Pro-Apoptotic Effect of An Anti-CD37 Scfv-Fc Fusion Protein, in Combination with the Anti-CD20 Antibody, Ofatumumab, on Tumor Cells From B-Cell Malignancies

SMIP-016, a mouse/human chimeric SMIP™ (mono-specific protein therapeutic) targeting human CD37, was shown to have preclinical antitumor activity against B-cell lymphoid malignancies. SMIP-016 demonstrated synergistic or additive activity in non-Hodgkin's lymphoma cells in combination with rituximab, rapamycin, doxorubicin and bendamustine. TRU-016, a humanized form of SMIP-016, is currently in phase 1 clinical trial in refractory or relapsed patients with CLL or small lymphocytic lymphoma. The aim: The goal of this study was to evaluate the antitumor activity of SMIP-016 against tumor cells from CLL and other B-cell lymphoid malignancies when combined with monoclonal anti-CD20 antibody, ofatumumab (OFA) or routine cytostatics. Material and Methods: Cytotoxic effects of SMIP-016 alone and in combinations with OFA or purine analogues, fludarabine (FA) or cladribine (2-chlodeoxyadenozine; 2-CDA), were assessed ex vivo in peripheral blood samples from 21 untreated CLL patients. Moreover, activity of SMIP-016 alone and combined with OFA was assessed in vitro on cell lines derived from B-cell lymphoblastic leukemia (NALM-6), Burkitt lymphoma (Raji) and diffuse large B-cell lymphoma (Toledo). Effects of that treatment on multiple myeloma (MM) cells, both in vitro (RPMI 8226 line) and ex vivo (bone marrow), were also studied. Cells were cultured with drugs for 24–72 hrs. SMIP-016 was used together with a secondary cross-linking Fc-specific IgG antibody (a-Fc). Antitumor effect was evaluated by the Annexin-V(Ann-V) flow cytometric assay. Apoptotic index (AI) was calculated as a percentage of Ann-V-positive cells. For assessing potential synergistic/additive effects of combinations the combination index (CI) was calculated. Additionally, mitochondrial potential and activation of caspases-8, -9 and -3 were assessed. Expression of several apoptosis-regulating proteins, including Bcl-2 protein and inhibitor of apoptosis protein (IAP) family, were investigated by flow cytometry and Western blot. Results: Both SMIP-016 and OFA were analyzed at the doses ranging from 0.1 to 100 mg/ml for in vitro studies. For ex vivo studies, 10mg/ml of SMIP-016 and 5mg/ml of OFA were chosen, as the lowest doses inducing significant pro-apoptotic effect compared to the control, whereas for in vitro studies, the dose of 1mg/ml of both SMIP-016 and OFA were used. 2-CdA and FA were used at concentrations 50ng/ml and 1mg/ml, respectively. After 48 hrs of incubation of ex vivo CLL cells with study drugs, SMIP-016 treated cells had median AI of 30.5% (vs. control – p<0.01) and OFA treated cells had median AI of 32.0%. Interestingly, in our experiments both SMIP-016 and OFA induced significant loss of mitochondrial potential, activation of caspases-9, -3, and overexpression of Bax protein. 2-CdA and FA induced median AI of 41.0% and 36.3%, respectively. Combination of SMIP-016 and OFA exerted additive effect (CI=0.95), whereas SMIP-016 combined with FA or 2-CdA showed synergistic interactions (CIs <0.8). In the in vitro models, SMIP-016 was significantly active against Raji (median AI 36.1%; vs. control p=0.007) and RPMI 8226 (median AI 40.5%; vs. control p<0.001), but did not affect NALM-6 or Toledo cell lines. In combination studies, SMIP-016 and OFA exerted a synergistic effect (median AI for Raji and RPMI 8226 cells - 58.0% and 70.2%, respectively; CI<0.8). Importantly, both agents induced cytotoxicity at very low concentrations which may decrease their potential side effects in clinical practice. Similar strong activity of these drug combinations were confirmed also in ex vivo cultures of MM patients cells. The mechanism of action for SMIP-016 cytotoxicity in all the examined models was likely apoptosis, triggered evidently by activation of both mitochondrial and external caspase pathways. Additionally, SMIP-016 induced drop of Bcl-2/Bax ratio (p<0.01), downregulation of cIAP1 (p<0.03) and overexpression of Smac/DIABLO (p<0.003) apoptosis-regulation proteins. Conclusion: Our study demonstrated strong pro-apoptotic activity of SMIP-016, especially in combination with CD20-targeting treatment in B cell tumor lines, CLL and MM patient samples. The data presented provides supporting evidence for further combination studies in preclinical models in vivo and may be a promising therapeutic concept for treatment of B-cell lymphoid malignancies, especially CLL, Burkitt lymphoma or MM.

Stromatt:Emergent Product Development Seattle, LLC, Seattle, WA: Employment.