CAR-expressing Cells Research Articles

Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting

BackgroundOn encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward...

Single-cell transcriptome analysis of CAR T-cell products reveals subpopulations, stimulation, and exhaustion signatures

ABSTRACT Chimeric antigen receptor (CAR) T-cell adoptive therapy is set to transform the treatment of a rapidly expanding range of malignancies. Although the activation process of normal T cells is well characterized, comparatively little is known about the activation of cells via the CAR. Here we have used flow cytometry together with single-cell transcriptome profiling to characterize the starting material (peripheral blood mononuclear cells) and CAR therapeutic products of 3 healthy donors in the presence and absence of antigen-specific stimulation. Analysis of 53,191 single-cell transcriptomes showed APRIL-based CAR products to contain several subpopulations of cells, with cellular composition reproducible from donor to donor, and all major cellular subsets compatible with CAR expression. Only 50% of CAR-expressing cells displayed transcriptional changes upon CAR-specific antigen exposure. The resulting molecular signature for CAR T-cell activation provides a rich resource for future dissection of underlying mechanisms. Targeted data interrogation also revealed that a small proportion of antigen-responding CAR-expressing cells displayed an exhaustion signature, with both known markers and genes not previously associated with T-cell exhaustion. Comprehensive single-cell transcriptomic analysis thus represents a powerful way to guide the assessment and optimization of clinical-grade CAR-T-cells, and inform future research into the underlying molecular processes.

Engineering CAR T Cells to Target the HIV Reservoir.

The HIV reservoir remains to be a difficult barrier to overcome in order to achieve a therapeutic cure for HIV. Several strategies have been developed to purge the reservoir, including the “kick and kill” approach, which is based on the notion that reactivating the latent reservoir will allow subsequent elimination by the host anti-HIV immune cells. However, clinical trials testing certain classes of latency reactivating agents (LRAs) have so far revealed the minimal impact on reducing the viral reservoir. A robust immune response to reactivated HIV expressing cells is critical for this strategy to work. A current focus to enhance anti-HIV immunity is through the use of chimeric antigen receptors (CARs). Currently, HIV-specific CARs are being applied to peripheral T cells, NK cells, and stem cells to boost recognition and killing of HIV infected cells. In this review, we summarize current developments in engineering HIV directed CAR-expressing cells to facilitate HIV elimination. We also summarize current LRAs that enhance the “kick” strategy and how new generation and combinations of LRAs with HIV specific CAR T cell therapies could provide an optimal strategy to target the viral reservoir and achieve HIV clearance from the body.

Electroporation of NKG2D RNA CAR Improves Vγ9Vδ2 T Cell Responses against Human Solid Tumor Xenografts

Vγ9Vδ2 T cell-based anticancer immunotherapy has shown some promise in early-phase clinical trials but there is still large room for improvement. Using the extracellular domain of the human NKG2D, a stimulatory receptor expressed by Vγ9Vδ2 T cells, we constructed NKG2D ligand-specific chimeric antigen receptors (CARs). We adopted a non-viral CAR approach via mRNA electroporation to modify Vγ9Vδ2 T cells and demonstrated that, upon interaction with the NKG2D ligand-positive cancer cells, the CARs substantially enhanced the cytotoxic activity of the modified cells toward multiple cultured solid tumor cell lines, including those resistant to Zometa treatment. Repeated doses of the CAR-expressing cells resulted in tumor regression in mice with established tumors, extending median survival time by up to 132% as compared to the PBS control group. The findings suggest clinical potential for RNA CAR-modified Vγ9Vδ2 T cells to treat a wide variety of NKG2D ligand-expressing cancers.

Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

Chimeric Antigen Receptor Modified Memory-like (CAR-ML) NK Cells Exhibit Potent Responses to NK-Resistant Tumors

NK cells are cytotoxic innate lymphoid cells that mediate anti-tumor responses and traffic to most tissues. NK cells activated briefly with IL-12, IL-15, and IL-18 differentiate into memory-like (ML) NK cells with enhanced anti-tumor effects, which has been translated into clinical trials for patients with leukemia. Adoptive cellular therapy with donor ML NK cells were safe and induced CR/CRi in >50% of rel/ref AML patients in a first-in-human phase 1 clinical trial at Washington University (PMID27655849). In preliminary results, donor ML NK cells expand and can persist for months in an immune-compatible recipients during or after allogeneic hematopoietic cell transplantation (HCT), and maintain potent effector function. However, NK cell recognition of many cancer types is limited, since they depend on expression of stress-induced activating receptor ligands. We hypothesized that ML NK cells engineered with chimeric antigen receptors (CAR) would demonstrate improved anti-tumor responses against classically NK-resistant targets. To test this idea, ML NK cells were engineered to express an anti-CD19-CAR, and responses against NK-resistant B-cell malignancies evaluated in vitro and in vivo. CAR-modified primary human ML NK cells (CAR-ML) were transduced with an anti-CD19-CD8a-41BB-CD3z-GFP (CD19-CAR-ML) lentivirus. This differentiation and transfection approach resulted in approximately 15-25% of ML NK cells transduced with the CAR construct, as determined flow cytometry staining for soluble CD19 and GFP. As an additional control, αCD33-CD8a-41BB-CD3z-GFP CAR-transduced ML NK cells and GFP- internal control ML NK cells were used. Here, in vitro functional assays were used to determine if CD19-CAR enhances ML NK cells in an antigen (CD19)-specific manner. CD19-CAR-ML (GFP+) and control ML NK (GFP-) cells were evaluated for functional responses to CD19-positive or CD19-negative tumor targets in vitro. CD19-CAR-ML NK cells demonstrated significantly increased IFN-γ production (44±4% vs. 15±3%, p<0.001); mean ± SEM) and degranulation (31±4% vs. 5±1%, p<0.001) against NK-resistant CD19+ Raji targets, compared to control GFP- and CD33-CAR ML NK cells. To understand the contributions of ML differentiation on the enhanced functionality of CD19-CAR modified NK cells, we compared CD19-CAR-ML NK cells to control CD19-CAR NK cells that were treated with IL-15 only. CD19-CAR-ML NK cells also exhibited significantly increased effector responses compared to control CAR NK cells against CD19+ targets (p<0.01). CD19-CAR-ML NK cells responded similarly to GFP- ML NK cells against CD19-negative Kasumi leukemia targets. Finally, CD19-CAR-ML NK cells also exhibited significantly enhanced killing, degranulation, and IFN-γ production against primary CD19+ follicular lymphoma targets from patient lymph nodes (p<0.01). To establish the translational utility of this approach, autologous CD19-CAR-ML NK cells generated from lymphoma patients demonstrated significantly increased IFN-γ production (p<0.05) and degranulation (p<0.01) against their own CD19+ lymphoma targets, compared to control ML NK cells (GFP-). These data confirm contributions of both ML differentiation and CAR expression in the enhanced antigen-specific, anti-tumor responses observed in CAR-ML NK cells. To test the expansion and persistence of ML NK cells transduced with CD19 or CD33-CAR were transferred into CD19+ Raji-bearing NSG mice and supported in vivo with IL-15. After three weeks, mice were sacrificed and NK (CD56) cell persistence and tumor (CD19) burden assessed by flow cytometry. CD19-CAR ML recipient mice had reduced tumor burden in the BM, spleen, and blood compared to CD33-CAR ML treated mice. Notably, CD19-CAR-ML (GFP+) were increased to >70% of human NK cells from 20% in the CD33-CAR-ML recipient mice, suggesting antigen-specific CAR-expressing NK cells expand or survive better in vivo than non-transduced ML or non-specific CAR-ML NK cells in vivo. Thus, combining CAR with ML differentiation results in NK cells with enhanced responses to NK resistant tumors. These studies warrant continued CAR-ML development and provide the pre-clinical rationale for translating this combination NK cell therapy approach to the clinic. Figure Disclosures Fehniger: Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).

Investigation of fatty aldehyde and alcohol synthesis from fatty acids by αDox- or CAR-expressing Escherichia coli

Fatty aldehydes are among the most important flavor and fragrance compounds. Most biotechnological production approaches make use of the one step conversion of fatty acids from renewable sources by the enzymes α-dioxygenase (αDox) or carboxylic acid reductase (CAR). Their reaction mechanisms and cofactor dependencies are very different. In contrast to heme-containing αDox which requires only oxygen as cosubstrate, CAR needs NADPH and ATP, which is a clear argument for the application of a whole cell catalyst. Therefore we compared fatty acid biotransformations with growing Escherichia coli cells expressing αDox or CAR to investigate their suitability for fatty aldehyde and also fatty alcohol production. Our results show the main product of fatty acid conversions with αDox-expressing cells to be the expected Cn-1 aldehyde. However, 14% of the products consist of the corresponding alcohol, but in addition, 17% of the products consist of further shortened aldehydes, alcohols and acids that result from the consecutive activity of αDox and a putative endogenous fatty aldehyde dehydrogenase activity in E. coli. Conversely, CAR-expressing cells produced only the unshortened fatty aldehyde and alcohol, whereby the latter surprisingly accounts for at least 80% of the products. The considerably higher extend of aldehyde reduction of CAR-expressing cells was shown to be causally connected to the CAR-mediated fatty acid conversion. Our study provides an overview about the applicability of αDox- or CAR-based whole cell catalysts and gives a detailed description of side products as well as suggestions for tailored strain engineering.

Engineering Precision Medicine to Increase Graft-Versus-Lymphoma Activity: Hematopoietic Stem Cells Modified with Chimeric Antigen Receptors

Background: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure. Trials using autologous T-cells engineered with anti-CD19 chimeric antigen receptors (CAR) have demonstrated complete remissions even in chemotherapy-resistant malignancies, but the persistence of the cells is transient, limiting efficacy. Our hypothesis is modification of hematopoietic stem cells (HSC) with CAR will lead to persistent production of target-specific immune cells in multiple lineages, enhancing graft-versus-tumor activity and development of immunological memory.Design/Methods: Using CD19 as target, we generated second-generation CD28- and 4-1BB-costimulated CAR constructs for modification of human HSC for assessment in vitro and in vivo, using third-generation lentiviral vectors. Additionally, co-delivery of suicide gene systems was tested to allow ablation of gene-modified cells.Results: Gene modification of HSC with anti-CD19 CAR using lentiviral vectors did not impair differentiation or proliferation, and led to functional CAR-expressing cell progeny, at 40-50% transduction efficiency and engineered antigen-dependent cytotoxicity in myeloid, NK and T-cells. In vivo studies using humanized NSG engrafted with CAR-modified HSC demonstrated similar levels of humanization to non-modified HSC, with multilineage CAR-expressing cells present in bone marrow, spleen, blood and thymus in stable levels up to 44 weeks of life. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. Ex vivo cells presented antigen-dependent cytotoxicity against targets. Mice engrafted with CAR-modified HSC had decreased CD19+ populations and successfully presented tumor growth inhibition and survival advantage at tumor challenge (55-60%). CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice humanized with gene-modified HSC presented significant ablation of gene-modified cells after treatment (p=0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues.Conclusions: CAR modification of HSC for cancer immunotherapy is feasible. This approach can be applied to different cancers by adjusting target specificity and could be employed in the context of HSC transplantation to augment the anti-lymphoma activity. It also bears the possibility of decreased morbidity and mortality and offers alternative treatment for patients with no available sources for bone marrow transplantation, benefiting ethnic minorities. These results also give proof of principle for CAR-modified HSC regulated by suicide gene; further studies are needed to enable clinical translation. DisclosuresNo relevant conflicts of interest to declare.

Providing a Homing Receptor for CAR Engineered NK Cells - Improving Cellular Immunotherapy for B-Cell Lymphoma

▪BackgroundDespite some robust initial responses, anti-CD19 Chimeric Antigen Receptor (CAR) T-cell therapy can be associated with significant short-term (cytokine release syndrome) and long-term (B-cell deficiency) toxicities. CAR-engineered natural killer (NK) cells potentially provide a safer alternative while maintaining efficacy.Activated Natural Killer (aNKTM) cells are a clinical grade cell line derived from the NK-92R cell line that has demonstrated potent cytotoxicity towards a broad spectrum of malignant cell lines as well as safety and efficacy in phase I trials. Variants of the aNKcell line are currently in Phase I/II clinical trials: a CAR-expressing aNK cell line and the haNKTM cell line, which have been engineered to carry a high-affinity version of the CD16/FcγRIII receptor to allow for combination therapy with monoclonal antibodies. haNK cells have also been genetically modified to express an endoplasmic reticulum-retained version of IL-2 (ERIL-2), which provides IL-2 independence and limits IL-2 secretion to sub-physiological, safe levels.A key factor for the efficacy of cellular immunotherapies against a given target is biodistribution, which affects the local effector to target ratio. Inability to reach the tumor cells, either by lack of homing or by the accumulation of extracellular matrix (ECM) surrounding a tumor, can be responsible for the clinical failure of even the most effective CAR. The chemokines CCL19 and CCL21 drive recruitment of CCR7-expressing immune cells to secondary lymphoid organs. Engineering aNK cells to express the CCR7 receptor is likely to improve their efficacy by increasing their targeted migration to lymphoma tumor sites.Methods and ResultsClinical grade aNK cells were electroporated with a non-viral vector containing the CCR7 receptor, an anti-CD19 CAR, and a high affinity CD16 receptor. To assay the migration of these engineered cell lines, a modified Boyden Chamber assay was performed using Matrigel coated Transwells. K562 cells or modified K562 cells engineered to express CCL19 (K-19) were placed in the destination chamber and CFSE-stained effector cells were placed in the top well. After 24 hours, cells in the bottom well were analyzed by flow cytometry to measure the number of effectors which had migrated through the Matrigel (Fig 1a). The excellent activity of the CAR in stably transfected cells was confirmed against SUP-B15 cells (aNK-resistant), while the ADCC activity was tested against a SUP-B15 variant expressing CD20, but engineered to lack the CD19 antigen (Sup-B15 CD19-, CD20+).Migration towards human lymph node chemokine CCL19 was also tested in vivo in NSG mice with bilateral subcutaneous tumors - with parental K562 in one flank and K-19 tumors on the contralateral flank. CFSE-stained effector cells were delivered via tail vein injection once average tumor size reached 100mm3 and following randomization. Tumors were then harvested at multiple time points, dissociated, and the number of infiltrating effectors in each tumor compared by flow cytometric analysis. (Fig 1b).In testing, monoclonal cell lines expressing all components of the polycistronic system displayed preferential migration towards CCR7 chemokines both in vitro and in vivo, as well as robust cytotoxicity vs. K562 (92.4% +/- 2.4% at 5:1 E:T), Sup-B15(97% +/- 0.6% at 5:1 E:T), and Sup-B15(CD19-, CD20+) when pre-incubated with Rituximab(83.2% +/- 2.8% at 5:1 E:T) but not with control antibody Trastuzumab (22.3% +/- 1.1% at 5:1 E:T) in standard cytotoxicity and ADCC assays.ConclusionWe show here that the incorporation of a CCR7 receptor into an off the shelf CAR engineered NK cell line improves their homing towards lymph node chemokines both in vitro and in vivo. This improved homing should result in a greater ratio of effector to target in lymphoid tissue, and maximize the immunogenic cell death. [Display omitted] DisclosuresSchomer:NantKwest, Inc.: Employment, Equity Ownership. Boissel:NantKwest, Inc.: Employment, Equity Ownership. Jiang:NantKwest, Inc.: Employment, Equity Ownership. Klingemann:NantKwest, INc.: Employment, Equity Ownership, Patents & Royalties. Lee:NantKwest, Inc.: Employment, Equity Ownership. Soon-Shiong:NantKwest, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Off-the-Shelf Natural Killer Cells with Multi-Functional Engineering Using a Novel Anti-CD19 Chimeric Antigen Receptor Combined with Stabilized CD16 and IL15 Expression to Enhance Directed Anti-Tumor Activity

▪T lymphocytes that express an anti-CD19 chimeric antigen receptor (CAR) to redirect target specificity exhibit remarkable remissions in B cell malignancies. However, these cells are typically produced in a patient-specific manner that is relatively inefficient and expensive. Additionally, CAR-T cell treatment can lead to severe adverse events (SAEs) such as cytokine release syndrome (CRS) and neurotoxicity, as well as graft versus host disease (GvHD) when given in allogeneic setting. To circumvent these safety issues while maintaining multi-faceted anti-tumor activity, we developed an off-the-shelf natural killer (NK) cell consisting of a novel CAR combined with other effector mechanisms to enhance targeted cytotoxicity.NK cells are potent anti-tumor effector cells that play an important role in innate and adaptive immunity. Multiple clinical studies have demonstrated that adoptive transfer of allogeneic NK cells can induce durable remissions in patients with cancers that have relapsed or are refractory to standard therapies without detection of SAEs such as CRS. However, NK cells are challenging to genetically engineer, and are dependent on cytokine support for persistence and exhibit donor-to-donor variability factors that make it difficult to create a consistent clinical product from NK cells.Here we report the use of human induced pluripotent stem cells (iPSCs) to produce a renewable source of precisely engineered NK cells. This iPSC platform was utilized to evaluate a combination of CARs comprised of distinct NK-cell specific signaling and transmembrane domains with an autonomous protein to create a persistent and targeted NK cell immunotherapy. The selected NK cell optimized CAR (NK-CAR) backbone contains an NKG2D transmembrane domain, a 2B4 co-stimulatory domain, and a CD3ζ signaling domain to mediate a potent NK cell activating signal. To provide directed anti-tumor activity, anti-CD19 scFv was added to the NK-CAR backbone, engineered at the iPSC stage and subsequently differentiated on-demand to produce a uniform population of CAR-expressing NK cells. In addition, an IL-15RF fusion transgene was introduced to provide self-stimulating signals to support NK cell function and persistence. The IL-15RF construct was created by fusing IL-15Rα to IL-15 at the C-terminus through a flexible linker. As a third modality, a metalloprotease ADAM17-resistant version of the high-affinity CD16a (hnCD16) 158V variant was introduced at the iPSC stage to augment antibody-dependent cellular-cytotoxicity (ADCC) when used in combination with monoclonal antibodies. The selected iPSC clone exhibits stable expression of all three modalities and represents a renewable source of starting material for the reproducible generation of NK cells consisting of NK-CAR, IL-15RF and hnCD16 with product purity that is greater than 95% CD45+CD56+ with a product expansion greater than one million-fold over the course of the manufacturing process. In preclinical studies, these multi-functional engineered NK cells demonstrated enhanced directed cytotoxicity against CD19+ tumor targets when compared to non-engineered NK cells or iPSC-derived NK cells engineered with other CAR constructs. Additionally, the multi-functional engineered iPSC-CAR-NK cells significantly reduced tumor burden in a xenograft model of B acute lymphoblastic leukemia (p<0.05 at day 28). NK cells with IL-15RF inclusion demonstrated improved proliferation in the absence of cytokine support, as well as improved potency when immediately thawed and tested for efficacy. NK-CAR + IL-15RF eliminated 94% and 86% of target cells in the presence and absence of IL-2, respectively, while NK-CAR efficacy was reduced from 98% target elimination in the presence of IL-2 to 56% in the absence of IL-2. In conclusion, these studies demonstrate iPSCs serve as an optimal platform to provide a renewable multi-engineered NK cell product suitable for an “off-the-shelf” approach and serve as preclinical proof of concept for program FT519, a standardized CAR-targeted NK cell immunotherapy against B cell malignancies. DisclosuresKaufman:Fate Therapeutics: Consultancy, Research Funding. Bjordahl:Fate Therapeutics Inc.: Employment. Mahmood:Fate Therapeutics Inc.: Employment. Bonello:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment. Cichocki:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

Chimeric antigen receptor T-cell approaches to HIV cure.

Combination antiretroviral therapy (ART) has enabled tremendous progress in suppressing HIV replication in infected patients. However, ART alone cannot eradicate HIV and its latent, persisting reservoirs. Novel approaches are needed to eradicate the virus or achieve functional cure in the absence of ART. Adoptive T-cell therapies were initially tested in HIV-infected individuals with limited efficiency. Benefiting from new and improved methodologies, an increasing array of CAR T-cell therapies has been successfully developed in the cancer immunotherapy field, demonstrating promising new avenues that could be applied to HIV. Numerous studies have characterized various HIV-specific CAR constructs, types of cytolytic effector cells, and CAR-expressing cells' trafficking to the reservoir compartments, warranting further in-vivo efforts. Notably, the ability of CAR cells to persist and function in low-antigen environments in vivo, that is, in ART-suppressed patients, remains unclear. Despite promising results in preclinical studies, only a handful of clinical trials have been initiated worldwide. Several obstacles remain prior to successful application of HIV-specific CAR T-cell therapies in patients. In this review, we survey the current state of the field, and address paths towards realizing the goal of an efficacious HIV CAR T-cell product.

Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors.

Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19- K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells. Cancer Immunol Res; 6(4); 467-80. ©2018 AACR.

Preclinical Optimization of a CD20-specific Chimeric Antigen Receptor Vector and Culture Conditions.

Chimeric antigen receptor (CAR)-based adoptive T-cell therapy is a highly promising treatment for lymphoid malignancies, and CD20 is an ideal target antigen. We previously developed a lentiviral construct encoding a third generation CD20-targeted CAR but identified several features that required additional optimization before clinical translation. We describe here several improvements, including replacement of the immunogenic murine antigen-binding moiety with a fully human domain, streamlining the transgene insert to enhance lentiviral titers, modifications to the extracellular IgG spacer that abrogate nonspecific activation resulting from binding to Fc receptors, and evaluation of CD28, 4-1BB, or CD28 and 4-1BB costimulatory domains. We also found that restimulation of CAR T cells with an irradiated CD20 cell line boosted cell growth, increased the fraction of CAR-expressing cells, and preserved in vivo function despite leading to a reduced capacity for cytokine secretion in vitro. We also found that cryopreservation of CAR T cells did not affect immunophenotype or in vivo antitumor activity compared with fresh cells. These optimization steps resulted in significant improvement in antitumor activity in mouse models, resulting in eradication of established systemic lymphoma tumors in 75% of mice with a single infusion of CAR T cells, and prolonged in vivo persistence of modified cells. These results provide the basis for clinical testing of a lentiviral construct encoding a fully human CD20-targeted CAR with CD28 and 4-1BB costimulatory domains and truncated CD19 (tCD19) transduction marker.

Long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor T cells in a nonhuman primate model of HIV/AIDS.

Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model.

Development of chimeric antigen receptors targeting T-cell malignancies using two structurally different anti-CD5 antigen binding domains in NK and CRISPR-edited T cell lines

ABSTRACTRelapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting. To circumvent this problem two approaches were investigated. First, a natural killer (NK) cell line, which does not express CD5, was used for CAR expression. Second, CRISPR-Cas9 genome editing technology was used to knockout CD5 expression in CD5-positive Jurkat T cells and in primary T cells, allowing for the use of CD5-negative T cells for CAR expression. Two structurally distinct anti-CD5 sequences were also tested, i) a traditional immunoglobulin-based single chain variable fragment (scFv) and ii) a lamprey-derived variable lymphocyte receptor (VLR), which we previously showed can be used for CAR-based recognition. Our results show i) both CARs yield comparable T-cell activation and NK cell-based cytotoxicity when targeting CD5-positive cells, ii) CD5-edited CAR-modified Jurkat T cells have reduced self-activation compared to that of CD5-positive CAR-modified T cells, iii) CD5-edited CAR-modified Jurkat T cells have increased activation in the presence of CD5-positive target cells compared to that of CD5-positive CAR-modified T cells, and iv) although modest effects were seen, a mouse model using the CAR-expressing NK cell line showed the scFv-CAR was superior to the VLR-CAR in delaying disease progression.

Clinical grade manufacturing of genetically modified, CAR-expressing NK-92 cells for the treatment of ErbB2-positive malignancies.

The NK-92/5.28.z cell line (also referred to as HER2.taNK) represents a stable, lentiviral-transduced clone of ErbB2 (HER2)-specific, second-generation CAR-expressing derivative of clinically applicable NK-92 cells. This study addresses manufacturing-related issues and aimed to develop a GMP-compliant protocol for the generation of NK-92/5.28.z therapeutic doses starting from a well-characterized GMP-compliant master cell bank. Commercially available GMP-grade culture media and supplements (fresh frozen plasma, platelet lysate) were evaluated for their ability to support expansion of NK-92/5.28.z. Irradiation sensitivity and cytokine release werealso investigated. NK-92/5.28.z cells can be grown to clinically applicable cell doses of 5×108 cells/L in a 5-day batch culture without loss of viability and potency. X-Vivo 10 containing recombinant transferrin supplemented with 5% FFP and 500IU/mL IL-2 in VueLife 750-C1 bags showed the best results. Platelet lysate was less suited to support NK-92/5.28.z proliferation. Irradiation with 10Gy completely abrogated NK-92/5.28.z proliferation and preserved viability and potency for at least 24h. NK-92/5.28.z showed higher baseline cytokine release compared to NK-92, which was significantly increased upon encountering ErbB2(+) targets [GZMB (twofold), IFN-γ (fourfold), IL-8 (24-fold) and IL-10 (fivefold)]. IL-6 was not released by NK cells, but was observed in some stimulated targets. Irradiation resulted in upregulation of IL-8 and downregulation of sFasL, while other cytokines were not impacted. Our concept suggests NK-92/5.28.z maintenance culture from which therapeutic doses up to 5×109 cells can be expanded in 10L within 5days. This established process is feasible to analyze NK-92/5.28.z in phase I/II trials.

Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity.

Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR-engineered NK-92 cells as off-the-shelf cellular therapeutics, with special emphasis on ErbB2 (HER2)-specific NK-92 cells that are approaching clinical application.

Abstract 2314: Different stimulation conditions affect the immune phenotype of GD2-specific chimeric antigen receptor-expressing T cells

Abstract The in vivo persistence of chimeric antigen receptor (CAR) modified T cells is a major prerequisite for their antitumor activity and was found to be associated with a less differentiated immune phenotype. Here, we compared two in vitro T cell stimulation conditions: coated anti-CD3/CD28 antibodies [3/28], and Dynabead stimulation of enriched CD3+ T cells [DB]. Peripheral blood T cells from three healthy donors were stimulated with either of the two methods, retrovirally transduced with the GD2-specific CAR GD2-BBz on day 2 or 3, and expanded in RPMI/AIMV medium with 50 IU/ml recombinant human interleukin-2 for 13 days. T cell expansion rates were comparable between the two stimulation conditions and independent of CAR gene expression. Transduction efficiencies, determined by staining with the GD2-CAR-specific antibody Ganglidiomab, were also comparable. The immune phenotype by expression of CD3, CD4, CD8, CD45RO and CD197 was determined by flow cytometry analysis on day 13 or 14 after initial stimulation. The proportions of central memory (TCM), effector memory (TEM) or naïve T cells (TN) within the two types of cultures were noticeably different (Table 1), with a higher proportion of non-transduced CD8+ T cells with a TCM phenotype after DB compared to CD3/CD28 stimulation (p = 0.005 for DB d2, p = 0.01 for DB d3). Compared to non-transduced T cells, CAR-expressing cells of all types of cultures had higher proportions of TCM cells (p = 0.02 for CD4+ T cells, p&amp;lt;0.01 for CD8+ T cells). In conclusion, we found that the stimulation conditions have a strong impact on the T cell phenotype and that retroviral CAR gene transduction can also affect T cell differentiation. The optimal T cell culture conditions for a product with sustained persistence in vivo will ultimately emerge from clinical trials. Table 1:Proportions of T cell subpopulations on day 13 or 14 (medians and ranges)xyTN: CD45RO-/CD197+TCM: CD45RO+/CD197+TEM: CD45RO+/CD197-3/28NT37.9% CD4+ (31.8-41.3) 53.0% CD8+ (20.9-72.9)20.2% CD4+ (20.2-27.0) 6.1% CD8+ (3.7-6.9)35.2% CD4+ (32.8-45.4) 35.5% CD8+ (17.0-66.3)CAR d224.4% CD4+ (15.1-28.8) 32.9% CD8+ (23.9-48.9)33.6% CD4+ (29.9-49.3) 27.7% CD8+ (17.7-38.8)37.3% CD4+ (22.4-47.2) 27.1% CD8+ (25.4-32.7)DBNT33.1% CD4+ (15.0-45.6) 53.8% CD8+ (46.8-53.8)32.6% CD4+ (29.9-45.2) 19.6% CD8+ (17.8-26.3)23.3% CD4+ (16.6-51.7) 12.1% CD8+ (10.6-15.2)CAR d211.5% CD4+ (10.9-27.7) 37.1% CD8+ (24.7-41.860.7% CD4+ (53.0-69.7) 48.0% CD8+ (34.4-65.2)16.5% CD4+ (16.1-26.6) 8.7% CD8+ (5.7-10.3)DBNT33.2% CD4+ (26.5-55.2) 51.6% CD8+ (34.2-62.5)23.0% CD4+ (21.9-40.2) 14.2% CD8+ (11.2-16.9)19.1% CD4+ (17.6-48.6) 15.2% CD8+ (13.0-19.6)CAR d317.5% CD4+ (13.4-34.5) 36.6% CD8+ (28.5-46.5)45.7% CD4+ (36.6-57.5) 38.0% CD8+ (22.2-55.1)22.0% CD4+ (21.4-32.6) 10.8% CD8+ (8.5-19.6) Citation Format: Laurin Ochs, Bianca Altvater, Sareetha Kailayangiri, Christian Spurny, Claudia Rossig, Silke Jamitzky. Different stimulation conditions affect the immune phenotype of GD2-specific chimeric antigen receptor-expressing T cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2314.

761. Targeted Killing of HIV Infected Cells Using CCR5-Disrupted Anti-HIV-CAR T Cells

A cure for HIV remains an important treatment goal for 30 million HIV-infected individuals worldwide. Long term control of HIV following a single treatment will require a mechanism to eradicate the latent reservoir of HIV infected cells that are capable of reactivating. A previous phase II clinical trial using an anti-HIV chimeric antigen receptor (αHIV-CAR) targeting the CD4-binding site on HIV envelope was partially effective. To optimize this approach we have developed a series of CARs based on the scFV of broadly neutralizing HIV antibodies targeting four different structural regions of the HIV envelope: the V1/V2 loop, the V3 loop, the CD4 binding site, and the membrane-proximal external region. αHIV-CAR T cells targeting different epitopes were compared and were able to kill > 80% of HIV-infected cells grown in the presence of ART. However, a limitation of αHIV-CAR T cells is that the αHIV-CAR also serves as a receptor for HIV and allows HIV infection of αHIV-CAR T cells. Therefore we disrupted the major co-receptor required for HIV cell entry, CCR5, using a megaTAL nuclease, as a means of protecting the CAR-expressing cells from HIV infection. We used two strategies for achieving these dual modifications in primary human T cells. For both strategies, the CCR5 megaTAL nuclease was delivered by mRNA electroporation, which has previously been shown to induce a high rate of bi-allelic NHEJ-mediated gene disruption. αHIV-CAR expression cassettes were delivered into the host genome via lentiviral vectors (LV), or were targeted to the megaTAL nuclease cleavage site in CCR5 using an adeno-associated virus (AAV) that included CCR5 homology arms. Both strategies resulted in stable expression of the CAR construct and specific activation of CAR+ T cells in the presence of an HIV+ cell line. In the presence of actively replicating virus, CCR5-megaTAL treated CAR+ T cells out-performed CAR+ T cells generated by LV delivery alone as measured by reduction in HIV capsid protein. To enable testing in non-human primates (NHP), we re-optimized the cell-editing protocol with NHP lymphocytes. Primary T cells from pigtail macaques were successfully transfected with CCR5 megaTAL mRNA and achieved a CCR5 disruption rate of 50%. Cells were subsequently transduced with αHIV-CAR LV resulting in ~70% of manipulated cells with αHIV-CAR. Expansion of the cells in vitro resulted in 60-fold expansion over 8 days. CAR+ NHP cells specifically killed HIV infected human cells demonstrating that NHP-derived αHIV-CAR+ T cells retained killing function. In conclusion, it is feasible to construct αHIV-CAR+ T cells that are protected from HIV infection in human and NHP cells, and warrants further study in vivo.

Abstract A175: Design and evaluation of novel natural killer cell chimeric antigen receptors

Abstract Chimeric Antigen Receptors (CARs) are used to direct and activate cytotoxic lymphocytes against virally infected or malignant cells. They have been heavily investigated in cytotoxic T cells, and, to a much lesser extent, in natural killer (NK) cells with promising results, especially for anti-CD19 CARs to mediate killing of B cell leukemias. To date, CARs that have been utilized in NK cells have primarily employed the same intracellular signaling components as T cell CARs. However, this may not be the optimal approach for CAR-expressing NK cells due to differences in key signaling pathways. To improve the activity of NK cell-CARs targeted against solid tumors, the intracellular signaling domains of an anti-mesothelin CAR were modified to express NK cell specific signaling molecules and evaluated for activity in NK-92 cells against ovarian cancer cell lines. CARs were assembled using gBlocks encoding various transmembrane, co-activating, and signaling domains. NK cell specific CARs were then transfected into NK-92 cells, a human NK cell tumor line, using Sleeping Beauty. Positive cells were selected for using a drug resistance marker and CAR surface expression was evaluated using flow cytometry. CAR expressing NK-92 cells were then evaluated for degranulation, as indicated by CD107a production, and interferon (INF)-γ release when challenged with mesothelin negative, MA148, and mesothelin positive, A1847, ovarian cell lines. In addition, beads conjugated to mesothelin were used to measure NK cell activation via the CAR alone. In total 10 novel CARs were assembled and transfected into NK-92 cells, 6 of which were properly expressed on the cell surface. All 6 properly expressed CARs enhanced both CD107a and INF-γ production. When CAR activity alone was evaluated using mesothelin conjugated protein A beads, non-transfected NK-92 cells or cells that did not properly express the CAR all had &amp;lt;3% positive cells for CD107a and INF-γ. In contrast, NK-92 cells expressing CARs had between 8 and 16% positive cells for CD107a, with 5-12% positive for INF-γ. Since CAR4, expressing an NKG2D transmembrane, 2B4 co-activation, and CD3ζ signaling domain had the greatest activity, individual domains were mutated to assess their function. Mutation of the key binding residue in NKG2D for DAP10 resulted in a drastic decrease in the percentage of INF-γ producing cells, while mutation of the tyrosine residues in the CD3ζ signaling domain greatly abrogated CD107a production. As expected, mutation of all domains resulted in an inactive CAR. These studies demonstrate that CARs containing NK cell receptor domains function properly and may lead to better NK-92 cell activation. In addition, mutating CAR domains allows for dissecting the different pathways leading to either degranulation or cytokine release in NK cells. To better assess CAR activation potential in non-transformed NK cells, we are currently expressing the most promising CAR constructs into induced pluripotent stem cells (iPSCs) followed by differentiation into mature NK cells that will be tested for activity against ovarian cancer cells both in vitro and in vivo. These iPSC-NK cells will have the potential to create an “off-the-shelf” immunotherapy with the capability of targeting multiple malignancies. Citation Format: David L. Hermanson, Branden Moriarity, Trevor Argall, Melissa A. Geller, David Largaespada, Dan S. Kaufman. Design and evaluation of novel natural killer cell chimeric antigen receptors. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A175.