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Supplementary figure legend

Supplementary Figure 1: Murine SAR expression and immunophenotyping of transduced T cells

Supplementary Figure 5: Effect of pre-activated SAR T cells on hepatocellular carcinoma and glioblastoma cells


Purpose:Genetically engineered T cells are powerful anticancer treatments but are limited by safety and specificity issues. We herein describe an MHC-unrestricted modular platform combining autologous T cells, transduced with a targetable synthetic agonistic receptor (SAR), with bispecific antibodies (BiAb) that specifically recruit and activate T cells for tumor killing.Experimental Design:BiAbs of different formats were generated by recombinant expression. T cells were retrovirally transduced with SARs. T-cell activation, proliferation, differentiation, and T-cell–induced lysis were characterized in three murine and human tumor models in vitro and in vivo.Results:Murine T cells transduced with SAR composed of an extracellular domain EGFRvIII fused to CD28 and CD3ζ signaling domains could be specifically recruited toward murine tumor cells expressing EpCAM by anti-EGFRvIII × anti-EpCAM BiAb. BiAb induced selective antigen-dependent activation, proliferation of SAR T cells, and redirected tumor cell lysis. Selectivity was dependent on the monovalency of the antibody for EGFRvIII. We identified FAS ligand as a major mediator of killing utilized by the T cells. Similarly, human SAR T cells could be specifically redirected toward mesothelin-expressing human pancreatic cancer cells. In vivo, treatment with SAR T cells and BiAb mediated antitumoral activity in three human pancreatic cancer cell xenograft models. Importantly, SAR activity, unlike CAR activity, was reversible in vitro and in vivo.Conclusions:We describe a novel ACT platform with antitumor activity in murine and human tumor models with a distinct mode of action that combines adoptive T-cell therapy with bispecific antibodies.

Supplementary Figure 4: Human SAR T cells are specifically stimulated by anti-human mesothelin x anti-EGFRv3 2 + 1 BiAb and redirect lysis to mesothelin+ target cells in vitro and in vivo

Supplementary Figure 3: In contrast to SAR T cells, CAR T cells rely on the release of perforin for efficient killing.

Supplementary Figure 2: Soluble and bound EGFR ligands do not trigger unwanted SAR T cell activation, while antibodies monovalent and bivalent for SAR can trigger T cell activation to the same extent.

Small molecule prodrug approaches that can activate cancer therapeutics selectively in tumors are urgently needed. Here, we developed the first antitumor prodrugs designed for activation by the thioredoxin (Trx) oxidoreductase system. This critical cellular disulfide redox axis is tightly linked to dysregulated redox/metabolic states in cancer, yet it cannot be addressed by current bioreductive prodrugs, which mainly cluster around oxidised nitrogen species. We instead harnessed Trx/TrxR-specific artificial dichalcogenides to gate the bioactivity of a series of 10 “off-to-on” reduction-activated duocarmycin prodrugs. The prodrugs were tested for cell-free and cellular activity dependent on reducing enzyme systems in 177 cell lines, to establish broad trends for redox-based cellular bioactivity of the dichalcogenides. They were well toleratedin vivoin mice, indicating low systemic release of their duocarmycin cargo, andin vivoanti-tumor efficacy trials in mouse models of breast and pancreatic cancer gave promising initial results indicating effective tumoral drug release, presumably byin situbioreductive activation. This work therefore presents a chemically novel class of bioreductive prodrugs against a previously unaddressed reductase type, validates its ability to accessin vivocompatible small-molecule prodrugs even of potently cumulative toxins, and so introduces carefully tuned dichalcogenides as a platform strategy for specific bioreduction-based release.

Novel photoswitches with features complementary to the well-established azobenzenes are increasingly driving high-precision research in photopharmacology in cells and in vivo. Styrylthiazolium (StyTz) and styrylbenzothiazolium (StyBtz) are cellularly untested E/Z-isomerisation photoswitches which are nearly isosteric to azobenzenes, but have distinct photophysical and physicochemical properties: including ca. 60 nm red-shifted π→π* absorption, self-reporting fluorescence, Z→E relaxation that matches typical biological timescales, and good solubility due to their permanent positive charge. We here tested StyTz and StyBtz for their potential as photopharmaceutical scaffolds, by applying them to photocontrol the dynamics of the microtubule cytoskeleton. We observed light-specific disruption of microtubule network architecture and antiproliferative activity, with a structure-activity relationship matching expectations for tubulin inhibitors. However, while testing the lead StyBtz2 for its molecular mechanism of action, we found that it did not inhibit microtubule dynamics. We tracked its localisation in live cells by relying on its self-reporting fluorescence, observing accumulation of E-StyBtz2 into mitochondria; after several minutes of illumination it was then released into the cytosol concomitant with blebbing and cell death. We interpret this as light-dependent catastrophic rupturing of mitochondria on acute timescales. We conclude that StyTz/StyBtz can be interesting photopharmaceutical scaffolds for addressing mitochondrial, rather than cytosolic, targets.

Helicenes are high interest synthetic targets with unique conjugated helical structures that have found important technological applications. Despite this interest, helicenes have had limited impact in chemical biology. Herein, we disclose a first-in-class antimitotic helicene, helistatin 1 (HA-1), where the helicene scaffold acts as a structural mimic of colchicine, a known antimitotic drug. The synthesis proceeds via sequential Pd-catalyzed coupling reactions and a π-Lewis acid cycloisomerization mediated by PtCl2. HA-1 was found to block microtubule polymerisation in both cell-free and live cell assays. Not only does this demonstrate the feasibility of using helicenes as bioactive scaffolds against protein targets, but also suggests wider potential for the use of helicenes as isosteres of biaryls or cis-stilbenes - themselves common drug and natural product scaffolds. Overall, this study further supports future opportunities for helicenes for a range of chemical biological applications.

Background T cell receptor fusion constructs (TRuC) consist of an antibody-based single chain variable fragment (scFv) fused to a T cell receptor chain (TCR) and allow recognition of cancer cells in an HLA-independent manner. Unlike chimeric antigen receptors (CAR), TRuC are integrated into the TCR complex resulting in a functional chimera with novel specificity, whilst retaining TCR signaling. To further enhance anti-tumor function, we expressed a PD-1-CD28 fusion receptor in TRuC T cells aiming to prevent tumor-induced immune suppression and T cell anergy. Methods The activation level of engineered T cells was investigated in co-culture experiments with tumor cells followed by quantification of released cytokines using ELISA. To study T cell-mediated tumor cell lysis in vitro, impedance-based real-time tumor cell killing and LDH release was measured. Finally, two xenograft mouse cancer models were employed to explore the therapeutic potential of engineered T cells. Results In co-culture assays, co-expression of PD-1-CD28 enhanced cytokine production of TRuC T cells. This effect was dependent on PD-L1 to PD-1-CD28 interactions, as blockade of PD-L1 amplified IFN-γ production in unmodified TRuC T cells to a greater level compared to TRuC-PD-1-CD28 T cells. In vivo, PD-1-CD28 co-expression supported the anti-tumor efficacy of TRuC T cells in two xenograft mouse cancer models. Conclusion Together, these results demonstrate the therapeutic potential of PD-1-CD28 co-expression in TRuC T cells to prevent PD-L1-induced T cell hypofunction.

Photoswitchable reagents to modulate microtubule stability and dynamics are an exciting tool approach towards micron- and millisecond-scale control over endogenous cytoskeleton-dependent processes. When these reagents are globally administered yet locally photoactivated in 2D cell culture, they can exert precise biological control that would have great potential forin vivotranslation across a variety of research fields and for all eukaryotes. However, photopharmacology’s reliance on the azobenzene photoswitch scaffold has been accompanied by a failure to translate this temporally- and cellularly-resolved control to 3D models or toin vivoapplications in multi-organ animals, which we attribute substantially to the metabolic liabilities of azobenzenes.Here, we optimised the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based instead on the styrylbenzothiazole (SBT) photoswitch scaffold, that are non-responsive to the major fluorescent protein imaging channels and so enable multiplexed imaging studies. We applied these reagents to 3D systems (organoids, tissue explants) and classic model organisms (zebrafish, clawed frog) with one- and two-protein imaging experiments. We successfully used systemic treatment plus spatiotemporally-localised illuminationsin vivoto photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in these systems with cellular precision and second-level resolution. These nanomolar,in vivo-capable photoswitchable reagents can prove a game-changer for high-precision cytoskeleton research in cargo transport, cell motility, cell division and development. More broadly, their straightforward design can also inspire the development of similarly capable optical reagents for a range of protein targets, so bringing generalin vivophotopharmacology one step closer to productive realisation.

Photoswitchably bioactive reagents, known as “photopharmaceuticals”, promise powerful applications in high-precision biological research. Yet most photoswitch scaffolds cannot be quantitatively bidirectionally photoisomerised, so they suffer from residual background activity that can confound experiments. We rationally designed photopharmaceuticals using the emerging near-quantitative photoswitch pyrrole hemithioindigo (PHTubs), to isomer-specifically inhibit the cytoskeletal protein tubulin. These PHTub reagents allow simultaneous visible-light imaging and photoswitching in live cells, where they could be used for cell-precise photomodulation of microtubule dynamics, and photocontrol over cell cycle progression and cell death. This is, as far as we know, the first use of a hemithioindigo photopharmaceutical for high-spatiotemporal-resolution biological control in live cells. This work opens up new horizons for high-precision microtubule research using PHTubs; and shows the cellular applicability of the near-quantitative photoswitch pyrrole hemithioindigo as a valuable scaffold for photocontrol of a range of other biological targets.

Forced expression of C-X-C chemokine receptor type 6 in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer.The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.

Purpose: Genetically engineered T cells are powerful anticancer treatments but are limited by safety and specificity issues. We herein describe an MHC-unrestricted modular platform combining autologous T cells, transduced with a targetable synthetic agonistic receptor (SAR), with bispecific antibodies (BiAb) that specifically recruit and activate T cells for tumor killing. Experimental Design: BiAbs of different formats were generated by recombinant expression. T cells were retrovirally transduced with SARs. T-cell activation, proliferation, differentiation, and T-cell–induced lysis were characterized in three murine and human tumor models in vitro and in vivo. Results: Murine T cells transduced with SAR composed of an extracellular domain EGFRvIII fused to CD28 and CD3ζ signaling domains could be specifically recruited toward murine tumor cells expressing EpCAM by anti-EGFRvIII × anti-EpCAM BiAb. BiAb induced selective antigen-dependent activation, proliferation of SAR T cells, and redirected tumor cell lysis. Selectivity was dependent on the monovalency of the antibody for EGFRvIII. We identified FAS ligand as a major mediator of killing utilized by the T cells. Similarly, human SAR T cells could be specifically redirected toward mesothelin-expressing human pancreatic cancer cells. In vivo, treatment with SAR T cells and BiAb mediated antitumoral activity in three human pancreatic cancer cell xenograft models. Importantly, SAR activity, unlike CAR activity, was reversible in vitro and in vivo. Conclusions: We describe a novel ACT platform with antitumor activity in murine and human tumor models with a distinct mode of action that combines adoptive T-cell therapy with bispecific antibodies.

Small molecule inhibitors provide a versatile method for studies in microtubule cytoskeleton research, since tubulin is not readily amenable to functional control using genetics. However, traditional chemical inhibitors do not allow spatiotemporally precise applications on the length and time scales appropriate for selectively modulating microtubule-dependent processes. We have synthesised a panel of taxane-based light-responsive microtubule stabilisers, whose tubulin hyperpolymerisation activity can be induced by photoisomerisation to their thermodynamically metastable state. These reagents can be isomerised in live cells, optically controlling microtubule network integrity, cell cycle repartition, and cell survival, and offering biological response on the timescale of seconds and spatial precision to the level of individual cells. These azobenzene-based microtubule stabilisers offer the possibility of noninvasive, highly spatiotemporally precise modulation of the microtubule cytoskeleton in live cells, and can prove powerful reagents for studies of intracellular transport, cell motility, and neurodegeneration.Abstract Figure

We here report the discovery of isoquinoline-based biaryls as a new scaffold for colchicine domain tubulin inhibitors. Colchicine domain inhibitors comprise a structurally diverse class of compounds offering highly desirable cytotoxic and vascular disrupting bioactivities. Current research on colchicine domain inhibitors is focused on improving in vivo robustness and tolerability: properties that are inextricably linked to the scaffold structure employed. The isoquinoline-based biaryl scaffold we now report offers high-potency tubulin inhibition with excellent robustness and druglikeness, allowing solubility, in vivo tolerability and facile late-stage structural diversification through a tolerant synthetic route. We have confirmed the tubulin-binding properties and mechanism of these isoquinoline-based biaryls through a series of cellular tests and established safe in vivo dosing parameters in mice. By addressing several problems facing the current families of inhibitors, we thus expect that this new scaffold will find a range of powerful in vivo applications towards translational use in cancer therapy.

BACKGROUND CD16-chimeric antigen receptors (CAR) T cells recognise the Fc-portion of therapeutic antibodies, which can enable the selective targeting of different antigens. Limited evidence exists as to which CD16-CAR design and antibody partner might be most effective. We have hypothesised that the use of high-affinity CD16 variants, with increased Fc-terminus antibody affinity, combined with Fc-engineered antibodies, would provide superior CD16-CAR T cell efficacy. METHODS CD16-CAR T (wild-type or variants) cells were co-cultured with Panc-1 pancreatic cancer, Raji lymphoma or A375 melanoma cells in the presence or absence of anti-CD20, anti-MCSP, wild-type or the glycoengineered antibody variants. The endpoints were proliferation, activation, and cytotoxicity in vitro. RESULTS The CD16 158 V variant of CD16-CAR T cells showed increased cytotoxic activity against all the tested cancer cells in the presence of the wild-type antibody directed against MCSP or CD20. Glycoengineered antibodies enhanced CD16-CAR T cell activity irrespective of CD16 polymorphisms as compared with the wild-type antibody. The combination of the glycoengineered antibodies with the CD16-CAR 158 V variant synergised as seen by the increase in all endpoints. CONCLUSION These results indicate that CD16-CAR with the high-affinity CD16 variant 158 V, combined with Fc-engineered antibodies, have high anti-tumour efficacy.

IL-22 has been identified as a cancer-promoting cytokine, but its regulation in cancer tissue has not been addressed. Using both murine and human models, we demonstrate that cancer cells directly induce IL-22 production. We prove that interleukin-1β induced by inflammasome activation is critical for IL-22 production. IL-1β increased the activity of the IL-22 transcription factors in lineage-committed T cells. We show the existence of IL-22–producing Th1, Th17, and Th22 cells in tumor tissue of patients. Use of the clinically approved IL-1 receptor antagonist anakinra in vivo reduced IL-22 production and reduced tumor growth in a breast cancer model. These data provide the basis for therapeutic interventions, particularly using anakinra, aiming at limiting IL-22 production in patients with cancer.

Adoptive T cell therapy, namely chimeric antigen receptor (CAR) T cell therapy has been a groundbreaking and effective treatment of relapsed or refractory haematological malignancies. Still, many patients do not respond or relapse with treatment-resistant disease. Additionally, toxicities such as cytokine release syndrome remain problematic. Cancer heterogeneity, beyond cancer types and inter-patient differences, is present within every individual patient. This heterogeneity, especially in the context of solid tumors, has meant that targeted immunotherapies have fared relatively poorly, creating a need for a modular platform with a capacity to target multiple antigens simultaneously and/or sequentially. Likewise, treatment-related toxicities have limited the therapeutic efficacy and breadth of patient selection. To tackle these caveats through a modular and controllable approach, we equipped T cells with synthetic agonistic receptors (SARs) that are only activated when a tumor-associated antigen and a cross-linking bispecific antibody (BiAb) specific for both SAR T cell and tumor cell are also present. The SAR itself is constituted of an inert extracellular domain in the form of EGFRvIII, that is fused to the T cell activating domains CD28 and CD3ζ. The BiAb employed is a trivalent CrossMab, with two binding arms (2 x Fab) for the tumor-associated antigen (mesothelin), and one binding arm for the SAR receptor (EGFRvIII). We showed that BiAb triggering of the SAR is conditional upon the binding of the second BiAb specificity. What is particularly advantageous with this approach is that T cell activation may only occur when the BiAb is present and in proximity to the antibody-targeted tumor cell. This conditional T cell activation is an inherent safety feature of the platform, whereby if unwanted levels of T cell activation are observed, depletion of the BiAb from the system could result in the reversal of said activation, thus managing the potential toxicity. This work was able to determine the validity and efficacy of the approach. Through the generation of several human pancreatic cancer models, and extensive in vitro and in vivo testing, the platform could be characterized. Its translational relevance and significance as a next-generation adoptive T cell therapy with the potential to plug some gaping pitfalls of current ACT approaches were also shown.

The combination of directed migration and immunosuppressive shielding enables effective T cell therapy in solid tumors., CAR T cell therapy remains ineffective in solid tumors, due largely to poor infiltration and T cell suppression at the tumor site. T regulatory (Treg) cells suppress the immune response via inhibitory factors such as transforming growth factor–β (TGF-β). Treg cells expressing the C-C chemokine receptor 8 (CCR8) have been associated with poor prognosis in solid tumors. We postulated that CCR8 could be exploited to redirect effector T cells to the tumor site while a dominant-negative TGF-β receptor 2 (DNR) can simultaneously shield them from TGF-β. We identified that CCL1 from activated T cells potentiates a feedback loop for CCR8+ T cell recruitment to the tumor site. This sustained and improved infiltration of engineered T cells synergized with TGF-β shielding for improved therapeutic efficacy. Our results demonstrate that addition of CCR8 and DNR into CAR T cells can render them effective in solid tumors.