Inotuzumab ozogamicin (CMC-544) Drug Description

Inotuzumab ozogamicin, (Besponsa® (Pfizer/Wyeth) also known as CMC-544 is an antibody-drug conjugate which consists of a semi-synthetic derivative of N-acetyl ɣ-calicheamicin 1, 2-dimethyl hydrazine dichloride (NAc ɣ-calicheamicin DMH), a potent DNA-binding cytotoxic antibiotic, covalently linked via an acid-labile 4-(4′-acetylphenoxy) butanoic acid (acetyl butyrate) linker to a humanized monoclonal IgG4 antibody, G544. The linker provides stability in physiologic pH and successful calicheamicin release inside the acidic environment of the lysosomes.[1]

The drug, which was approved by the U.S. Food and Drug Administration in August 2017, is directed against the CD22+ antigen present on B cells in all patients with mature B-ALL and most patients (>90%) with precursor B-ALL. [2][3] Inotuzumab ozogamicin is based on an antibody-drug conjugation platform that is similar to gemtuzumab ozogamicin (Mylotarg®; Pfizer/Wyeth-Ayerst Laboratories), a humanized anti-CD33 monoclonal antibody attached to calicheamicin for treatment of patients with relapsed CD33+ acute myeloid leukemia (AML).

While, in pre-clinical models, both inotuzumab ozogamicin and unconjugated G5/44 bind human CD22 with subnanomolar affinity, only inotuzumab ozogamicin exerted potent cytotoxicity against CD22+ B-cell lymphoma cell lines (inhibitory concentration of 50%: 6-600 pM N-acetyl ɣ-calicheamicin dimethyl hydrazide).[4]

In pre-clinical models, the anti-CD22 monoclonal antibody without conjugated cytotoxic drug has shown to have no antitumor activity. Instead, only conjugation with the cytotoxic agent, a derivative of calicheamicin, provided potent dose-depending cellular damage [3]. Furthermore, IgG4 antibodies alone poorly fix complement and therefore cannot cause apoptosis via complement-mediated and antibody-dependent cytotoxicity.[4]

Being an antibody-drug conjugate, inotuzumab ozogamicin preferentially delivers the cytotoxic agent (calicheamicin) to tumor cells and minimizes the exposure of normal tissues resulting in an improved therapeutic index.

Calicheamicin (LL-E33288 complex) is natural product of Micromonospora echinospora ssp. calichensis, a bacterium isolated from chalky soil or caliche. It is considered to be intolerantly toxic when not bound to the antibody [3]. Calicheamicin is linked to the anti-CD22 antibody through 4-(4-acetylphenoxy) butanoic acid (acetyl butyrate), which provides stability in physiologic pH and successful calicheamicin release inside the acidic environment of the lysosomes.

Mechanism of Action
After inotuzumab ozogamicin binds to the CD22 receptor on the surface of B-cells, the CD22 receptor–Inotuzumab ozogamicin complex is internalized forming an endosome. Subsequently, the CD22 receptor–Inotuzumab ozogamicin complex containing endosome fuses with lysosomes, which results in a lower internal pH of the vesicle, leading to the degradation of the acid labile linker and intracellular release of calicheamicin. In turn, calicheamicin, after being released, binds to the minor groove of DNA in a sequence specific manner and breaks double-stranded DNA. This results in cell death because it is not repairable by the cell.

Although CD22 expression is required for inotuzumab ozogamicin activity, scientists observed that efficacy was not dependent on cell surface CD22 expression levels. Cell death, induced by inotuzumab ozogamicin, is completely mediated via calicheamicin-induced apoptosis and not by CD22 signaling.

In xenograft models, inotuzumab ozogamicin showed a greater single-agent therapeutic benefit than either CVP (cyclophosphamide, vincristine, and prednisone) or CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), and it induced superior antitumor activity when coadministered with standard chemotherapeutic regimens. Tumor growth inhibition (>100 days) lasted longer than that by CVP or CHOP [4]

Maximum tolerated dose
Based on a first-in-human clinical trial evaluating the safety of inotuzumab ozogamicin, the maximum tolerated dose (MTD) was determined as 1.8 mg/m2. The trial, which followed dose escalation schedules evaluated 0.4, 0.8, 1.34, 1.8 and 2.4 mg/m2 intravenously (as a 1-hour infusion) once every 3 week included 79 relapsed and refractory NHL patients (the MTD lead-in cohort included 36 patients) . [5]

The study results showed that escalation stop criteria were met as 2 of 6 cohort patients had dose-limiting toxicities (one grade 4 neutropenia, one grade 4 thrombocytopenia) at 2.4 mg/m2. Based on this outcome, 1.8 mg/m2 was established as the MTD.

Although inotuzumab ozogamicin targets CD22+ cells, some observed toxicities reflect possibly nontarget effects. Grade 3 or 4 thrombocytopenia was considered clinically manageable with no major hemorrhages reported.

The pharmacokinetics of inotuzumab ozogamicin shows a nonlinear disposition, with increases in drug exposure with increasing dose or number of doses.  The nonlinearities in drug disposition is a common characteristic for antibodies and thought to be a result of target-mediated disposition in which distribution and elimination are influenced by interaction of antibody with target cells and with binding to neonatal Fc receptor (FcRn), in turn, protecting antibodies from intracellular catabolism. Scientist believe that inotuzumab ozogamicin, at low concentrations, is a substrate for FcRn, which protects the conjugate from systemic elimination. However, at higher inotuzumab ozogamicin concentrations, FcRn may become saturated, rendering a greater proportion of drug susceptible to systemic elimination processes and a more rapid elimination half-life.

Various studies shows that antibodies engineered to have increased binding at acidic pH but retain low binding at neutral pH, exhibit a greater physiologic affinity for FcRn. This in turn may results in a longer half-life in serum and improved pharmacokinetics.

Clinical use
Inotuzumab ozogamicin can be used alone or in combination with other bioactive (chemothjerapeutic) agents to treat patients with B-cell malignancies.

Role of CD22

  • CD22, a terminal α2,6-linked sialic acid-binding cellular lectin (a 135 kDa type I transmembrane sialoglycoprotein)  and a member of the Ig superfamily, is an important modulator of B-cell lymphocyte function and survival.[6][7]
  • CD22 is expressed on the surface of mature B lymphocytes (cells) and their malignant counterparts but not on other non-B lineages including hematopoietic stem cells. This allows for targeted delivery of the cytotoxic agent. Therefore, CD22 targeted chemotherapy is not expected to affect other tissue and should not impact the ability to generate new B-cells. [3]
  • Studies have shown that adding an antibody-drug conjugate targeting CD22, such as inotuzumab ozogamicin, to existing treatments for B-cell non-Hodgkin lymphoma (NHL) may provide additional anti-tumor activity. Currently, approximately 50% of patients with aggressive NHL relapse following treatment with standard of care treatment.[8]
  • Additionally, there is preclinical evidence that an antibody-drug conjugate targeting CD22, such as inotuzumab ozogamicin, may provide antitumor activity against CD22 positive acute lymphoblastic leukemia (ALL). [9]

B-cell and the immune system
B-cells, responsible for antibody production (humoral immunity), are a critical component of the immune response in mammals. Their diversity is critical to the immune system. In humans, each B cell can produce a large number of antibody molecules. Maturation of B-cells, and as a result antibody production, typically ceases or substantially decreases when the foreign antigen has been neutralized. However, occasionally, proliferation of a particular B-cell or plasma cell will continue unabate, which can result in B cell lymphoma or multiple myeloma.

B cell lymphomas include both Hodgkin’s lymphoma (HL) and a broad class of non-Hodgkin’s lymphoma (NHL). Cell lymphomas, such as the B-cell subtype of non-Hodgkin’s lymphoma, are significant contributors to cancer mortality. The response of B-cell malignancies to various forms of treatment is mixed. For example, in cases in which adequate clinical staging of non-Hodgkin’s lymphoma is possible, field radiation therapy can provide satisfactory treatment. Still, nearly half of all patients die from the disease. [10]

Trial results
One clinical trial with inotuzumab ozogamicin found the agent to be highly active in patients with refractory-relapsed acute lymphocytic leukemia (ALL), with an overall response rate of 58% and a median survival of 6.3 months. [11]

Pediatric use
Inotuzumab ozogamicin has shown significant activity in adult patients with relapsed or refractory acute B-cell lymphoblastic leukemia (ALL). In pediatric patients, survival of ALL is poor and the therapeutic options are limited.  In a phase II non-randomized trial clinical trial, researchers investigated inotuzumab ozogamicin in pediatric patients with multiply relapsed ALL. The trial results showed that single agent inotuzumab ozogamicin, given at the single dose of 1.8 mg/m2 every 3 weeks or given as a split, weekly dose was generally well tolerated considering the inherent risks in this population of patients and showed promising activity in pediatric patients with relapsed and refractory ALL.[12]

Clinical studies with Inotuzumab ozogamicin
An overview of clinical trials contains all current and past trials/


Structure of inotuzumab ozogamicin
Fig. 1.0: Structural representation of inotuzumab ozogamicin; N-acetyl ɣ-calicheamicin 1, 2-dimethyl hydrazine dichloride (NAc ɣ-calicheamicin DMH). Inotuzumab ozogamicin has an average loading of 65 to 80 μg ɣ-calicheamicin DMH/mg antibody protein (5-7 molesɣ-calicheamicin DMH/mol antibody). Less than 10% of the antibody protein in inotuzumab ozogamicin is unconjugated. Furthermore, the conjugate preparation contains less than 1 μg unconjugated ɣ-calicheamicin DMH/mg conjugated antibody. (Source: U.S. Patent Application No. 10/428894)  [13]
Manufacturing considerations
Inotuzumab ozogamicin can undergo degradation via a number of degradation pathways. Hence, physical instabilities need to be considered in the development of formulations. One of the main considerations in the development of inotuzumab ozogamicin is that the rate of hydrolysis of calicheamicin from the antibody must be minimized while, at the same time, the physical and chemical integrity of the anti-CD-22 antibody must be maintained. [14]

Precipitation of the calicheamicin-antibody conjugate, which can occur under certain pH and concentration conditions, is a concern and needs to be minimized. In developing a monomeric calicheamicin derivative-antibody conjugate, the pH of the formulation is critical, as this minimizes degradation and physical instability. Scientists have established that a pH of 8.0 minimizes hydrolysis of calicheamicin and maintain adequate solubility of the conjugate.  A pH of 8.0 also guarantees that a significant structural integrity and specificity of the antibody are maintained.  To maintain a pH of 8.0, tromethamine has been selected as a buffering agent. Alternatively, dibasic sodium or potassium phosphate can be used as buffer. The range of buffer concentration can be 5 to 50 mM. A preferred pH range of 7.5 to 8.5 is suggested for optimum stability/solubility. The current pH specification for the finished product is 7.0-9.0.[14]

While the stability of the buffered inotuzumab ozogamicin solution is adequate for the short time, long-term stability is poor.  Lyophilization is used to improve and extend shelf life. However,  problems associated with lyophilization of a protein solution and the loss of secondary, tertiary and quaternary structure may occur during freezing and drying processes. Scientists address this by including sucrose  in the formulation to act as an amorphous stabilizer and maintain the structural integrity of the antibody during freezing and drying. Formulations with a concentrations of 1.5-5.0% w/v sucrose are common.[14]

To enhance the appearance and physical rigidity of the lyophilized cakes at a concentration of 0.5-1.5% by weight, a polymeric bulking agent, such as Dextran 40 or hydroxyethyl starch can be incorporated. These materials form lyophilized cakes at relatively low concentrations and can be used to minimize the overall solids content of the final, lyophilized, formula. In turn, this results more rapid freeze drying. In various formulation studies a Dextran 40 concentration of 0.9% by weight has been used.[14]

Polysorbate 80 is included in the formulation to enhance the solubility. A preferred concentration of 0.01% is used with a potential range of 0.005-0.05%. Tween is also added to the formulation at a concentration of 0.91-0.05% by volume.[14]

To improve the efficiency of the final purification process, an electrolyte may be present in the formula. Typically sodium chloride is used at a concentration of 0.01M to 0.1 M. Additional electrolytes such as sodium sulfate can be used as a replacement for sodium chloride, making lyophilization easier. The final   inotuzumab ozogamicin solution generally comprises 1.5% sucrose (by weight), 0.9% Dextran 40 (by weight), 0.01% tween 80, 50 mM sodium chloride, 0.01% polysorbate 80 (by weight) and 20 mM tromethamine.[13]

[1] Kantarjian H., Thomas D., Wayne A., O’Brien S. (2012b) Monoclonal antibody-based therapies: a new dawn in the treatment of acute lymphoblastic leukemia. J Clin Oncol. 2012 Nov 1;30(31):3876-83. doi: 10.1200/JCO.2012.41.6768. Epub 2012 Aug 13.
[2] Piccaluga P., Arpinati M., Candoni A., Laterza C., Paolini S., Gazzola A., et al. Surface antigens analysis reveals significant expression of candidate targets for immunotherapy in adult acute lymphoid leukemia. Leuk Lymphoma. 2011 Feb;52(2):325-7. doi: 10.3109/10428194.2010.529206. Epub 2010 Nov 15.
[3] Dijoseph J., Armellino D., Boghaert E., Khandke K., Dougher M., Sridharan L., Kunz A, et al. Antibody-targeted chemotherapy with CMC-544: a CD22-targeted immunoconjugate of calicheamicin for the treatment of B-lymphoid malignancies. Blood. 2004 Mar 1;103(5):1807-14. Epub 2003 Nov 13.
[4] DiJoseph JF, Dougher MM, Evans DY, Zhou BB, Damle NK. Preclinical anti-tumor activity of antibody-targeted chemotherapy with CMC-544 (inotuzumab ozogamicin), a CD22-specific immunoconjugate of calicheamicin, compared with non-targeted combination chemotherapy with CVP or CHOP. Cancer Chemother Pharmacol. 2011 Apr;67(4):741-9. doi: 10.1007/s00280-010-1342-9. Epub 2010 Jun 3.
[5] Advani A., Coiffier B., Czuczman M., Dreyling M., Foran J., Gine E., et al. Safety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of B-cell non-Hodgkin’s lymphoma: results of a phase I study. J Clin Oncol. 2010 Apr 20;28(12):2085-93. doi: 10.1200/JCO.2009.25.1900. Epub 2010 Mar 22.
[6] Dörner T, Goldenberg DM. Targeting CD22 as a strategy for treating systemic autoimmune diseases. Ther Clin Risk Manag. 2007 Oct;3(5):953-9.
[7] Crocker PR, Varki A. Siglecs, sialic acids and innate immunity. Trends Immunol. 2001 Jun;22(6):337-42.
[8] Hagemeister FB. Maintenance and consolidation strategies in non-Hodgkin’s lymphoma: A review of the data. Curr Oncol Rep. 2010 Nov;12(6):395-401. doi: 10.1007/s11912-010-0128-x.
[9] Dijoseph JF, Dougher MM, Armellino DC, Evans DY, Damle NK. Therapeutic potential of CD22-specific antibody-targeted chemotherapy using inotuzumab ozogamicin (CMC-544) for the treatment of acute lymphoblastic leukemia. Leukemia. 2007 Nov;21(11):2240-5. Epub 2007 Jul 26.
[10] Devesa SS, Silverman DT, Young JL Jr, Pollack ES, Brown CC, Horm JW, Percy CL, et al. Cancer incidence and mortality trends among whites in the United States, 1947-84. J Natl Cancer Inst. 1987 Oct;79(4):701-70.
[11] Jabbour E, O’Brien S, Huang X, Thomas D, Rytting M, Sasaki K, Cortes J, et al. Prognostic factors for outcome in patients with refractory and relapsed acute lymphocytic leukemia treated with inotuzumab ozogamicin, a CD22 monoclonal antibody. Am J Hematol. 2015 Mar;90(3):193-6. doi: 10.1002/ajh.23901. Epub 2015 Jan 16.
[12] Rytting M, Triche L, Thomas D, O’Brien S, Kantarjian H. Initial experience with CMC-544 (inotuzumab ozogamicin) in pediatric patients with relapsed B-cell acute lymphoblastic leukemia. Pediatr Blood Cancer. 2014 Feb;61(2):369-72. doi: 10.1002/pbc.24721. Epub 2013 Sep 2.
[13] Combination of inotuzumab ozogamicin and torisel for the treatment of cancer. US Patent Office. WO 2013088304 A1
[14] Calicheamicin derivative-carrier conjugates US Patent Office US 8153768 B2