Overcoming CD19-Negative Relapses in Patients with B-Cell Lymphomas Treated with Tisagenlecleucel

Anti-CD19 chimeric antigen receptor T cell (CART) immunotherapy has revolutionized the treatment of large B-cell lymphomas (LBCL). However, up to 70% of LBCL patients (pts) ultimately fail CART treatment. Several mechanisms of relapse have been described, such as T cell exhaustion, an immunosuppressive tumor microenvironment, and antigen-negative escape. While antigen-negative relapse after anti-CD19 CART (CART19) is well studied in acute B lymphoblastic leukemia, less data are available for LBCL, especially in pts treated with the 4-1BB costimulated CART19, tisagenlecleucel (tisa-cel, formerly CTL019). In this study, we evaluated the frequency of CD19 loss in LBCL pts treated with tisa-cel and identified CD79 as an antigen highly expressed by immunohistochemistry (IHC) in CD19-negative biopsies. We then generated and optimized an anti-CD79 CART (CART79) and subsequently cloned a dual anti-CD79b/anti-CD19 CART that showed high potency against CD19-neg models.

We first studied an exploratory cohort of 38 pts treated at the University of Pennsylvania on the first single-center clinical trial of CTL019 for B-cell lymphomas (NCT02030834 and Chong, NEJM, 2021). Among relapsed or refractory pts (n=23, 60.5%), 17 pts had a biopsy at disease progression (PD). We analyzed CD19 and CD79a expression in these biopsies by IHC. Four biopsies (23.5%) showed no CD19 expression by neoplastic cells. Also, 12 pts had available matched biopsies pre-CTL019 infusion (performed within 1 year before infusion) and at PD. We observed that 3/12 (25%) pts converted from a CD19-positive to a CD19-negative tumor after CTL019. Notably, 100% of biopsies at PD showed high CD79a expression. We then studied a confirmatory cohort including 110 LCBL pts treated with commercial tisa-cel. In this retrospective cohort, 63 pts had PD, and 31 had an available biopsy at PD. In line with previous observations, 10/26 (38%) showed loss of CD19 at PD, and, of note, 5/23 (22%) loss of CD20, a B-cell marker targeted by previous therapies. Again, 100% of pts maintained CD79a expression. (Figure A). These data confirm that loss of detection of CD19 at the level of sensitivity of IHC is frequently observed at PD and may be a cause of resistance in LBCL pts treated with tisa-cel. In addition, persistence of CD79 expression suggests that it may be an optimal therapeutic target for potentially preventing CD19-negative PD.

Therefore, we aimed to generate and optimize a CART79 product. We cloned and screened a large panel of anti-CD79 CAR constructs (n=32) generated using 6 different scFvs (targeting either CD79a or CD79b), two scFv chain orientations (H to L vs. L to H), three hinges (derived from CD8a, IgG4, or truncated IgG4). We used the CD8 transmembrane and 4-1BB co-stimulatory domains of tisa-cel, given its established clinical safety and efficacy profile. The constructs were initially screened in-vitro against LBCL cells (OCI-Ly18), evaluating cytotoxicity, proliferation, and cytokine production. The six top candidates were then tested in-vivo using human xenograft models (NSG mice subcutaneously engrafted with OCI-Ly18 cells). When the tumor was palpable, mice were randomized to receive one dose (2.5x10⁶/mouse) of CART79 candidates, CART19, or untransduced T cells (UTD) intravenously. An anti-CD79b CAR construct (CART79#3: H-L orientation, truncated IgG4 hinge), demonstrated the strongest anti-tumor effect.

This construct was incorporated into a bicistronic vector also containing anti-CD19 CAR (FMC63-based). We tested the activity of this novel anti-CD19/CD79b dual CART in-vitro, using CRISPR/Cas9 gene editing to knock out (KO) either CD19 or CD79b in HBL-1 LBCL cells. CD19 KO and CD79b KO HBL-1 cells were mixed 1:1 to simulate the heterogeneity of lymphomas and tumor targeting was tested for CART19, CART79b, dual CART79b/19, and UTD. In both in-vitro and in-vivo models, dual CART19/79b demonstrated a strong anti-tumor effect, possibly preventing or treating CD19-negative escape (Figure B).

In conclusion, our study demonstrates that CD19 loss by IHC is a marker of treatment failure in tisa cel-treated LBCL pts. We also demonstrate that CD79, as opposed to CD20, is expressed in virtually all cases of PD and is an ideal target for next-generation CART immunotherapy. Finally, we developed a dual CART19/79b that shows high activity against CD19-negative disease progression. This construct will be taken to phase I clinical studies.

Ghilardi:ViTToria Biotherapeutics: Consultancy. Patel:viTToria biotherapeutics: Consultancy, Research Funding. Chong:KITE: Consultancy; Tessa: Consultancy; Juno/BMS: Consultancy; Novartis: Consultancy; Beigene: Consultancy. Dwivedy Nasta:Roche: Research Funding; Pharmacyclics: Research Funding; FortySeven/Gilead: Research Funding; Rafael: Research Funding. Barta:Affimed: Consultancy; Kyowa Kirin: Consultancy, Honoraria; Seagen: Honoraria; Daiichi Sankyo: Consultancy; Janssen: Other: Independent Data Monitoring Committee member; Acrotech: Honoraria. Garfall:Janssen, Novartis, Tmunity, CRISPR Therapeutics: Research Funding; Janssen: Other: Independent data monitoring committee; Janssen, GSK, Amgen, Legend: Consultancy. Landsburg:Curis, Inc: Research Funding; Epizyme: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Calithera: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Triphase: Research Funding. Svoboda:TG: Research Funding; SEAGEN: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Merck: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Consultancy; BMS: Consultancy, Research Funding; Atara: Consultancy; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; ADCT: Consultancy. Schuster:AbbVie: Research Funding; Adaptive Biotechnologies: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; DTRM: Research Funding; Genentech: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Juno Therapeutics: Consultancy, Research Funding; Merck: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Research Funding; Pharmacyclics: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Consultancy; BeiGene: Consultancy; Genmab: Consultancy; Fate Therapeutics: Consultancy; Legend Biotech: Consultancy; Loxo Oncology: Consultancy; Morphosys: Consultancy; Mustang Biotech: Consultancy; Nordic Nanovector: Consultancy; Regeneron: Consultancy; N/A: Patents & Royalties: Related to CD19 CAR T cells and autologous co-stimulated T cells. Ruella:NanoString: Consultancy, Research Funding; viTToria Biotherapeutics: Other: Scientific Founder, Patents & Royalties: related to CD19 CAR T cells; Bayer: Consultancy; GlaxoSmithKline: Consultancy; BMS: Consultancy; AbClon: Consultancy, Research Funding; Beckman Coulter: Research Funding.