55 days. CMC-544, presence of human CD45. Bone marrow from CMC-544 treated mice was also collected at the corresponding times for analysis. administered at a dose of 80 µg/kg of calichDMH (Q4Dx3) produced 100% survival of the treated mice over the 127 day observa- tion period. At the dose of 4 µg/kg Calich- tional chemotherapeutic agents. DMH (Q4Dx3), 20 fold lower than the cura- To determine the serum levels of CMC-544 tive dose of 80 µg/kg, 90% of the mice still that were associated with the anti-tumor survived throughout the observation period. effects described above, tumor-bearing (RL B- In this disseminated disease model, four lymphoma xenograft) or non-tumor-bearing vehicle-treated mice were killed when they ini- nude mice received a single dose of CMC-544 tially exhibited hind-limb paralysis (days 35, ip (160 µg of conjugated CalichDMH/kg) and 41, 41, and 43). At equivalent time points serum samples from these mice were assayed (days 36, 41 and 43), 3 CMC-544-treated mice for the presence of CMC-544 using an were randomly selected and killed. Flow cyto- enzyme-linked immunoassay.3 The immunoas- metric analysis of bone marrow cells collected say was specifically designed and validated to from the femur of these vehicle-treated mice detect antibody conjugates of CalichDMH.12 demonstrated the presence of human CD45+ The mean (± SE) Cmax values for non-tumor- leukemic cells (Figure 3). CMC-544 (80 bearing and RL BCL-bearing nude mice, µg/kg, Q4Dx3) potently inhibited the engraft- respectively, were 2.6±0.1 and 2.4±0.2 µg/mL ment of the human CD45+ cells. (CalichDMH equivalents of CMC-544), the t1/2 In summary, CMC-544 is a potent antibody- were 34.2 and 35 h, and the corresponding targeted chemotherapeutic agent that has mean AUC0-∞ values were 145 and 93 µghr/mL demonstrated potent anti-tumor activity in pre- in non-tumor-bearing and RL BCL-bearing clinical models of B-cell lymphoma and mice, respectively. The difference between the leukemia. CMC-544 should be considered as a AUC0-∞ values for non-tumor-bearing mice and targeted chemotherapeutic agent since the RL tumor-bearing mice was statistically signif- unconjugated targeting antibody G5/44 is inac- icant (p<0.05). The lower systemic exposure tive as a therapeutic agent. CMC-544 is | 76 | Hematology Meeting Reports 2008;2(5) New Drugs in Hematology expected to have a better safety profile than py of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22. Cancer non-targeted chemotherapeutics due to the Immunol Immunother 2005;54:11-2. 5. Lee MD, Dunne TS, Chang CC, Siegal MM, Morton preferential delivery of the potent cytotoxic GO, Ellestadt G et al. Calicheamicins, a novel family of agent calicheamicin to the tumor associated antibiotics. 4. Structural elucidations of calicheamicins. J Am Chem Soc 1992;114:985-7. antigen via the targeting antibody G5/44. 6. Zein N, Sinha A, McGahren W, Ellestad G. Calicheamicin γI: an antitumor antibiotic that cleaves Phase I clinical data demonstrated that CMC- double-stranded DNA site specifically. Science 1988; 544 is active in non-Hodgkin’s B-cell lym- 240:1198-201. 7. Thorson J, Sievers E, Ahlert J, Sheperd E, Whitwam RE, phoma with manageable toxicity. Expanded Onweume KC et al. Understanding and exploiting Nature’s chemical arsenal: The past, present and future of clinical trials (phase 2/3) are currently being calicheamicin research. Current Pharmaceut Design conducted with CMC-544 in both follicular 2000; 6:1841-79. 8. Hamann P, Hinman L, Hollander I, Beyer C, Lindh D, non-Hodgkin’s lymphoma and diffuse large B- Holcomb R et al.