Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling.

Proximity-induction of cell-cell interactions via small molecules represents an emerging field in basic and translational sciences. Covalent anchoring of these small molecules represents a useful chemical strategy to enforce proximity; however, it remains largely unexplored for driving cell-cell interactions. In immunotherapeutic applications, bifunctional small molecules are attractive tools for inducing proximity between immune effector cells like T cells and tumor cells to induce tumoricidal function. We describe a two-component system composed of electrophilic bifunctional small molecules and paired synthetic antigen receptors (SARs) that elicit T cell activation. The molecules, termed covalent immune recruiters (CIRs), were designed to affinity label and covalently engage SARs. We evaluated the utility of CIRs to direct anti-tumor function of human T cells engineered with three biologically distinct classes of SAR. Irrespective of the electrophilic chemistry, tumor-targeting moiety, or SAR design, CIRs outperformed equivalent non-covalent bifunctional adapters, establishing a key role for covalency in maximizing functionality. We determined that covalent linkage enforced early T cell activation events in a manner that was dependent upon each SARs biology and signaling threshold. These results provide a platform to optimize universal SAR-T cell functionality and more broadly reveal new insights into how covalent adapters modulate cell-cell proximity-induction.

Pyroptosis activates conventional type I dendritic cells to mediate the priming of highly functional anticancer T cells.

BACKGROUND: Initiation of antitumor immunity is reliant on the stimulation of dendritic cells (DCs) to present tumor antigens to naïve T cells and generate effector T cells that can kill cancer cells. Induction of immunogenic cell death after certain types of cytotoxic anticancer therapies can stimulate T cell-mediated immunity. However, cytotoxic therapies simultaneously activate multiple types of cellular stress and programmed cell death; hence, it remains unknown what types of cancer cell death confer superior antitumor immunity. METHODS: Murine cancer cells were engineered to activate apoptotic or pyroptotic cell death after Dox-induced expression of procell death proteins. Cell-free supernatants were collected to measure secreted danger signals, cytokines, and chemokines. Tumors were formed by transplanting engineered tumor cells to specifically activate apoptosis or pyroptosis in established tumors and the magnitude of immune response measured by flow cytometry. Tumor growth was measured using calipers to estimate end point tumor volumes for Kaplan-Meier survival analysis. RESULTS: We demonstrated that, unlike apoptosis, pyroptosis induces an immunostimulatory secretome signature. In established tumors pyroptosis preferentially activated CD103+ and XCR1+ type I conventional DCs (cDC1) along with a higher magnitude and functionality of tumor-specific CD8+ T cells and reduced number of regulatory T cells within the tumor. Depletion of cDC1 or CD4+ and CD8+ T cells ablated the antitumor response leaving mice susceptible to a tumor rechallenge. CONCLUSION: Our study highlights that distinct types of cell death yield varying immunotherapeutic effect and selective activation of pyroptosis can be used to potentiate multiple aspects of the anticancer immunity cycle.

Endogenous CD28 drives CAR T cell responses in multiple myeloma.

Recent FDA approvals of chimeric antigen receptor (CAR) T cell therapy for multiple myeloma (MM) have reshaped the therapeutic landscape for this incurable cancer. In pivotal clinical trials B cell maturation antigen (BCMA) targeted, 4-1BB co-stimulated (BBζ) CAR T cells dramatically outperformed standard-of-care chemotherapy, yet most patients experienced MM relapse within two years of therapy, underscoring the need to improve CAR T cell efficacy in MM. We set out to determine if inhibition of MM bone marrow microenvironment (BME) survival signaling could increase sensitivity to CAR T cells. In contrast to expectations, blocking the CD28 MM survival signal with abatacept (CTLA4-Ig) accelerated disease relapse following CAR T therapy in preclinical models, potentially due to blocking CD28 signaling in CAR T cells. Knockout studies confirmed that endogenous CD28 expressed on BBζ CAR T cells drove in vivo anti-MM activity. Mechanistically, CD28 reprogrammed mitochondrial metabolism to maintain redox balance and CAR T cell proliferation in the MM BME. Transient CD28 inhibition with abatacept restrained rapid BBζ CAR T cell expansion and limited inflammatory cytokines in the MM BME without significantly affecting long-term survival of treated mice. Overall, data directly demonstrate a need for CD28 signaling for sustained in vivo function of CAR T cells and indicate that transient CD28 blockade could reduce cytokine release and associated toxicities.

The SGLT2 inhibitor canagliflozin suppresses growth and enhances prostate cancer response to radiotherapy.

Radiotherapy is a non-invasive standard treatment for prostate cancer (PC). However, PC develops radio-resistance, highlighting a need for agents to improve radiotherapy response. Canagliflozin, an inhibitor of sodium-glucose co-transporter-2, is approved for use in diabetes and heart failure, but is also shown to inhibit PC growth. However, whether canagliflozin can improve radiotherapy response in PC remains unknown. Here, we show that well-tolerated doses of canagliflozin suppress proliferation and survival of androgen-sensitive and insensitive human PC cells and tumors and sensitize them to radiotherapy. Canagliflozin blocks mitochondrial respiration, promotes AMPK activity, inhibits the MAPK and mTOR-p70S6k/4EBP1 pathways, activates cell cycle checkpoints, and inhibits proliferation in part through HIF-1α suppression. Canagliflozin mediates transcriptional reprogramming of several metabolic and survival pathways known to be regulated by ETS and E2F family transcription factors. Genes downregulated by canagliflozin are associated with poor PC prognosis. This study lays the groundwork for clinical investigation of canagliflozin in PC prevention and treatment in combination with radiotherapy.

HDACi-dependent Microenvironmental Normalization Overcomes Tumor Burden-induced T-cell Exhaustion.

PURPOSE: T-cell exhaustion limits immunotherapy for the treatment of solid tumors. Although immune checkpoint blockade and adoptive T-cell therapy (ACT) can mediate tumor regression, their potency is often determined by tumor burden. Here, we identified tumor burden-related pathway changes that are conducive to T-cell exhaustion. We then determined whether microenvironmental reprogramming via epigenetic modulation could reverse T-cell exhaustion and improve immunotherapeutic responsiveness. EXPERIMENTAL DESIGN: We developed a murine syngeneic tumor model wherein an increased burden ablated therapeutic responsiveness to ACT, which corresponded with systemic induction of T-cell exhaustion. Transcriptome analysis of these large tumors allowed us to characterize changes to immunosuppressive pathway expression during class I histone deacetylase inhibitor MS-275 treatment. We then measured the therapeutic impact of MS-275 during ACT and assessed T-cell exhaustion by transcriptome/phenotypic analysis. RESULTS: ACT durably regressed small tumors but failed to control large tumors, which were associated with systemic T-cell exhaustion and ablation of T-cell responses. Large tumors were defined by an immunosuppressive pathway signature. MS-275 reversed this pathway signature and promoted durable regression of large tumors during ACT. Prototypical exhaustion marker Tim-3 was selectively upregulated in transferred T cells despite displaying a reduced exhaustion signature. Instead, we observed enhanced activation-dependent signaling correlating with enrichment of the IL2-STAT5 signaling axis. Activated CD8+ T-cell responses were predominantly skewed toward terminal effector cell-like CD44+ Tim-3hi TCF1- CD127- KLRG1+ differentiation. CONCLUSIONS: Tumor burden-induced pathway changes can be reversed through epigenetic reprogramming, enabling the conversion from T-cell exhaustion to effector lineage differentiation.

Canagliflozin mediates tumor suppression alone and in combination with radiotherapy in non-small cell lung cancer (NSCLC) through inhibition of HIF-1α.

Non-small cell lung cancer (NSCLC) has a poor prognosis, and effective therapeutic strategies are lacking. The diabetes drug canagliflozin inhibits NSCLC cell proliferation and the mammalian target of rapamycin (mTOR) pathway, which mediates cell growth and survival, but it is unclear whether this drug can enhance response rates when combined with cytotoxic therapy. Here, we evaluated the effects of canagliflozin on human NSCLC response to cytotoxic therapy in tissue cultures and xenografts. Ribonucleic acid sequencing (RNA-seq), real-time quantitative PCR (RT-qPCR), metabolic function, small interfering ribonucleic acid (siRNA) knockdown, and protein expression assays were used in mechanistic analyses. We found that canagliflozin inhibited proliferation and clonogenic survival of NSCLC cells and augmented the efficacy of radiotherapy to mediate these effects and inhibit NSCLC xenograft growth. Canagliflozin treatment alone moderately inhibited mitochondrial oxidative phosphorylation and exhibited greater antiproliferative capacity than specific mitochondrial complex-I inhibitors. The treatment downregulated genes mediating hypoxia-inducible factor (HIF)-1α stability, metabolism and survival, activated adenosine monophosphate-activated protein kinase (AMPK) and inhibited mTOR, a critical activator of hypoxia-inducible factor-1α (HIF-1α) signaling. HIF-1α knockdown and stabilization experiments suggested that canagliflozin mediates antiproliferative effects, in part, through suppression of HIF-1α. Transcriptional regulatory network analysis pinpointed histone deacetylase 2 (HDAC2), a gene suppressed by canagliflozin, as a key mediator of canagliflozin's transcriptional reprogramming. HDAC2 knockdown eliminated HIF-1α levels and enhanced the antiproliferative effects of canagliflozin. HDAC2-regulated genes suppressed by canagliflozin are associated with poor prognosis in several clinical NSCLC datasets. In addition, we include evidence that canagliflozin also improves NSCLC response to chemotherapy. In summary, canagliflozin may be a promising therapy to develop in combination with cytotoxic therapy in NSCLC.

Constitutive expression of interleukin-27 diminishes proinflammatory cytokine production without impairing effector function of engineered T cells.

Immunomodulatory cytokines can alter the tumor microenvironment and promote tumor eradication. Interleukin (IL)-27 is a pleiotropic cytokine that has potential to augment anti-tumor immunity while also facilitating anti-myeloma activity. We engineered human T cells to express a recombinant single-chain (sc)IL-27 and a synthetic antigen receptor targeting the myeloma antigen, B-cell maturation antigen, and evaluated the anti-tumor function of T cells bearing scIL-27 in vitro and in vivo. We discovered that T cells bearing scIL-27 sustained anti-tumor immunity and cytotoxicity yet manifested a profound reduction in pro-inflammatory cytokines granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha. IL-27-expressing T cells therefore present a potential avenue to avert treatment-related toxicities commonly associated with engineered T-cell therapy due to the reduced pro-inflammatory cytokine profile.

LCL161 enhances expansion and survival of engineered anti-tumor T cells but is restricted by death signaling.

Background: The genesis of SMAC mimetic drugs is founded on the observation that many cancers amplify IAP proteins to facilitate their survival, and therefore removal of these pathways would re-sensitize the cells towards apoptosis. It has become increasingly clear that SMAC mimetics also interface with the immune system in a modulatory manner. Suppression of IAP function by SMAC mimetics activates the non-canonical NF-κB pathway which can augment T cell function, opening the possibility of using SMAC mimetics to enhance immunotherapeutics. Methods: We have investigated the SMAC mimetic LCL161, which promotes degradation of cIAP-1 and cIAP-2, as an agent for delivering transient costimulation to engineered BMCA-specific human TAC T cells. In doing so we also sought to understand the cellular and molecular effects of LCL161 on T cell biology. Results: LCL161 activated the non-canonical NF-κB pathway and enhanced antigen-driven TAC T cell proliferation and survival. Transcriptional profiling from TAC T cells treated with LCL161 revealed differential expression of costimulatory and apoptosis-related proteins, namely CD30 and FAIM3. We hypothesized that regulation of these genes by LCL161 may influence the drug's effects on T cells. We reversed the differential expression through genetic engineering and observed impaired costimulation by LCL161, particularly when CD30 was deleted. While LCL161 can provide a costimulatory signal to TAC T cells following exposure to isolated antigen, we did not observe a similar pattern when TAC T cells were stimulated with myeloma cells expressing the target antigen. We questioned whether FasL expression by myeloma cells may antagonize the costimulatory effects of LCL161. Fas-KO TAC T cells displayed superior expansion following antigen stimulation in the presence of LCL161, suggesting a role for Fas-related T cell death in limiting the magnitude of the T cell response to antigen in the presence of LCL161. Conclusions: Our results demonstrate that LCL161 provides costimulation to TAC T cells exposed to antigen alone, however LCL161 did not enhance TAC T cell anti-tumor function when challenged with myeloma cells and may be limited due to sensitization of T cells towards Fas-mediated apoptosis.

T-cell engineered with a fully humanized B-cell maturation antigen-specific T-cell antigen coupler receptor effectively target multiple myeloma.

B-cell maturation antigen (BCMA) is a clinically validated target for multiple myeloma. T-cell engineered with chimeric antigen receptors (CARs) directed against BCMA have demonstrated robust therapeutic activity in clinical trials, but toxicities remain a significant concern for a subset of patients, supporting continued investigation of other engineered T-cell platforms that may offer equal efficacy with an improved toxicity profile. The authors recently described a BCMA-specific, T-cell-centric synthetic antigen receptor, the T-cell antigen coupler (TAC) receptor, that can be used to engineer T-cell with robust anti-myeloma activity. Here the authors describe the creation of a fully humanized BCMA-specific TAC receptor. Single-chain variable fragments (scFvs) were developed from BCMA-specific F(ab)s that were identified in a fully human phage display library. Twenty-four configurations of the F(ab)s were evaluated in a medium-throughput screening using primary T-cell, and a single F(ab), TRAC 3625, emerged as the most robust following in vitro and in vivo evaluation. An optimized BCMA-specific TAC receptor was developed through iterations of the BCMA-TAC design that evaluated a next-generation TAC scaffold sequence, different domains connecting the TAC to the 3625 scFv and different orientations of the TRAC 3625 heavy and light variable regions.

Abstracts of the Cell Therapy Transplant Canada 2022 Annual Conference.

On behalf of Cell Therapy Transplant Canada (CTTC), we are pleased to present the Abstracts of the CTTC 2022 Annual Conference. The conference was held in-person 15-18 June 2022, in Niagara Falls, Ontario. Poster authors presented their work during a lively and engaging welcome reception on Thursday, 16 June, and oral abstract authors were featured during the oral abstract session in the afternoon on Friday, 17 June 2022. Thirty-three (33) abstracts were selected for presentation as posters and six (6) as oral presentations. The top abstracts in each of four (4) categories, (1) Basic/Translational sciences, (2) Clinical Trials/Observations, (3) Laboratory/Quality, and (4) Pharmacy/Nursing/Other Transplant Support, received awards for both the oral and poster presentations. All of these were marked as "Award Recipient" with the relevant category. We congratulate all the presenters on their research and contribution to the field.

The histologic effects of neoadjuvant stereotactic body radiation therapy (SBRT) followed by pulmonary metastasectomy-rationale and protocol design for the Post SBRT Pulmonary Metastasectomy (PSPM) trial.

Background: For the local management of pulmonary malignancies, surgical resection and stereotactic body radiation therapy (SBRT) are mutually exclusive treatments. This study aims to assess the effectiveness of SBRT on reducing tumor viability at a histologic level in the context of pulmonary metastases. Methods: This protocol describes an open-label unblinded single-arm prospective Phase 2 trial to determine the effects of dual treatment of pulmonary metastasis amenable to curative resection using neoadjuvant SBRT followed by surgical resection, the Post SBRT Pulmonary Metastasectomy (PSPM) trial. Sample size require 39 patients, with an anticipated study duration of 30-36 months. Following completion of SBRT, eligible patients will be assessed at the 4-6-week mark by the treating radiation oncologist and thoracic surgeon with a post-treatment computed tomography (CT) of the chest. Patients with no disease progression will undergo scheduled surgical resection of all metastatic tumors at 8-12 weeks post SBRT. Patients will then be evaluated postoperatively at 30 days, and every 6 months for a total of 36 months with surveillance CT scans. Patients will also undergo sequential serologic evaluation of circulating tumor DNA (ctDNA) levels throughout their respective treatment pathway. The primary outcome of this study is the rate of complete pathologic response (pCR) following SBRT, assess using the International Association for the Study of Lung Cancer (IASLC) multidisciplinary recommendations for pathologic assessment of lung cancer resection specimens after neoadjuvant therapy. Secondary outcomes include overall survival (OS), disease free survival (DFS), local recurrence rates, cancer histology effects on pCR and treatment related complications, and treatment effect on ctDNA levels. Primary and secondary outcomes will be analyzed using Fisher's Exact test and Student's t-test based on data type. Cox-proportional hazard ratios will be used to evaluate OS and DFS, using the log rank test. Discussion: In evaluating the effect of SBRT on pulmonary metastasis at a histologic level, this trial may increase the use of this modality in selected patients who would otherwise only undergo surgery for disease that has already metastasized. Also, the trial provides secondary benefits of evaluating the abscopal effects of radiation on pulmonary metastatic disease, and serves as a platform for more comprehensive large-scale research in this field. Trial Registration: ClinicalTrials.gov identifier: NCT04160143 (HiREB: 7925).

Metformin-induced reductions in tumor growth involves modulation of the gut microbiome.

BACKGROUND/PURPOSE: Type 2 diabetes and obesity increase the risk of developing colorectal cancer. Metformin may reduce colorectal cancer but the mechanisms mediating this effect remain unclear. In mice and humans, a high-fat diet (HFD), obesity and metformin are known to alter the gut microbiome but whether this is important for influencing tumor growth is not known. METHODS: Mice with syngeneic MC38 colon adenocarcinomas were treated with metformin or feces obtained from control or metformin treated mice. RESULTS: We find that compared to chow-fed controls, tumor growth is increased when mice are fed a HFD and that this acceleration of tumor growth can be partially recapitulated through transfer of the fecal microbiome or in vitro treatment of cells with fecal filtrates from HFD-fed animals. Treatment of HFD-fed mice with orally ingested, but not intraperitoneally injected, metformin suppresses tumor growth and increases the expression of short-chain fatty acid (SCFA)-producing microbes Alistipes, Lachnospiraceae and Ruminococcaceae. The transfer of the gut microbiome from mice treated orally with metformin to drug naïve, conventionalized HFD-fed mice increases circulating propionate and butyrate, reduces tumor proliferation, and suppresses the expression of sterol response element binding protein (SREBP) gene targets in the tumor. CONCLUSION: These data indicate that in obese mice fed a HFD, metformin reduces tumor burden through changes in the gut microbiome.

Manufacturing T cells in hollow fiber membrane bioreactors changes their programming and enhances their potency.

Engineered T cell therapies have revolutionized modern oncology, however processes for manufacturing T cell therapies vary and the impact of manufacturing processes On the cell product is poorly understood. Herein, we have used a commercially available hollow fiber membrane bioreactor (HFMBR) operated in a novel mode to demonstrate that T cells can be engineered with lentiviruses, grown to very high densities, and washed and harvested in a single, small volume bioreactor that is readily amenable to automation. Manufacturing within the HFMBR dramatically changed the programming of the T cells and yielded a product with greater therapeutic potency than T cells produced using the standard manual method. This change in programming was associated with increased resistance to cryopreservation, which is beneficial as T cell products are typically cryopreserved prior to administration to the patient. Transcriptional profiling of the T cells revealed a shift toward a glycolytic metabolism, which may protect cells from oxidative stress offering an explanation for the improved resistance to cryopreservation. This study reveals that the choice of bioreactor fundamentally impacts the engineered T cell product and must be carefully considered. Furthermore, these data challenge the premise that glycolytic metabolism is detrimental to T cell therapies.

Development of a B-cell maturation antigen-specific T-cell antigen coupler receptor for multiple myeloma.

BACKGROUND AIMS: T cells engineered with synthetic receptors have delivered powerful therapeutic results for patients with relapsed/refractory hematologic malignancies. The authors have recently described the T-cell antigen coupler (TAC) receptor, which co-opts the endogenous T-cell receptor (TCR) and activates engineered T cells in an HLA-independent manner. Here the authors describe the evolution of a next-generation TAC receptor with a focus on developing a TAC-engineered T cell for multiple myeloma. METHODS: To optimize the TAC scaffold, the authors employed a bona fide antigen-binding domain derived from the B-cell maturation antigen-specific monoclonal antibody C11D5.3, which has been used successfully in the clinic. The authors first tested humanized versions of the UCHT1 domain, which is used by the TAC to co-opt the TCR. The authors further discovered that the signal peptide affected surface expression of the TAC receptor. Higher density of the TAC receptor enhanced target binding in vitro, which translated into higher levels of Lck at the immunological synapse and stronger proliferation when only receptor-ligand interactions were present. RESULTS: The authors observed that the humanized UCHT1 improved surface expression and in vivo efficacy. Using TAC T cells derived from both healthy donors and multiple myeloma patients, the authors determined that despite the influence of receptor density on early activation events and effector function, receptor density did not impact late effector functions in vitro, nor did the receptor density affect in vivo efficacy. CONCLUSIONS: The modifications to the TAC scaffold described herein represent an important step in the evolution of this technology, which tolerates a range of expression levels without impacting therapeutic efficacy.

Expanded human NK cells armed with CAR uncouple potent anti-tumor activity from off-tumor toxicity against solid tumors.

Despite the remarkable success of chimeric antigen receptor (CAR)-T cells against hematologic malignancies, severe off-tumor effects have constrained their use against solid tumors. Recently, CAR-engineered natural killer (NK) cells have emerged as an effective and safe alternative. Here, we demonstrate that HER2 CAR-expression in NK cells from healthy donors and patients with breast cancer potently enhances their anti-tumor functions against various HER2-expressing cancer cells, regardless of MHC class I expression. Moreover, HER2 CAR-NK cells exert higher cytotoxicity than donor-matched HER2 CAR-T cells against tumor targets. Importantly, unlike CAR-T cells, HER2 CAR-NK cells do not elicit enhanced cytotoxicity or inflammatory cytokine production against non-malignant human lung epithelial cells with basal HER2 expression. Further, HER2 CAR-NK cells maintain high cytotoxic function in the presence of immunosuppressive factors enriched in solid tumors. These results show that CAR-NK cells may be a highly potent and safe source of immunotherapy in the context of solid tumors.

De novo necroptosis creates an inflammatory environment mediating tumor susceptibility to immune checkpoint inhibitors.

Cancer immunotherapies using monoclonal antibodies to block inhibitory checkpoints are showing durable remissions in many types of cancer patients, although the majority of breast cancer patients acquire little benefit. Human melanoma and lung cancer patient studies suggest that immune checkpoint inhibitors are often potent in patients that already have intratumoral T cell infiltrate; although it remains unknown what types of interventions can result in an intratumoral T cell infiltrate in breast cancer. Using non-T cell-inflamed mammary tumors, we assessed what biological processes and downstream inflammation can overcome the barriers to spontaneous T cell priming. Here we show a specific type of combination therapy, consisting of oncolytic virus and chemotherapy, activates necroptosis and limits tumor growth in autochthonous tumors. Combination therapy activates proinflammatory cytokines; intratumoral influx of myeloid cells and cytotoxic T cell infiltrate in locally treated and distant autochthonous tumors to render them susceptible to immune checkpoint inhibitors.

A rational relationship: Oncolytic virus vaccines as functional partners for adoptive T cell therapy.

Tumours employ a variety of immune-evasion and suppression mechanisms to impair development of functional tumor-specific T cells and subvert T cell-mediated immunity in the tumour microenvironment. Adoptive T cell therapy (ACT) aims to overcome these barriers and overwhelm tumor defenses with a bolus of T cells that were selectively expanded ex vivo. Although this strategy has been effective in liquid tumors and melanomas, many tumors appear to be resistant to ACT. Several factors are thought to play into this resistance, including poor engraftment and persistence of transferred cells, tumour cell heterogeneity and antigen loss, poor immune cell recruitment and infiltration into the tumour, and susceptibility to local immunosuppression in the tumor microenvironment. Oncolytic viruses (OV) have been identified as powerful stimulators of the anti-tumour immune response. As such, OVs are inherently well-positioned to act in synergy with ACT to bolster the anti-tumour T cell response. Further, OV vaccines, wherein tumour-associated antigens are encoded into the viral backbone, have proven to be remarkable in boosting antigen-specific T cell response. Pre-clinical studies have revealed remarkable therapeutic outcomes when OV vaccines are paired with ACT. In this scenario, OV vaccines are thought to function in a "push and pull" manner, where push refers to expanding T cells in the periphery and pull refers to recruiting those cells into the tumour that has been rendered amenable to T cell attack by the actions of the OV. In this review, we discuss barriers that limit eradication of tumors by T cells, highlight attributes of OVs that break down these barriers and present strategies for rational combinations of ACT with OV vaccines.

A Cross-Reactive Small Protein Binding Domain Provides a Model to Study Off-Tumor CAR-T Cell Toxicity.

Tumor-targeted chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR-T cells) have demonstrated striking clinical success, but their use has been associated with a constellation of toxicities. A better understanding of the pathogenesis of these toxicities is required to improve the safety profile of CAR-T cells. Herein, we describe a xenograft model of off-tumor CAR-T cell-associated toxicity. Human CAR-T cells targeted against HER2 using a small-protein binding domain induced acute, dose-dependent toxicities in mice. The inclusion of a CD28 or 4-1BB co-stimulatory domain in the CAR was required to produce toxicity; however, co-stimulation through CD28 was most toxic on a per-cell basis. CAR-T cell activation in the lungs and heart was associated with a systemic cytokine storm. The severity of observed toxicities was dependent upon the peripheral blood mononuclear cell (PBMC) donor used as a T cell source and paralleled the CD4+-to-CD8+ T cell ratio in the adoptive transfer product. CD4+ CAR-T cells were determined to be the primary contributors to CAR-T cell-associated toxicity. However, donor-specific differences persisted after infusion of a purified CD4+ CAR-T cell product, indicating a role for additional variables. This work highlights the contributions of CAR-T cell-intrinsic variables to the pathogenesis of off-tumor toxicity.

The Rational Development of CD133-Targeting Immunotherapies for Glioblastoma.

CD133 marks self-renewing cancer stem cells (CSCs) in a variety of solid tumors, and CD133+ tumor-initiating cells are known markers of chemo- and radio-resistance in multiple aggressive cancers, including glioblastoma (GBM), that may drive intra-tumoral heterogeneity. Here, we report three immunotherapeutic modalities based on a human anti-CD133 antibody fragment that targets a unique epitope present in glycosylated and non-glycosylated CD133 and studied their effects on targeting CD133+ cells in patient-derived models of GBM. We generated an immunoglobulin G (IgG) (RW03-IgG), a dual-antigen T cell engager (DATE), and a CD133-specific chimeric antigen receptor T cell (CAR-T): CART133. All three showed activity against patient-derived CD133+ GBM cells, and CART133 cells demonstrated superior efficacy in patient-derived GBM xenograft models without causing adverse effects on normal CD133+ hematopoietic stem cells in humanized CD34+ mice. Thus, CART133 cells may be a therapeutically tractable strategy to target CD133+ CSCs in human GBM or other treatment-resistant primary cancers.

Endogenous T cells prevent tumor immune escape following adoptive T cell therapy.

While the outcome of adoptive T cell therapy (ACT) is typically correlated with the functionality of the inoculated T cells, the role of the endogenous T cells is unknown. The success of checkpoint blockade therapy has demonstrated the potentially curative value of preexisting tumor-primed T cells in cancer treatment. Given the results from checkpoint blockade therapy, we hypothesized that endogenous T cells contribute to long-term survival following ACT. Here, we describe a therapeutic approach combining ACT with an oncolytic vaccine that allows simultaneous analysis of antitumor immunity mediated by transferred and endogenous T cells. We found that, in addition to promoting the expansion and tumor infiltration of the transferred T cells, oncolytic vaccines boosted tumor-primed host T cells. We determined that transferred T cells contributed to rapid destruction of large tumor masses while endogenous T cells concurrently prevented the emergence of antigen-loss variants. Moreover, while transferred T cells disappeared shortly after tumor regression, endogenous T cells secured long-term memory with a broad repertoire of antigen specificity. Our findings suggest that this combination strategy may exploit the full potential of ACT and tumor-primed host T cells to eliminate the primary tumor, prevent immune escape, and provide long-term protective memory.