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918 | CANCER DISCOVERY August 2018 www.aacrjournals.org

IN THE SPOTLIGHT

CAR T-cell Integration of Multiple Input signals Allows for Precise Targeting of Cancer Daniela Achkova 1 and Martin Pule 1 , 2

1 Autolus Ltd., White City, London, UK. 2 Cancer Institute, University College London, London, UK. Corresponding Author: Martin Pule, Cancer Institute, University College London, 72 Huntley Street, London WC1E 6BT, UK. Phone: 44-20-3829-6232; E-mail: m.pule@ucl.ac.uk doi: 10.1158/2159-8290.CD-18-0672 ©2018 American Association for Cancer Research.

summary: Chimeric antigen receptor (CAR)–mediated adoptive T-cell therapy has achieved unprecedented success in the treatment of relapsed and refractory hematologic malignancies. However, this success may be more diffi cult to recapitulate in the treatment of metastatic solid tumors, where the lack of costimulatory signals and cytokine support as well as the strongly inhibitory microenvironment pose a substantial challenge to unleashing the antitumor potential of CAR T cells. Furthermore, nearly all described target antigens are expressed on normal tissue. In this issue of Cancer Discovery , Sukumaran and colleagues address these challenges by engineering T cells to recognize a specifi c expression pattern unique to the tumor site using independent chimeric molecules that cooperatively deliver a fully functional T-cell response selectively in the tumor microenvironment. Cancer Discov; 8(8); 918–20. ©2018 AACR.

See related article by Sukumaran et al., p. 972 (1).

T cells transduced with chimeric antigen receptors (CAR) can be effectively redirected to and activated by a cell-surface native target antigen of choice. The adoptive transfer of CD19 (a pan B-cell antigen)-targeting CAR T cells has demonstrated sustained clinical responses in patients with hematologic malignancies, leading to the recent FDA approvals of tisa-genlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta) for relapsed/refractory cases of acute B-cell lymphoblastic leukemia and adult large B-cell lymphoma, respectively. CAR T-cell targeting of solid cancers faces several obstacles. First, almost every proposed cancer antigen has some expression on normal tissue, which risks “on-target, off-tumor” toxicity. Another obstacle is the strongly immunosuppressive micro-environment characteristic for solid tumors, which impairs the effector function, proliferative capacity, and in vivo persis-tence of adoptively transferred T cells.

Most engineering solutions reported to date address indi-vidual problems posed by target specifi city or the tumor immunosuppressive microenvironment. In this issue of Can-cer Discovery , Sukumaran and colleagues describe an engineer-ing approach that not only increases CAR T-cell specifi city to the tumor through pattern recognition of three ligands but in doing so renders the CAR T cells resistant to immunosup-pressive components (IL4) and TGFβ) of the tumor milieu ( 1 ). The authors devised a three-component split CAR system, comprising of chimeric receptors that recognize prostate stem cell antigen (PSCA), TGFβ, and IL4 and whose endodo-mains recapitulate a physiologic T-cell signaling (providing signals 1, 2, and 3; Fig. 1 ). Using pancreatic cancer as a model

system, they demonstrated that a functional T-cell response is reconstituted in tumor-mimicking conditions (target cells expressing PSCA, TGFβ, and IL4) leading to PSCA + target cell lysis accompanied by survival and expansion of genetically modifi ed T cells in TGFβ- and IL4-rich milieu. Conversely, the presence of antigen alone or either cytokine alone was insuffi cient to promote expansion of the transgenic T cells, highlighting their dependence on all three input signals. This system, termed SmarT cells (tumor-specifi c molecular-pattern activated and regulated T cells), showed increased tumor selectivity both in vitro and in vivo . The authors gener-ated an animal model recapitulating both healthy and tumor-mimicking conditions by injecting NSG mice subcutaneously in opposite fl anks with cells expressing PSCA only (mimick-ing normal tissue) and tumor cells expressing PSCA, TGFβ, and IL4, a signature present in pancreatic tumors. Upon administration, SmarT cells “sensed” the tumor site, distin-guishing it from healthy antigen-positive tissue, resulting in selective elimination of PSCA + TGFβ + IL4 + tumors. Further-more, substantial tumor site–restricted SmarT-cell expan-sion was observed, attesting to the reliance of the genetically modifi ed T cells on the presence of all three input signals for adequate T-cell response. In subsequent rechallenge experi-ments, SmarT cells reexpanded only at the tumor site in the presence of PSCA, IL4, and TGFβ and led to effective tumor eradication, demonstrating retained selectivity and potency of this approach.

Over the last few years, several strategies to overcome “on-target, off-tumor” toxicities by more refi ned discrimi-nation between normal and malignant tissues have been described. Among the proposed strategies, the most prom-ising ones involve splitting of the conventional single-input CAR into two independent receptors, capable of Boolean logic signal transduction (e.g., OR gate, AND gate, and AND–NOT gate). An early example of the AND strategy involves separating CD3ζ chain (signal 1) and costimula-tion (signal 2) into two independent chimeric receptors, whose signaling is complementary upon recognition of

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August 2018 CANCER DISCOVERY | 919

distinct targets expressed by tumor cells (MUC1 and HER2 CARs, ref. 2; and later PSMA and PSCA CARs, ref. 3). A more sophisticated strategy to engineer multi-input con-trol of T cells involves the building of two antigen tumor recognition circuits based on a synthetic Notch (synNotch) receptor-inducible system, whereby engagement with one tumor antigen induces expression of a second CAR, result-ing in biphasic signaling response (4).

Although Boolean-gated CAR approaches are appealing in their refinement of tumor selectivity, they do not address any of the hurdles that the immunosuppressive microenviron-ment poses for adoptively transferred CAR T cells. Most solid tumors have insufficient amounts of activating costimula-tory ligands and immunostimulatory cytokines, which con-tributes to the lack of expansion and persistence of adoptively transferred CAR T cells that have trafficked to the tumor site. Preclinical studies aiming at improving persistence of T cells at the tumor site have shown that restoration of signal 3 through genetic modification of T cells to express secreted or tethered cytokines enhances T-cell antitumor activity (5). However, systemic or locally provided cytokines exhibit sub-stantial toxicities. To circumvent this effect, a constitutively active IL7 cytokine receptor has been described, which deliv-ers an IL7 signal in the absence of the secreted cytokine (6). An alternative cytokine support system is one provided by chimeric cytokine receptors that translate a negative signal produced by an immunosuppressive cytokine (IL4) within the tumor microenvironment to an immunostimulatory sig-nal (IL2 receptor or IL7 receptor; ref. 7). Similarly, a soluble

immunosuppressive signal (TGFβ) has been successfully used to stimulate CAR T-cell effector functions, demonstrating that CARs can be specifically engineered to respond to freely soluble ligands (8).

Building onto these concepts, the authors of this paper have engineered T cells to recognize a pattern exclusive to the tumor site using three independent receptors that rec-ognize the tumor cells (PSCA+) but also invert the effects of two soluble immunosuppressive molecules (IL4 and TGFβ) into immunostimulatory signals, thereby recapitulating fully functional physiologic T-cell signaling (Fig. 1). Furthermore, by using a three-input CAR design incorporating soluble as well as membrane-bound antigens, Sukumaran and col-leagues sought to minimize the risk of immune escape due to antigen loss, an event already observed in the clinic (9).

In summary, the current study by Sukumaran and col-leagues demonstrates that T cells can be engineered to rec-ognize a specific pattern present exclusively at the tumor site, thereby enhancing both the antitumor efficacy and safety profile of the transgenic T cells. By limiting T-cell activity exclusively to the tumor site, this genetic approach reduces “on-target, off-tumor” toxicity. In addition, this strategy not only has the benefit of rendering genetically engineered T cells resistant to immunosuppressive cytokines (TGFβ and IL4) present in the tumor milieu, but also allows SmarT cells to engraft within the tumor microenvironment. The described system can readily be extended to other soluble and membrane-bound inhibitory molecules present at the tumor site (e.g., IL10, PD-1, and CTLA4).

Figure 1.  Schematic of SmarT cells that recognize the pancreatic tumor microenvironment. SmarT cells are engineered with a three-component split CAR system, which recognizes PSCA, TGFβ, and IL4 and whose endodomains recapitulate a physiologic T-cell signaling by delivering signal 1, activation (CD3ζ); signal 2, costimulation (41BB); and signal 3, cytokine (IL7). Incorporating a combination of receptors in SmarT cells allows for complex multisignal computation that confers engineered T cells responsive to an expression pattern present exclusively at the tumor site.

SmarT cell

PSCA

TGFβIL4

Tumor cell

T cell

PSCA-specific chimeric antigen receptor

TGFβ-specific chimeric costimulatory receptor

IL4-specific chimeric cytokine receptor

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920 | CANCER DISCOVERY August 2018 www.aacrjournals.org

Increasingly complex synthetic biology approaches as described by Sukumaran and colleagues represent the “killer apps” of T-cell engineering. It is inconceivable that such complexity could be incorporated into a small molecule or a protein therapeutic; consequently, T-cell engineering will find increasing application in cancer therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Published online August 3, 2018.

RefeRenCes1. Sukumaran S, Watanabe N, Bajgain P, Raja K, Mohammed S, Fisher

WE, et al. Enhancing the potency and specificity of engineered T cells for cancer treatment. Cancer Discov 2018;8:972–87.

2. Wilkie S, van Schalkwyk MCI, Hobbs S, Davies DM, van der Stegen SJC, Pereira ACP, et al. Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide comple-mentary signaling. J Clin Immunol 2012;32:1059–70.

3. Kloss CC, Condomines M, Cartellieri M, Bachmann M, Sadelain M. Com-binatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol 2013;31:71–5.

4. Roybal KT, Rupp LJ, Morsut L, Walker WJ, McNally KA, Park JS, et al. Precision tumor recognition by T cells with combinatorial antigen-sensing circuits. Cell 2016;164:770–9.

5. Hurton LV, Singh H, Najjar AM, Switzer KC, Mi T, Maiti S, et  al. Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Proc Natl Acad Sci U S A 2016; 113:E7788–E97.

6. Shum T, Omer B, Tashiro H, Kruse RL, Wagner DL, Parikh K, et al. Consti-tutive signaling from an engineered IL7 receptor promotes durable tumor elimination by tumor-redirected T cells. Cancer Discov 2017;7:1238–47.

7. Wilkie S, Burbridge SE, Chiapero-Stanke L, Pereira ACP, Cleary S, van der Stegen SJC, et al. Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4. J Biol Chem 2010;285:25538–44.

8. Chang ZL, Lorenzini MH, Chen X, Tran U, Bangayan NJ, Chen YY. Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nat Chem Biol 2018;14:317–24.

9. Sotillo E, Barrett DM, Black KL, Bagashev A, Oldridge D, Wu G, et al. Con-vergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov 2015;5:1282–95.

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