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314 Development of bi-specific chimeric antigen receptor (CAR) T cell therapy for the treatment of AIDS-related B cell malignancies
  1. Ryan Urak,
  2. Saghar Pahlavanneshan,
  3. Ryotaro Nakamura,
  4. John Zaia,
  5. John Baird,
  6. Mary C Clark,
  7. Stephen J Forman and
  8. Xiuli Wang
  1. City of Hope, Duarte, CA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background People living with human immunodeficiency virus (HIV) are at significantly higher risk than the general population of developing cancer, with a lifetime prevalence of cancer diagnosis of 25–40%. Non-Hodgkin lymphomas (NHL) are the most common type of AIDS-defining malignancy and predominantly manifests as Burkitt lymphoma (25%) or diffuse large B cell lymphoma (75%). Although CD19-directed (CD19) CAR T cell therapies are promising in treating B cell NHL, clinical trials have excluded HIV-positive patients due to concerns about safety (infectious complications), viral control, and limited CAR efficacy.

Recent reports have demonstrated the safety and feasibility of using CD19CAR T cell therapy to treat NHL in patients with HIV, indicating a move toward addressing the unmet need of this patient population. We previously designed mono-specific CAR constructs targeting either lymphoma (CD19) or HIVgp120 (N6) that show efficacy against their respective diseases in clinical and preclinical testing, respectively. We hypothesized that a dual construct targeting both antigens could simultaneously target both lymphoma cells and HIV-infected cells in the same individual. Thus, we developed a bi-specific CD19/N6 CAR T cell platform that can target both antigens in a single therapeutic product.

Methods We generated 3 bi-specific CAR constructs (2 tandem and 1 loop) (figure 1A) that incorporate a humanized (hu)CD19 single-chain variable fragment (scFv) and an N6 scFv from an anti-HIV broadly neutralizing antibody (bNAb) into a 2nd-generation CAR backbone. The tandem CARs consisted of N6 and huCD19 scFvs fused with a G4S linker in either huCD19:N6 or N6:huCD19 orientation. The loop CAR was generated by fusing huCD19(VL):N6(VH):N6(VL):huCD19(VH) with a Whitlow linker. All constructs included the CD4 transmembrane domain, a double-mutated IgG4 Fc spacer, 4–1BB co-stimulatory and CD3ζ signaling domains, and EGFRt separated by a T2A ribosomal skip sequence. We tested the 3 bi-specific constructs in healthy-donor T cells using cytotoxicity co-culture assay (figure 1B) against either Raji (CD19+) or 8E5 (gp120+) target cells and confirmed functionality in HIV-positive donors.

Results Although all 3 bi-specific CAR constructs were functional against both CD19 and HIVgp120 antigens, the N6:huCD19CAR tandem CAR demonstrated equivalent or better efficacy against both antigens and could serially target single or alternating antigens (figure 1C,D). Moreover, we successfully generated N6:huCD19CAR tandem CAR T cells using HIV-positive donors and confirmed functionality.

Conclusions The development of the N6:huCD19CAR bi-specific CAR represents a novel CAR T cell therapy that could potentially provide life-saving treatment for patients with HIV-associated NHL through simultaneous targeting of tumor and viral suppression.

Ethics Approval This study was approved by the City of Hope IRB 09025.

Abstract 314 Figure 1

N6:huCD19 bi-specific CAR-mediated elimination of HIVgp120- and CD19-expressing cells. (A) Schema of three tandem designs: N6-huCD19 scfv, huCD19-N6 scfv, huCD19-N6 loop design. (B) Serial killing assay schema. On day 0, healthy donor N6-huCD19 CAR bi-specific T cells were co-cultured with gp120-expressing cells (8E5). After a 48-hour incubation period, the cells were examined to determine the survival of gp120-expressing cells. Simultaneously, CD19-expressing cells were introduced into the co-culture. Following an additional 48-hour incubation, the survival of CD19-expressing cells was quantified. (C) Cytotoxic analysis was performed on Day 2 to evaluate the targeting of HIV gp120. (D) On Day 4, the cytotoxic analysis was conducted to assess CD19 targeting. huCD19 and N6 monospecific CARs were included as control groups.

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