Probiotic-guided CAR-T cells for solid tumor targeting

Abstract

A major challenge facing tumor-antigen targeting therapies such as chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. By contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be engineered as antigen-independent platforms for therapeutic delivery. To bridge these approaches, we developed a platform of probiotic-guided CAR-T cells (ProCARs), in which tumor-colonizing probiotics release synthetic targets that label tumor tissue for CAR-mediated lysis in situ. This system demonstrated CAR-T cell activation and antigen-agnostic cell lysis that was safe and effective in multiple xenograft and syngeneic models of human and mouse cancers. We further engineered multifunctional probiotics that co-release chemokines to enhance CAR-T cell recruitment and therapeutic response.

Fig. 1.. Synthetic CAR targets “Tag” tumor cells for lysis by ProCAR-T cells in situ.

(A) Schematic demonstrating the ProCAR platform, in which synthetic CAR targets (Tags) are produced and released in situ by tumor-colonizing probiotic bacteria (E. coli Nissle 1917) to label ubiquitous components of solid tumors for de novo lysis by GFP-CARs (GFP28z). Tags are designed as dimers of sfGFP fused to an HBD (PlGF_123–144) that broadly bind to cell surface and matrix proteins found in the TME. (B) Representative flow cytometry histograms demonstrating GFP28z surface expression through binding purified sfGFP (left) and coexpression of mScarlet (right) in primary human T cells. (C) Flow cytometric quantification of CD25 surface expression after exposure to 100 ng/ml of GFP-based CAR targets for 16 hours—monomeric sfGFP (GFP), soluble diGFP, collagen-bound GFP (Tag)—shown relative to MDA-MB-468 cells expressing mbGFP at a 1:1 ratio. Data represent mean ± SD of n = 3 biological replicates. MFI, mean fluorescence intensity. (D) Representative flow cytometry histograms of surface-bound GFP after incubation with 500 ng/ml Tag-GFP or diGFP for 20 min. (E) Confocal microscopy images of Jurkats expressing GFP28z-msScarlet fusion receptors for subcellular visualization. CARs are shown in orange and cocultured with unlabeled MDA-MB-468 target cells; images were acquired every 2 to 5 min after addition of 100 ng/ml of purified Tag. White arrows indicate CAR clusters. (F) Overnight killing assay against ffLuc⁺ HEK293T target cells at defined E:T ratios. CAR-T cells were cocultured with target cells ± 100 ng/ml of CAR targets for 20 hours. Specific lysis (%) was determined by normalizing relative light units (RLU) to cocultures with UT T cells. Data represent mean ± SD of n = 3 biological replicates. (G) Overnight killing assay of ffLuc⁺ HEK293T target cells at a 3:1 E:T with half log dilutions of purified Tag. Data represent mean ± SD of n = 3 biological replicates. (H) Overnight killing assays against a panel of ffLuc⁺ target cells at a fixed E:T ratio (3:1) and treated as in (F). (I) Overnight killing assay of ffLuc⁺ MDA-MB-468 in the presence of 20 ng/ml human HSPE (hHSPE) at a 1:1 E:T ratio, ± 100 ng/ml Tag. Data represent mean ± SD of n = 3 biological replicates. ***P < 0.001; ****P < 0.0001; two-way analysis of variance (ANOVA) [(C), (F), (H), and (I)] or one-way ANOVA (G), Holm–Sidak multiple comparison correction. ns, not significant; 468, MDA-MB-468.

Fig. 2.. ProCAR-T cells yield antigen-agnostic therapeutic efficacy in response to Tags and bacterial adjuvants provided by probiotics in situ.

(A to C) Nalm6 cells (5 × 10⁶) were implanted subcutaneously (“s.c.”) into the hind flank of NSG mice. When tumor volumes reached ~100 mm³, mice were intratumorally injected with 1 × 10⁵ CFU of engineered probiotic strains (Pro^X) producing diGFP (Pro^diGFP) or Tag (Pro^Tag) targets or an empty control (Pro⁻) (A). Then, 2.5 × 10⁶ GFP28z⁺ ProCAR-T cells were delivered 48 hours post bacteria treatment (pbt), with tumor growth monitored by caliper measurements every 3 to 4 days. Mean tumor trajectories (B) and survival curves (C) are shown. Data represent mean ± SEM of n > 4 biological replicates. (D) ELISA quantification of sfGFP levels from tumor homogenates (left) and serum (right) on day 14 pbt; data represent SEM of n = 3 biological replicates. (E and F) MDA-MB-468 cells (5 × 10⁶) were subcutaneously implanted into the hind flank of NSG mice. When tumors reached palpable volume, mice were intratumorally injected with 1 × 10⁵ CFU of Pro^Tag or control Pro⁻ strains or PBS. On days 2 and 15 pbt, tumors were treated with 2.5 × 10⁶ GFP28z⁺ ProCAR-T cells, and tumor growth was measured as in (A). Mean tumor trajectories are shown (F). Data represent mean ± SEM of n > 3 biological replicates. (G to K) Nalm6 tumors were established and treated as in (A) and resected on day 2 after T cell treatment (day 4 pbt) for analysis by flow cytometry. (H) Frequency of IT hCD45⁺CD3⁺CD8⁺ T cell memory and effector populations determined by CD62L and CD45RO expression patterns. Data represent mean ± SEM of n > 3 biological replicates. (I) Flow cytometric quantification of CD69 surface expression on IT hCD45⁺CD3⁺ CD8⁺ cells in each treatment group. Data represent mean ± SEM of n = 3 biological replicates. (J and K) Luminex quantification of IT IFN-γ (J) and TNF-α (K) concentrations. Data represent mean ± SD of biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; two-way ANOVA [(B), (F), and (H)], log-rank test (C), or one-way ANOVA [(D), (I) to (K)], with Holm–Sidak multiple comparison correction.

Fig. 3.. Treatment with the ProCAR platform provides a systemic therapeutic benefit in an immune-competent model of colorectal cancer.

(A) CT26 cells (5 × 10⁵) were implanted subcutaneously into the hind flank of BALB/c mice. When tumor volumes reached ~100 mm³, mice were treated with 5 × 10⁶ CFU of engineered probiotic strains (Pro^X) producing Tag (Pro^Tag) or an empty control (Pro⁻). On days 2 and 5 pbt, tumors were treated with 2.5 × 10⁶ mGFP28z⁺ T cells, and growth was monitored by caliper measurements every 3 to 4 days. Mean tumor trajectories are shown. Data represent mean ± SEM of n > 10 biological replicates. (B) A20 cells (5 × 10⁵) were implanted subcutaneously into the hind flank of BALB/c mice. When tumor volumes reached 200 to 300 mm³, mice were treated with probiotics as in (A). On day 2 pbt, tumors were treated with 1 × 10⁶ mGFP28z⁺ T cells. Mean tumor trajectories are shown. Data represent mean ± SEM of n > 3 biological replicates. (C to E) MC38 cells (5 × 10⁵) were implanted subcutaneously into both hind flanks of C57BL/6 mice. When tumor volumes reached ~150 mm³, the left tumor received a single injection of 2 × 10⁶ CFU Pro^Tag, Pro⁻, or a PBS control (C). On days 2 and 5 pbt, tumors on the left flank were treated with 1.5 × 10⁶ mGFP28z⁺ T cells. Tumors on the right flank were left untreated. Mean tumor trajectories of the treated tumors (D) and untreated tumors (E) are shown. Data represent mean ± SEM of n > 4 biological replicates. (F to J) C57BL/6 mice were grafted and treated as in (C). On day 9 pbt, treated tumors and tumor-draining lymph nodes were retrieved for analysis by flow cytometry. (F) Frequency of IT CD69⁺CAR⁺ T cells; representative flow cytometry histograms (left) and quantification are shown (right). Data represent mean ± SEM of n > 3 biological replicates. (G) Frequencies of CD69⁺ tumor infiltrating CD8⁺ and CD4⁺Foxp3⁻ (T_conv.) CAR⁻ T cells. Data represent mean ± SEM of n > 3 biological replicates. (H) Frequency of Ki-67⁺ tumor-infiltrating CAR⁻CD8⁺ T cells from each treatment group. (I and J) Frequency of activated (CD40⁺MHCII⁺) (I) and PD-L1⁺ (J) proinflammatory monocytes (CD11b⁺Ly6C⁺) in the lymph nodes of treated and control mice. Data represent mean ± SEM of n > 3 biological replicates. *P < 0.05; **P < 0.01; ****P < 0.0001; two-way ANOVA [(A), (B), (D), (E), and (G)] or one-way ANOVA [(F), (H), (I), and (J)]; ANOVAs performed with Holm–Sidak multiple comparison correction.

Fig. 4.. Multifunctional probiotics produce combinations of TME-modulating factors to facilitate systemic delivery and delay the growth of orthotopic breast tumors.

(A) Probiotics are engineered to release an activating mutant of the human chemokine, CXCL16^K42A (“CXCL16”), to recruit CXCR6⁺ ProCAR-T cells directly to the tumor site. (B) MDA-MB-468 cells (5 × 10⁶) were subcutaneously implanted into the hind flank of NSG mice. Palpable tumors were then either injected with 1 × 10⁵ CFU of a strain engineered to produce both CXCL16 and Tag in combination (Pro^Combo), control strains producing single agents (Pro^Tag*, Pro^CXCL16), or a PBS control. On days 2 and 15 pbt, mice were intravenously treated with 6 × 10⁶ GFP28z⁺ T cells. Mean tumor growth trajectories are shown. Data represent mean ± SEM of n > 3 biological replicates per group. (C) Counts of infiltrating hCD45⁺CD3⁺ cells per milligram of tumor. MDA-MB-468 tumors were established and treated as in (A). On day 7 after treatment, tumors from Pro^Tag* and Pro^Combo treatment groups were retrieved and homogenized for analysis by flow cytometry. Data represent mean ± SEM of n = 4 biological replicates per group. (D and E) MDA-MB-468 tumors were established and measured as in (B). When tumors reached palpable size, mice were intravenously treated with 5 × 10⁶ CFU of Pro^X strains or PBS. On days 3 and 17 pbt, mice were intravenously treated with 6 × 10⁶ GFP28z⁺ ProCAR-T cells. Mean tumor trajectories are shown (E). Data represent mean ± SEM of n > 5 biological replicates per group. I.V., intravenously. (F to I) An orthotopic model of TNBC was established through the surgical implantation of 5 × 10⁶ MDA-MB-468 cells into the mammary fat pad (“m.f.p.”) of female NSG mice. When tumor volumes reached ~100 mm³, mice were treated as in (D); mean tumor growth trajectories are shown (G). Data represent mean ± SEM of n > 6 biological replicates per group. (H and I) Biodistribution assessment of Pro^X (H) and sfGFP (I) in tumor, lung, kidney, spleen, and liver homogenates. On day 42 pbt, tumor and matched healthy tissue were digested and plated with the appropriate antibiotics for colony quantification or assessed by ELISA for sfGFP concentration. Data represent mean ± SEM of n = 5 (H) or n = 3 (I) biological replicates per group. Limit of detection (LOD), 200 CFU/g. *P < 0.05; **P < 0.01; ****P < 0.0001; two-way or one-way (C) ANOVA, with Holm–Sidak multiple comparison correction.