Chimeric Antigen Receptor Natural Killer Research Articles

Off-the-Shelf Prostate Stem Cell Antigen–Directed Chimeric Antigen Receptor Natural Killer Cell Therapy to Treat Pancreatic Cancer

Pancreatic cancer (PC) is the third leading cause of cancer-related death with a 5-year survival rate of approximately 10%. It typically presents as a late-stage incurable cancer and chemotherapy provides modest benefit. Here, we demonstrate the feasibility, safety, and potency of a novel human natural killer (NK) cell-based immunotherapy to treat PC. The expression of prostate stem cell antigen (PSCA) was evaluated in primary PC at messenger RNA and protein levels. The processes of retroviral transduction, expansion, activation, and cryopreservation of primary human NK cells obtained from umbilical cord blood were optimized, allowing us to develop frozen, off-the-shelf, allogeneic PSCA chimeric antigen receptor (CAR) NK cells. The safety and efficacy of PSCA CAR NK cells also expressing soluble (s) interleukin 15 (PSCA CAR_s15 NK cells) were evaluated invitro and invivo. PSCA was elevated in primary human PC compared with the adjacent or other normal tissues. PSCA CAR_s15 NK cells displayed significant tumor-suppressive effects against PSCA(+) PC invitro before and after 1 cycle of freeze-thaw. The viability of frozen PSCA CAR_s15 NK cells persisted more than 90 days invivo after their last infusion and significantly prolonged the survival of mice engrafted with human PC. PSCA CAR_s15 NK cells showed therapeutic efficacy in human metastatic PC models without signs of systematic toxicity, providing a strong rationale to support clinical development.

Adoptive Cellular Therapy in Acute Myeloid Leukemia: Current Scope and Challenges.

Adoptive cellular therapies have revolutionized the management of hematologic malignancies, particularly lymphoma and multiple myeloma. These therapies targeting disease-specific antigens, such as CD19 in lymphoma and B cell maturation antigen in multiple myeloma, are efficacious and well-tolerated compared with conventional chemotherapies. Unfortunately, their potential remains unrealized in acute myeloid leukemia (AML). This is because most targetable antigens on AML cells are also expressed on healthy myeloid hematopoietic stem cells (HSC). Therefore, targeting them results in severe myeloablative effects and pancytopenia. Several strategies have been devised to overcome this barrier, including identifying AML-specific antigens, limiting CAR-T cell persistence to prevent prolonged myeloablation, and creating AML-specific antigens through manipulating HSCs prior to allogenic transplant. In this review, we discuss these strategies and the ongoing clinical trials on adoptive cellular therapies in AML, limiting our focus to chimeric antigen receptor-T cells (CAR-T) and chimeric antigen receptor-natural killer cells (CAR-NK).

Engineered CAR-T and novel CAR-based therapies to fight the immune evasion of glioblastoma: gutta cavat lapidem

Introduction The field of cancer immunotherapy has achieved great advancements through the application of genetically engineered T cells with chimeric antigen receptors (CAR), that have shown exciting success in eradicating hematologic malignancies and have proved to be safe with promising early signs of antitumoral activity in the treatment of glioblastoma (GBM). Areas covered : We discuss the use of CAR T cells in GBM, focusing on limitations and obstacles to advancement, mostly related to toxicities, hostile tumor microenvironment, limited CAR T cells infiltration and persistence, target antigen loss/heterogeneity and inadequate trafficking. Furthermore, we introduce the refined strategies aimed at strengthening CAR T activity and offer insights in to novel immunotherapeutic approaches, such as the potential use of CAR NK or CAR M to optimize anti-tumor effects for GBM management. Expert opinion With the progressive wide use of CAR T cell therapy, significant challenges in treating solid tumors, including central nervous system (CNS) tumors, are emerging, highlighting early disease relapse and cancer cell resistance issues, owing to hostile immunosuppressive microenvironment and tumor antigen heterogeneity. In addition to CAR T cells, there is great interest in utilizing other types of CAR-based therapies, such as CAR natural killer (CAR NK) or CAR macrophages (CAR M) cells for CNS tumors.

Engineering CAR-NK cells to secrete IL-15 sustains their anti-AML functionality but is associated with systemic toxicities

BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via...

NK Cells Armed with Chimeric Antigen Receptors (CAR): Roadblocks to Successful Development.

In recent years, cell-based immunotherapies have demonstrated promising results in the treatment of cancer. Chimeric antigen receptors (CARs) arm effector cells with a weapon for targeting tumor antigens, licensing engineered cells to recognize and kill cancer cells. The quality of the CAR-antigen interaction strongly depends on the selected tumor antigen and its expression density on cancer cells. CD19 CAR-engineered T cells approved by the Food and Drug Administration have been most frequently applied in the treatment of hematological malignancies. Clinical challenges in their application primarily include cytokine release syndrome, neurological symptoms, severe inflammatory responses, and/or other off-target effects most likely mediated by cytotoxic T cells. As a consequence, there remains a significant medical need for more potent technology platforms leveraging cell-based approaches with enhanced safety profiles. A promising population that has been advanced is the natural killer (NK) cell, which can also be engineered with CARs. NK cells which belong to the innate arm of the immune system recognize and kill virally infected cells as well as (stressed) cancer cells in a major histocompatibility complex I independent manner. NK cells play an important role in the host’s immune defense against cancer due to their specialized lytic mechanisms which include death receptor (i.e., Fas)/death receptor ligand (i.e., Fas ligand) and granzyme B/perforin-mediated apoptosis, and antibody-dependent cellular cytotoxicity, as well as their immunoregulatory potential via cytokine/chemokine release. To develop and implement a highly effective CAR NK cell-based therapy with low side effects, the following three principles which are specifically addressed in this review have to be considered: unique target selection, well-designed CAR, and optimized gene delivery.

Targeting HLA-DR loss in hematologic malignancies with an inhibitory chimeric antigen receptor

Chimeric antigen receptor natural killer (CAR-NK) cells have remarkable cytotoxicity against hematologic malignancies; however, they may also attack normal cells sharing the target antigen. Since human leukocyte antigen DR (HLA-DR) is reportedly lost or downregulated in a substantial proportion of hematologic malignancies, presumably a mechanism to escape immune surveillance, we hypothesize that the anti-cancer specificity of CAR-NK cells can be enhanced by activating them against cancer antigens while inhibiting them against HLA-DR. Here, we report the development of an anti-HLA-DR inhibitory CAR (iCAR) that can effectively suppress NK cell activation against HLA-DR-expressing cells. We show that dual CAR-NK cells, which co-express the anti-CD19 or CD33 activating CAR and the anti-HLA-DR iCAR, can preferentially target HLA-DR-negative cells over HLA-DR-positive cells invitro. We find that the HLA-DR-mediated inhibition is positively correlated with both iCAR and HLA-DR densities. We also find that HLA-DR-expressing surrounding cells do not affect the target selectivity of dual CAR-NK cells. Finally, we confirm that HLA-DR-positive cells are resistant to dual CAR-NK cell-mediated killing in a xenograft mouse model. Our approach holds great promise for enhancing CAR-NK and CAR-T cell specificity against malignancies with HLA-DR loss.

Therapeutic Targeting of Mesothelin with Chimeric Antigen Receptor Natural Killer Cell Therapy in Acute Myeloid Leukemia

Effective immunotherapy for acute myeloid leukemia (AML) has been limited by the lack of AML-restricted targets (expression in AML but silent in normal hematopoiesis). Current immunotherapies targeting lineage markers CD33 and CD123 (if effective) would lead to prolonged myelosuppression or myeloablation, requiring stem cell transplantation to restore hematopoiesis after treatment. In search for AML-restricted targets, we interrogated the AML transcriptome of nearly 3000 cases in pediatric and young adults and contrasting it to normal bone marrow and peripheral blood CD34+ samples. This extensive discovery effort has identified over 200 AML-restricted targets with mesothelin (MSLN) emerged as one of the highest expressing AML-restricted targets and highly enriched in the KMT2A-rearranged AML subtype. We previously validated cell surface expression of MSLN on both AML blasts and leukemic stem cells (Le et. al. 2021). We further showed efficacy targeting MSLN in AML using antibody-drug conjugates (Kaeding et. al. 2021) and chimeric antigen receptor (CAR) T cells (Le et. al. 2021). Given that natural killer (NK) cells are potent immune effector cells and generally have a more favorable toxicity profile than CAR T cells (i.e without cytokine release syndrome), we developed CAR NK cells targeting MSLN and evaluated their efficacy in AML preclinical models.From primary patient samples, we verified MSLN cell surface expression and showed high correlation between cell surface expression (mean fluorescence intensity, MFI) and transcript expression (TPM, R = 0.72, p = 2.1x10 -8, Figure 1A) . Importantly, MSLN expression was restricted to AML blasts and entirely absent in normal lymphocytes and myeloid cells in individual patients (Figure 1B, C). Having confirmed cell surface and AML-restricted expression of MSLN, we developed CAR NK cells targeting MSLN. The VH and VL sequences from immunotoxin SS1P were used to create the single-chain variable fragment domain of the standard CAR (41-BB and CD3Zeta, Figure 1D). CAR NK cells were generated by transducing NK-92 cells with the MSLN CAR construct. Cytotoxicity of CAR NK cells was evaluated against Nomo-1 AML cell line, which expresses endogenous level of MSLN; MV4;11 and Kasumi-1 cell lines engineered to express MSLN with a lentivirus construct (MV4;11 MSLN+ and Kasumi-1 MSLN+).We initially tested the target specificity of MSLN-directed CAR NK cells against MSLN-positive (Nomo-1, MV4;11 MSLN+ and Kasumi-1 MSLN+) and MSLN-negative (Nomo-1 MSLN KO, MV4;11 and Kasumi-1) cells. CAR NK cells exhibited enhanced cytolytic activity against MSLN-positive but not MSLN-negative AML cells after 12 hours of co-incubation at the indicated effector: target (E:T) ratios (Figure 1E). We next assessed the in vivo efficacy of CAR NK cells. Nomo-1 cells transduced with GFP/Luciferase were transplanted into NSG mice at 1x10 6 cells/mouse. Unmodified or CAR NK cells were infused 1 week following leukemic cell injection at 1x10 7 cells/mouse. Monitoring leukemia burden by bioluminescence IVIS imaging showed that after 4 days post NK injection, the leukemia was significantly reduced in Nomo-1 xenograft mice treated with CAR NK cells compared to mice that received unmodified NK cells (Figure 1F), suggesting highly potent anti-leukemia activity of CAR NK cells.In this study, we demonstrate that the cell surface expression of MSLN is restricted to AML blasts but is entirely silent in normal hematopoietic subsets in individual patients. Previous and current clinical trials utilizing NK-92 cells have demonstrated safety and efficacy in variety of cancers, including AML. Here, we demonstrate that NK-92 cells genetically modified with a CAR to redirect their specificity against MSLN-positive AML cells exhibit potent, target-dependent anti-leukemia activity in vitro and in vivo. These results provide compelling data to evaluate MSLN-directed CAR NK cell therapy in clinical trials for refractory/relapsed AML, especially for high-risk KMT2A-rearranged leukemia where majority of patients express MSLN at diagnosis and relapse. [Display omitted] DisclosuresPardo: Hematologics, Inc.: Current Employment.

Recruitment of NK Cells during Autologous Apheresis Allows Efficient NK Cell Collection for Immunotherapy

Background:Natural killer (NK) cell-based immunotherapies are emerging as a new, cutting-edge, promising cancer treatment. NK cells have been used to generate autologous as well as allogenic chimeric antigen receptor NK (CAR-NK) cells, and are currently being tested in multiple clinical trials. The advantage of these cells over CAR-T cells is that they show a superior toxicity profile, less on-target/off-tumor effects, and their toxicity mechanism could be independent of CAR. Additionally, allogenic CAR-NK cells are less immunogenic and cause less graft versus host disease in comparison to CAR-T cells. NK-cell collection during apheresis is the first step in the production of CAR-NK-cells. Efficient collection of NK-cells is paramount to successful manufacture of a CAR-NK cell product. Here, we present our single-center experience on individualized high-flow autologous lymphocyte apheresis in heavily pre-treated patients who qualified for a CAR-T treatment. In this abstract, we demonstrate data on NK-cell collections that derived from our research on T-cell collections.Study design and methods:In this retrospective, descriptive study, we compared collection efficiencies (CE%), absolute efficiencies (AE; absolute yield) and relative efficiencies (RE; AE/total number of absolute circulating pre-apheresis cells) for NK- and T-cell collections. Recruitment of cells was assumed if RE>1. The analysis was performed for 9 heavily pre-treated diffuse large B-cell lymphoma patients who underwent autologous T-cell collections for CAR-T treatment between October 2018 and November 2019. These 9 patients were chosen because the data on pre-apheresis NK- and T-cell concentrations, and harvest NK- and T-cell yield, were available for them. For calculation of CE% a standard formula was used (Figure 1, A).Results:A total of 9 high-flow lymphocyte collections achieved the cell yield of 1x10 9 cells for T- cells and 88% (n=8) achieved the cell yield of 1x10 9 cells for NK-cells. The median pre-apheresis cell concentration for NK-cells was 0.21 x10 9/L (range 0.05 - 0.29) and for T-cells was 0.69 x10 9/L (0.11 - 2.09). The median average blood flow was 77 ml/min (49 - 132), the median processed blood volume was 13.51L (7.35 - 31.17), while the median ratio of processed blood volume to total blood volume was 2.57 (1.46 - 6.67). The median AE for NK-cells with 1.58x10 9 (0.55 - 5.9) was markedly lower but not statistically significant (p=0.14) than that of T-cells with 5.5x10 9 (1.88 - 18.41). The CE of NK-collections was median 89% (29 - 125), while the CE of T-cell collections was median 68% (31 - 93) and they were not statistically different (p=0.605). The same was observed for RE which was 3.04 (0.63 - 4.31) for NK-cells and 1.91 (0.68 - 4.52) for T-cells with no statistically significant difference (Independent-Samples Mann-Whitney U Test, p=0.836). Figure 1 presents the differences in AE (B), CE (C) and RE (D) between NK- and T-cell collections.Conclusions:During high-flow lymphocyte apheresis, NK- as well as T-cells are recruited, as in the majority of collections the cell yield is greater than the absolute circulating pre-apheresis cell count (when RE was > 1). Thus, the origin and immunophenotype of these NK- and T-cells should be subject of further investigation. CE for NK- and T-cells are similar and high enough to perform efficient cell collections of both cell types for immunotherapy. [Display omitted] DisclosuresJalowiec: Kite Pharma Gilead Sciences': Honoraria, Speakers Bureau. Daskalakis: Amgen: Other: Travel grant; Roche: Other: Travel grant; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; Celgene/BMS: Consultancy, Other: Travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; NovoNordisk: Other: Travel grant.

A Phase 1 Study of NKX019, a CD19 Chimeric Antigen Receptor Natural Killer (CAR NK) Cell Therapy, in Subjects with B-Cell Malignancies

A Phase 1 Study of NKX019, a CD19 Chimeric Antigen Receptor Natural Killer (CAR NK) Cell Therapy, in Subjects with B-Cell Malignancies

New Orders to an Old Soldier: Optimizing NK Cells for Adoptive Immunotherapy in Hematology.

NK (Natural Killer) cell-mediated adoptive immunotherapy has gained attention in hematology due to the progressing knowledge of NK cell receptor structure, biology and function. Today, challenges related to NK cell expansion and persistence in vivo as well as low cytotoxicity have been mostly overcome by pioneering trials that focused on harnessing NK cell functions. Recent technological advancements in gene delivery, gene editing and chimeric antigen receptors (CARs) have made it possible to generate genetically modified NK cells that enhance the anti-tumor efficacy and represent suitable “off-the-shelf” products with fewer side effects. In this review, we highlight recent advances in NK cell biology along with current approaches for potentiating NK cell proliferation and activity, redirecting NK cells using CARs and optimizing the procedure to manufacture clinical-grade NK and CAR NK cells for adoptive immunotherapy.

Chimeric Antigen Receptor-Engineered Natural Killer (CAR NK) Cells in Cancer Treatment; Recent Advances and Future Prospects.

Natural Killer (NK) cells are critical members of the innate immunity lymphocytes and have a critical role in host defense against malignant cells. Adoptive cell therapy (ACT) using chimeric antigen receptor (CAR) redirects the specificity of the immune cell against a target-specific antigen. ACT has recently created an outstanding opportunity for cancer treatment. Unlike CAR-armored T cells which hadnsome shortcomings as the CAR-receiving construct, Major histocompatibility complex (MHC)-independency, shorter lifespan, the potential to produce an off-the-shelf immune product, and potent anti-tumor properties of the NK cells has introduced NK cells as a potent alternative target for expression of CAR. Here, we aim to provide an updated overview on the current improvements in CAR NK design and immunobiology and describe the potential of CAR-modified NK cells as an alternative “off-the-shelf” carrier of CAR. We also provide lists for the sources of NK cells in the process of CAR NK cell production, different methods for transduction of the CAR genetic sequence to NK cells, the differences between CAR T and CAR NK, and CAR NK-targeted tumor antigens in current studies. Additionally, we provide data on recently published preclinical and clinical studies of CAR NK therapy and a list of finished and ongoing clinical trials. For achieving CAR NK products with higher efficacy and safety, we discuss current challenges in transduction and expansion of CAR NK cells, CAR NK therapy side effects, and challenges that limit the optimal efficacy of CAR NK cells and recommend possible solutions to enhance the persistence, function, safety, and efficacy of CAR NK cells with a special focus on solid tumors.Graphical abstract

Selective Cytotoxicity of Single and Dual Anti-CD19 and Anti-CD138 Chimeric Antigen Receptor-Natural Killer Cells against Hematologic Malignancies.

Natural killer (NK) cells are part of the first line of defense that rapidly respond to malignant transformed cells. Chimeric antigen receptor- (CAR-) engineered NK cells, although are still at the preliminary stage, have been shown to be alternative to CAR-T cells, mainly due to the absence of graft-versus-host disease and safer clinical profile. Allogeneic human NK cell line NK-92 cells, equipped by CAR, are being developed for clinical applications. Herein, we designed third-generation CARs, optimized the production protocol, and generated CAR-NK-92 cells, targeting CD19 and/or CD138 antigens that employ CD28, 4-1BB, and CD3ζ signaling, with >80% CAR expression, designated as CD19-NK-92, CD138-NK-92, and dual-NK-92 cells. The generated CAR-NK-92 cells displayed high and selective cytotoxicity toward various corresponding leukemia, lymphoma, and multiple myeloma cell lines in vitro. Multitargeting approach using a mixture of CD19-NK-92 and CD138-NK-92 cells was also evaluated at various ratios to test the idea of personalized formulation to match the patients' antigen expression profile. Our data indicate that increasing the ratio of CD19-NK-92 to CD138-NK-92 could improve NK cytotoxicity in leukemia cells with a relatively higher expression of CD19 over CD138, supporting the personalized proof of concept. This information represents the basis for further in vivo studies and future progress to clinical trials.

Abstract LB154: A potent human CAR NK cell therapy directed against pancreatic cancer

Abstract Pancreatic cancer (PC) is one of the most lethal forms of cancer with a 5-year overall survival rate of 9%, routinely presenting as a late-stage incurable cancer that responds only modestly to standard chemotherapy. It is the third leading cause of cancer-related deaths. Approximately 60-80% of PC express prostate stem cell antigen (PSCA), as do gastric, bladder, prostate and some lung cancers. We therefore developed a chimeric antigen receptor (CAR) directed against PSCA for transduction into human natural killer (NK) cells. NK cells spontaneously kill both liquid and solid tumors without regards to expression of MHC self-antigens. In vivo, human NK cells utilize endogenous IL-15 to develop, survive, expand and activate against tumor cells. We therefore incorporated a soluble (s), secretable form of IL-15 into the PSCA CAR construct itself, followed by transduction into human NK cells obtained from umbilical cord blood with ~50% transduction efficiency. Transduced NK cells expressed the CAR directed against PSCA and secreted measurable amounts of human IL-15 protein in vitro. The PSCA CAR NK cells could be expanded ex vivo over 1,000-fold in approximately 16 days and retain their expression and their capacity to specificity kill PSCA(+) tumor cell targets in vitro. Indeed, when directed against the PSCA(+) human pancreatic tumor cell line (Capan-1), PSCA CAR NK cells produced significantly greater TNFα (P < 0.0001), IFNγ (P < 0.0001), and CD107a (P < 0.05), when compared to PSCA CAR NK cells against the PSCA(-) human pancreatic tumor cell line PANC-1. Moreover, we showed that the co-expression of sIL-15 with PSCA CAR NK cells significantly enhances their cytotoxic function against pancreatic tumor cells compared to PSCA CAR NK cells without expression of sIL-15 (P < 0.01) determined by a real-time cytolysis assay (RTCA) over 3.5 days. Encouraged by these in vitro data, we developed a model of metastatic human pancreatic cancer in immunodeficient mice using the PSCA(+) Capan-1 cell line. Compared to NK cells only expressing sIL-15, repeated infusions of human PSCA CAR NK cells from a viably frozen source resulted in a significantly prolonged survival (P < 0.001), including clearance of metastatic disease. In summary, our in vitro and in vivo studies utilizing viably frozen human PSCA CAR NK cells co-expressing sIL-15 demonstrate significant efficacy in prolonging survival against a human pancreatic tumor cell line without evidence of systemic toxicity, providing a rationale to move this novel form of cell therapy into the clinic for PSCA(+) solid tumors. Citation Format: Kun-Yu Teng, Anthony Mansour, Zhu Zheng, Lei Tien, Yi Zheng, Zhiyao Li, Jianying Zhang, Saul J. Priceman, Michael A. Caligiuri, Jianhua Yu. A potent human CAR NK cell therapy directed against pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB154.

CS1-Specific Chimeric Antigen Receptor Drives Cord Blood NK Cell Maturation and Expansion In Vivo

Chimeric antigen receptors (CAR) have been used extensively to redirect the specificity of T cells against leukemic cells, with remarkable clinical responses in patients with lymphoid malignancies. CAR-transduced natural killer (NK) cells might serve as an alternative to CAR T cells as they lack the potential to cause GVHD, and could be made available as an off-the-shelf allogeneic product for immediate clinical use. Moreover, unlike CAR-T cells, CAR NK cells retain their intrinsic capacity to recognize and target tumor cells through their native receptors, in theory making disease escape through downregulation of the CAR target antigen less likely. We have identified cord blood (CB) as a readily available source of allogeneic NK cells.Thus, in this study, we reprogrammed CB-NK cells to recognize multiple myeloma (MM) using a retroviral construct encoding a CAR against CS1, a coreceptor expressed on MM cells, IL-15 to enhance their in vivo proliferation and survival, and inducible caspase-9, a suicide gene that can be activated as needed. We consistently achieved a median CAR positivity of >90%, 14 days post transduction and viability of >95% with only limited CS1-mediated fratricide (since CS1 is also expressed on NK cells) during long-term ex vivo culture. CS1-CAR NK cells killed the CS1-expressing MM.1S cell line significantly better than ex vivo expanded non-transduced (NT) CB-NK cells at all effector:target ratios tested (p<0.001). We next tested the in vivo trafficking and antitumor activity of iC9/CAR.CS1/IL-15+ CB-NK cells in a xenograft mouse model of MM. Mice engrafted with MM1.S cells received saline (control) or 1 x107 ex vivo expanded CB-NK cells that were either unmodified or transduced with iC9/CAR.CS1/IL-15; iC9/CAR.CS1/IL15+ CB-NK cells controlled tumor expansion and prolonged survival of mice significantly better than NT CB-NK cells.This was associated withsignificantly higher frequencies of circulating NK cells in the CAR+ CB-NK cell treated mice (median=19%, range 13.6-31.1%) compared to the NT CB-NK group (median=1.26%, range 0.98-2.49%, p=0.0003). We next used mass cytometry (CyTOF) to interrogate the features that shape NK cell diversity, their maturation and their trafficking in vivo. The expression of CAR.CS1 on NK cells infiltrating tumor sites remained stable over time in vivo (median 86.7%, range 81-89.8% compared to the infused product). This was associated with significantly less tumor infiltration in liver (0.013% vs 33.8%), spleen (0.01% vs 23.4%) and BM (4.3% vs 79.8%) harvested on day 21 post-infusion, compared to mice treated with NT NK cells. Interestingly, the residual MM cells in the BM of iC9/CAR.CS1/IL-15 treated mice lost CS1 expression on their surface, suggesting that target antigen downregulation may also play a part in escape from CAR-mediated immune recognition in MM, as also described with CD19 CAR T cell therapy.Using a panel of 40 monoclonal antibodies, we profiled the global signature of NK cells harvested from organs of animals sacrificed on day 21 after infusion and compared them to CAR+ CB-NK cells prior to infusion. The diversity of iC9/CAR.CS1.IL-15-transduced CB-NK cells, measured using the inverse Simpson index, was similar to that of ex vivo expanded NT CB-NK cells (167 vs 114). Additionally, using t-SNE maps to visualize the high-dimensional data generated by CyTOF, we observed complete overlap of clusters for ex vivo expanded NT CB-NK cells and CS1-CAR-transduced CB-NK cells, indicating, that any potential CS1-related fratricide does not significantly impact on the repertoire of the CAR-transduced CB-NK cells (Figure 1).Interestingly, there was a profound and significant increase in the diversity of the infiltrating CS1-CAR CB-NK cells in the liver (830), spleen (843) and BM (965) on day 21 after infusion, suggesting in vivo maturation and expansion of NK subpopulations in response to the tumor. t-SNE analysis revealed a clear separation between iC9/CAR.CS1.IL-15 transduced CB-NK cells prior to infusion and tissue-infiltrating CAR+ CB-NK cells, indicating significant shifts in NK subpopulations, with an increase in diversity and maturation after adoptive transfer in vivo (Figure 1). The data from this analysis will have important implications for future studies as we seek to generate CAR+ CB-NK cells with enhanced antitumor potential. [Display omitted] DisclosuresNeelapu:Karus: Research Funding; Poseida Therapeutics, Inc: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Cellectis Inc.: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Orlowski:BioTheryX: Consultancy, Membership on an entity's Board of Directors or advisory committees.

An Oncolytic Virus Expressing IL15/IL15Rα Combined with Off-the-Shelf EGFR-CAR NK Cells Targets Glioblastoma.

IL15 is a pleiotropic cytokine with multiple roles that improve immune responses to tumor cells. Oncolytic viruses (OV) specifically lyse tumors and activate immune responses. Systemic administration of IL15 or its complex with the IL15Rα and chimeric antigen receptor (CAR) natural killer (NK) cells are currently being tested in the clinic. Here, we generated a herpes simplex 1-based OV-expressing human IL15/IL15Rα sushi domain fusion protein (named OV-IL15C), as well as off-the-shelf EGFR-CAR NK cells, and studied their monotherapy and combination efficacy in vitro and in multiple glioblastoma (GBM) mouse models. In vitro, soluble IL15/IL15Rα complex was secreted from OV-IL15C-infected GBM cells, which promoted GBM cytotoxicity and improved survival of NK and CD8+ T cells. Frozen, readily available off-the-shelf EGFR-CAR NK cells showed enhanced killing of tumor cells compared with empty vector-transduced NK cells. In vivo, OV-IL15C significantly inhibited tumor growth and prolonged survival of GBM-bearing mice in the presence of CD8+ T cells compared with parental OV. OV-IL15C plus EGFR-CAR NK cells synergistically suppressed tumor growth and significantly improved survival compared with either monotherapy, correlating with increased intracranial infiltration and activation of NK and CD8+ T cells and elevated persistence of CAR NK cells in an immunocompetent model. Collectively, OV-IL15C and off-the-shelf EGFR-CAR NK cells represent promising therapeutic strategies for GBM treatment to improve the clinical management of this devastating disease. SIGNIFICANCE: The combination of an oncolytic virus expressing the IL15/IL15Rα complex and frozen, ready-to-use EGFR-CAR NK cells elicits strong antitumor responses in glioblastoma.

Chimeric Antigen Receptor T Cells for Glioblastoma: Current Concepts, Challenges, and Future Perspectives.

Glioblastoma is the most common malignant primary brain tumor and is associated with a poor prognosis even after multimodal therapy. Chimeric antigen receptor (CAR) T cells have emerged as a promising therapeutic avenue in glioblastoma. CARs incorporate antigen-recognition moieties that endow autologous T cells with specificity against antigens expressed on glioblastoma (e.g., interleukin [IL]-13Rα2, epidermal growth factor receptor variant III [EGFRvIII], and human epidermal growth factor receptor 2 [HER2]). Compelling antitumor effects of such therapy have been shown in murine glioblastoma models. In humans, 5 phase I/II studies on IL-13Rα2-, EGFRvIII-, and HER2-directed CAR T cells for the treatment of glioblastoma have been published suggesting an acceptable safety profile. However, antitumor effects fell short of expectations in these initial clinical studies. Tumor heterogeneity, antigen loss, and the immunosuppressive tumor microenvironment are among the most important factors to limit the efficacy of CAR T-cell therapy in glioblastoma. Novel target antigens, modification of CAR T-cell design, the combination of CAR T-cell therapy with other therapeutic approaches, but also the use of CAR natural killer cells or CAR macrophages may optimize antitumor effects. Numerous clinical trials studying such approaches are ongoing, as well as several preclinical studies. With an increasing understanding of immune-escape mechanisms of glioblastoma and novel manufacturing techniques for CARs, CAR T cells may provide clinically relevant activity in glioblastoma. This review focuses on the use of CAR T cells in glioblastoma, but also introduces the basic structure, mechanisms of action, and relevant side effects of CAR T cells.

GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells.

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A− and HLA-B− K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.

Immunotherapeutic Strategies for Neuroblastoma: Present, Past and Future.

Neuroblastoma is the most common extracranial pediatric solid tumor with a heterogeneous clinical course, ranging from spontaneous regression to metastatic disease and death, irrespective of intensive chemotherapeutic regimen. On the basis of several parameters, children affected by neuroblastoma are stratified into low, intermediate and high risk. At present, more than 50% of high-risk patients with metastatic spread display an overall poor long-term outcome also complicated by devastating long-term morbidities. Thus, novel and more effective therapies are desperately needed to improve lifespan of high-risk patients. In this regard, adoptive cell therapy holds great promise and several clinical trials are ongoing, demonstrating safety and tolerability, with no toxicities. Starting from the immunological and clinical features of neuroblastoma, we here discuss the immunotherapeutic approaches currently adopted for high-risk patients and different innovative therapeutic strategies currently under investigation. The latter are based on the infusion of natural killer (NK) cells, as support of consolidation therapy in addition to standard treatments, or chimeric antigen receptor (CAR) T cells directed against neuroblastoma associated antigens (e.g., disialoganglioside GD2). Finally, future perspectives of adoptive cell therapies represented by γδ T lymphocyes and CAR NK cells are envisaged.

Optimization and Validation of CAR Transduction into Human Primary NK Cells Using CRISPR and AAV

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity, but genetic modification of primary human NK cells has been problematic compared to the ease in which primary human T cells are transduced. Despite some progress made in using lentiviral and retroviral vectors, the efficiency of viral transduction in NK cells remains relatively low. Additionally, using these viral vectors results in random integration events which can potentially affect the safety or efficacy of transduced immune cells. Recently, we described an efficient method for gene targeting in primary human NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined that approach with Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus, and optimized transduction efficiency using various AAV pseudotypes, single-stranded (ss) or self-complementary (sc) with a wide variety of homology arms, and homology repair (HR) andor non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For initial optimization we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms for HR resulted in optimal transduction efficiency. To validate the process for generating large numbers of CAR-NK cells needed for clinical application, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered by the transgene, and the NK cells maintained high CAR expression (mean 68% CAR+) during expansion. The expanded CD33-CAR-NK cells showed enhanced activation and antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML). Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome and low off-target insertion. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

Initial Clinical Activity of FT596, a First-in-Class, Multi-Antigen Targeted, Off-the-Shelf, iPSC-Derived CD19 CAR NK Cell Therapy in Relapsed/Refractory B-Cell Lymphoma

Induced pluripotent stem cell (iPSC)-derived immune effector cells offer distinct advantages over existing patient- and donor-derived therapeutic approaches, including the use of a clonal master engineered iPSC line as a renewable source for the mass production of immune cells, which are available off-the-shelf for broad patient access. FT596 is an investigational, off-the-shelf, multi-antigen targeting, chimeric antigen receptor (CAR) natural killer (NK) cell therapy derived from a human clonal master iPSC line engineered with three anti-tumor modalities: (1) a proprietary CD19-targeting CAR; (2) a novel high-affinity, non-cleavable CD16 Fc receptor that enables tumor targeting and enhanced antibody-dependent cell cytotoxicity in combination with a therapeutic monoclonal antibody (mAb); and (3) interleukin (IL)-15/IL-15 receptor fusion promoting cytokine-autonomous persistence. Preclinical in vivo models demonstrate potent multi-antigen targeting activity of FT596 against both CD19+ and CD19- tumor cell lines when combined with the anti-CD20 agent rituximab (Goodridge et al. 2019). FT596 is currently being investigated as a monotherapy and in combination with the anti-CD20 mAbs rituximab and obinutuzumab in a multicenter, Phase I clinical trial for the treatment of relapsed/refractory B-cell lymphoma and chronic lymphocytic leukemia. The trial will test up to four FT596 dose levels ranging from 30 to 900 million cells. We describe the demonstration of early clinical benefit of FT596 in a patient who received a single administration of FT596 as a monotherapy at the first dose level in the Phase I trial. The patient is a 76-year-old female with diffuse large B-cell lymphoma, germinal center B-cell subtype, who was initially diagnosed in January 2014. The patient received eight prior treatment regimens, including autologous stem cell transplant, rituximab in combination with engineered autologous T cells expressing antibody-coupled T-cell receptor, and, most recently, was refractory to an experimental combination therapy comprised of lymphodepleting chemotherapy followed by ex vivo expanded allogeneic NK cells, IL-2, and rituximab. The patient received fludarabine and cyclophosphamide lympho-conditioning followed by a single administration of 30 million cells of FT596 as monotherapy. During the 28-day follow-up period for safety, no dose-limiting toxicities were observed. No cytokine release syndrome, neurologic toxicity, or graft-versus-host disease of any grade was observed. Grade ≥3 treatment-emergent adverse events (AEs) included decreased white blood cell count, decreased neutrophil count, anemia, urinary tract infection with neutropenic fever, and hypertension. Notably, none of these AEs were considered related to FT596 by the treating investigator physician, except for decreased neutrophil count, which was considered possibly related to FT596 and resolved. Evidence of hematologic recovery was observed by the end of the 28-day follow-up safety period. On Study Day 29, the patient's tumor response assessment showed partial response by Lugano 2014 criteria, with a greater than 70% decrease in 18F-fluorodeoxyglucose uptake and greater than 50% reduction in tumor size. The patient remains on study and, per protocol and in collaboration with the U.S. Food and Drug Administration, is being considered for retreatment with a second cycle of FT596 monotherapy. This is the first-ever demonstration of clinical activity following treatment with an off-the-shelf, iPSC-derived CAR immune cell therapy and provides direct evidence that low doses of FT596 can induce short-term responses, opening the opportunity for multi-dosing strategies to deepen response and duration. Additional clinical, pharmacokinetic, and pharmacodynamic data from this patient will be provided at the time of the meeting. The Phase I trial is ongoing and is registered on clinicaltrials.gov: NCT04245722. Disclosures Bachanova: FATE: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Karyopharma: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding. Janakiram:Takeda, Fate, Nektar: Research Funding. Payne:Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wong:Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cooley:Fate Therapeutics: Current Employment, Current equity holder in publicly-traded company. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Chu:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company; Roche Holding AG: Current equity holder in publicly-traded company. Miller:GT Biopharma: Consultancy, Patents &amp; Royalties, Research Funding; Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents &amp; Royalties, Research Funding. OffLabel Disclosure: Cyclophosphamide and fludarabine will be used as lympho-conditioning therapy prior to FT596 administration.