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1411 Novel BAFF-R CAR T-Cell Therapy for CD19 Antigen-Loss Relapsed B Cell Tumors

Program: Oral and Poster Abstracts
Session: 614. Acute Lymphoblastic Leukemia: Therapy, excluding Transplantation: Poster I
Hematology Disease Topics & Pathways:
Diseases, Leukemia, ALL, Biological, Lymphoma (any), Therapies, CAR-Ts, Lymphoid Malignancies
Saturday, December 1, 2018, 6:15 PM-8:15 PM
Hall GH (San Diego Convention Center)

Hong Qin, MD, PhD1*, Zhenyuan Dong, PhD1*, Xiuli Wang, PhD2, Wesley Cheng, BS1*, Diane Lynne Smith, PhD1*, Joo Y Song, MD3*, Ibrahim Aldoss, MD4, Markus Muschen, MD5,6,7, Stephen J Forman8 and Larry W Kwak, MD, PhD9

1Toni Stephenson Lymphoma Center, Department of Hematology and Hematopoietic Stem Cell Transplantation, Beckman Research Institute of the City of Hope, City of Hope National Medical Center, Duarte, CA
2Center for CAR T Cell Therapy, Department of Hematology and Hematopoietic Stem Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA
3Department of Hematology/HCT, City of Hope National Medical Center, Duarte, CA
4Gehr Family Center for Leukemia Research, City of Hope, Duarte, CA
5Department of Systems Biology, Beckman Research Institute, City of Hope Medical Center, Monrovia, CA
6Department of Systems Biology, City of Hope Comprehensive Cancer Center, Monrovia CA, Monrovia, CA
7Department of Systems Biology, City of Hope Comprehensive Cancer Center, Monrovia CA, San Francisco, CA
8Department of Hematology/HCT, City of Hope, Duarte, CA
9Toni Stephenson Lymphoma Center and Department of Hematology and Hematopoietic Stem Cell Transplantation, City of Hope National Medical Center, Duarte, CA

Background: Chimeric antigen receptor (CAR) T cells against CD19 have shown great potential in treatment of B cell malignancies. However, tumor relapse from antigen loss can limit efficacy. B-cell activating factor-receptor (BAFF-R), a tumor necrosis factor receptor superfamily protein (TNFRSF13C), is another potential B-cell specific target of B cell malignancies. BAFF-R is an especially interesting alternative to CD19 as BAFF-R signaling is a driver of B-cell survival, which may limit the capacity of clonal B-cell tumors to escape therapy by down-regulation of antigen expression. However, while the BAFF/BAFF-R axis has been successfully targeted for autoimmune diseases, the promise for cancer therapy has not yet been fulfilled.

Methods and Results: A humanized, single-chain variable fragment (scFv) derivative of an anti-human BAFF-R antibody (Qin et al. Clin Can Res 2018;24:1114-1123) was engineered onto a second generation CAR construct containing 4-1BB costimulatory and CD3ζ intracellular signaling domains. BAFF-R-CAR T cells demonstrated cytotoxicity against human lymphoma and acute lymphocytic leukemia (ALL) lines. Adoptively transferred BAFF-R-CAR T cells eradicated 10-day pre-established tumor xenografts after a single treatment and were superior to CD19-CAR T cells (not shown) and retained efficacy against xenografts deficient in CD19 expression, including one primary patient-derived xenograft (PDX). Specifically, we modeled disease relapse due to the loss of CD19 by generating CRISPR CD19 gene knock-out of the ALL (Nalm-6) cell line and a gRNA-silenced CD19 gene knock-down of an ALL PDX. We confirmed the absence of CD19 and presence BAFF-R expression, which was unaffected, on the resulting cell lines by surface staining (Fig. 1A). Using transduced CD8 TN cells, we found that CD19-CAR T cells demonstrated cytotoxicity only against wild-type tumor cells, while BAFF-R-CAR T cells maintained significant cytotoxicity against both wild-type and CD19-negative tumors in vitro (Fig. 1B). The therapeutic efficacy of BAFF-R-CAR T cells was tested against human ALL Nalm-6-CD19 deficient xenografts established in NSG mice following IV tumor challenge on day 0 with luciferase-expressing cells. A single dose of 2.5 x 106 CD4 TN + 106 CD8 TN BAFF-R- or CD19-CAR T cells/mouse infused IV on day 11 post tumor implantation completely eliminated established Nalm-6-CD19KO ALL tumors and conferred long-term survival. In contrast, treatment with PBS or identical mixtures of CD19-CAR T cells or non-transduced T cells from the same donor were associated with progressive tumor growth and 100% mortality by Day 60 (Fig. 1C).

Finally, four relapsed, antigen loss primary ALLs obtained after CD19-directed therapy retained BAFF-R expression and activated BAFF-R-, but not CD19-CAR T cells. Specifically, cell surface staining demonstrated CD19 and BAFF-R expression in tumors obtained prior to CD19-targeted therapy. However, post-treatment samples exhibited clear down-regulation of CD19, while retaining positive BAFF-R expression (Fig. 1D, results from a single representative patient shown). The ability of the primary tumor samples to activate either CD19- or BAFF-R-CAR T cells was determined by expression of the degranulation marker CD107a on the CAR T cells. Cryopreserved ALL samples were co-cultured with BAFF-R or CD19 CAR-T cells derived from the same healthy donor in the presence of anti-CD107a antibody for 6 h. Non-transduced T cells (non-CAR) from the same donor were used as a negative control. Activation of CD19-CAR T cells by all four CD19-negative post-blinatumomab therapy tumors was significantly reduced, compared with BAFF-R-CAR T cells and with corresponding available CD19-positive pre-therapy tumors, while BAFF-R-CAR T cells were equally activated by pre- and post-CD19-targeted therapy tumors (Fig.1E-F). We observed similar trends for both CD19- and BAFF-R-CAR T cell activation by pre- and post-CD19-targeted therapy tumors, as measured by specific intracellular CAR T-cell TNF-α and IFN-γ production (not shown).

Conclusion: Taken together, our data suggest that BAFF-R is amenable to CAR T-cell therapy and that targeting it may add to existing alternative strategies to overcome relapse from CD19 antigen loss, such as CD22 CAR T cells. Future strategies combining dual targeting of CD19 and BAFF-R may also be effective.

Disclosures: Forman: Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

*signifies non-member of ASH