A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor

Abstract

Adoptive immunotherapies composed of T cells engineered to express a chimeric antigen receptor (CAR) offer an attractive strategy for treatment of human cancer. However, CARs have a fixed antigen specificity such that only one tumor-associated antigen (TAA) can be targeted, limiting the efficacy that can be achieved because of heterogeneous TAA expression. For this reason, a more generalized and effective application of CAR therapy would benefit from the capability to produce large panels of CARs against many known TAAs. In this study, we show a novel strategy to extend the recognition specificity potential of a bioengineered lymphocyte population, allowing flexible approaches to redirect T cells against various TAAs. Our strategy employs a biotin-binding immune receptor (BBIR) composed of an extracellular-modified avidin linked to an intracellular T-cell signaling domain. BBIR T cells recognized and bound exclusively to cancer cells pretargeted with specific biotinylated molecules. The versatility afforded by BBIRs permitted sequential or simultaneous targeting of a combination of distinct antigens. Together, our findings show that a platform of universal T-cell specificity can significantly extend conventional CAR approaches, permitting the tailored generation of T cells of unlimited antigen specificity for improving the effectiveness of adoptive T-cell immunotherapies for cancer.

Figure 1

Generation and specific immune recognition by BBIR transduced human T cells in vitro. A. Schematic representation of avidin based Immune Receptor gene constructs containing extracellular avidin as a monomer (mcAV) or dimer (dcAv) fused to the human CD3z cytosolic domain alone (BBIR-z) or in combination with the CD28 co-stimulatory module (BBIR-28z). B. BBIR expression (open histograms) was detected via GFP expression for mcAv constructs, or anti-avidin antibody for dcAV constructs. Staining was performed 5 days after transduction with lentivirus and compared to untransduced T cells (grey filled histograms). Percent CAR transduction is indicated. C. Biotin re-directed dcAV but not mcAV.BBIR T cells secrete IFNγ in response to plate-bound biotinylated, but not non-biotinylated, antibody or scFv (10ng) in overnight culture. Concentration of IFNγ was expressed as mean ± SEM in pg/ml from triplicate wells. D. dcAv.BBIR-z and dcAv.BBIR-28z transduced T cells specifically react against immobilized biotinylated-IgG1. Biotin re-directed dcAv.BBIR-z and dcAv.BBIR-28z T cells secrete IFNγ in response to plate-bound biotinylated antibody in overnight culture at the lowest concentration of 1ng/well. dcAv.BBIR-z, dcAv.BBIR-28z T cells or control GFP cells (10⁵ cells/well) were incubated with plate-immobilized antibody at a concentration range 0 – 100ng/well. Concentration of IFNγ is expressed in pg/ml (means ± SEM; n = 3).

Figure 2

BBIR⁺ T cells exhibit specific effector functions. A. BBIRs respond against immobilized human mesothelin protein when redirected with biotinylated anti-mesothelin scFv or antibody (P4 Biobody and Bio-K1 Ab, respectively). dcAv.BBIR-z, dcAv.BBIR-28z T cells or control GFP cells (10⁵ cells/well) were incubated with 10ng of plate-immobilized mesothelin and with either biotinylated or not, anti-mesothelin antibodies or scFvs (0.1 μg/ml). Overnight culture supernatants were analyzed for human IFNγ cytokine by ELISA. Data represent the means ± SD for 3 different experiments. B. Biotinylated specific molecules retention on the BBIR T cell surface was assessed by flow cytometry. BBIR⁺ T cells were incubated with 10 ng biotinylated reagents Biotin-APC or P4 Biobody (open histograms), and compared to untransduced control T cells (grey). C. BBIRs exhibit effector functions in the presence of free biotin at physiological concentration. BBIR T cells were incubated overnight with Bio-K1 Ab or P4 Biobody painted immobilized mesothelin protein or only with plate-bound biotinylated Abs in the presence of the indicated concentration of biotin. Concentration of IFNγ is expressed as mean ± SEM in pg/ml from triplicate wells. D. BBIR⁺ T cells exhibit effector functions against painted cell surface tumor antigens in the presence of antigen-specific biotinylated antibodies. Left, BBIR T cells respond against painted EpCAM on A1847 cancer cell surface. dcAv.BBIR-28z⁺ or control GFP⁺ T cells (10⁵) were cultured with an equal number of human A1847 unlabeled or labeled with biotinylated anti-EpCAM Ab (0 up to 1000 ng). After overnight incubation, cell-free supernatants were analyzed for human IFNγ by ELISA. Results depict the mean ± SEM of triplicate wells. Upper Right, Detectable surface EpCAM expression (open histograms) after labeling with different concentrations of biotinylated EpCAM Ab was evaluated by flow cytometry. Lower Right, Correlation of detectable Bio-EpCAM mean fluorescence intensity (MFI) on EpCAM⁺ tumors was plotted vs. the production of IFNγ by BBIR-28z T cells when co-cultured with labeled cancer cells.

Figure 3

BBIR⁺ T cells exhibit effector functions against various painted cell surface tumor antigens in the presence of antigen-specific biotinylated antibodies. A. BBIR⁺ T cells exhibit effector functions against multiple antigen specificities. BBIR or GFP transduced T cells were cultured overnight with an equal number of antigen-negative AE17, AE17/mesothelin⁺, AE17/Folate binding protein (FRa)⁺, or A1847 cancer cells. Cell-free supernatant from three independent cultures was harvested after overnight incubation and IFNγ levels were measured by ELISA. Mean IFNγ concentration ± SEM (pg/ml) is shown. B. BBIR T cells can be redirected towards different antigens sequentially. BBIR T cells were cultured with GFP transduced EpCAM⁺ A1847 and AE17/FRa⁺ cell lines at a 1:1:1 ratio. After addition of Bio-EpCAM Ab to cultures for 10 hours, CD3-negative cells were analyzed by FACS to detect for the presence of GFP transduced EpCAM⁺ A1847 cells. A second Bio-MOV18Ab (anti-FRa) was then added to culture for an additional 10 hours, and FACS was repeated to measure for remaining CD3-negative, GFP-negative AE17/FRa⁺ cells. Left, Histograms are shown. Right, Results of tumor cell count analysis of pretreated cultures (pre) and after sequential Bio-EpCAM Ab and Bio-MOV18 Ab targeting of A1847 and AE17/FRa⁺ cells, respectively.

Figure 4

Activity of dcAv.BBIR-28z engineered T cells. A. dcAv.BBIR-28z⁺ T lymphocytes produce inflammatory cytokines in response to painted A1847 tumor cells with biotinylated antibodies: anti-mesothelin (Bio-K1) and/or anti-EpCAM (Bio-EpCAM). BBIR⁺ T cells produced equal levels of (Right) IFNγ, and (Left) Th1 cytokines in response to painted A1847 cells compared with conventional anti-mesothelin P4-28z CAR⁺ T cells. Left, Overnight culture supernatants were analyzed for human IFNγ cytokine by ELISA. Concentration of IFNγ is expressed as mean ± SEM in pg/ml from triplicate wells. Right, Cytokine bead-array analysis of cytokine production by dcAv.BBIR-28z⁺ T cells or P4-28z CAR⁺ T cells. Supernatants from three independent cultures were pooled and assessed after 16h. B. Antigen-specific tumor killing by mesothelin or EpCAM redirected BBIRs. Primary human T cells transduced to express P4-28z CAR or dcAv.BBIR-28z were co-cultured with Cr⁵¹-labeled A1847 cells with painted mesothelin (Bio-K1) or (C) EpCAM (Bio-EpCAM) for 17hrs at the indicated effector to target ratio. Percent specific target cell lysis was calculated as (experimental - spontaneous release) ÷ (maximal -spontaneous release) × 100. Data represent the means ± SD for 3 different experiments. *P ≤ .005 comparing BBIR⁺/Bio-K1 and BBIR⁺/Bio-IgG1 T cells. **P ≤ 0.005 comparing BBIR⁺ and P4 CAR⁺ T cells and ***P ≤ .005 comparing BBIR⁺/Bio-EpCAM and BBIR⁺/Bio-IgG1 T cells. The difference between the cytotoxic activity was statistically significant at given E:T ratio.

Figure 5

dcAv.BBIR-28z⁺ T cells control tumor growth in an ovarian cancer xenograft model. 5×10⁶ A1847 tumor cells were inoculated subcutaneously in the flank of NSG mice. To test the therapeutic efficacy of BBIR⁺ T cells, mice bearing an established tumor (≥150mm³) were inoculated IT with 6×10⁶ BBIR⁺ T cells and Bio-EpCAM Ab (100ng) or BBIR⁺ T cells and Bio-IgG1 Ab (100ng) on day 45, 48 and 51. Additional antibody only injections (100ng) were performed on day 56 and 60. Tumor growth was then monitored as tumor diameter per day. Data represent the means ± SD of 4 mice for each panel presented. P ≤ .005 comparing BBIR⁺/Bio-EpCAM and BBIR⁺/Bio-IgG1 group.