A novel antibody-TCR (AbTCR) platform combines Fab-based antigen recognition with gamma/delta-TCR signaling to facilitate T-cell cytotoxicity with low cytokine release

Abstract

The clinical use of genetically modified T-cell therapies has led to unprecedented response rates in leukemia and lymphoma patients treated with anti-CD19 chimeric antigen receptor (CAR)-T. Despite this clinical success, FDA-approved T-cell therapies are currently limited to B-cell malignancies, and challenges remain with managing cytokine-related toxicities. We have designed a novel antibody-T-cell receptor (AbTCR) platform where we combined the Fab domain of an antibody with the γ and δ chains of the TCR as the effector domain. We demonstrate the ability of anti-CD19-AbTCR-T cells to trigger antigen-specific cytokine production, degranulation, and killing of CD19-positive cancer cells in vitro and in xenograft mouse models. By using the same anti-CD19 binding moiety on an AbTCR compared to a CAR platform, we demonstrate that AbTCR activates cytotoxic T-cell responses with a similar dose-response as CD28/CD3ζ CAR, yet does so with less cytokine release and results in T cells with a less exhausted phenotype. Moreover, in comparative studies with the clinically validated CD137 (4-1BB)-based CAR, CTL019, our anti-CD19-AbTCR shows less cytokine release and comparable tumor inhibition in a patient-derived xenograft leukemia model.

Conflict of interest statement

Y.X., Z.Y., L.H.H., P.Z., L.L., B.Z., S.G., J.L., J.F.M., V.W.C., H.L., and C.L. are employees of and have equity ownership and/or stock options in Eureka Therapeutics, Inc. S.A.G. is a scientific advisor to Eureka Therapeutics with stock options in the company. D.M.B. is a consultant to Eureka Therapeutics. Other associations: S.A.G. has received research and/or clinical trial support from Novartis, Servier and Kite. He consults for, has participated in ad boards, or serves on study steering committees or scientific advisory boards for the following companies: Novartis, Adaptimmune, TCR2, Juno, GlaxoSmithKline, Cellectis, Vertex, Roche and Janssen. He is listed on a patent related to toxicity management in cell therapy managed by University of Pennsylvania and CHOP policies.

Figures

Fig. 1. The molecular design, expression, and cellular characterization of our antibody-TCR.

a Schematic of our antibody-TCR (AbTCR) platform compared to a TCR and a second-generation CAR platform. b ET190L1-AbTCR is expressed on the surface of primary T cells following lentiviral transduction. Anti-human Fab antibody was used to detect AbTCR expression. Representative flow cytometry plots and images (40×) of T cells stained with anti-human Fab antibody are shown. c AbTCR expression stabilizes the cell surface presentation of CD3ɛ on TCRβ negative J.RT3-T3.5 cells. d The CD3 complex co-immunoprecipitates with the AbTCR. Cell lysates and anti-FLAG immunoprecipitates from primary T cells expressing a FLAG-tagged ET190L1-AbTCR (or untransduced; Mock) were subjected to western blot analysis for CD3δ, ε, γ, and ζ chains

Fig. 2. AbTCR-T cells have a less exhausted surface phenotype and a higher percentage of naïve and stem cell memory T cells compared to CAR-T cells.

a ET190L1-AbTCR T cells (AbTCR) and ET190L1-CAR-T cells (CAR) were cultured and the number of cells determined at the indicated time points. b–e Flow cytometry analysis of ET190L1-AbTCR-T cells and ET190L1-CAR-T cells at day 10 of in vitro expansion prior to antigen stimulation (b) Proportions of CD4/CD8 within receptor+ cells. c Frequency of naïve (CCR7+ CD45RA+), central memory (CM; CCR7+ CD45RA−), effector memory (EM; CCR7- CD45RA-) and effector (E; CCR7- CD45RA+) T cells within CD8+ receptor+ cells. d Frequency of stem cell memory (SCM; CCR7+ CD45RO- CD95+ CD122+) T cells within CD8+ receptor+ cells. e Expression of T cell differentiation markers CD28, CCR7, and granzyme B (GranB). f Expression of T cell exhaustion markers PD-1, LAG-3, and TIM-3. **P < 0.01

Fig. 3. AbTCR-T cells mediate antigen-specific responses in vitro.

a Expression of activation markers CD69 and CD25 on ET190L1-AbTCR-T cells (AbTCR) and ET190L1-CAR-T cells (CAR) following a 16 h co-incubation with Raji or Raji K/O cells. Raji K/O: Raji cells in which CD19 was knocked out using CRISPR technology. Gated on CD3+ receptor+ cells. b ET190L1-AbTCR-T cells selectively degranulate in the presence of CD19+ target cells. Representative flow cytometry plot showing CD107a expression on ET190L1-AbTCR incubated with target cells for 4 h at an E:T of 1:1. Degranulation in AbTCR+ CD8+ T cells is shown. c ET190L1-AbTCR-T cells selectively kill CD19+ tumor cells and are comparable to ET190L1-CAR-T cells. T cells were incubated with target cells for 16 h at an E:T ratio of 2:1. Cytotoxicity was measured by LDH release assay (n = 3 technical replicates). Error bars, SEM. d ET190L1-AbTCR-T cells and ET190L1-CAR-T cells proliferate at similar rates upon antigen stimulation in vitro. T cells were labeled with CFSE and co-cultured with CD19+ Raji cells and the degree of T cell proliferation was assessed by flow cytometry. e ET190L1-AbTCR CD4+ T cells express lower levels of exhaustion markers following co-incubation with CD19+ tumor cells

Fig. 4. AbTCR-T cells release less CRS-related cytokines than CAR-T cells, including monocyte-lineage-derived IL-6.

a ET190L1-AbTCR-T cells (AbTCR) release less cytokines than ET190L1-CAR-T cells (CAR) during in vitro killing assays. T cells were incubated with target cells for 16 h at an E:T ratio of 5:1. Secreted cytokine levels in the supernatant were measured. Data shown are representative of three independent experiments. b Cytokine expression after trans-well co-culture. T cells, targets (CD19+ NALM-6 cells) were placed in plate wells, and monocyte-lineage cells (APCs) were place in transwell inserts. Supernatants were collected after 18 h of co-cultures

Fig. 5. Robust tumor rejection and reduced cytokine release with ET190L1-AbTCR-T cells in pre-clinical xenograft tumor models.

a ET190L1-AbTCR-T cell therapy shows efficacy in a Raji lymphoma model. Bioluminescent images (left panel) and total flux (right panel) over time of Raji-luc implanted mice intravenously administered with 5 × 106 (1) un-transduced donor-matched T cells (Mock), (2) ET190L1-CAR-T cells (CAR), or (3) ET190L1-AbTCR-T cells (AbTCR). Doses were based on number of receptor-positive cells; n = 6–8 mice/group. b ET190L1-AbTCR treated mice resist Raji lymphoma and NALM-6 B-ALL tumor re-challenge. ET190L1-AbTCR and ET190L1-CAR-treated mice that showed no detectable Raji lymphoma were re-challenged with Raji cells (left panel) or NALM-6 cells (right panel). As controls, Raji-naïve mice were implanted with Raji cells (left panel) or NALM-6 cells (right panel) following an injection of Mock T cells; n = 2–3 mice/group. c ET190L1-AbTCR shows anti-tumor activity in a PDX model of CD80/86 negative primary B-ALL (CHP105R1). Total flux overtime of CHP105R1-bearing mice treated with either un-transduced T cells (Mock), CTL019 T cells, or ET190L1-AbTCR T cells; n = 5 mice/group. d ET190L1-AbTCR-T cells release significantly less cytokines than ET190L1-CAR-T cells in vivo. Serum cytokine levels (pg/ml) collected from Raji-bearing mice 24 h after T cell dosing. IL-2, IL-10, IFN-γ and TNF-α levels are shown. e ET190L1-AbTCR-T cells express significantly less PD-1 levels than ET190L1-CAR-T cells in vivo. PD-1 expression levels (mean fluorescent intensity; MFI) on CAR+ CD4+ and AbTCR+ CD4+ T cells at select times from Raji-bearing mice after T cell infusion. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.