Summary The efficacy of engineered T cell therapies in solid tumors remains limited by T cell dysfunction, driven by complex processes that cannot be easily manipulated via genetic knockouts or overexpression of individual genes. Protein design can create new biological functions that can rewire these consequential cell fate decisions. Here, we introduce OUTLAST Regulators, designed proteins that reprogram critical T cell signaling pathways to enhance functional persistence. These proteins are capable of regulating diverse groups of proteins such as the NR4A family of pro-exhaustion transcription factors, E3 ligases Cbl-b and c-Cbl, and SOCS family proteins. Our designs markedly improve CAR-T and TCR-T performance in vitro and in vivo in stringent solid tumor preclinical models. OUTLAST Regulators are implemented as compact genetic modules compatible with standard viral vectors and cell therapy manufacturing processes, creating a powerful platform for programming new functions into enhanced cell and gene therapies.

Abstract T cell therapies have had modest efficacy in solid tumors due to their failure to proliferate following infusion. We have designed a mesothelin (MSLN) CAR T product (OPB-101) which includes a novel promoter (OP1), an optimized CAR, a safety switch, and a CD8α-targeted IL-2/15 designed cytokine to promote T cell expansion and improve efficacy in solid tumors. Human anti-MSLN binders were optimized for the target using our OUTSPACERTM library. CAR evaluation was conducted using in vitro coculture assays and validated NSG tumor xenograft models. OUTSMARTTM IL-2/15 was designed to avoid regulatory T cell activation by ablating IL-2Rα binding and re-targeted to CD8α to enhance CD8+ T cell and NK stimulation. EGFRoptTM was engineered for improved sensitivity to EGFR-targeted immunotherapies. Using 2A polypeptides, all transgenes were expressed with an antigen-dependent, inducible “stim-on” promoter (OP1) to regulate CAR expression, cytokine production, and resistance to exhaustion. The efficacy of the OPB-101 construct was tested in stringent exhaustion assays. In vivo efficacy was assessed with mice engrafted with s.c. H1650 tumors that were treated by i.v. injection of mock, CAR only or OPB-101 T cells at doses ranging from 0.25 × 106 to 2 × 106 cells. Tumor volume and weight were measured for 80 days. T cell levels and phenotype were measured by peripheral blood (PB) draws and intratumoral analysis (IT; day 14). OPB-101 efficiently killed MSLN+ tumor cell lines in repeat challenge and spheroid assays, and inducibly produced the IL-2/15 cytokine. IL-2/15 expression did not result in antigen-independent CAR T cell proliferation but extended survival in conditions without exogenous cytokine support. In vivo efficacy studies showed tumor control at 2 × 106 cells in the CAR only condition and modest anti-tumor efficacy at the 1 × 106 dose. In contrast, OPB-101 demonstrated complete and durable tumor elimination at the lowest dose of 0.25 × 106 cells (OPB-101 versus CAR only at 1 × 106 cells, p < 0.0001 and p < 0.0005 for donor 1 and 2, respectively). OPB-101 efficacy was associated with robust expansion in the PB (>100-fold) within IT (>5-fold) compared to the CAR only condition. OPB-101 PB and IT T cells showed reduced levels of PD-1, TIGIT and CD39 compared to CAR only suggesting resistance to exhaustion. Finally, studies conducted in tumor-free mice showed that OPB-101 expansion in vivo was dependent on the presence of tumor. In summary, OPB-101 using a regulated promoter (OP1) to dynamically express an optimized MSLN CAR, EGFRoptTM, and a CD8-targeted IL-2/15 cytokine enhances anti-tumor efficacy against solid tumors by promoting CAR expansion and persistence while limiting T cell exhaustion. These technologies allow for complete elimination of solid tumors at a low treatment dose (i.e., <250K cells) and may pave the way to more effective CAR T cell therapies for solid tumors. Citation Format: Rupesh Amin, Howell Moffett, Kevin Haworth, Jerry Chen, Jason Yokoyama, Allan Wang, Leah Tait, Maria Steele, John Crowl, Thaddeus Davenport, Joseph DeSautelle, Jared Hammer, Willimark Obenza, Vanessa Montoya, Robin Kirkpatrick, Laura Baker, Tina Tan, Kristie Shirley, Bradley Hammerson, Robert Langan, David Clausen, Paul Sample, Brian Weitzner, Shujun Yuan, Marc Lajoie, Scott Boyken, Aaron Foster. OPB-101: An optimized mesothelin-specific CAR T cell product expressing CD8-targeted IL-2/15 and engineered to resist T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 51.

Abstract A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB070.

Abstract IL-12 is an immune-stimulatory cytokine that can modulate the tumor microenvironmnent (TME) to enhance the cytotoxic activity of T and NK cells; however, IL-12 expression by T cells caused severe toxicity in a previous clinical trial. Using Outpace’s OutSmart™ technology, we designed a tumor-restricted IL-12 (trIL-12) that is under control of an activation-inducible promoter and auto-inactivates within minutes after secretion. T cells were engineered via lentiviral vectors (LVV) to express wild-type single-chain IL-12 (WT scIL-12) or trIL-12 under the control of an activation-inducible promoter; a second LVV introduces an NY-ESO-1 TCR. Kinetics of IL-12 expression was measured by qPCR and MSD technology; kinetics of IL-12p70 heterodimer half-life was measured using Octet bio-layer interferometry (BLI). T-cell cytotoxicity and cytokine production was evaluated in vitro after repeated stimulation with NY-ESO-1 expressing target cells using Incucyte and MSD. IL-12 activity in bystander cells was measured by detection of IFN-γ using flow cytometry. In vivo T-cell function of trIL-12-engineered NY-ESO-1 TCR T cells was measured in NSG MHCI/II KO mice bearing A375 xenografts. trIL-12 activity in a fully immune-competent mouse model was measured in B6 mice implanted with B16F10 tumor cells engineered to express murine surrogates of trIL-12. Expression of WT scIL-12 under the control of an activation-inducible promoter peaks within 6 hours after activation and produces sufficient IL-12 to improve T-cell function in vitro and in vivo. trIL-12 was created by inclusion of a cleavable linker and elimination of the covalent disulfide bond between the p35 and p40 subunits of IL-12p70, resulting in dissociation of the functional cytokine within 10 minutes post-cleavage. Additional mutations further reduce the IL-12p70 half-life. trIL-12 activates the IL-12-producing T cells, as well as proximal bystander T-cells in direct co-culture, but it does not have the ability to activate distal bystander T-cells separated by Transwell membranes. In xenograft mouse models, trIL-12-expressing T cells have improved proliferation upon antigen recognition and display potent anti-tumor activity and cytokine production without showing systemic IL-12 accumulation. Furthermore, in fully immune competent mice, expression of a murine trIL-12 surrogate by tumor cells led to generation of potent anti-tumor responses without systemic trIL-12 accumulation and reduced systemic IFN-γ. trIL-12-engineered T cells generate potent anti-tumor activity in vitro and in vivo. Unlike WT scIL-12, trIL-12 activity is localized to the region around the producing T cell and systemic IL-12 exposure is not observed in vivo. Collectively, these preclinical data suggest that trIL-12 may enable the development of potent T-cell therapeutics while maintaining an acceptable safety profile. Citation Format: Szu-Han Huang, Thaddeus M. Davenport, Howell F. Moffett, Brian D. Weitzner, Luke Cassereau, Laura E. Baker, Bradley Hammerson, Summer Zhuang, Christine Saechao, Lisa Song, Jade Mimms, David Chian, Candace Sims, Hajime Hiraragi, Marc J. Lajoie, Scott E. Boyken, Bijan Boldajipour. Development of tumor-restricted IL-12 with antigen-dependent expression and localized IL-12 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4067.

Introduction: Mesothelin (MSLN) is an attractive target for chimeric antigen receptor T cell (CAR-T) immunotherapy as it is highly expressed on mesothelioma, ovarian, lung cancer, and some forms of leukemia, including approximately 30% of acute myeloid leukemia (AML); however, MSLN directed CAR-T cells are prone to exhaustion, and soluble proteins, such as shed MSLN and MUC16 can inhibit anti-tumor efficacy. To address these deficiencies, we developed a novel, fully-human MSLN-specific CAR using our OutSpacer™ technology to optimize the immune synapse geometry. Further, this MSLN CAR is expressed using a novel regulatory response element to dynamically express the CAR molecule to enhance CAR-T potency and durability. Methods: A set of fully-human single domain binders (VHH) were produced and screened against a spacer library to identify high-performing CARs. The spacer library is composed of human extracellular polypeptides selected for optimal biophysical properties (e.g., flexibility), enabling systematic sampling to identify CAR constructs with optimal immune synapse geometry. More than 300 MSLN-directed CAR candidates were evaluated in vitro using 2D or 3D (spheroid) culture assays to measure tumor killing and cytokine production against several MSLN + cell lines that express different levels of the target protein. These functional assays were also performed in the presence of recombinant MSLN (10ug/mL) or MUC16 (1ug/mL) to assess potential inhibition by shed target or ligand. Top performing MSLN CARs were subsequently tested in NSG mice (2e6, 4e6 and 8e6 cells/animal; 2 donors) subcutaneously engrafted with MSLN + H1650 tumor cells to identify the best CAR candidate. To further improve the durability of T cell response, the MSLN CAR was expressed under the control of an engineered promoter (OP1) to dynamically regulate expression, and compared to CARs expressed constitutively using the MND promoter. CAR-T cells were repetitively stimulated (4x) by plate-bound antigen in combination with CD58 and ICAM1, and subsequently tested in a tumor spheroid assay for killing, cytokine production, CAR expression and T cell activation/ exhaustion markers (i.e., PD-1, TIGIT, CD39 and TIM3). Results: Anti-MSLN VHH domains targeting region 1 or 3 (RI and RIII) of the MLSN extracellular domain were paired with a defined spacer library to define the optimal immune synapse. Contrary to the conclusions of prior studies, RI (membrane-distal) binders outperformed RIII (membrane-proximal) binders when paired with their optimal spacer. Based on tumor killing and cytokine production, RI targeted CARs preferred a spacer length of approximately 75 Å, consistent with our previously-reported optimal CAR synapse of ~200 Å. All spacer optimized MSLN-specific CARs resisted inhibition by soluble MSLN and MUC16. In vivo studies with two donors in NSG mice engrafted with MSLN + tumors showed that one CAR (B8S8) eliminated tumors at a dose of 2e6 and 4e6 CAR-T cells per animal, where other CAR constructs, including a clinically-used MSLN-specific CAR, failed to control tumors even at 8e6 cells per animal. Secondly, we assessed whether a novel “stim-on” promoter (OP1) could enhance CAR-T activity by preserving T cell function. Here, B8S8 CAR was expressed via OP1 or the constitutive MND promoter (n=3 donors). Following chronic antigen exposure by plate-bound antigen, OP1 regulated MSLN CAR T cells retained the capacity to eliminate MSLN + H226 tumor spheroids compared to MND-driven CAR T constructs. OP1-regulated CAR T cells also continued to produce high levels of cytokines (IFN-γ and IL-2) whereas MND CAR-T cells lost cytokine production ability. Further, phenotypic analyses showed that MND CAR-T cells expressed elevated levels of PD-1 and TIGIT compared to OP1 CAR-T cells. Conclusions: Contrary to the conclusions of prior studies with MSLN-targeted CARs, membrane distal epitopes were superior to membrane proximal epitopes when binders were paired with the correct spacer, and shed MSLN and MUC16 were not inhibitory for the best-performing CARs, regardless of target epitope. Furthermore, regulated expression enhanced the potency and durability of response compared to constitutive expression. This approach may improve CAR activity in patients with MSLN + solid tumors or leukemia.

Background IL-12 is a pleiotropic immune-stimulatory cytokine that can modulate the tumor microenvironment to promote innate and adaptive immune responses and support cytotoxic activity of T and NK cells. However, systemic delivery of IL-12 recombinant protein or T cells engineered to secrete IL-12 causes severe toxicity in patients. To overcome these limitations, we leveraged OutSmart™ technology to design a tumor-restricted IL-12 (trIL-12) that rapidly auto-inactivates after secretion from engineered T cells under the control of an inducible promoter. The resulting design achieves safe, local delivery of IL-12 activity from engineered tumor-specific T cells. Methods trIL-12 was designed via sequence alterations that remove the intermolecular disulfide bond between the p40 (C177S) and p35 (C74S) subunits, and the addition of Furin-cleavable sites on the linker between p40 and p35 in a single-chain (scIL-12) format. The dissociation rate of the cleaved IL-12 heterodimer was measured using bio-layer interferometry. T cells were engineered with lentiviral vectors expressing either wild-type (wt) scIL-12 or trIL-12 under the control of an engineered inducible promoter and a constitutively expressed NY-ESO-1 TCR. IL-12 activity in proximal or distal bystander immune cells was measured by IFN-γ production in T cells co-cultured with engineered T cells producing IL-12, either directly or separated by a transwell membrane. Next, engineered T cells were functionally assessed in vitro by repeat challenge with NY-ESO-1+ A375 tumor cells. Lastly, T-cell efficacy and systemic IL-12 exposure in vivo were evaluated using NSG MHCI/II KO mice engrafted with A375 tumors and treated i.v. with engineered T cells. Results The inducible promoter enabled minimal basal IL-12 production from T cells and substantially increased IL-12 production after T-cell activation. trIL-12 dissociated into an inactive state post-cleavage with a half-life of ~10 minutes, and it activated proximal but not distal bystander T cells, demonstrating that function is restricted to the site of induced expression. trIL-12 and wt scIL-12 both enhanced T-cell cytotoxicity similarly in vitro, and both exhibited potent and comparable anti-tumor efficacy in vivo; however, only wt scIL-12, but not trIL-12, was detectable in an active state in peripheral blood, thus demonstrating trIL-12's localized activity that may improve the safety profile. Conclusions These data demonstrate that trIL-12 can deliver potent IL-12 stimulation at the tumor site while avoiding systemic exposure, potentially improving efficacy for T-cell therapies while maintaining a favorable safety profile that may finally allow effective administration of IL-12.

Introduction: Effective treatment with chimeric antigen receptor T (CAR-T) cells against solid and some liquid tumors will require the safe production of immune-modulating cytokines to promote CAR-T cell expansion and to stimulate endogenous anti-tumor immune responses within the tumor microenvironment. OutSmart™ IL-2/15 is a computationally designed, CD8α-targeted, IL-2-based cytokine produced by engineered T cells via a T cell activation-dependent promoter. Here, we demonstrate that inducible production of CD8-targeted IL-2/15 drives proliferation and anti-tumor efficacy of ROR1-specific CAR-T cells, stimulates bystander CD8 + T and NK cells, and minimizes activation of immune suppressive T regulatory cells (Tregs). Methods and Results: Wild-type (wt) IL-2 was modified using Rosetta protein design software package to eliminate IL-2Rα binding while retaining native IL-2Rβγ binding interfaces. Out of 10,355 designs, 38 were prioritized based on in silico analysis and ML-guided structure prediction, and evaluated for IL-2R binding as recombinant proteins. Consistent with the design criteria, none of the designs bound IL-2Rα, and 33 (87%) retained IL-2Rβγ binding and signaling activity. pSTAT5 analysis showed wt IL-2 highly activated CD4 +CD25 +FoxP3 + Treg cells, whereas the novel IL-2 designs showed a >3-log reduction Treg stimulation. These designs were iteratively optimized to generate a version exhibiting improved thermostability and IL-2Rβγ binding affinity equivalent to wt IL-2. To increase the potency against immune effector cells, but not Tregs, an anti-CD8α VHH was tethered to the modified IL-2 molecules (CD8-IL-2/15) to mimic high-affinity IL-2Rα binding and selectively activate CD8 + T and NK cells. This prosthetic binding interaction dramatically increased CD8 + T cell stimulation and CD8α + NK cell activation (>2-logs and 1-log by pSTAT5, respectively), while retaining its reduced capacity to stimulate Tregs. CD8-IL-2/15 gene expression was subsequently placed under the control of a T cell activation-dependent promoter and integrated into a lentiviral construct with a constitutively expressed ROR1-specific CAR. T cells transduced with a lentivirus expressing CD8-IL-2/15 and a ROR1-targeting CAR showed inducible cytokine production following exposure to ROR1 + tumor cells (H1975) leading to enhanced CAR-T cell proliferation and anti-tumor killing during repeat tumor challenge assays in vitro. Using immune-deficient mice (NSG) engrafted with H1975 tumors, inducible CD8-IL-2/15 enhanced ROR1-specific CAR-T cells showed durable tumor control at low doses (1e6 - 4e6 CAR T cells/animal), which was comparable to wt IL-2, whereas mock T cells and ROR1 CAR only T cells failed to control tumor growth ( Figure 1). In addition, CAR-T expansion with CD8-IL-2/15 increased >4-fold over wt IL-2 (p<0.0001), measured by cell counts in peripheral blood. Conclusions: In summary, OutSmart™ IL-2/15 is a genetic module that produces CD8-IL-2/15 in response to T cell activation, promoting robust CAR-T expansion and enhanced anti-tumor efficacy. Furthermore, local production of a CD8-IL-2/15 enhances the effector function of bystander CD8 + T cells and NK cells, but only minimally activates Treg cells due to removing the IL-2Rα binding interface. Inducible production of CD8-IL-2/15 dramatically improves the potency of a ROR1 CAR for treating ROR1 + solid and liquid cancers. More generally, OutSmart™ protein design methods and control technologies can be applied to create other designed cytokines for oncology and beyond.

Chimeric antigen receptor (CAR) T-cell therapy has been shown to produce profound results in the treatment of certain hematologic malignancies, however treatment of solid tumors with CAR T cells has not been as successful. Studies have suggested that T-cell exhaustion plays a role in limiting the ability of CAR T cells to eradicate solid tumors. Additionally, stem-like qualities of T cells have been associated with better outcomes in patients treated with cellular therapies, including CAR T cells. Therefore, maintaining stem-like qualities and overcoming T-cell exhaustion may be key to improving clinical efficacy of CAR T cells in patients with solid tumors. ROR1 is a cell surface antigen expressed in several solid tumor types and chronic lymphocytic leukemia (CLL). ROR1 expression has been reported in 57% of triple-negative breast cancer (TNBC), as well as 42% of adenocarcinoma and 12% of squamous cell carcinoma subtypes of non-small cell lung cancer (NSCLC). These expression data of ROR1 in TNBC and NSCLC provide support for anti-ROR1 agents as a therapeutic strategy for these cancers. LYL797 is a novel, ROR1-targeted chimeric antigen receptor (CAR) T-cell product that incorporates genetic and epigenetic reprogramming technologies, Gen-R and Epi-R, to overcome barriers of CAR T-cell therapies in solid tumors. The ROR1-specific CAR contains a single-chain variable fragment (scFv) derived from an R12 rabbit monoclonal antibody that recognizes and binds with high specificity to human ROR1. Gen-R is ex vivo genetic reprogramming technology that engineers CAR T cells to overexpress c-Jun. Dysregulation of activator protein 1 (AP-1) has been implicated in CAR T-cell exhaustion, and studies have demonstrated that overexpression of c-Jun renders CAR T cells less susceptible to exhaustion, enhancing both anti-tumor efficacy and persistence in preclinical models of hematologic and solid tumors. Epi-R is a proprietary optimized manufacturing process that results in maintenance of stem-like phenotype and function of T-cell products. In preclinical studies LYL797 cells reprogrammed with Gen-R and Epi-R led to improved functional activity in the presence of ROR1+ tumor cells compared to conventional ROR1 CAR T cells. Additional studies are underway to determine the mechanisms by which antitumor activity of LYL797 in ROR1-positive solid tumor xenograft models is enhanced. LYL797 is anticipated to enter into Phase 1 clinical trials for TNBC and NSCLC in 2022. Citation Format: Spencer Park, Courtney Simianer, Sydney Spadinger, Xiao Wang, Purnima Sundar, Shobha Potluri, Rachel Lynn, Bijan Boldajipour, Grace Wang, Neeraj Sharma, Hajime Hiraragi, Veena Krishnamoorthy, Suman Kumar Vodnala, E-Ching Ong, Chang-Chih Wu, Martin Wohlfahrt, Byoung Ryu, Lisa Song, Brian D. Weitzner, Howell Moffett, Marc Lajoie, Scott Boyken, Tamer Shabaneh, Shivani Srivastava, Tina Albertson, Blythe Sather. LYL797, a ROR1 CAR T-cell therapy with genetic and epigenetic reprogramming for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2754.

The de novo design of three protein chains that associate to form a heterotrimer (but not any of the possible two-chain heterodimers) and that can drive the assembly of higher-order branching structures is an important challenge for protein design. We designed helical heterotrimers with specificity conferred by buried hydrogen bond networks and large aromatic residues to enhance shape complementary packing. We obtained ten designs for which all three chains cooperatively assembled into heterotrimers with few or no other species present. Crystal structures of a helical bundle heterotrimer and extended versions, with helical repeat proteins fused to individual subunits, showed all three chains assembling in the designed orientation. We used these heterotrimers as building blocks to construct larger cyclic oligomers, which were structurally validated by electron microscopy. Our three-way junction designs provide new routes to complex protein nanostructures and enable the scaffolding of three distinct ligands for modulation of cell signaling.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

Background Chimeric antigen receptors (CARs) are synthetic receptors that target engineered immune cell effector functions against target cells expressing specific antigens. CAR activity can be significantly improved by optimization of multiple parameters, including geometry of the immunologic synapse, biophysical properties of the extracellular domains, and signaling properties of the intracellular domains. Native T cell acti-vation is driven by adhesion molecules and T cell Receptors (TCRs) binding to peptides displayed on the Major Histocom-patibility Complex (MHC) of target cells, with a well-defined cell-cell distance of 14 – 15 nm in synaptic contact areas. How-ever, the optimal synaptic distance, and the importance of cell-cell synaptic distance for CAR activity has not been systematically determined. Here we investigate the role of spacer length on the recognition of clinically relevant tumor antigens using a panel of 4 previously-published spacers and 41 novel spacers derived from human extracellular proteins and ranging in length from 3.6 – 30.6 nm. Methods CAR targeting at from the three CARs and one HER2 CAR (herceptin). we developed a model system in which a single linear epitope could be systematically presented at different from the target cell membrane using our spacer sequences. this an anti-HA scFv (clone was used as the CAR binding domain and an HA peptide (YPYDVPDYA) was used as a model epitope. We tested all possible CAR constructs in vitro by evaluating cyto-kine production and target cell killing kinetics (primary and serial restimulation).. Addtionally, we tested whether our in vitro observations are predictive of in vivo performance by choosing five spacers that cover a wide range of performance for two CARs with different predicted optimal spacer lengths in the in vitro study (R12, FMC63) and tested them in mouse xenograft models. Results We demonstrate that both in vitro and in vivo CAR activity is dependent on spacer length, with optimal activity observed at a synaptic distance of about 20 nm, substantially longer than the 14 – 15 nm TCR:MHC complex. Furthermore, the optimal range of synaptic distances is far narrower than previously appreciated.

Natural molecular machines contain protein components that undergo motion relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein machinery from designed axle and rotor components with internal cyclic or dihedral symmetry. We find that the axle-rotor systems assemble in vitro and in vivo as designed. Using cryo-electron microscopy, we find that these systems populate conformationally variable relative orientations reflecting the symmetry of the coupled components and the computationally designed interface energy landscape. These mechanical systems with internal degrees of freedom are a step toward the design of genetically encodable nanomachines.

Naturally occurring allosteric protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications 1, but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of allosteric protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest 2. Using broadly applicable design principles, we allosterically couple binding of protein analytes of interest to the reconstitution of luciferase activity and a bioluminescent readout through the association of designed lock and key proteins. Because the sensor is based purely on thermodynamic coupling of analyte binding to switch activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the hIgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-19 3, we use the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder, has a limit of detection of 15pM with an up to seventeen fold increase in luminescence upon addition of RBD. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. One-Sentence Summary We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

Natural nanomachines like the F 1 /F 0 -ATPase contain protein components that undergo rotation relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein rotary machinery from designed axle and ring components. Using cryoelectron microscopy, we find that axle-ring systems assemble as designed and populate diverse rotational states depending on symmetry match or mismatch and the designed interface energy landscape. These mechanical systems with internal rotational degrees of freedom are a step towards the systematic design of genetically encodable nanomachines. One-Sentence Summary Computationally designed self-assembling protein rotary machines sample internal degrees of freedom sculpted within the energy landscape.