Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice.

BACKGROUND AND AIMS: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties. METHODS: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgcnull (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgcnull (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice. RESULTS: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active ß-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. CONCLUSIONS: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.

CAR-T cells and combination therapies: What's next in the immunotherapy revolution?

Cancer immunotherapies are dramatically reshaping the clinical management of oncologic patients. For many of these therapies, the guidelines for administration, monitoring, and management of associated toxicities are still being established. This is especially relevant for adoptively transferred, genetically-modified T cells, which have unique pharmacokinetic properties, due to their ability to replicate and persist long-term, following a single administration. Furthermore, in the case of CAR-T cells, the use of synthetic immune receptors may impact signaling pathways involved in T cell function and survival in unexpected ways. We, herein, comment on the most salient aspects of CAR-T cell design and clinical experience in the treatment of solid tumors. In addition, we discuss different possible scenarios for combinations of CAR-T cells and other treatment modalities, with a special emphasis on kinase inhibitors, elaborating on the strategies to maximize synergism. Finally, we discuss some of the technologies that are available to explore the molecular events governing the success of these therapies. The young fields of synthetic and systems biology are likely to be major players in the advancement of CAR-T cell therapies, providing the tools and the knowledge to engineer patients' T lymphocytes into intelligent cancer-fighting micromachines.

Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma.

Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

Expression profiling of TCR-engineered T cells demonstrates overexpression of multiple inhibitory receptors in persisting lymphocytes.

Despite significant progress in the development of adoptive cell-transfer therapies (ACTs) using gene-engineered T cells, little is known about the fate of cells following infusion. To address that, we performed a comparative analysis of gene expression between T-cell receptor-engineered lymphocytes persisting in the circulation 1 month after administration and the product that was infused. We observed that 156 genes related to immune function were differentially expressed, including underexpression of stimulators of lymphocyte function and overexpression of inhibitory genes in postinfusion cells. Of genes overexpressed postinfusion, the product of programmed cell death 1 (PDCD1), coinhibitory receptor PD-1, was expressed at a higher percentage in postinfusion lymphocytes than in the infusion product. This was associated with a higher sensitivity to inhibition of cytokine production by interaction with its ligand PD-L1. Coinhibitory receptor CD160 was also overexpressed in persisting cells, and its expression was associated with decreased reactivity, which surprisingly was found to be ligand-independent. These results contribute to a deeper understanding of the properties of transgenic lymphocytes used to treat human malignancies and may provide a rationale for the development of combination therapies as a method to improve ACT.

Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells.

Background: Glioblastoma (GBM) is both the most common and aggressive type of primary brain tumor, associated with high mortality rates and resistance to conventional therapy. Despite recent advancements in knowledge and molecular profiling, recurrence of GBM is nearly inevitable. This recurrence has been attributed to the presence of glioma stem cells (GSCs), a small fraction of cells resistant to standard-of-care treatments and capable of self-renewal and tumor initiation. Therefore, targeting these cancer stem cells will allow for the development of more effective therapeutic strategies against GBM. We have previously identified several 7-amino acid length peptides which specifically target GSCs through in vitro and in vivo phage display biopanning. Methods and results: We have combined two of these peptides to create a dual peptide construct (EV), and demonstrated its ability to bind GSCs in vitro and target intracranial GBM in mouse models. A peptide pull-down performed with peptide EV followed by mass spectrometry determined N-cadherin as the binding partner of the peptide, which was validated by enzyme-linked immunosorbent assay and surface plasmon resonance. To develop cytotoxic cellular products aimed at specifically targeting GSCs, chimeric antigen receptors (CARs) were engineered containing the peptide EV in place of the single-chain variable fragment (scFv) as the antigen-binding domain. EV CAR-transduced T cells demonstrated specific reactivity towards GSCs by production of interferon-gamma when exposed to GSCs, in addition to the induction of GSC-specific apoptosis as illustrated by Annexin-V staining. Conclusion: These results exemplify the use of phage display biopanning for the isolation of GSC-targeting peptides, and their potential application in the development of novel cytotoxic therapies for GBM.

γδ-Enriched CAR-T cell therapy for bone metastatic castrate-resistant prostate cancer.

Immune checkpoint blockade has been largely unsuccessful for the treatment of bone metastatic castrate-resistant prostate cancer (mCRPC). Here, we report a combinatorial strategy to treat mCRPC using γδ-enriched chimeric antigen receptor (CAR) T cells and zoledronate (ZOL). In a preclinical murine model of bone mCRPC, γδ CAR-T cells targeting prostate stem cell antigen (PSCA) induced a rapid and significant regression of established tumors, combined with increased survival and reduced cancer-associated bone disease. Pretreatment with ZOL, a U.S. Food and Drug Administration-approved bisphosphonate prescribed to mitigate pathological fracture in mCRPC patients, resulted in CAR-independent activation of γδ CAR-T cells, increased cytokine secretion, and enhanced antitumor efficacy. These data show that the activity of the endogenous Vγ9Vδ2 T cell receptor is preserved in CAR-T cells, allowing for dual-receptor recognition of tumor cells. Collectively, our findings support the use of γδ CAR-T cell therapy for mCRPC treatment.

SARS-CoV-2 antibody response duration and neutralization following natural infection.

Background: The role of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) neutralizing antibody response from natural infection and vaccination, and the potential determinants of this response are poorly understood. Characterizing this antibody response and the factors associated with neutralization can help inform future prevention efforts and improve clinical outcomes in those infected. Objectives: The goals of this study were to prospectively evaluate SARS-CoV-2 antibody levels and the neutralizing antibody responses among naturally infected adults and to determine demographic and behavioral factors independently associated with these responses. Methods: Serum was collected from seropositive individuals at baseline, four-weeks, and three-months following their first study visit to be evaluated for antibody levels. Detection of neutralizing antibodies was performed at baseline. Participant demographic and behavioral information was collected via web questionnaire prior to their first visit. Results: At baseline, higher antibody levels were associated with better neutralization capacity, with 83% of participants having detectable neutralizing antibodies. We found an age-dependent effect on antibody level and neutralization capacity with participants over 65 years having significantly higher levels. Ethnicity, heart disease, autoimmune disease, and COVID symptoms were associated with higher antibody levels, but not with increased neutralization capacity. Work environment during the pandemic correlated with increased neutralization capacity, while kidney or liver disease and traveling out of state after February 2020 correlated with decreased neutralization capacity, however neither correlated with antibody levels. Conclusions: Our data show that natural infection by SARS-CoV-2 can induce a humoral response reflected by high antibody levels and neutralization capacity.

Neoantigen-specific CD4+ tumor-infiltrating lymphocytes are potent effectors identified within adoptive cell therapy products for metastatic melanoma patients.

BACKGROUND: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a promising immunotherapeutic approach for patients with advanced solid tumors. While numerous advances have been made, the contribution of neoantigen-specific CD4+T cells within TIL infusion products remains underexplored and therefore offers a significant opportunity for progress. METHODS: We analyzed infused TIL products from metastatic melanoma patients previously treated with ACT for the presence of neoantigen-specific T cells. TILs were enriched on reactivity to neoantigen peptides derived and prioritized from patient sample-directed mutanome analysis. Enriched TILs were further investigated to establish the clonal neoantigen response with respect to function, transcriptomics, and persistence following ACT. RESULTS: We discovered that neoantigen-specific TIL clones were predominantly CD4+ T cells and were present in both therapeutic responders and non-responders. CD4+ TIL demonstrated an effector T cell response with cytotoxicity toward autologous tumor in a major histocompatibility complex class II-dependent manner. These results were validated by paired TCR and single cell RNA sequencing, which elucidated transcriptomic profiles distinct to neoantigen-specific CD4+ TIL. CONCLUSIONS: Despite methods which often focus on CD8+T cells, our study supports the importance of prospective identification of neoantigen-specific CD4+ T cells within TIL products as they are a potent source of tumor-specific effectors. We further advocate for the inclusion of neoantigen-specific CD4+ TIL in future ACT protocols as a strategy to improve antitumor immunity.

γδ T Cell-Based Adoptive Cell Therapies Against Solid Epithelial Tumors.

ABSTRACT: Conventionally, adoptive cell therapies have been developed and optimized using αß T cells. However, the understudied and less abundant γδ T cells offer unique advantages to the immunotherapy field especially for therapies against solid tumors. Recently, γδ T-cell potential against a broad spectrum of malignant cells has been demonstrated in the preclinical setting. In the clinic, γδ T-cell-based immunotherapies have proven to be safe; however, their efficacy needs improvement. Considering the growing body of literature reflecting the increasing interest in γδ T cells, we sought to capture the current topics of discussion in the field, pertaining to their use in adoptive immunotherapy. We aimed to compile information about γδ T-cell enhancement in terms of expansion, phenotype, and inhibitory receptors, in addition to the latest advances in preclinical and clinical research using γδ T cells specifically against solid epithelial tumors.

Allogeneic double-negative CAR-T cells inhibit tumor growth without off-tumor toxicities.

The development of autologous chimeric antigen receptor T (CAR-T) cell therapies has revolutionized cancer treatment. Nevertheless, the delivery of CAR-T cell therapy faces challenges, including high costs, lengthy production times, and manufacturing failures. To overcome this, attempts have been made to develop allogeneic CAR-T cells using donor-derived conventional CD4+ or CD8+ T cells (Tconvs), but severe graft-versus-host disease (GvHD) and host immune rejection have made this challenging. CD3+CD4-CD8- double-negative T cells (DNTs) are a rare subset of mature T cells shown to fulfill the requirements of an off-the-shelf cellular therapy, including scalability, cryopreservability, donor-independent anticancer function, resistance to rejection, and no observed off-tumor toxicity including GvHD. To overcome the challenges faced with CAR-Tconvs, we evaluated the feasibility, safety, and efficacy of using healthy donor-derived allogeneic DNTs as a CAR-T cell therapy platform. We successfully transduced DNTs with a second-generation anti-CD19-CAR (CAR19) without hampering their endogenous characteristics or off-the-shelf properties. CAR19-DNTs induced antigen-specific cytotoxicity against B cell acute lymphoblastic leukemia (B-ALL). In addition, CAR19-DNTs showed effective infiltration and tumor control against lung cancer genetically modified to express CD19 in xenograft models. CAR19-DNT efficacy was comparable with that of CAR19-Tconvs. However, unlike CAR19-Tconvs, CAR19-DNTs did not cause alloreactivity or xenogeneic GvHD-related mortality in xenograft models. These studies demonstrate the potential of using allogeneic DNTs as a platform for CAR technology to provide a safe, effective, and patient-accessible CAR-T cell treatment option.

Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors.

BACKGROUND: Neuroendocrine tumors (NETs) overexpress somatostatin receptors (SSTRs). METHODS: We developed a second-generation, ligand-based, anti-SSTR chimeric antigen receptor (CAR) incorporating the somatostatin analog octreotide in its extracellular moiety. RESULTS: Anti-SSTR CAR T cells exerted antitumor activity against SSTR+NET cell linesin vitro. The killing activity was highly specific, as demonstrated by the lack of CAR T cell reactivity against NET cells engineered to express mutated variants of SSTR2/5 by CRISPR/Cas9. When adoptively transferred in NSG mice, anti-SSTR CAR T cells induced significant antitumor activity against human NET xenografts. Although anti-SSTR CAR T cells could recognize the murine SSTRs as shown by their killing ability against murine NET cells, no obvious deleterious effects on SSTR-expressing organs such as the brain or the pancreas were observed in mice. CONCLUSIONS: Taken together, our results establish anti-SSTR CAR T cells as a potential candidate for early phase clinical investigations in patients with NETs. More broadly, the demonstration that a known peptide drug can direct CAR T cell targeting may streamline the potential utility of multiple peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers.

Cell cycle control pathways act as conditioning factors for TK/GCV sensitivity in pancreatic cancer cells.

The suicide system TK/GCV is an enzyme/prodrug therapy that involves the transfer of the cDNA for the herpes simplex virus thymidine kinase gene (TK) into tumor cells which then sensitizes the cells to the non-toxic antiviral drug ganciclovir. Although extensively characterized, the suicide system TK/GCV conceals the details of its mechanism of action. In order to shed some light on this issue, we conducted experiments designed to identify key features of sensitive cells, as compared to cells that displayed reduced sensitivity to TK/GCV. Cell lines displaying different degrees of sensitivity underwent apoptotic cell death upon treatment with TK/GCV. S-phase delay, however, was almost exclusively restricted to sensitive cells and was impaired in a model of treatment-induced resistance. In this model genes with differential expression associated to induced resistance were identified. Noteworthy, two cell cycle-related genes (CCNE1 and GADD45) were functionally validated as conditioners of cellular sensitivity to TK/GCV. The relevance of cell cycle control was further demonstrated by experiments showing the association of Chk1 activation with greater TK/GCV cytotoxicity. Combination treatment with Chk1 inhibitor UCN-01 induced, in sensitive cells, an antagonistic effect on TK/GCV cytotoxicity highlighting the relevance of Chk1's activity on TK/GCV mechanism of action. These results reveal the relevance of cell cycle control pathways in the cytotoxicity induced by the TK/GCV system identifying candidate genes as conditioners of TK/GCV sensitivity. Moreover it points out, for the first time at Chk1 activation as a key factor to mediate TK/GCV cytotoxicity.

Adoptive cellular therapy in solid tumor malignancies: review of the literature and challenges ahead.

While immune checkpoint inhibitors (ICIs) have ushered in major changes in standards of care for many solid tumor malignancies, primary and acquired resistance is common. Insufficient antitumor T cells, inadequate function of these cells, and impaired formation of memory T cells all contribute to resistance mechanisms to ICI. Adoptive cellular therapy (ACT) is a form of immunotherapy that is rapidly growing in clinical investigation and has the potential to overcome these limitations by its ability to augment the number, specificity, and reactivity of T cells against tumor tissue. ACT has revolutionized the treatment of hematologic malignancies, though the use of ACT in solid tumor malignancies is still in its early stages. There are currently three major modalities of ACT: tumor-infiltrating lymphocytes (TILs), genetically engineered T-cell receptors (TCRs), and chimeric antigen receptor (CAR) T cells. TIL therapy involves expansion of a heterogeneous population of endogenous T cells found in a harvested tumor, while TCRs and CAR T cells involve expansion of a genetically engineered T-cell directed toward specific antigen targets. In this review, we explore the potential of ACT as a treatment modality against solid tumors, discuss their advantages and limitations against solid tumor malignancies, discuss the promising therapies under active investigation, and examine future directions for this rapidly growing field.

Adoptive T-cell immunotherapy in digestive tract malignancies: Current challenges and future perspectives.

Multiple systemic treatments are currently available for advanced cancers of the digestive tract, but none of them is curative. Adoptive T-cell immunotherapy refers to the extraction, modification and re-infusion of autologous or allogenic T lymphocytes for therapeutic purposes. A number of clinical trials have investigated either non-engineered T cells (i.e., lymphokine-activated killer cells, cytokine induced killer cells, or tumor-infiltrating lymphocytes) or engineered T cells (T cell receptor-redirected T cells or chimeric antigen receptor T cells) in patients with digestive tract malignancies over the past two decades, with variable degrees of success. While the majority of completed trials have been primarily aimed at assessing the safety of T-cell transfer strategies, a new generation of studies is being designed to formally evaluate the antitumor potential of adoptive T-cell immunotherapy in both the metastatic and adjuvant settings. In this review, we provide an overview of completed and ongoing clinical trials of passive T-cell immunotherapy in patients with cancers of the digestive tract, focusing on present obstacles and future strategies for achieving potential success.

Mathematical Modeling of Oncolytic Virotherapy.

Mathematical modeling in biology has a long history as it allows the analysis and simulation of complex dynamic biological systems at little cost. A mathematical model trained on experimental or clinical data can be used to generate and evaluate hypotheses, to ask "what if" questions, and to perform in silico experiments to guide future experimentation and validation. Such models may help identify and provide insights into the mechanisms that drive changes in dynamic systems. While a mathematical model may never replace actual experiments, it can synergize with experiments to save time and resources by identifying experimental conditions that are unlikely to yield favorable outcomes, and by using optimization principles to identify experiments that are most likely to be successful. Over the past decade, numerous models have also been developed for oncolytic virotherapy, ranging from merely theoretic frameworks to fully integrated studies that utilize experimental data to generate actionable hypotheses. Here we describe how to develop such models for specific oncolytic virotherapy experimental setups, and which questions can and cannot be answered using integrated mathematical oncology.

Estimation of probability distributions of parameters using aggregate population data: analysis of a CAR T-cell cancer model.

In this effort we explain fundamental formulations for aggregate data inverse problems requiring estimation of probability distribution parameters. We use as a motivating example a class of CAR T-call cancer models in mice. After ascertaining results on model stability and sensitivity with respect to parameters, we carry out first elementary computations on the question how much data is needed for successful estimation of probability distributions.

CAR-T Engineering: Optimizing Signal Transduction and Effector Mechanisms.

The adoptive transfer of genetically engineered T cells expressing a chimeric antigen receptor (CAR) has shown remarkable results against B cell malignancies. This immunotherapeutic approach has advanced and expanded rapidly from preclinical models to the recent approval of CAR-T cells to treat lymphomas and leukemia by the Food and Drug Administration (FDA). Ongoing research efforts are focused on employing CAR-T cells as a therapy for other cancers, and enhancing their efficacy and safety by optimizing their design. Here we summarize modifications in the intracellular domain of the CAR that gave rise to first-, second-, third- and next-generation CAR-T cells, together with the impact that these different designs have on CAR-T cell biology and function. Further, we describe how the structure of the antigen-sensing ectodomain can be enhanced, leading to superior CAR-T cell signaling and/or function. Finally we discuss how tissue-specific factors may impact the clinical efficacy of CAR-T cells for bone and the central nervous system, as examples of specific indications that may require further CAR signaling optimization to perform in such inhospitable microenvironments.

An immunoproteomic approach to characterize the CAR interactome and signalosome.

Adoptive transfer of T cells that express a chimeric antigen receptor (CAR) is an approved immunotherapy that may be curative for some hematological cancers. To better understand the therapeutic mechanism of action, we systematically analyzed CAR signaling in human primary T cells by mass spectrometry. When we compared the interactomes and the signaling pathways activated by distinct CAR-T cells that shared the same antigen-binding domain but differed in their intracellular domains and their in vivo antitumor efficacy, we found that only second-generation CARs induced the expression of a constitutively phosphorylated form of CD3ζ that resembled the endogenous species. This phenomenon was independent of the choice of costimulatory domains, or the hinge/transmembrane region. Rather, it was dependent on the size of the intracellular domains. Moreover, the second-generation design was also associated with stronger phosphorylation of downstream secondary messengers, as evidenced by global phosphoproteome analysis. These results suggest that second-generation CARs can activate additional sources of CD3ζ signaling, and this may contribute to more intense signaling and superior antitumor efficacy that they display compared to third-generation CARs. Moreover, our results provide a deeper understanding of how CARs interact physically and/or functionally with endogenous T cell molecules, which will inform the development of novel optimized immune receptors.

Melanoma central nervous system metastases: An update to approaches, challenges, and opportunities.

In February 2018, the Melanoma Research Foundation and the Moffitt Cancer Center hosted the Second Summit on Melanoma Central Nervous System (CNS) Metastases in Tampa, Florida. In this white paper, we outline the current status of basic science, translational, and clinical research into melanoma brain metastasis development and therapeutic management. We further outline the important challenges that remain for the field and the critical barriers that need to be overcome for continued progress to be made in this clinically difficult area.