Genetic Manipulation Approaches to Enhance the Clinical Application of NK Cell-Based Immunotherapy

Natural killer (NK) cells are a subset of cytotoxic lymphocytes within the innate immune system. While they are naturally cytotoxic, genetic modifications can enhance their tumor-targeting capability, cytotoxicity, persistence, tumor infiltration, and prevent exhaustion. These improvements hold the potential to make NK-cell-based immunotherapies more effective in clinical applications. Currently, several viral and non-viral technologies are used to genetically modify NK cells. For nucleic acid delivery, non-viral methods such as electroporation, lipid nanoparticles, lipofection, and DNA transposons have gained popularity in recent years. On the other hand, viral methods including lentivirus, gamma retrovirus, and adeno-associated virus, remain widely used for gene delivery. Furthermore, gene editing techniques such as clustered regularly interspaced short-palindromic repeats-based, zinc finger nucleases, and transcription activator-like effector nucleases are the pivotal methodologies in this field. This review aims to provide a comprehensive overview of chimeric antigen receptor (CAR) arming strategies and discuss key gene editing techniques. These approaches collectively aim to enhance NK cell/NK cell CAR-based immunotherapies for clinical translation.publishersversionpublishe

Utilizing cytokines to function-enable human NK cells for the immunotherapy of cancer

Natural killer (NK) cells are innate lymphoid cells important for host defense against pathogens and mediate antitumor immunity. Cytokine receptors transduce important signals that regulate proliferation, survival, activation status, and trigger effector functions. Here, we review the roles of major cytokines that regulate human NK cell development, survival, and function, including IL-2, IL-12, IL-15, IL-18, and IL-21, and their translation to the clinic as immunotherapy agents. We highlight a recent development in NK cell biology, the identification of innate NK cell memory, and focus on cytokine-induced memory-like (CIML) NK cells that result from a brief, combined activation with IL-12, IL-15, and IL-18. This activation results in long lived NK cells that exhibit enhanced functionality when they encounter a secondary stimulation and provides a new approach to enable NK cells for enhanced responsiveness to infection and cancer. An improved understanding of the cellular and molecular aspects of cytokine-cytokine receptor signals has led to a resurgence of interest in the clinical use of cytokines that sustain and/or activate NK cell antitumor potential. In the future, such strategies will be combined with negative regulatory signal blockade and enhanced recognition to comprehensively enhance NK cells for immunotherapy

Expanding and Improving Natural Killer Cells for Adoptive Cell Therapy

Funding Information: This work was funded by the Fundação da Ciência e Tecnologia (FCT) with reference number SFRH/BD/144965/2019 and by the European Hematology Association (EHA) Research Mobility Grant 2023. Publisher Copyright: © 2024 by the authors.Natural killer (NK) cells have gained attention as a promising adoptive cell therapy platform for their potential to improve cancer treatments. NK cells offer distinct advantages over T-cells, including major histocompatibility complex class I (MHC-I)-independent tumor recognition and low risk of toxicity, even in an allogeneic setting. Despite this tremendous potential, challenges persist, such as limited in vivo persistence, reduced tumor infiltration, and low absolute NK cell numbers. This review outlines several strategies aiming to overcome these challenges. The developed strategies include optimizing NK cell expansion methods and improving NK cell antitumor responses by cytokine stimulation and genetic manipulations. Using K562 cells expressing membrane IL-15 or IL-21 with or without additional activating ligands like 4-1BBL allows “massive” NK cell expansion and makes multiple cell dosing and “off-the-shelf” efforts feasible. Further improvements in NK cell function can be reached by inducing memory-like NK cells, developing chimeric antigen receptor (CAR)-NK cells, or isolating NK-cell-based tumor-infiltrating lymphocytes (TILs). Memory-like NK cells demonstrate higher in vivo persistence and cytotoxicity, with early clinical trials demonstrating safety and promising efficacy. Recent trials using CAR-NK cells have also demonstrated a lack of any major toxicity, including cytokine release syndrome, and, yet, promising clinical activity. Recent data support that the presence of TIL-NK cells is associated with improved overall patient survival in different types of solid tumors such as head and neck, colorectal, breast, and gastric carcinomas, among the most significant. In conclusion, this review presents insights into the diverse strategies available for NK cell expansion, including the roles played by various cytokines, feeder cells, and culture material in influencing the activation phenotype, telomere length, and cytotoxic potential of expanded NK cells. Notably, genetically modified K562 cells have demonstrated significant efficacy in promoting NK cell expansion. Furthermore, culturing NK cells with IL-2 and IL-15 has been shown to improve expansion rates, while the presence of IL-12 and IL-21 has been linked to enhanced cytotoxic function. Overall, this review provides an overview of NK cell expansion methodologies, highlighting the current landscape of clinical trials and the key advancements to enhance NK-cell-based adoptive cell therapy.publishersversionpublishe

dynamics of immune cell reconstitution in allogeneic hematopoietic cell transplant patients receiving post transplant cyclophosphamide ptcy

In the setting of haploidentical hematopoietic cell transplantation (haplo-HCT), post-transplant cyclophosphamide (PTCy) selectively eliminates alloreactive T cells in-vivo, resulting in favorable graft versus host disease (GVHD), non-relapse mortality (NRM) and relapse outcomes. However, few studies have examined the impact of PTCy on immune reconstitution (IR). We quantified IR in 63 patients after haplo-HCT with PTCy, mofetil mycophenolate and tacrolimus (TAC) and compared results to 93 patients with 8/8 HLA matched related or unrelated donors (MD) receiving TAC, methotrexate and sirolimus for GVHD prophylaxis. Both groups received reduced intensity conditioning for hematologic malignancies. The median age of the Haplo-PTCy and MD cohorts was 55 and 57 years. Patient samples were analyzed using multi-color flow cytometry panels to characterize distinct lymphocyte populations. All IR values are expressed as median absolute cell count per μL. One month after HCT, recovery of all T cell subsets (CD3, CD4Tcon, Treg, CD8) was significantly reduced in the PTCy cohort compared to MD (Figure A, B, C). Recovery of CD4Tcon was also reduced at 2 and 3 months after PTCy (p NK cells were lower 1 month after PTCy (52.7 vs 91.1, p=0.08), but were significantly higher at 2, 3 and 6 months (153.4 vs 94.8, p=0.001, 153.7 vs 87.5, p=0.008, 180 vs 102, p=0.01, respectively, Figure D) compared to the MD cohort. Delayed NK cell recovery at 1 month after PTCy was due entirely to reduced numbers of CD56dim NK cells (Figure E). Subsequently recovery of CD56dim NK cells was similar in both cohorts. Recovery of CD56bright NK cells was significantly increased in the PTCy cohort (p Consistent with prior reports, 1 year cumulative incidence of extensive cGvHD was lower in the PTCy cohort compared to the MD cohort, 13% (5-26%, 95% CI) and 40% (30-50%, 95% CI) respectively, p=0.003, without increased NRM (p=0.28) or relapse (p=0.17). In summary, the effect of PTCy on IR was most pronounced 1 month after transplant with significantly delayed recovery of CD3, CD4Tcon, Treg, CD8 and CD56dim NK cells. Slow recovery of CD4Tcon persisted for 3 months and delayed recovery of Treg persisted for 1 year. Beginning 2 months after HCT, recovery of both CD56dim and CD56bright NK cells was more rapid in the PTCy cohort. Further studies will examine the effects of these differences in IR on clinical outcomes such as relapse, infections and GVHD

Mobilized Peripheral Blood Stem Cells Versus Unstimulated Bone Marrow As a Graft Source for T-Cell-Replete Haploidentical Donor Transplantation Using Post-Transplant Cyclophosphamide.

Purpose T-cell-replete HLA-haploidentical donor hematopoietic transplantation using post-transplant cyclophosphamide was originally described using bone marrow (BM). With increasing use of mobilized peripheral blood (PB), we compared transplant outcomes after PB and BM transplants. Patients and Methods A total of 681 patients with hematologic malignancy who underwent transplantation in the United States between 2009 and 2014 received BM (n = 481) or PB (n = 190) grafts. Cox regression models were built to examine differences in transplant outcomes by graft type, adjusting for patient, disease, and transplant characteristics. Results Hematopoietic recovery was similar after transplantation of BM and PB (28-day neutrophil recovery, 88% v 93%, P = .07; 100-day platelet recovery, 88% v 85%, P = .33). Risks of grade 2 to 4 acute (hazard ratio [HR], 0.45; P \u3c .001) and chronic (HR, 0.35; P \u3c .001) graft-versus-host disease were lower with transplantation of BM compared with PB. There were no significant differences in overall survival by graft type (HR, 0.99; P = .98), with rates of 54% and 57% at 2 years after transplantation of BM and PB, respectively. There were no differences in nonrelapse mortality risks (HR, 0.92; P = .74) but relapse risks were higher after transplantation of BM (HR, 1.49; P = .009). Additional exploration confirmed that the higher relapse risks after transplantation of BM were limited to patients with leukemia (HR, 1.73; P = .002) and not lymphoma (HR, 0.87; P = .64). Conclusion PB and BM grafts are suitable for haploidentical transplantation with the post-transplant cyclophosphamide approach but with differing patterns of treatment failure. Although, to our knowledge, this is the most comprehensive comparison, these findings must be validated in a randomized prospective comparison with adequate follow-up

BK virus-specific T-cell immune reconstitution after allogeneic hematopoietic cell transplantation

© 2020 by The American Society of HematologyClinical disease caused by BK virus reactivation is a frequent complication of allogeneic hematopoietic cell transplantation (HCT). Because of the lack of effective antiviral agents, BK virus-specific T cells are emerging as a potential therapy for BK virus disease, but the immune response to BK virus after allogeneic HCT has not been well characterized. Our study describes reconstitution of BK virus-specific T-cell immunity in 77 adult patients after HCT. All patients had urinary symptoms, and urine was tested for BK virus replication; 33 patients were positive for BK virus (cases), and 44 were negative (controls). In BK virus cases, the median time to first positive test was 75 days (range, 2-511). BK virus cases had lower CD4 T-cell counts 3 to 9 months after transplant, but CD8 T-cell counts were similar in cases and controls. BK virus-specific T cells were identified by cytokine flow cytometry in cryopreserved samples collected prospectively. BK virus-specific CD4 T cells producing T helper 1 (Th1) cytokines recovered quickly after HCT. BK virus-specific T cells were detected more frequently in patients with BK virus reactivation at most time points, and CD4 T cells producing Th1 cytokines were more frequent than BK virus-specific cytolytic CD8 T cells. Early detection of interferon-γ+ and cytolytic BK virus-specific CD4 T cells was associated with lower rates of hematuria among cases. Overall, our study describes recovery of BK virus-specific T cells after HCT and the distinct roles for BK virus-specific T cells in the development and resolution of clinical symptoms.This work was supported by a Collaborative Research Grant from the Harvard Medical School–Portugal Program in Translational Research HMSP-ICT/0001/201, National Institutes of Health, National Cancer Institute grants CA183559, CA183560, and CA229092, and the Pasquarello Tissue Bank in Hematologic Malignancies. E.E. is a PhD candidate at Universidade de Lisboa, and this work is submitted in partial fulfillment of the requirement for a PhD and was supported by a grant for medical fellows enrolled in a PhD program (Subsídios aos Internos Doutorandos–SINTD) from Fundação para a Ciência e Tecnologia, number SFRH/SINTD/135312/2017info:eu-repo/semantics/publishedVersio

Allogeneic Hematopoietic Cell Transplantation Provides Effective Salvage Despite Refractory Disease or Failed Prior Autologous Transplant in Angioimmunoblastic T-Cell Lymphoma: A CIBMTR Analysis

Background: There is a paucity of data on the role of allogeneic hematopoietic cell transplantation (allo-HCT) in patients with angioimmunoblastic T-cell lymphoma (AITL). Using the CIBMTR registry, we report here the outcomes of AITL patients undergoing an allo-HCT. Methods: We evaluated 249 adult AITL patients who received their first allo-HCT during 2000–2016. Results: The median patient age was 56 years (range = 21–77). Majority of the patients were Caucasians (86%), with a male predominance (60%). Graft-versus-host disease (GVHD) prophylaxis was predominantly calcineurin inhibitor-based approaches while the most common graft source was peripheral blood (97%). Median follow-up of survivors was 49 months (range = 4–170 months). The cumulative incidence of grade 2–4 and grade 3–4 acute GVHD at day 180 were 36% (95% CI = 30–42) and 12 (95% CI = 8–17), respectively. The cumulative incidence of chronic GVHD at 1 year was 49% (95%CI 43–56). The 1-year non-relapse mortality (NRM) was 19% (95% CI = 14–24), while the 4-year relapse/progression, progression-free survival (PFS), and overall survival (OS) were 21% (95% CI = 16–27), 49% (95% CI = 42–56), and 56% (95% CI = 49–63), respectively. On multivariate analysis, chemoresistant status at the time of allo-HCT was associated with a significantly higher risk for therapy failure (inverse of PFS) (RR = 1.73 95% CI = 1.08–2.77), while KPS \u3c 90% was associated with a significantly higher risk of mortality (inverse of OS) (RR = 3.46 95% CI = 1.75–6.87). Conclusion: Our analysis shows that allo-HCT provides durable disease control even in AITL patients who failed a prior auto-HCT and in those subjects with refractory disease at the time of allografting