An APRIL Based Chimeric Antigen Receptor to Simultaneously Target BCMA and TACI in Multiple Myeloma (MM) Has Potent Activity in Vitro and in Vivo

Immune based strategies have shown early promise in multiple myeloma (MM), and are likely to prove a valuable addition to the therapeutic platforms for these patients. B cell maturation antigen (BCMA) is a promising therapeutic target with selective expression on plasma cells, including MM cells. In the first report of BCMA targeting therapies, an anti-BCMA scFv-based chimeric antigen receptor (CAR) had clinical activity, albeit with short lived responses (Ali et al, 2016). Observed challenges included low expression level of BCMA, shedding of BCMA and potential loss of expression.

In addition to BCMA, MM cells also express transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) which functions in plasma cell survival and differentiation. TACI is selectively expressed within lymphoid tissue and at reduced levels compared to BCMA, as measured by RT-PCR on normal tissues. TACI and BCMA share a common ligand called a proliferating ligand (APRIL), a compact self-protein forming a trimer and binding with nanomolar affinity to TACI and BCMA. The natural trimerization of APRIL would serve to enhance cell binding, especially important in the case of low levels of target antigen. Concurrent targeting of two tumour antigens, by increasing antigen density, would improve efficacy as well as reducing the risk of antigen negative disease escape.

We quantified APRIL targets on primary MM cells by flow cytometry and observed BCMA expression in all of 41 patients tested (median 1051, range 105-8323 antibodies bound per cell, ABC) with most patients (78%) also expressing TACI (333, 0-21301 ABC). We designed an APRIL based 3^rd generation chimeric antigen receptor (APRIL-CAR) comprising an engineered APRIL protein as antigen-binding domain. Peripheral blood mononuclear cells (PBMCs) were activated, transduced with APRIL-CAR and CD56-depleted before testing against cell lines expressing a wide range of BCMA and TACI (1005-1.5x10^5 and 2200-1.6x10^5 ABC respectively). We confirmed the cytolytic capability of APRIL-CAR against engineered targets, even at the low antigen levels observed in primary cells. Using standard chromium release, there was significant cytolysis of the lowest antigen expressers, with 40.9±3.8% (mean±SEM) and 42.6±5.7% cell death in SUPT1^BCMAlo and SUPT1^TACIlo respectively cf 15.0±5.8% for control SUPT1^NT targets, at an E:T ratio of 4:1 (n=4, p<0.01 and p<0.05). When we lowered the E:T ratio, APRIL-CAR continued to show potent activity, with cytolysis of SUPT1^BCMAlo/ SUPT1^TACIlo targets at 1:10 (71.7±3.8% and 70.4±6.3% cell death respectively cf 21.6±5.8% for SUPT1^NT, n=4, p<0.001 for both) and human myeloma cell lines at 1:32 (MM.1s and U266, 56.2±3.9% and 25.1±3.9% target cytolysis by APRIL-CAR and 4.7±11.6% and 6.6±3.9% by PBMCs NT respectively, n=5, p<0.01 for both).

We confirmed cytolytic activity of APRIL-CAR against primary MM cells. Allogeneic PBMCs transduced with APRIL-CAR and CD56-depleted were co-cultured 1:1 with CD138+ bone marrow MM cells from 5 patients. Despite variable BCMA and TACI expression between patient samples (BCMA 1224-7728 and TACI 563-1213 ABC), tumour cytolysis was evident in all cases (APRIL-CAR 72.9±12.2% specific cell death cf media control 2.8±15.3%; p<0.05).

Finally we tested the in vivo activity of APRIL-CAR in an intramedullary model established with tail vein injection of MM.1s.Fluc cells into NOD scid gamma (NSG) mice. Animals were analysed by bioluminescent imaging and FACS analysis of bone marrow for tumour cells. Complete clearance of established intramedullary disease was seen 5 days after infusion of 5x10^6 APRIL-CAR positive PBMCs in 12/12 animals. Elimination of tumour cells was confirmed in the bone marrow (p<0.01) with concurrent detection of APRIL-CAR T cells by FACS analysis.

In summary we report for the first time a 3^rd generation CAR utilising a natural ligand to simultaneously target two antigens found on MM cells. Our APRIL-based CAR demonstrated potent activity at the low levels of BCMA and TACI found in primary MM cells, at low (physiological) E:T ratios, and in an intramedullary MM model. This APRIL based CAR holds particular promise and deserves testing in clinical studies.