Abstract
Introduction
Despite the development of potent therapeutics, multiple myeloma (MM) remains an incurable disease due to high instances of relapse and resistance (RRMM). Monoclonal antibodies (mAbs), which function to activate immune cells against target cells, have been extensively explored for the treatment of MM. Daratumumab (Dara), an FDA-approved anti-CD38 mAb, has significantly increased the lifespan of MM patients; however, most patients inevitably develop resistance. CD38 has proven to be a desirable immunotherapy target, as it is highly overexpressed on the surface of MM cells. Dara binds CD38 on MM cells with robust affinity, and then engages immune effector cells, such as natural killer (NK) cells and macrophages, through binding to Fc gamma receptors (FcγRs). This interaction elicits antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), resulting in strong anti-tumor efficacy. Unfortunately, mechanisms of acquired resistance due to depletion and loss of function of immune effector cells remain a major challenge, highlighting the necessity for improved therapeutic interventions. Analysis of patient samples after relapse from Dara-based treatments has revealed that MM cells retain targetable CD38 surface expression, while CD38 is downregulated on non-cancerous immune cells. As such, CD38 remains an excellent target to treat RRMM with CD38 mAbs, albeit invoking a different mechanism of action. Antibody-drug conjugates (ADCs) are an attractive alternative for treating RRMM patients with diminished immune systems, as they utilize the specificity of an antibody to deliver a highly toxic payload directly to tumor cells. While Dara has been investigated as an ADC, it has shown insufficient efficacy, highlighting the need to explore novel anti-CD38 antibodies.
Methods
We have discovered a panel of unique anti-CD38 antibodies and performed extensive biophysical and cellular analysis by surface plasmon resonance (SPR), differential scanning fluorimetry (DSF), X-ray crystallography, and flow cytometry. Advanced protein engineering, such as affinity maturation, Fc modification, and site-specific conjugation, was performed to develop enhanced ADCs. In vitro anti-MM efficacy of these ADCs was evaluated via MTT viability assays against several MM cell lines with varied CD38 expression levels. In vivo efficacy was also investigated in NSG mice engrafted with MM.1S GFP/Luc+ cells. Tumor burden was monitored weekly via bioluminescence imaging of mice.
Results
We have engineered an anti-CD38 ADC with optimized affinity, abolished Fc gamma interaction, and site-specific conjugation to the tubulin polymerization inhibitor, monomethyl auristatin F (MMAF). Biophysical analysis has elucidated favorable binding kinetics, thermostability, and binding epitope of the fragment antigen binding (Fab) domain of our lead candidate, mAb 1. While SPR reveals the Fab of mAb 1 has a reduced affinity to recombinant CD38 compared to the Fab of Dara, flow cytometry shows mAb 1 binds specifically to CD38+ cells at similar levels to Dara. Furthermore, the crystal structure illustrates mAb 1 and Dara bind non-overlapping epitopes, although these mAbs cannot bind simultaneously due to steric hindrance. In vitro studies demonstrate mAb 1 conjugated to MMAF has CD38+ cell-specific killing activity with superior potency compared to Dara using the same conjugation strategy. Additionally, Trastuzumab (Tras), which targets an antigen not expressed on MM cells, had no killing activity when conjugated to MMAF. Finally, preliminary in vivo results suggest mice treated with mAb 1 conjugated to MMAF has elicits potent and dose-dependent anti-tumor responses compared to mice treated with Tras conjugated to MMAF or unconjugated mAb 1.
Conclusions
Patients who have relapsed from CD38 targeted therapy are often transferred to B-cell maturation antigen (BCMA)-directed treatments, but many patients continue to experience relapse. The targeted delivery of a toxic payload represents an orthogonal approach to current treatment options for RRMM. These preclinical data establish that mAb 1 has enhanced efficacy compared to Dara when used as an ADC, which can be attributed to its distinct kinetics and binding epitope. This strategy addresses a critical unmet need and may represent a valuable addition to the evolving treatment landscape for RRMM.
