Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens.

Clinical response to adoptive T cell therapies is associated with the transcriptional and epigenetic state of the cell product. Thus, discovery of regulators of T cell gene networks and their corresponding phenotypes has potential to improve T cell therapies. Here we developed pooled, epigenetic CRISPR screening approaches to systematically profile the effects of activating or repressing 120 transcriptional and epigenetic regulators on human CD8+ T cell state. We found that BATF3 overexpression promoted specific features of memory T cells and attenuated gene programs associated with cytotoxicity, regulatory T cell function, and exhaustion. Upon chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. Moreover, BATF3 enhanced the potency of CAR T cells in both in vitro and in vivo tumor models and programmed a transcriptional profile that correlates with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens that defined cofactors and downstream mediators of the BATF3 gene network.

Orthogonal CRISPR screens to identify transcriptional and epigenetic regulators of human CD8 T cell function.

The clinical response to adoptive T cell therapies is strongly associated with transcriptional and epigenetic state. Thus, technologies to discover regulators of T cell gene networks and their corresponding phenotypes have great potential to improve the efficacy of T cell therapies. We developed pooled CRISPR screening approaches with compact epigenome editors to systematically profile the effects of activation and repression of 120 transcription factors and epigenetic modifiers on human CD8+ T cell state. These screens nominated known and novel regulators of T cell phenotypes with BATF3 emerging as a high confidence gene in both screens. We found that BATF3 overexpression promoted specific features of memory T cells such as increased IL7R expression and glycolytic capacity, while attenuating gene programs associated with cytotoxicity, regulatory T cell function, and T cell exhaustion. In the context of chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. CAR T cells overexpressing BATF3 significantly outperformed control CAR T cells in both in vitro and in vivo tumor models. Moreover, we found that BATF3 programmed a transcriptional profile that correlated with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens with and without BATF3 overexpression to define co-factors and downstream factors of BATF3, as well as other therapeutic targets. These screens pointed to a model where BATF3 interacts with JUNB and IRF4 to regulate gene expression and illuminated several other novel targets for further investigation.

Immunotoxin-αCD40 therapy activates innate and adaptive immunity and generates a durable antitumor response in glioblastoma models.

D2C7-immunotoxin (IT), a dual-specific IT targeting wild-type epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII) proteins, demonstrates encouraging survival outcomes in a subset of patients with glioblastoma. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. To improve the response rate and reverse immunosuppression, we combined D2C7-IT tumor cell killing with αCD40 costimulation of antigen-presenting cells. In murine glioma models, a single intratumoral injection of D2C7-IT+αCD40 treatment activated a proinflammatory phenotype in microglia and macrophages, promoted long-term tumor-specific CD8+ T cell immunity, and generated cures. D2C7-IT+αCD40 treatment increased intratumoral Slamf6+CD8+ T cells with a progenitor phenotype and decreased terminally exhausted CD8+ T cells. D2C7-IT+αCD40 treatment stimulated intratumoral CD8+ T cell proliferation and generated cures in glioma-bearing mice despite FTY720-induced peripheral T cell sequestration. Tumor transcriptome profiling established CD40 up-regulation, pattern recognition receptor, cell senescence, and immune response pathway activation as the drivers of D2C7-IT+αCD40 antitumor responses. To determine potential translation, immunohistochemistry staining confirmed CD40 expression in human GBM tissue sections. These promising preclinical data allowed us to initiate a phase 1 study with D2C7-IT+αhCD40 in patients with malignant glioma (NCT04547777) to further evaluate this treatment in humans.

Ethanol Ablation Therapy Drives Immune-Mediated Antitumor Effects in Murine Breast Cancer Models.

Ethanol ablation is a minimally invasive, cost-effective method of destroying tumor tissue through an intratumoral injection of high concentrations of cytotoxic alcohol. Ethyl-cellulose ethanol (ECE) ablation, a modified version of ethanol ablation, contains the phase-changing polysaccharide ethyl-cellulose to reduce ethanol leakage away from the tumor. Ablation produces tissue necrosis and initiates a wound healing process; however, the characteristic of the immunologic events after ECE ablation of tumors has yet to be explored. Models of triple-negative breast cancer (TNBC), which are classically immunosuppressive and difficult to treat clinically, were used to characterize the immunophenotypic changes after ECE ablation. In poorly invasive TNBC rodent models, the injury to the tumor induced by ECE increased tumor infiltrating lymphocytes (TILs) and reduced tumor growth. In a metastatic TNBC model (4T1), TILs did not increase after ECE ablation, though lung metastases were reduced. 4T1 tumors secrete high levels of granulocytic colony stimulating factor (G-CSF), which induces a suppressive milieu of granulocytic myeloid-derived suppressor cells (gMDSCs) aiding in the formation of metastases and suppression of antitumor immunity. We found that a single intratumoral injection of ECE normalized tumor-induced myeloid changes: reducing serum G-CSF and gMDSC populations. ECE also dampened the suppressive strength of gMDSC on CD4 and CD8 cell proliferation, which are crucial for anti-tumor immunity. To demonstrate the utility of these findings, ECE ablation was administered before checkpoint inhibitor (CPI) therapy in the 4T1 model and was found to significantly increase survival compared to a control of saline and CPI. Sixty days after tumor implant no primary tumors or metastatic lung lesions were found in 6/10 mice treated with CPI plus ECE, compared to 1/10 with ECE alone and 0/10 with CPI and saline.

The In Vitro Differentiation of Human CD141+CLEC9A+ Dendritic Cells from Mobilized Peripheral Blood CD34+ Hematopoietic Stem Cells.

As shown in various preclinical studies, conventional type-1 dendritic cells, or cDC1s, play a critical role in the immunological rejection of tumors and in the defense against pathogens. This indispensability stems from their potent capacity to activate cytotoxic T cells, especially via the cross-presentation of exogenous antigens. For this reason, cDC1s have become an attractive target for immunotherapy. Here we report a simplified method for generating large numbers of cDC1-like cells in vitro from mobilized human peripheral blood CD34+ hematopoietic stem cells using FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF). An important aspect of this Protocol is the growth of cells on a non-tissue culture-treated surface rather than on a tissue culture-treated surface since the latter suppresses cDC1-marker expression. The resulting CD11c+ DCs express high levels of cDC1-specific markers such as CD141, CLEC9A, TLR3, and several DC maturation markers. Compared to alternative differentiation methods, this method generates large numbers of cDC1-like cells without the need for immortalized feeder cells and should prove useful for studying cDC1 immunobiology and clinical applications of this DC subset. © 2022 Wiley Periodicals LLC. Basic Protocol: Generation of human CD141+CLEC9A+ dendritic cells from mobilized peripheral blood CD34+ hematopoietic stem cells Support Protocol: Flow cytometric immunophenotyping of CD141+ dendritic cells.

Generation of Tumor Targeted Dendritic Cell Vaccines with Improved Immunogenic and Migratory Phenotype.

Our group has employed methodologies for effective ex vivo generation of dendritic cell (DC) vaccines for patients with primary malignant brain tumors. In order to reliably produce the most potent, most representational vaccinated DC that will engender an antitumor response requires the ability to orchestrate multiple methodologies that address antigen cross-presentation, T-cell costimulation and polarization, and migratory capacity. In this chapter, we describe a novel method for augmenting the immunogenicity and migratory potential of DCs for their use as vaccines. We have elucidated methodologies to avoid the phenomenon known as immunodominance in generating cancer vaccines. We have found that culturing DC progenitors in serum-free conditions for the duration of the differentiation protocol results in a more homogeneously mature population of DCs that exhibit enhanced immunogenicity compared to DCs generated in serum-containing culture conditions. Furthermore, we demonstrate our method for generating high mobility DCs that readily migrate toward lymphoid organ chemoattractants using CCL3 protein. The combination of these two approaches represents a facile and clinically tractable methodology for generating highly mature DCs with excellent migratory capacity.

A conjoined universal helper epitope can unveil antitumor effects of a neoantigen vaccine targeting an MHC class I-restricted neoepitope.

Personalized cancer vaccines targeting neoantigens arising from somatic missense mutations are currently being evaluated for the treatment of various cancers due to their potential to elicit a multivalent, tumor-specific immune response. Several cancers express a low number of neoantigens; in these cases, ensuring the immunotherapeutic potential of each neoantigen-derived epitope (neoepitope) is crucial. In this study, we discovered that therapeutic vaccines targeting immunodominant major histocompatibility complex (MHC) I-restricted neoepitopes require a conjoined helper epitope in order to induce a cytotoxic, neoepitope-specific CD8+ T-cell response. Furthermore, we show that the universally immunogenic helper epitope P30 can fulfill this requisite helper function. Remarkably, conjoined P30 was able to unveil immune and antitumor responses to subdominant MHC I-restricted neoepitopes that were, otherwise, poorly immunogenic. Together, these data provide key insights into effective neoantigen vaccine design and demonstrate a translatable strategy using a universal helper epitope that can improve therapeutic responses to MHC I-restricted neoepitopes.

Antigen-loaded monocyte administration induces potent therapeutic antitumor T cell responses.

Efficacy of dendritic cell (DC) cancer vaccines is classically thought to depend on their antigen-presenting cell (APC) activity. Studies show, however, that DC vaccine priming of cytotoxic T lymphocytes (CTLs) requires the activity of endogenous DCs, suggesting that exogenous DCs stimulate antitumor immunity by transferring antigens (Ags) to endogenous DCs. Such Ag transfer functions are most commonly ascribed to monocytes, implying that undifferentiated monocytes would function equally well as a vaccine modality and need not be differentiated to DCs to be effective. Here, we used several murine cancer models to test the antitumor efficacy of undifferentiated monocytes loaded with protein or peptide Ag. Intravenously injected monocytes displayed antitumor activity superior to DC vaccines in several cancer models, including aggressive intracranial glioblastoma. Ag-loaded monocytes induced robust CTL responses via Ag transfer to splenic CD8+ DCs in a manner independent of monocyte APC activity. Ag transfer required cell-cell contact and the formation of connexin 43-containing gap junctions between monocytes and DCs. These findings demonstrate the existence of an efficient gap junction-mediated Ag transfer pathway between monocytes and CD8+ DCs and suggest that administration of tumor Ag-loaded undifferentiated monocytes may serve as a simple and efficacious immunotherapy for the treatment of human cancers.

Preventing Lck Activation in CAR T Cells Confers Treg Resistance but Requires 4-1BB Signaling for Them to Persist and Treat Solid Tumors in Nonlymphodepleted Hosts.

PURPOSE: Chimeric antigen receptor (CAR) T cells have shown promise against solid tumors, but their efficacy has been limited, due in part, to immunosuppression by CD4+FoxP3+ regulatory T cells (Tregs). Although lymphodepletion is commonly used to deplete Tregs, these regimens are nonspecific, toxic, and provide only a narrow window before Tregs repopulate hosts. Importantly, CARs have also been shown to inadvertently potentiate Tregs by providing a source of IL2 for Treg consumption. We explored whether disruption of the IL2 axis would confer efficacy against solid tumors without the need for lymphodepletion. EXPERIMENTAL DESIGN: We developed second- (CD28z) and third- (CD28-4-1BBz) generation CARs targeting EGFRvIII. To eliminate secretion of IL2, 2 amino acid substitutions were introduced in the PYAP Lck-binding motif of the CD28 domain (ΔCD28). We evaluated CARs against B16 melanomas expressing EGFRvIII. RESULTS: CD28z CARs failed to engraft in vivo. Although 4-1BB addition improved expansion, CD28-4-1BBz CARs required lymphodepletion to treat solid tumors. CARs deficient in Lck signaling, however, significantly retarded tumor growth without a need for lymphodepletion and this was dependent on inclusion of 4-1BB. To evaluate CAR vulnerability to Tregs, we lymphodepleted mice and transferred CARs alone or with purified Tregs. Cotransfer with Tregs abrogated the efficacy of CD28-4-1BBz CARs, whereas the efficacy of ΔCD28-4-1BBz CARs remained unperturbed. CONCLUSIONS: In the absence of lymphodepletion, CARs targeting solid tumors are hindered by Treg immunosuppression and poor persistence. Here, CARs were modified to circumvent Treg suppression and to simultaneously improve in vivo engraftment. Modified CARs treated solid tumors without a need for lymphodepletion.

Promising vaccines for treating glioblastoma.

INTRODUCTION: Conventional therapies for glioblastoma  (GBM) typically fail to provide lasting antitumor benefits, owing to their inability to specifically eliminate all malignant cells. Cancer vaccines are currently being evaluated as a means to direct the adaptive immune system to target residual GBM cells that remain following standard-of-care treatment. AREAS COVERED: In this review, we provide an overview of the more noteworthy cancer vaccines that are under investigation for the treatment of GBM, as well as potential future directions that may enhance GBM-vaccine effectiveness. EXPERT OPINION: To date, no cancer vaccines have been proven effective against GBM; however, only a few have reached phase III clinical testing. Clinical immunological monitoring data suggest that GBM vaccines are capable of stimulating immune responses reactive to GBM antigens, but whether these responses have an appreciable antitumor effect on GBM is still uncertain. Nevertheless, there have been several promising outcomes in early phase clinical trials, which lend encouragement to this area of study. Further studies with GBM vaccines are, therefore, warranted.

A simple and enzyme-free method for processing infiltrating lymphocytes from small mouse tumors for ELISpot analysis.

The ELISpot assay prevails as one of the most sensitive and meaningful assays for the detection of antigen-specific, effector immune responses. Acquisition of cellular analyte for ELISpot analysis is typically not problematic when derived from tissues enriched in lymphocytes (e.g., lymphoid organs and blood); however, cell processing becomes more difficult when lymphocytes represent only a very minor population relative to the source tissue, especially when the source tissue is in limited supply (e.g., small mouse tumors). Traditional enzymatic-based methods for dissociating tumors often result in poor yields, inconsistent lymphocyte enrichment, and can have deleterious effects on lymphocyte phenotype and function. To address these limitations, we have developed an enzyme-free protocol for processing tumor infiltrating lymphocytes (TILs) from small mouse tumors, which enables the enumeration of antigen-specific effector lymphocytes using ELISpot analysis. This procedure is predicated on the dissociation of tumor tissue using gentle agitation with a paddle blender followed by a brief in vitro culture period to remove adherent cells, as well as to revive lymphocytes from a non-responsive state. Although this method is demonstrated with mouse intracerebral tumors, we have found that this protocol is applicable to peripheral tumors and may likely extend to alternative tissue sources wherein lymphocytes exist in low numbers.

Dendritic Cells Enhance Polyfunctionality of Adoptively Transferred T Cells That Target Cytomegalovirus in Glioblastoma.

Median survival for glioblastoma (GBM) remains <15 months. Human cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T-cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However, ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated (R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256-64. ©2017 AACR.

Preconditioning Vaccine Sites for mRNA-Transfected Dendritic Cell Therapy and Antitumor Efficacy.

Messenger RNA (mRNA)-transfected dendritic cell (DC) vaccines have been shown to be a powerful modality for eliciting antitumor immune responses in mice and humans; however, their application has not been fully optimized since many of the factors that contribute to their efficacy remain poorly understood. Work stemming from our laboratory has recently demonstrated that preconditioning the vaccine site with a recall antigen prior to the administration of a dendritic cell vaccine creates systemic recall responses and resultantly enhances dendritic cell migration to the lymph nodes with improved antitumor efficacy. This chapter describes the generation of murine mRNA-transfected DC vaccines, as well as a method for vaccine site preconditioning with protein antigen formulations that create potent recall responses.

Enhancing dendritic cell-based vaccination for highly aggressive glioblastoma.

INTRODUCTION: Patients with primary glioblastoma (GBM) have a dismal prognosis despite standard therapy, which can induce potentially deleterious side effects. Arming the immune system is an alternative therapeutic approach, as its cellular effectors and inherent capacity for memory can be utilized to specifically target invasive tumor cells, while sparing collateral damage to otherwise healthy brain parenchyma. AREAS COVERED: Active immunotherapy is aimed at eliciting a specific immune response against tumor antigens. Dendritic cells (DCs) are one of the most potent activators of de novo and recall immune responses and are thus a vehicle for successful immunotherapy. Currently, investigators are optimizing DC vaccines by enhancing maturation status and migratory potential to induce more potent antitumor responses. An update on the most recent DC immunotherapy trials is provided. EXPERT OPINION: Targeting of unique antigens restricted to the tumor itself is the most important parameter in advancing DC vaccines. In order to overcome intrinsic mechanisms of immune evasion observed in GBM, the future of DC-based therapy lies in a multi-antigenic vaccine approach. Successful targeting of multiple antigens will require a comprehensive understanding of all immunologically relevant oncological epitopes present in each tumor, thereby permitting a rational vaccine design.

Peptide vaccines for the treatment of glioblastoma.

Glioblastoma multiforme (GBM) is an extremely malignant brain tumor for which current therapies do little to remedy. Despite aggressive treatment with surgery, radiation therapy, and chemotherapy, tumors inevitably recur as a direct consequence of the infiltrative nature of GBM. The poor prognosis of patients with GBM underscores the clear and urgent need for more precise and potent therapies. Immunotherapy is emerging as a promising means to treat GBM based on the immune system's capacity to mediate tumor-specific cytotoxicity. In this review, we will discuss the use of peptide vaccines for the treatment of GBM. The simplicity of peptide vaccines and their ability to elicit tumor antigen-specific immune responses make them an invaluable tool for the study of brain tumor immunotherapy.

Rindopepimut: a promising immunotherapeutic for the treatment of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive glial cell-derived primary tumor. Current standard of care for patients with GBM includes maximal tumor resection plus adjuvant radiotherapy and temozolomide chemotherapy, increasing median overall survival to a mere 15 months from diagnosis. Because these therapies are inherently nonspecific, there is an increased likelihood of off-target and incomplete effects; therefore, targeted modalities are required for enhanced safety and efficacy. Rindopepimut is emerging as a safe and potentially effective drug for the treatment of GBM. Rindopepimut consists of a 14-mer peptide that spans the length of EGF receptor variant III, a mutant variant of EGF receptor found on approximately 30% of primary GBM, conjugated to the carrier protein keyhole limpet hemocyanin. Vaccination with rindopepimut has been shown to specifically eliminate cells expressing EGF receptor variant III. Phase II clinical trials have suggested that vaccination of newly diagnosed GBM patients with rindopepimut plus adjuvant granulocyte-macrophage colony-stimulating factor results in prolonged progression-free and overall survival with minimal toxicity. This review will outline the development of rindopepimut, as well as the current status of this vaccine.