Antigen Receptor Natural Killer Cells Research Articles

Chimeric antigen receptor-natural killer cell therapy: current advancements and strategies to overcome challenges.

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is a novel immunotherapy targeting cancer cells via the generation of chimeric antigen receptors on NK cells which recognize specific cancer antigens. CAR-NK cell therapy is gaining attention nowadays owing to the ability of CAR-NK cells to release potent cytotoxicity against cancer cells without side effects such as cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GvHD). CAR-NK cells do not require antigen priming, thus enabling them to be used as "off-the-shelf" therapy. Nonetheless, CAR-NK cell therapy still possesses several challenges in eliminating cancer cells which reside in hypoxic and immunosuppressive tumor microenvironment. Therefore, this review is envisioned to explore the current advancements and limitations of CAR-NK cell therapy as well as discuss strategies to overcome the challenges faced by CAR-NK cell therapy. This review also aims to dissect the current status of clinical trials on CAR-NK cells and future recommendations for improving the effectiveness and safety of CAR-NK cell therapy.

Abstract 1327: Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a highly prevalent blood and bone marrow cancer characterized by the uncontrolled growth of abnormal myeloblasts. Current treatments for AML often result in systemic toxicities for patients, many of whom will still experience relapse. There is thus an unmet need to develop safer and more effective therapies targeting AML. Chimeric antigen receptor (CAR) T cells, which are engineered to express a cancer-targeting extracellular antibody single-chain variable fragment (scFv) linked to an intracellular T cell-activating domain, have shown promise in treating B cell leukemias, but their success has been limited in myeloid leukemias. This is due in part to the lack of AML-specific target antigens. Moreover, impaired T cell function, a hallmark of AML, is also problematic when relying on autologous T cell activation upon reinfusion. We overcame the limitations of CAR T therapy in AML by developing CAR-expressing natural killer (NK) cells targeting CD123. While present on both healthy and AML cells, CD123 is expressed at >10-fold higher levels on malignant cells. Furthermore, use of NK allows use of fully functional allogeneic cells from healthy donors, since NK cells do not induce graft-versus-host-disease. To optimize the tumor cell selectivity of CAR NK cells, we employed the yeast display directed evolution platform to isolate anti-CD123 scFvs with varying affinities and are identifying and characterizing clones that maximize the interaction with CD123high AML cells over healthy CD123low cells, as determined by binding, cytotoxicity, cytokine secretion, and intracellular signaling studies. Collectively, our efforts introduce a new paradigm for engineered cell therapeutics with significant promise for AML treatment. Citation Format: Monika Kizerwetter, Huilin Yang, Ruyan Rahnama, Challice Bonifant, Jamie Spangler. Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1327.

An Injectable Puerarin Depot Can Potentiate Chimeric Antigen Receptor Natural Killer Cell Immunotherapy Against Targeted Solid Tumors by Reversing Tumor Immunosuppression.

Chimeric antigen receptor natural killer (CAR-NK) cell therapy represents a potent approach to suppressing tumor growth because it has simultaneously inherited the specificity of CAR and the intrinsic generality of NK cells in recognizing cancer cells. However, its therapeutic potency against solid tumors is still restricted by insufficient tumor infiltration, immunosuppressive tumor microenvironments, and many other biological barriers. Motivated by the high potency of puerarin, a traditional Chinese medicine extract, in dilating tumor blood vessels, an injectable puerarin depot based on a hydrogen peroxide-responsive hydrogel comprising poly(ethylene glycol) dimethacrylate and ferrous chloride is concisely developed. Upon intratumoral fixation, the as-prepared puerarin depot (abbreviated as puerarin@PEGel) can activate nitrogen oxide production inside endothelial cells and thus dilate tumor blood vessels to relieve tumor hypoxia and reverse tumor immunosuppression. Such treatment can thus promote tumor infiltration, survival, and effector functions of customized epidermal growth factor receptor (HER1)-targeted HER1-CAR-NK cells after intravenous administration. Consequently, such puerarin@PEGel-assisted HER1-CAR-NK cell treatment exhibits superior tumor suppression efficacy toward both HER1-overexpressing MDA-MB-468 and NCI-H23 human tumor xenografts in mice without inducing obvious side effects. This study highlights a potent strategy to activate CAR-NK cells for augmented treatment of targeted solid tumors through reprogramming tumor immunosuppression.

Targeted Integration of a Chimeric Antigen Receptor in Key Selected Loci in Primary Natural Killer Cells

Despite promising preclinical studies and early clinical data (Liu et al., NEJM, 2020), chimeric antigen receptor natural killer cells (CAR NK) are not yet on par with CAR T cells for the treatment of malignant diseases. Much effort has gone into developing NK-optimized CAR architectures (Li et al., Cell Stem Cell, 2018) and to increasing the persistence of CAR NK cells. We hypothesized that CAR NK cells could also benefit from the targeted integration of a CAR transgene, as compared with semi-random integration obtained using gammaretroviral vectors. Precise targeting has proven highly advantageous for TRAC CAR T cells owing to the optimal regulation and homogeneous expression conferred by the endogenous TCR alpha promoter (Eyquem et al., Nature, 2017). We first optimized an editing protocol for primary NK cells that uses electroporation of a CRISPR/Cas9 ribonucleoprotein followed by transduction with AAV6 bearing a homology-directed repair template. With this protocol we were able to knock out (KO) genes (including CD38, B2M, and different NK receptors), knock in transgenes (GFP-fusion protein, truncated EGFR, CAR), or both, at high efficiency. There was only moderate cell toxicity, and proliferation and cytotoxicity were conserved. Because of the high constitutive expression of B2M, we initially chose to focus on this locus and targeted a 1928z CAR transgene for regulation under the endogenous B2M promoter; our template design also rescues the B2M gene upon knock-in, enabling intermediate B2M expression (Figure 1). This strategy conferred high expression of the CAR and significantly more homogeneity as compared with gammaretroviral delivery (Figure 1). Furthermore, the rescued B2M expression protected B2M CAR NK cells from fratricide killing, whereas the B2M KO NK cells and the CAR NK cells with disrupted B2M expression were susceptible. B2M CAR NK cells also displayed higher in vitro cytotoxicity as compared with WT NK cells against two different CD19-expressing tumor cell lines (NALM6, SupB15). We tested B2M CAR NK cells in an NSG mouse xenograft model with an intraperitoneally injected human ovarian cancer cell line engineered to express CD19 (CD19 + SKOV3). Unexpectedly, compared to retroviral CAR-NK cells, B2M CAR NK cells conferred less tumor control (assessed by bioluminescence imaging) and less of a survival benefit. Therefore, we screened a panel of other loci for CAR targeting: five for NK activating receptors ( CD16A, CD56, DNAM1, NKp30, NKp46) and four for NK inhibitory receptors ( CD161, LILRB1, NKG2A, TIGIT). For activating receptors, the transgene was designed to use the endogenous promoter and to maintain expression of the targeted locus. For inhibitory receptors, the transgene was placed under the control of an EF1a exogenous promoter and designed to disrupt expression of the targeted locus. We compared these constructs and the initial B2M CAR NK design for CAR expression (by flow cytometry) and in vitro functionality (cytotoxicity assays) and observed wide variation in knock-in efficiency and CAR expression (Figure 2). For the top constructs based on knock-in efficiency, we conducted in vitro cytotoxicity assays against NALM6 and SupB15 cell lines. We found that when normalized for CAR percentage using the respective KO conditions, all tested knock-in loci performed better than B2M CAR NK cells, despite the lower CAR expression. Our current focus is on validating these top loci for the targeted integration of a 1928z CAR using in vivo models for comparison with B2M CAR NK and retroviral CAR NK. In summary, we have identified advantageous in vitro characteristics for the targeted integration of a CAR transgene in primary NK cells and screened 10 loci for CAR expression and in vitro cytotoxicity against tumor target cells. Confirmatory in vivo studies are ongoing and will be presented.

Anti-TIM3 chimeric antigen receptor-natural killer cells from engineered induced pluripotent stem cells effectively target acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)—the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR.MethodsA clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells.ResultsWe successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs.ConclusionsiPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications.

Application of chimeric antigen receptor-natural killer cells for the treatment of type 1 diabetes

For the past 100 years, insulin supplementation has been the mainstay of treatment for type 1 diabetes (T1D), which is characterized by progressive autoimmune-mediated loss of insulin-producing β cells in the islets of Langerhans over the last decades, technological advances in glucose monitoring and therapeutics have greatly improved the care and management of these patients. However, morbidity, mortality, and quality of life remain challenges for patients with T1D. Islet transplantation has been successfully performed, but there are several limiting factors, such as the lack of cadaveric donors and the need for lifelong immunosuppressive therapy. Therefore, there is a great medical need for alternative therapeutic approaches. In the current review, the current knowledge on novel approaches for the treatment of T1D with a focus on the potential of using chimeric antigen receptor (CAR)-T cells and natural killer (NK) cells is summarized.

Chimeric antigen receptor-natural killer cells: a promising sword against insidious tumor cells.

Natural killer (NK) cells are a critical component of innate immunity, particularly in initial cancer recognition and inhibition of additional tumor growth or metastasis propagation. NK cells recognize transformed cells without prior sensitization via stimulatory receptors and rapidly eradicate them. However, the protective tumor microenvironment facilitates tumor escaping via induction of an exhaustion state in immune cells, including NK cells. Hence, genetic manipulation of NK cells for specific identification of tumor-associated antigens or a more robust response against tumor cells is a promising strategy for NK cells' tumoricidal augmentation. Regarding the remarkable achievement of engineered CAR-T cells in treating hematologic malignancies, there is evolving interest in CAR-NK cell recruitment in cancer immunotherapy. Innate functionality of NK cells, higher safety, superior in vivo maintenance, and the off-the-shelf potential move CAR-NK-based therapy superior to CAR-T cells treatment. In this review, we have comprehensively discussed the recent genetic manipulations of CAR-NK cell manufacturing regarding different domains of CAR constructs and their following delivery systems into diverse sources of NK cells. Then highlight the preclinical and clinical investigations of CAR-NK cells and examine the current challenges and prospects as an optimistic remedy in cancer immunotherapy.

Nonviral technologies can pave the way for CAR-NK cell therapy.

Natural killer cells are a promising platform for cancer immunotherapy. Natural killer cells have high intrinsic killing capability, and the insertion of a chimeric antigen receptor can further enhance their antitumor potential. In first-in-human trials, chimeric antigen receptor-natural killer cells demonstrated strong clinical activity without therapy-induced side effects. The applicability of natural killer cells as an "off-the-shelf" product makes them highly attractive for gene-engineered cell therapies. Traditionally, viral transduction has been used for gene editing; however, the use of viral vectors remains a safety concern and is associated with high costs and regulatory requirements. Here, we review the current landscape of nonviral approaches for chimeric antigen receptor-natural killer cell generation. This includes transfection of vector particles and electroporation of mRNA and DNA vectors, resulting in transient modification and chimeric antigen receptor expression. In addition, using nonviral transposon technologies, natural killer cells can be stably modified ensuring long-lasting chimeric antigen receptor expression. Finally, we discuss CRISPR/Cas9 tools to edit key genes for natural killer cell functionality.

Generation and Functional Characterization of Anti-CD19 Chimeric Antigen Receptor-Natural Killer Cells from Human Induced Pluripotent Stem Cells.

Natural killer (NK) cells are a part of innate immunity that can be activated rapidly in response to malignant transformed cells without prior sensitization. Engineering NK cells to express chimeric antigen receptors (CARs) allows them to be directed against corresponding target tumor antigens. CAR-NK cells are regarded as a promising candidate for cellular immunotherapy alternatives to conventional CAR-T cells, due to the relatively low risk of graft-versus-host disease and safer clinical profile. Human induced pluripotent stem cells (iPSCs) are a promising renewable cell source of clinical NK cells. In the present study, we successfully introduced a third-generation CAR targeting CD19, which was validated to have effective signaling domains suitable for NK cells, into umbilical cord blood NK-derived iPSCs, followed by a single-cell clone selection and thorough iPSC characterization. The established single-cell clone of CAR19-NK/iPSCs, which is highly desirable for clinical application, can be differentiated using serum- and feeder-free protocols into functional CAR19-iNK-like cells with improved anti-tumor activity against CD19-positive hematologic cancer cells when compared with wild-type (WT)-iNK-like cells. With the feasibility of being an alternative source for off-the-shelf CAR-NK cells, a library of single-cell clones of CAR-engineered NK/iPSCs targeting different tumor antigens may be created for future clinical application.

Successful targeting of PD-1/PD-L1 with chimeric antigen receptor-natural killer cells and nivolumab in a humanized mouse cancer model.

In recent decades, chimeric antigen receptor (CAR)-engineered immune effector cells have demonstrated promising antileukemic activity. Nevertheless, their efficacy remains unsatisfactory on solid cancers, plausibly due to the influence of tumor microenvironments (TME). In a novel mouse cancer model with a humanized immune system, tumor-infiltrating immunosuppressive leukocytes and exhausted programmed death protein-1 (PD-1)high T cells were found, which better mimic patient TME, allowing the screening and assessment of immune therapeutics. Particularly, membrane-bound programmed death ligand 1 (PD-L1) level was elevated on a tumor cell surface, which serves as an attractive target for natural killer (NK) cell-mediated therapy. Hematopoietic stem cell-derived CAR-NK (CAR pNK) cells targeting the PD-L1 showed enhanced in vitro and in vivo anti-solid tumor function. The CAR pNK cells and nivolumab resulted in a synergistic anti-solid tumor response. Together, our study highlights a robust platform to develop and evaluate the antitumor efficacy and safety of previously unexplored therapeutic regimens.

Umbilical cord blood: an undervalued and underutilized resource in allogeneic hematopoietic stem cell transplant and novel cell therapy applications.

The purpose of this review is to primarily discuss the unwarranted decline in the use of umbilical cord blood (UCB) as a source of donor hematopoietic stem cells (HSC) for hematopoietic cell transplantation (HCT) and the resulting important implications in addressing healthcare inequities, and secondly to highlight the incredible potential of UCB and related birthing tissues for the development of a broad range of therapies to treat human disease including but not limited to oncology, neurologic, cardiac, orthopedic and immunologic conditions. When current best practices are followed, unrelated donor umbilical cord blood transplant (CBT) can provide superior quality of life-related survival compared to other allogeneic HSC donor sources (sibling, matched or mismatched unrelated, and haploidentical) through decreased risks of relapse and chronic graft vs. host disease. Current best practices include improved UCB donor selection criteria with consideration of higher resolution human leukocyte antigen (HLA) typing and CD34+ cell dose, availability of newer myeloablative but reduced toxicity conditioning regimens, and rigorous supportive care in the early posttransplant period with monitoring for known complications, especially related to viral and other infections that may require intervention. Emerging best practice may include the use of ex vivo expanded single-unit CBT rather than double-unit CBT (dCBT) or 'haplo-cord' transplant, and the incorporation of posttransplant cyclophosphamide as with haploidentical transplant and/or incorporation of novel posttransplant therapies to reduce the risk of relapse, such as NK cell adoptive transfer. Novel, non-HCT uses of UCB and birthing tissue include the production of UCB-derived immune effector cell therapies such as unmodified NK cells, chimeric antigen receptor-natural killer cells and immune T-cell populations, the isolation of mesenchymal stem cells for immune modulatory treatments and derivation of induced pluripotent stem cells haplobanks for regenerative medicine development and population studies to facilitate exploration of drug development through functional genomics. The potential of allogeneic UCB for HCT and novel cell-based therapies is undervalued and underutilized. The inventory of high-quality UCB units available from public cord blood banks (CBB) should be expanding rather than contracting in order to address ongoing healthcare inequities and to maintain a valuable source of cellular starting material for cell and gene therapies and regenerative medicine approaches. The expertise in Good Manufacturing Practice-grade manufacturing provided by CBB should be supported to effectively partner with groups developing UCB for novel cell-based therapies.

Abstract 6225: Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models

Abstract Chimeric antigen receptor (CAR) - engineered natural killer (NK) cell therapy has emerged as a promising platform for adoptive immunotherapy for cancer. However, CAR-NK or CAR-T therapy so far has achieved limited efficacy in solid tumors compared with hematologic malignancies. One of the challenges is the lack of prevalent tumor-specific surface antigen target in solid tumors. Hence, we are developing a novel oncolytic vaccinia virus (VV) to deliver a dual functional glycol-immune checkpoint inhibitor and universal tumor cell marker combined with CAR-NK cell therapy to increase anti-tumor efficacy. Using the vaccinia virus deleted in both viral TK and VGF genes (vvDD) to drive the expression of a membrane-bound sialidase derived from actinomyces viscosus (avSial) under the viral late promoter, we observed efficient desialylation of both VV-infected and non-infected tumor cells, which would alleviate the suppression of sialic acid on NK and other immune cells within TME. Further, the surface bound avSial on VV-infected tumor cells can also serve as a universal target for avSial-CAR NK cells, with less concern for cross-reactivity to normal human tissues and antigen loss. We developed anti-avSial antibodies through mice immunization study. The selected single chain variable fragments (scFV) were constructed in CAR format containing CD28 CD3 ζ for co-stimulation and human IL-15 gene for better NK persistence and function. The binding affinity of anti-avSial scFv to avSialidase were further screened in CAR constructs-transfected 293T cells. The selected anti-avSial scFv CAR were packaged into gamma retroviral vectors to transduce activated and expanded NK cells derived from healthy donor peripheral blood. For proof-of-concept, target tumor cell lines A375, A549 and HT-29 expressing transmembrane sialidase and GFP were also generated. Compared with CD19 CAR NK and none transduced (NT) NK cells, avSial CAR NK had markedly increased cytotoxicity against avSial-expressing tumor cells in co-culture assays at low E:T ratios <= 1:2. Upon tumor cell rechallenge, avSial CAR NK also controlled the tumor growth significantly better than CD19 CAR NK or NT NK cells. Furthermore, avSial CAR NK completely eliminated A375 tumor spheroids expressing transmembrane sialidase whereas CD19 CAR NK or NT NK cells only transiently controlled the tumor spheroids growth. Taken together, we have developed an effective avSial targeting CAR IL15 NK that will be used to demonstrate enhanced efficacy and versatility in combination with avSial-armed VV for treatment of solid tumors. Citation Format: Xiaomei Wang, Guowei Wei, Keshav B. Karki, Winnie Chan, Mariya Viskovska, Andrew Williams, Nancy Chang, Haiyan Jiang. Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6225.

Anti-CD123 Chimeric Antigen Receptor Natural Killer cell therapy to treat Acute Myeloid Leukemia.

Abstract Acute myeloid leukemia (AML) is a highly prevalent blood and bone marrow cancer characterized by the uncontrolled growth of abnormal myeloblasts. Current treatments for AML often result in systemic toxicities for patients, many of whom will still experience relapse. There is thus an unmet need to develop safer and more effective therapies targeting AML. Chimeric antigen receptor (CAR) T cells, which are engineered to express a cancer-targeting extracellular antibody single-chain variable fragment (scFv) linked to an intracellular T cell-activating domain, have shown promise in treating B cell leukemias, but their success has been limited in myeloid leukemias. This is due in part to the lack of AML-specific target antigens. Moreover, impaired T cell function, a hallmark of AML, is also problematic when relying on autologous T cell activation upon reinfusion. We overcame the limitations of CAR T therapy in AML by developing CAR-expressing natural killer (NK) cells targeting CD123. While present on both healthy and AML cells, CD123 is expressed at >10-fold higher levels on malignant cells. Furthermore, use of NK cells allows use of fully functional allogeneic cells from healthy donors, since NK cells do not induce graft-versus-host-disease. To optimize the tumor cell selectivity of CAR NK cells, we employed the yeast display directed evolution platform to isolate anti-CD123 scFvs with varying affinities and are identifying clones that maximize the interaction with CD123high AML cells over healthy CD123low cells, as determined by binding, cytotoxicity, and cytokine secretion studies. Collectively, our efforts introduce a new paradigm for engineered cell therapeutics with significant promise for AML treatment.

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).

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.

130 Engineered natural killer cells reactively block TIGIT and CD73 in the GBM microenvironment

BackgroundNatural killer (NK) cells have emerged as promising effectors to target GBM and other solid tumors through genetic modifications and ex vivo manipulation. However, immunosuppressive conditions within the tumor microenvironment (TME) and interactions between NK cell activating and inhibitory receptors further complicate NK cell-based treatments. In particular, the T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed on NK cells and interacts with CD155 to induce immunosuppression of NK cell cytolytic functions.1 2 Although CD155 also binds with activating receptors DNAM-1 and CD96 on NK cells, spurring NK cell activity, TIGIT has predominantly been reported as having an inhibitory effect on NK cells.3–5 Further, tumor cells release of high levels of ATP extracellularly. While intracellular ATP is necessary for cell metabolism, extracellular ATP is converted into adenosine (ADO) by ectonucleotidases CD39 and CD73, both overexpressed on GBM and other solid tumors.6 Extracellular ADO induces immunometabolic suppression of NK cells through binding with A2A adenosine receptors (A2ARs) on NK cells, suppressing cytokine secretion, proliferation, and other functional activities.7–9 We found that TIGIT and CD73 are effective combination targets in GBM for both primary and iPSC-derived NK cells.MethodsIn order to effectively target immunometabolic reprogramming induced by CD73-produced adenosine and the immunosuppressive TIGIT-CD155 axis, we have engineered NK cells to concomitantly target CD155 and CD73-induced immunosuppression on GBM using a tumor-responsive genetic construct based on the synNotch signaling system. The construct is capable of blocking the immunosuppressive CD155/TIGIT interaction, and, upon binding, release a CD73-blocking scFv to inhibit the accumulation of extracellular ADO and mitigate immunosuppression of NK cells. Such localized response enhances specificity and reduces off-target effects of NK-based targeting.ResultsPrimary NK cells and iPSC-derived NK cells were successfully engineered to express the synthetic TIGIT-synNotch construct, measured through expression of TIGIT. To evaluate the functionality of engineered NK cells against GBM targets, we tested the cytotoxicity of our engineered NK cells against a primary, patient-derived GBM cell line, GBM43. Overall, cytolytic function of engineered NK cells against GBM was significantly higher than that of non-engineered NK cells, with or without CD73 (10 ug/mL) and TIGIT (50 ug/mL) antibodies, for E:T ratios of 5:1 and 10:1, demonstrating the functional efficacy of our genetic construct.ConclusionsOverall, we have shown that co-targeting CD155 and CD73 in a localized, responsive manner can dampen immunosuppression and significantly enhance the killing potential of engineered NK cells against aggressive patient-derived GBM tumors.ReferencesZhang B, et al. Immunoreceptor TIGIT inhibits the cytotoxicity of human cytokine-induced killer cells by interacting with CD155. Cancer Immunol Immunother 2016;65:305–314.Lupo KB & Matosevic S. CD155 immunoregulation as a target for natural killer cell immunotherapy in glioblastoma. J Hematol Oncol 2020;13:76.Hung AL, et al. TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM. OncoImmunology 2018; e1466769. doi:10.1080/2162402X.2018.1466769.Mahnke K & Enk, AH. TIGIT-CD155 Interactions in Melanoma: A Novel Co-Inhibitory Pathway with Potential for Clinical Intervention. Journal of Investigative Dermatology 2016; 136, 9–11.Stanietsky N, et al. Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR: Innate immunity. Eur J Immunol 2013; 43:2138–2150.Chambers AM, et al. Adenosinergic Signaling Alters Natural Killer Cell Functional Responses. Front Immunol 2018;9:2533.Chambers AM, Lupo KB & Matosevic S. Tumor Microenvironment-Induced Immunometabolic Reprogramming of Natural Killer Cells. Front Immunol 2018;9:2517.Chambers AM. et al. Adenosinergic Signaling Alters Natural Killer Cell Functional Responses. Front Immunol 2018;9:2533.Wang J, Lupo KB, Chambers AM & Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J Immunotherapy Cancer 2018;6:136.Ethics ApprovalPrimary human NK cells were obtained from healthy adult donors approved under Purdue University’s Institutional Review Board (IRB) (IRB-approved protocol #1804020540). Donors gave written informed consent prior to taking part in the study.

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.

Recent advances in chimeric antigen receptor natural killer cell therapy for overcoming intractable hematological malignancies.

Natural killer (NK) cells have a potent cytotoxic activity against leukemia and lymphoma without recognition of human leukocyte antigen (HLA) molecules. Chimeric antigen receptor-engineered NK cells (CAR-NK cells) can be produced from the NK92 cell line, peripheral blood, cord blood, and induced pluripotent stem cells for immunotherapy of malignant tumor cells. Recently, the safety and efficacy of HLA-mismatched allogeneic cord blood-derived CD19 CAR-NK cell therapy for CD19-positive hematological malignancies have been reported. However, the durability of clinical effects has not been clarified. The characteristics of CAR-NK cells with a strong antileukemia/lymphoma effect and better proliferative capacity without severe adverse effects may be promising for overcoming intractable hematological malignancies as an off-the-shelf allogeneic cellular therapy.

Engineering chimeric antigen receptor-natural killer cells for cancer immunotherapy.

Adoptive cell transfer has attracted considerable attention as a treatment for cancer. The success of chimeric antigen receptor (CAR)-engineered T (CAR-T) cells for the treatment of haematologic tumors has demonstrated the potential of CAR. In this review, we describe the current CAR-engineered natural killer (CAR-NK) cell construction strategies, including the design principles and structural characteristics of the extracellular, transmembrane and intracellular regions of the CAR structure. In addition, we review different cellular carriers used to develop CAR-NK cells, highlighting existing problems and challenges. We further discuss possible ways to optimize CAR from the perspective of the tumor microenvironment to harness the strength of CAR-NK cells and provided rationales to combine CAR-NK cells with other treatment regimens to enhance antitumor effects.