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243 Efficient generation of CD8+ TSCM CD19 chimeric antigen receptor T-cells (CAR-T cells) by treatment with novel cytokine-based scaffolds
  1. Erin Cole1,
  2. Moriah Rabin1,
  3. Niraj Shrestha2,
  4. Jian Hua Zheng1,
  5. Hing C Wong2 and
  6. Harris Goldstein1
  1. 1Albert Einstein College of Medicine, New York, NY, USA
  2. 2HCW Biologics, Miramar, FL, 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 Despite the success of CAR-T therapy in treating relapsed/refractory B-cell malignancies, >40% of patients experience relapse in part due to reduction of CAR-T function and poor persistence. Traditional αCD3/αCD28-based activation of T-cells prior to CAR transduction is associated with production of more differentiated CAR-Ts, corresponding to reduced functional persistence. This may be overcome by targeting early memory T-cells. TSCM-cells are the least differentiated T-cell population with self-renewal capacity and a crucial role in the diversification of immune memory.1 We hypothesize that treatment of T-cells with specific cytokines will drive cell-cycle transitioning to enable lentiviral transduction and expansion of CAR-Ts displaying the TSCM phenotype. Using cytokine/protein scaffolds, we evaluated the impact that IL-7, IL-15, IL-21 and the TGF-β sink (TGFβRII) had on the generation of TSCM-like CAR-Ts.

Methods HCW Biologics has developed a bifunctional fusion molecule (HCW9218), which incorporates the extracellular domain of TGFβRII and an IL-15/IL-15 receptor α complex into a tissue factor-based scaffold.2 A similar scaffold comprised of IL-7, IL-15 and IL-21 (HCW9206) was also produced (figure 1). To generate CAR-Ts, primary T-cells isolated from donor-derived PBMCs are treated with HCW scaffolds prior to transduction with a lentivirus expressing an αCD19-CAR and a GFP reporter. 6-days post transduction we compared T-memory differentiation of HCW-treated CAR-T cells to αCD3/αCD28-activated cells by flow cytometry (figure 2). The functional activity of HCW-treated CAR-Ts was determined by a B-cell leukemia cytotoxicity assay.

Results After stimulation with HCW scaffolds for 3-days prior to transduction, the population of T-cells were enriched with a TSCM-like phenotype, which mediated the generation of CAR-TSCMs (figure 3). HCW-stimulated CAR-Ts also displayed more potent dose-responsive cytotoxic function towards a B-cell leukemia cell line than CAR-T cells generated using traditional aCD3/aCD28 activation (figure 4). Finally, these TSCM-like CAR-Ts will be assessed in future experiments for their longevity in vivo and their functional persistence to a relapse event in a humanized mouse model.

Conclusions HCW scaffolds are a novel class of immunotherapeutic that are currently being evaluated in Phase I clinical trials. We have demonstrated that activation with cytokine scaffolds significantly increases the number of TSCM-like CAR-Ts, while also retaining cytotoxic function. Our results indicated that treatment with cytokine scaffolds generate a large population of CD19 CAR-Ts with an early memory T-cell phenotype which should enhance the persistence of CAR-Ts in patients. This strategy will likely enhance long-term in vivo survival of CAR-T therapy and enable sustained suppression of relapsed/refractory B-cell malignancies.


  1. Arcangeli S, et al. CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome. JCI. 2022;132(12).

  2. Liu B, et al. Bifunctional TGF-β trap/IL-15 protein complex elicits potent NK cell and CD8+ T cell immunity against solid tumors. Molecular Therapy. 2021;29(10):2949–2962.

Ethics Approval This study was approved by Albert Einstein College of Medicine institution’s IRB Ethics Board; approval number 2017–8116.

Abstract 243 Figure 1

2nd generation CD19 CAR and HCW heterodimeric protein constructs. (A) Schematic of the 2nd generation CD19 CAR construct including CDɀ, CD28 and 4–1BB stimulatory domains under the control of an SFFV promoter. A GFP reporter gene is used to assess transduction efficiency. (B) Schematic of the HCW heterodimeric fusion molecules. We will evaluate scaffolds consisting of a TGFßRII extracellular domain linked to a human IL-15Rα sushi domain (HCW9218), a scaffold with the same TGFßRII domain but carrying a D8N nonfunctional mutation in the IL-15 domain (HCW9228), as well as a molecule consisting of IL-15, IL-7 and IL-21 cytokine domains (HCW9206).

Abstract 243 Figure 2

Enhanced transduction of CD8+ T-cells following stimulation with HCW protein scaffolds. (A) Schematic timeline of HCW activation and CD19 CAR transduction of donor-derived T-cells. (B) Donor-derived (HGLK0086) CD4/8 T-cell activation with 100nM HCW protein scaffolds prior to CAR LV transduction, which mediated increased transduction of the CD8+ T-cell subtype.

Abstract 243 Figure 3

Stimulation and expansion of TSCM CD19 specific CAR-T cells. (A) Donor-derived (HGLK0086) CD4/8 T-cell activation with HCW protein scaffolds prior to CAR LV transduction mediates larger expansion of TSCM-like (CD45RO-CCR7+) CAR-Ts compared to αCD3/28 activated CD19 CAR-T cells 6-days post transduction. Gating Strategy: Lymphocytes, Single Cells, CD4-, CD8+, GFP+, CD45RO- CCR7+, CD45RA+ CD95+. (B) Flow cytometric analysis of CD19 CAR-Ts showed that approximately 100% of the CD45RO-CCR7+ CAR-Ts express CD27 and CD95/Fas, which are commonly associated with TSCM memory. Gating Strategy: Lymphocytes, Single Cells, CD4-, CD8+, GFP+, CD45RO- CCR7+, CD95+ CD27+. (C) Percentage of TSCM-like (CD45RO-CCR7+) CD19 CAR-T cells. (D) Average cell count of CD45RO-CCR7+CD45RA+CD95+ TSCM-like CAR-Ts per sample. Data from one donor tested in duplicate are represented as mean ± SEM and statistical significance is calculated by 1-way ANOVA.

Abstract 243 Figure 4

HCW-stimuIated CAR-T cells demonstrated potent cytotoxic function against B-cell leukemic cell line. Through a fluorescence-based PKH26 cytotoxicity assay we compared the cytoxic function of cytokine activated CAR-T cells to αCD3/αCD28 activated CAR-T cells. In this assay, cytotoxic function of CD19 CAR-Ts was measured through fluorescence expression triggered by lysis of a stained CD19 expressing ALL cell line (NALM6) at effector:target ratios of 1:1 (A) and 0.5:1 (B). Data from one donor tested in triplicate are represented as mean ± SEM and statistical significance is calculated by 1-way ANOVA.

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