Construction and Testing of Two Distinct Humanized CD19-Specific Chimeric Antigen Receptors (CARs) for the Treatment of B-Cell Malignancies
Authors: Gopalakrishnan Ramakrishnan , Matta Hittu , Choi Sunju , Han Xu , Prakash Rekha , Narasappa Nell , Gong Songjie , Chitnis Saurabh , Kahn Michael , Sernas Jennifer , Khan Prottasha , Chaudhary Preet 
Published: October 2019

Abstract
Introduction: Modificiation of T cells using CD19-specific chimeric antigen receptor (CAR) therapy has produced dramatic responses against a number of hematologic malignancies in multiple clinical trials. To date, most of the CARs studied in clinical trials are derived from mouse single chain fragment variable (scFv), which can elicit an immune response when infused into human patients and thereby can limit the persistency of CAR-T cells. Indeed, a subset of patients with limited persistency of infused CAR-Ts has been observed in clinical trials. However, this can be overcome by utilizing the humanized scFv in CAR design. Here, we constructed two new CD19-specific CARs, which are derived from the scFv of two distinct humanized CD19 antibody clones and compared them with the widely used CD19-CAR derived from a mouse scFv (FMC63).
Methods: Lentiviral constructs encoding FMC63-BBZ CAR was generated based on the published sequence of FMC63 scFV and cloned in to a lentiviral vector driven by an EF1α promoter. The FMC63-BBZ CAR sequence was fused in frame with T2A ribosomal skip sequence followed by a puromycin resistance gene (PAC). Humanized CD19-Specific CARs were generated by replacing the FMC63 sequence with two humanized CD19-specific scFv sequences that were derived from two distinct humanized monoclonal antibodies. 293FT cells were used to make lentiviruses and the concentrated viruses were used to infect T-cells from healthy donors, NK92MI and Jurkat-NFAT-GFP (JNG) cells. Surface expressions of CARs were detected by protein-L staining using flow cytometry. Cytolytic activity of CAR-engineered T and NK cells was measured using a 4 hour co-culture assay. Cytokines (IL-2, IFN-γ and TNF-α) were measured from the co-cultured supernatants by ELISA.
Results: We were able to infect primary-T cells, NK92MI and JNG cells using the FMC63 and humanized CAR constructs, and the surface expression of CARs was comparable between them. Co-culture of JNG cells stably expressing the FMC63 or humanized CARs with CD19+ve Raji (Burkitt Lymphoma), Nalm6 (Pre- B-cell acute lymphoblastic leukemia) and BV173 (Chronic Myeloid Leukemia) cancer cells lead to robust expression of GFP (indicative of activated NFAT signaling), CD69 (an activation marker for cytotoxic cells), and a rapid increase in the secretion of IL-2. None of these effects were observed in unmodified JNG cells or with the co-culture of JNG cells stably expressing FMC63 or humanized CARs with CD19-ve HL60 (Acute Promyelocytic Leukemia) and U937 (Histiocytic Lymphoma) cancer cells. Next, we made stable NK92MI cells expressing FMC63, humanized CD19-specific CARs, and a control CAR, followed by cellular cytotoxicity assay. Both FMC63-CAR and humanized CD19-CARs expressing NK92MI cells readily eliminated CD19+ve cancer cells, whereas they had no discernible effect on CD19-ve cancer cells. Furthermore, parental or a control CAR expressing NK92MI cells are without any effect, either with CD19+ve or CD19-ve cancer cells, thereby strictly highlighting the comparable specificity of our humanized CD19-CARs with FMC63-CAR against CD19+ve cells. More importantly, co-culture of NK92MI cells stably expressing FMC63-CAR and humanized CD19-CARs with direct patient samples [CD19+ve B-ALLs and CD19-ve Acute Myelogenous Leukemia's (AMLs)] eliminated only the patient B-ALLs, but without any effect on patient AMLs. Also, increased surface expression of CD69 and robust secretion of cytokines (IFN-γ and TNF-α) were observed only when FMC63 or humanized CD19-CARs expressing NK92MI cells were incubated with CD19+ve patient B-ALLs. Finally, expression of FMC63and humanized CD19-CARs in primary donor T-cells also resulted in the elimination CD19+ve cancer cells along with a significant increase in the secretion of cytokines. Most importantly, both humanized CD19-specific CARs significantly increased the survival of CD19+ve cancer cells bearing NSG mice, whereas the unmodified T-cells or the T-cells expressing a non-specific CAR were without any effect.
Conclusion: Our extensive pre-clinical study clearly shows that humanized CD19-specific CAR-T or NK cells exhibit comparable potency with the widely used FMC63-CAR, which provides the basis for the clinical testing of these humanized CARs in CD19+ve malignancies, thereby allaying the possible concern on immune mediated rejection of mouse scFv based CD19-CAR therapy.

Disclosures
No relevant conflicts of interest to declare.