TGFb inhibition via CRISPR promotes the long- term efficacy of CAR-T cells against solid tumors

In recent years, CAR-T cell therapy has proven to be a promising approach against cancer. Nonetheless, this approach still faces multiple challenges in eliminating solid tumors, one of which being the immunosuppressive tumor microenvironment (TME). Here we demonstrated that knocking out the endogenous TGFβ receptor II (TGFBR2) in CAR-T cells with CRISPR/Cas9 technology could reduce the induced regulatory T-cell (iTreg) conversion and prevent the exhaustion of CAR-T cells. Meanwhile, TGFBR2 edited CAR-T cells had better in vivo tumor elimination efficacy, both in cell line derived xenograft (CDX) and patient derived xenograft (PDX) solid tumor models, whether administered locally or systemically. In addition, the TGFBR2 edited CAR-T cells could eliminate contralaterally re-inoculated xenografts in mice effectively with an increased proportion of central memory and effector memory subsets. In conclusion, we greatly improved the in vitro and in vivo function of CAR-T cells in TGFβ-rich tumor environments by knocking out endogenous TGFBR2, proposing a new method to improve the efficacy of CAR-T cell therapy for treating solid tumors.


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In recent years, chimeric antigen receptor modified T (CAR-T) cells have 44 emerged as a promising class of cancer therapeutics (1-3). Anti-CD19 CAR-T cells 45 have achieved remarkable therapeutic efficacy in treating hematological malignancy, 46 and two products were approved by FDA in 2017 (4, 5). However, despite many 47 attempts, CAR-T cells targeting various tumor associated antigens (TAA) still fail to 48 yield favorable clinical response in patients with solid tumors in a reproducible 49 manner (2, 6-8). One of the major challenges that CAR-T cells face after infiltrating 50 into the solid tumor is the suppressive tumor microenvironment (TME). The solid 51 tumor TME contains numerous cell types in addition to cancer cells, as well as 52 extracellular matrix components and inflammatory mediators. In this complex 53 microenvironment, T cells encounter many inhibitory cells and molecules that can 54 impair their survival, activation, proliferation and effector functions (9, 10). 55 TGFβ, one of the most important regulators in TME, can be secreted by stromal 56 cells, such as cancer-associated fibroblasts (CAFs), blood endothelial cells (BECs), 57 mesenchymal stem cells (MSCs), lymphatic epithelial cells (LECs) and pericytes (9).

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Upon binding to TGFβR II, TGFβR I is recruited, phosphorylated and activated to 62 phosphorylate the downstream mediators, and it then modulates gene transcription 63 (13,14). TGF-β plays a very important role in tumor initiation and progression. 64 Meanwhile, TGF-β has an adverse effect on anti-tumor immunity and significantly 65 inhibits host tumor immune surveillance (12,15). It has been reported that TGF-β 66 markedly suppresses the cytotoxic function of CD8 + T cells, through transcriptional 67 down-regulation of genes encoding key proteins, such as perforin, granzymes and 68 cytotoxins (16,17). TGF-β affects CD4 + T cell differentiation and function by 69 inducing T regulatory cell (Treg) conversion (17,18). In addition, inhibiting TGFβ 70 signaling with a TGF-β1 antibody (19), TGFβR I inhibitor (20), or over-expressing 71 4 4 dominant negative TGFβR II have all been shown to enhance the tumor elimination 72 ability of T cells (21,22). 73 In this study, we characterized the effects of TGF-β on CAR-T cells, and found 74 that CAR-T cells could be induced into a Treg-like phenotype that was FOXP3-75 dependent. In addition, we found that the presence of TGF-β1 accelerated the 76 exhaustion of CAR-T cells by up-regulating PD1. We completely eliminated the 77 negative effect of TGFβ by knocking out TGFBR2 in CAR-T cells using the 78 CRISPR/Cas9 system, and observed dramatically improved function both in vitro and 79 in cell-line-derived (CDX) and patient tumor-derived xenograft (PDX) models. 80 Impressively, after PDX tumor being eradicated by TGFBR2 knockout CAR-T cells, 81 the contralaterally re-inoculated patient-derived tumor was again fast cleared, 82 demonstrating the potent and persistent in vivo anti-tumor efficacy.

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TGFβ1 negatively regulates CAR-T cell cytotoxic function via TGFβ receptor 86 To study CAR-T cells targeting solid tumors, we designed anti-mesothelin 87 CAR. The scFV was derived from an anti-mesothelin monoclonal antibody P4 (23), 88 and CD28 co-stimulatory domain and CD3z was used as intercellular signaling 89 components. We generated CAR-T cells (M28z) via lentiviral transduction and used 90 the mesothelin positive tumor cell lines CRL5826 and OVCAR-3 as target cells. 91 We first confirmed the low expression level of endogenous TGFβ1 in CRL5826 92 cells and generated clonal cell lines expressing TGFβ1 at different levels (CRL5826-93 TGFβ1-low, intermedia, high). While these cell lines released different amounts of 94 TGFβ1, there was no difference of cell lysis when they were co-cultured with M28z 95 CAR-T cells ( Figure S1), suggesting that the TGFβ1 released by these cell lines could 96 not inhibit CAR-T function, likely being the latent form.

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Then we added different concentrations of human recombinant TGFβ1 to the 98 M28z CAR-T and CRL5826 co-culture system, and observed its effect on the 99 5 5 cytotoxic function of CAR-T cells. As shown in Figure 1A, the lysis of CRL5826 by 100 M28z CAR-T cells at 1:1 effector to target (E: T) ratio was reduced to a similar level 101 when 2.5, 5, or 10 ng/ml of TGFβ1 was added. Subsequently, we used 5ng/ml TGFβ1 102 in our following in vitro experiments. The release of IL-2 and IFN-γ by CAR-T cells 103 was also significantly reduced in the presence of TGFβ1 ( Figure 1B).

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Considering the number of T cells is much lower than the tumor cells upon 105 infiltration into the solid tumor microenvironment, we evaluated the effect of TGFβ1 106 at lower E:T ratio. Impressively, as we lowered the E:T ratio, the inhibitory effect 107 became more pronounced. At the E:T ratio of 0.25:1, the CAR-T-mediated tumor 108 lysis in the presence of TGFβ1 was only about a quarter of that in the absence of 109 TGFβ1 ( Figure 1C). These data indicate that TGFβ1 negatively regulates the 110 cytotoxic function of CAR-T cells, and the inhibition level corresponds to the E:T 111 ratio.

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To rescue the CAR-T cells from this immune suppression effect of TGFβ1, we 113 sought to eliminate TGFβ receptor by knocking out the TGFBR2 gene in CAR-T 114 cells. Upon optimization, we achieved KO efficiency of 50-80% ( Figure S2).

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Knocking out TGFBR2 did not affect the proliferation, CAR expression and T cell 116 subtype of M28z CAR-T cells ( Figure S3). Using three different E:T ratios, we 117 compared the specific lysis ability of control (M28z) and TGFBR2 knocked out CAR-118 T cells , and found that TGFBR2 knockout could completely rescue the 119 negative effect of TGFβ1 on tumor lysis ( Figure 1D    To confirm this effect was due to FOXP3 up-regulation, we performed a 161 proliferation suppression assay using TGFBR2 and FOXP3 ( Figure S6 improved CAR-T cell proliferation upon multiple tumor challenges, while it was still 204 worse than TKO and DKO ( Figure 4F). In the presence of TGFβ1 and PDL1 over-205 expression, the tumor lysis capability of M28z was reduced at round 2 and completely 206 lost at round 3. M28z-PKO was able to achieve over 90% tumor lysis at round 3, and 207 lost efficacy at round 4. In contrast, M28z-TKO was able to maintain about 60% 208 tumor lysis capability at round 4 ( Figure 4G). All these data showed that PD1 up-209 regulation partially contributed to the negative regulation effect of TGFβ. On the 210 other hand, TGFβ signaling was only partially responsible for PD1 expression, as a 211 portion of TKO cells can still express PD1, therefore subjected to PDL1 suppression. The DKO CAR-T cells had the best performance, eliminating about 90% of tumor at 213 round 4 ( Figure 4G), suggesting that simultaneous blocking both TGFβ and PD1 214 signaling can further improve the CAR-T's resistance to suppressive TME.  Figure 5A). We suspected that these TGFβ1 in TME

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These results suggest that TGFBR2 edited CAR-T cells function effectively in 363 different primary tumor environments. However, we also want to point out that TGFβ 364 signals through many cell types at TME to induce T cell suppression, and our CDX 365 and PDX models were only recapitulating some aspects of the TME. It is possible that 366 the lack of TGFβR II on T cells will allow for greater signaling through other cell 367 subsets, which is an important issue to be addressed using more sophisticated tumor 368 models.

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In the complex suppressive TME, different inhibitory cells and molecules inhibit 370 T cell function via multiple pathways (9). In the CRL5826 CDX model, which weakly  PDCD1was performed by CFX96 real-time detection system (Bio-Rad).

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Housekeeping gene GAPDH was used as an internal control. The qPCR primers used 471 in this study were listed in Supplementary Table 1. Vitalstar) were also randomly divided into above referred 3 groups when the tumor 529 sizes were between 200-300 mm 3 . 5×10 6 CAR-T cells (CAR + was 50%) were 530 administrated intratumoraly or intravenously twice with a one-week-interval. Body 531 weight, tumor size and the onset of GvHD were monitored weekly. Mice peripheral 532 bloods were collected, and the proportion and subsets of human cells was analyzed.

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Contralateral re-inoculation of same PDX was done when the primary PDX was 534 removed completely. Body weight, tumor size and the onset of GvHD were also 535 monitored weekly. Mice peripheral bloods were also collected, and the proportion and 536 subsets of human cells was analyzed.