CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation

Abstract

Background: The adoptive transfer of T cells redirected to tumor via chimeric antigen receptors (CARs) has produced clinical benefits for the treatment of hematologic diseases. To extend this approach to breast cancer, we generated CAR T cells directed against mucin1 (MUC1), an aberrantly glycosylated neoantigen that is overexpressed by malignant cells and whose expression has been correlated with poor prognosis. Furthermore, to protect our tumor-targeted cells from the elevated levels of immune-inhibitory cytokines present in the tumor milieu, we co-expressed an inverted cytokine receptor linking the IL4 receptor exodomain with the IL7 receptor endodomain (4/7ICR) in order to transform the suppressive IL4 signal into one that would enhance the anti-tumor effects of our CAR T cells at the tumor site.

Methods: First (1G - CD3ζ) and second generation (2G - 41BB.CD3ζ) MUC1-specific CARs were constructed using the HMFG2 scFv. Following retroviral transduction transgenic expression of the CAR±ICR was assessed by flow cytometry. In vitro CAR/ICR T cell function was measured by assessing cell proliferation and short- and long-term cytotoxic activity using MUC1+ MDA MB 468 cells as targets. In vivo anti-tumor activity was assessed using IL4-producing MDA MB 468 tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells.

Results: In the IL4-rich tumor milieu, 1G CAR.MUC1 T cells failed to expand or kill MUC1+ tumors and while co-expression of the 4/7ICR promoted T cell expansion, in the absence of co-stimulatory signals the outgrowing cells exhibited an exhausted phenotype characterized by PD-1 and TIM3 upregulation and failed to control tumor growth. However, by co-expressing 2G CAR.MUC1 (signal 1 - activation + signal 2 - co-stimulation) and 4/7ICR (signal 3 - cytokine), transgenic T cells selectively expanded at the tumor site and produced potent and durable tumor control in vitro and in vivo.

Conclusions: Our findings demonstrate the feasibility of targeting breast cancer using transgenic T cells equipped to thrive in the suppressive tumor milieu and highlight the importance of providing transgenic T cells with signals that recapitulate physiologic TCR signaling - [activation (signal 1), co-stimulation (signal 2) and cytokine support (signal 3)] - to promote in vivo persistence and memory formation.

Keywords: Breast cancer; Chimeric antigen receptor; Genetic engineering; Inverted cytokine receptor; T cell therapy.

Fig. 1

4/7ICR improves the cytolytic function and proliferation of CAR.MUC1 T cells in presence of IL4. a Schematic representation of 1st generation CAR.MUC1 (1G) construct. b CAR.MUC1 expression on activated T cells measured 3 days post-transduction (representative donor on the left, summary data on the right). Data represents mean ± SEM (n = 6). c Phenotypic analysis of MUC1 expression on different cell lines (top panel) and in vitro cytolytic function of control (NT) and CAR T cells assessed in a 5 hr ⁵¹Cr-release assay at E:Ts of 1.25:1 to 40:1, using MUC1+ targets (293T/MUC1, MDA MB 468, MCF-7) and MUC1- target (293T) (bottom). Data represents mean ± SEM (n = 5). d Schematic of 4/7ICR vector map. e Transgenic expression of both 4/7ICR and CAR.MUC1 in T cells as detected by mOrange and anti-IgG, respectively. Right panel shows summary data representing the percentage of double-positive cells (1G.4/7ICR) (mean ± SEM, n = 4). f Cytolytic function of transgenic (1G or 1G.4/7ICR) T cells pre-exposed to IL4 as assessed in a 4 hr ⁵¹Cr-release assay using MDA MB 468 as a target at the indicated E:T ratios. Statistical significance was calculated between 1G and 1G.4/7ICR using One-way ANOVA, p < 0.05. g Cell expansion of 1G or 1G.4/7ICR T cells (1 × 10⁶) stimulated weekly with irradiated MDA MB 468 cells (0.5 × 10⁶) with IL4 (400 U/mL) added twice weekly. T cell expansion was quantified by cell counting using trypan blue exclusion to assess cell viability. Statistical significance was calculated between 1G and 1G.4/7ICR using One-way ANOVA, p < 0.01

Fig. 2

Transgenic expression of 4/7ICR is insufficient to overcome tumor-mediated T cell dysfunction. a Schematic of co-culture experimental setup (left panel) and bioluminescence data tracking tumor expansion (in the absence of T cell treatment) over time (right panel). b Representative dot plots (top) and summary quantitative data (bottom) showing T cells and tumor cell numbers on day 0 and day 21 of coculture (mean ± SEM, n = 6 independent experiments). Significance was determined by an unpaired two-tailed t-test, p < 0.05, 1G.4/7ICR compared with 1G. c Representative images of bioluminescent tumor cells (top) and summarized quantitative bioluminescence signal from tumor cells treated with either NT, 1G or 1G.4/7ICR T cells over time (mean ± SEM, n = 6). d Phenotypic analysis of MUC1 expression on MDA MB 468 cells on day 0 and day 21 of co-culture (representative donor). e CAR expression on 1G.4/7ICR cells after co-culture with tumor cells. f CD25 expression on 1G versus 1G.4/7ICR T cells on day 0 and day 21 of co-culture (light grey: isotype control, dark grey: 1G T cells, green: 1G.4/7ICR T cells). g Surface expression of PD-1 (representative donor - left, summary data - right) on 1G.4/7ICR cells gated on CD3⁺TIM3⁺cells and analyzed on days 0, 7, 14 and 21 of co-culture (mean ± SEM, n = 6, p < 0.05). h Cytolytic activity of 1G.4/7/ICR prior to and day 21 post-coculture using MDA MB 468 cells as targets (E:T 10:1; mean ± SEM, n = 4, p < 0.01)

Fig. 3

Combining 4/7ICR with a 2G CAR preserves T cell function even under suppressive conditions. a Serial bioluminescence imaging of eGFP-FFLuc⁺ MDA MB 468 cells co-cultured with 1G, 1G.4/7ICR, 2G, or 2G.4/7ICR T cells in the presence of IL4 [representative images - left, quantitative data - right (n = 6)]. b Representative dot plots and summary FACS data quantifying tumor cells and T cells after 21 days of co-culture. c Surface expression of PD-1 and TIM3 on transgenic T cells analyzed on day 21 after co-culture (representative data - left, summary data - right). d CD25 expression on 1G.4/7ICR, 2G, and 2G.4/7ICR cells on day 21 of coculture. e In vitro cytolytic function of 1G.4/7ICR, 2G, and 2G.4/7ICR cells isolated on day 21 after co-culture (mean ± SEM, n = 4–6). Significance was determined by two-way ANOVA. p < 0.05, p < 0.01, p < 0.001

Fig. 4

Combined expression of 4/7ICR and 2G CAR augments anti-tumor activity in vivo. a Schematic of in vivo experiment where NSG mice with IL4-producing MDA MB 468 cells and treated i.v. with eGFP-FFLuc+1G, 1G.4/7ICR, 2G, or 2G.4/7ICR T cells. b Representative animal images (left) and summary bioluminescence data (right, mean ± SEM, n = 3–5/group) indicating T cell localization and expansion. c Tumor volume measured by calipers (mean ± SEM, n = 3–5/group). Significance was determined by two-way ANOVA. p < 0.05 on day 35. d Representative animal images (left) and summary bioluminescence data (right, mean ± SEM, n = 3–5/group). e Tumor volume measured by calipers. Significance was determined by two-way ANOVA

Fig. 5

2G.4/7ICR T cells persist long term and retain their anti-tumor activity and tumor selectivity. a Schema of tumor rechallenge model. b T cell expansion over time as assessed by quantitative bioluminescence imaging at the site of MDA MB 468 (left) and MDA MB 468/IL4 (right) tumor cell injection. c Tumor volume as measured by calipers