A Rational Strategy for Reducing On-Target Off-Tumor Effects of CD38-Chimeric Antigen Receptors by Affinity Optimization

Chimeric antigen receptors (CARs) can effectively redirect cytotoxic T cells toward highly expressed surface antigens on tumor cells. The low expression of several tumor-associated antigens (TAAs) on normal tissues, however, hinders their safe targeting by CAR T cells due to on-target/off-tumor effects. Using the multiple myeloma (MM)-associated CD38 antigen as a model system, here, we present a rational approach for effective and tumor-selective targeting of such TAAs. Using “light-chain exchange” technology, we combined the heavy chains of two high-affinity CD38 antibodies with 176 germline light chains and generated ∼124 new antibodies with 10- to >1,000-fold lower affinities to CD38. After categorizing them into three distinct affinity classes, we incorporated the single-chain variable fragments of eight antibodies from each class into new CARs. T cells carrying these CD38-CARs were extensively evaluated for their on-tumor/off-tumor cytotoxicity as well as CD38-dependent proliferation and cytokine production. We identified CD38-CAR T cells of ∼1,000- fold reduced affinity, which optimally proliferated, produced Th1-like cytokines, and effectively lysed CD382+ MM cells, but spared CD38+ healthy hematopoietic cells in vitro and in vivo. Thus, this systematic approach is highly suitable for the generation of optimal CARs for effective and selective targeting of TAAs

CD38 as a therapeutic target for adult acute myeloid leukemia and T-cell acute lymphoblastic leukemia.

Acute leukemia is a clonal expansion of malignant hematopoietic cells leading to impaired production of normal blood cells in the bone marrow (BM). Acute leukemia can be classified, according to the lineage involved, into acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL).1 AML is the most common form of acute leukemia in adults, accounting for over 80% of all diagnosed acute leukemias. Despite advances in the understanding of disease pathogenesis, the standard treatment protocols for adult acute leukemia have remained largely unchanged for the past decades. As a result, the overall 5-year survival in AML patients over 65 years of age is less than 5%,2 and, although the initial responses are better in ALL, the overall outcome of relapsed ALL in adults is poor, with an overall 5-year survival of less than 7%.3 Hence, more and novel treatment options are warranted for both types of adult acute leukemia. Targeted immunotherapies, including monoclonal or bispecific antibodies and chimeric antigen receptor (CAR) T cells, are currently being developed and clinically tested in a wide variety of hematologic malignancies. CD38 is a transmembrane glycoprotein that can function as an adhesion partner for CD31 or as multifunctional ectoenzyme involved in the catabolism of NAD+ and NADP. In the hematopoietic system, CD38 is expressed by lymphoid and myeloid cells, and is also expressed on red blood cells and platelets, with the highest expression found on plasma cells. This uniform, high expression on healthy and malignant plasma cells makes CD38 an ideal target for targeted immunotherapy in multiple myeloma (MM). Indeed, CD38 monoclonal antibodies (daratumumab, isatuximab, and MOR202) have been shown to be effective in relapsed or refractory MM patients and their use is gradually moving towards front-line therapy.