Preclinical characterization of a novel investigational monoclonal antibody CM313 with potent CD38-positive cell killing activity

Abstract

Introduction: CM313 is currently under clinical investigation for treatments of multiple myeloma, systemic lupus erythematosus, and immune thrombocytopenia. We aimed to report the preclinical profile of the novel therapeutic anti-CD38 monoclonal antibody (mAb) CM313, with an emphasis on the difference with other CD38-targeting mAb.

Methods: The binding of CM313 to CD38 recombinant protein across species was assessed using ELISA. The binding of CM313 to CD38-positive (CD38⁺) cells was detected using flow cytometry assays. CM313-induced complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis on different CD38⁺ cells were assessed by LDH release assays or flow cytometry assays. The effect of CM313 on CD38 enzymatic activity was measured using fluorescence spectroscopy. CM313 immunotoxicity in human blood was assessed using flow cytometry assays, ELISA, and LDH release assays. Anti-tumor activity of CM313 was assessed in multiple mouse xenograft models. Safety profile of CM313 were evaluated in cynomolgus monkeys and human CD38 transgenic (B-hCD38) mice.

Results: There exist unique sequences at complementarity-determining regions (CDR) of CM313, which facilitates its affinity to CD38 is consistently higher across a spectrum of CD38⁺ cell lines than daratumumab. In vitro studies showed that CM313 induces comparable killing activity than daratumumab, including ADCC, CDC, ADCP, apoptosis induced by Fc-mediated cross-linking, and effectively inhibited the enzymatic activity of CD38. However, CM313 showed more potent CDC than isatuximab. In vivo, CM313 dose-dependently inhibited xenograft tumor growth, both as a monotherapy and in combination with dexamethasone or lenalidomide. Furthermore, CM313 was well tolerated with no drug-related clinical signs or off-target risks, as evidenced by 4-week repeat-dose toxicology studies in cynomolgus monkeys and B-hCD38 mice, with the later study showing no observed adverse effect level (NOAEL) of 300mg/kg once weekly.

Discussion: CM313 is a novel investigational humanized mAb with a distinct CDR sequence, showing comparable killing effects with daratumumab and stronger CDC activity than isatuximab, which supports its clinical development.

Keywords: CD38; CM313; monoclonal antibody; novel; preclinical.

Figure 1

Binding affinity of CM313 to CD38. (A) Binding of CM313 to CD38 from different species was assessed using ELISA. EC⁵⁰, Half-maximal effective concentration. ND, Not detected. (B) Binding of CM313 and daratumumab to different CD38 mutants. (C) 3D visualization of epitope differences on the human CD38 molecule recognized by CM313 (DMFolD) and daratumumab. (D) Binding of CM313 to CD38 from different species. (E) Binding of CM313 to CD38-positive cells. CD38-positive (CD38⁺) cell lines were incubated with CM313 or Anti-KLH hIgG1, followed by flow cytometry analysis. Binding intensity was presented as geometric mean fluorescence intensity (gMFI).

Figure 2

Multiple tumor cell killing activity of CM313 in vitro. (A) Dose-dependent ADCC induced by CM313. (B) Dose-dependent CDC induced by CM313. (C) Dose-dependent ADCP induced by CM313. The percentage of phagocytosis was detected by measurement of the percentage of CD14⁺CFSE⁺ macrophages by flow cytometry and presented as mean and standard deviation. (D) Apoptosis induced by Fc-mediated cross-linking. The percentage of apoptosis was detected by measurement of the percentage of annexin V⁺PI^- cells and annexin V⁺PI⁺ cells by flow cytometry and presented as mean and standard deviation. **p <0.01, between the indicated groups. (E) Inhibition of CD38 enzymatic activity.

Figure 3

CM313 induces tumor regression as monotherapy and in combination with dexamethasone or lenalidomide in mouse models. (A) Daudi cell xenograft. CM313 was intravenously injected as indicated. ^** p <0.01, ^*** p <0.001, compared with Anti-KLH hIgG1 group on day 28. (B, C) MM.1R cell xenograft. CM313 was injected intravenously alone (B) or in combination with dexamethasone (C). Dexamethasone was injected intraperitoneally from day 0 to day13. ^* p <0.05, ^** p <0.01, ^*** p <0.001, compared with Anti-KLH hIgG1 group on day 19. (D) RPMI 8226 cell xenograft. CM313 was injected intravenously in combination with lenalidomide. Lenalidomide was injected intragastrically from day 0 to day 20. ^* p <0.05, ^** p <0.01, ^*** p <0.001, compared with Anti-KLH hIgG1 group on day 21; ^### p <0.001, compared with 3.0 mg/kg CM313 + 10.0 mg/kg group on day 21. Dara, daratumumab; Dex, dexamethasone; Len, lenalidomide; TGI%, tumor growth inhibition (%). Tumor volume was presented as mean and standard error. Anti-KLH hIgG1 was used as an isotype control.

Figure 4

Immunotoxicity of CM313 in human blood. (A, B) Binding of CM313 to human blood cells (A) and platelets (B). RT, room temperature. (C) CDC activity of CM313 on human blood erythrocytes. Ramos cells and cell lysis were used as positive controls. (D) Concentrations of IL-4, IL-5, IL-6, IL-1β, TNF-α and IFN-γ after co-incubation of CM313 and healthy human blood. Anti-CD3 was used as positive control and Anti-KLH hIgG1 was used as isotype control. PBS, Phosphate buffered saline.