Allogenic Memory-Like Natural Killer Cell Therapy

Abstract

Adoptive cell therapy has witnessed significant progress with the success of chimeric antigen receptor (CAR) T cells for treating cancer. However, their autologous nature limits scalability, and increases production time and manufacturing costs. Additionally, CAR-T cell administration can result in severe toxicities, including cytokine release syndrome (CRS) and neurotoxicity. To address these issues, allogeneic, natural killer (NK) cells are being explored as an alternative. NK cells are cytotoxic lymphocytes that play a pivotal role in tumor surveillance and eradication. Unlike T cells, NK cells can identify and eliminate targets without MHC restriction or prior sensitization. Furthermore, NK cells exhibit enhanced responses after exposure to virus infections or cytokine activation (cytokine induced memory-like). Allogeneic NK cell therapies offer a promising alternative to autologous cell therapies, with reduced risk of graft-versus-host disease and rapid availability. This review summarizes the current landscape of allogeneic memory-like NK cell therapies, including clinical applications and challenges.

Introduction

Natural killer (NK) cells are cytotoxic lymphocytes, critical to the innate immune system, that can attack and eliminate virally infected cells or cancerous cells without prior sensitization.¹ NK cells are the founding members to a rapidly expanding family of innate lymphoid cells (ILCs) and represent about 5–20% of all circulating lymphocytes in humans.^2,3 NK cells can respond immediately and independently of antigen presentation, making them valuable for off-the-shelf therapies.⁴ While chimeric antigen receptor (CAR) T cells have revolutionized the field of adoptive cell therapy for treating cancer, especially the hematological malignancies including multiple myeloma and lymphoma, their use is associated with severe adverse events such as cytokine release syndrome (CRS), neurotoxicity, and graft versus host disease (GVHD).⁵ Current CAR-T therapies are autologous (e.g. patient-derived), thus limiting scalability and increasing manufacturing time and cost. A safer and scalable allogeneic (unrelated donor-derived) alternative approach is vital for expanding the patient pool for cell therapies. Recent advances have been made in our understanding of NK cell biology, including their memory-like (ML) properties. Additionally, improved techniques for NK cell isolation, expansion, and engineering have been developed. These discoveries have led to advances in both preclinical and clinical research into NK cell-based therapies. In this context, CAR NK and CAR ML NK cells represent a promising, safer, allogeneic, off-the-shelf alternative to CAR T cells with lower risk of CRS, broader tumor recognition, and improved activity in immunosuppressive conditions.⁴

NK Cell Biology

Development

NK cells are innate immune cells that develop from common lymphoid progenitors in the bone marrow.⁶ NK cell development requires IL-15 and transcription factors like T-bet and eomesodermin.^7,8 Human NK cells are phenotypically identified by CD56 expression and the absence of CD3. They are mainly classified into two major subsets based on the expression levels of CD56: CD56^bright and CD56^dim NK cells.³ CD56^dim NK cells are mature, express the activating receptor CD16 that is critical in mediating antibody induced cellular cytotoxicity,⁹ and comprise most circulating NK cells.³ CD56^dim NK cells mainly mediate cytotoxic responses due to their high content of cytolytic granules containing perforin and granzymes.¹⁰ CD56^bright NK cells are less differentiated and give rise to 56^dim NK cell subsets; they constitute a minority of circulating NK cells while representing the predominant NK subset in tissues.³ CD56^bright NK cells are highly responsive to cytokine stimulation and produce cytokines robustly in response to activation. Recent studies reveal that CD56^bright NK cells can be primed by IL-15 allowing for increased cytotoxicity, challenging the paradigm that immature CD56^bright NK cells do not kill.¹¹

NK Receptors

Human NK cells express a variety of activating and inhibitory receptors that regulate their activity against target cells.¹² They are a complex group of receptors using opposing signaling motifs to either stimulate or inhibit activation. NK cell activating receptors (e.g. NKp46, Nkp30) detect stress-induced ligands, viral antigens and altered self-cells like infected or cancerous cells. NK cell activating receptors associate with adaptor molecules (e.g. DAP10/12), which contain immunoreceptor tyrosine-based activation motifs (ITAMs).¹² Upon binding to ligand, adaptor associated ITAMs start a cascade of intracellular signaling which leads to the release of cytotoxic granules containing perforin and granzymes, and cytokine production (e.g., IFN-γ). It is reported that stimulation of one activation receptor is not sufficient for cytokine secretion and cytotoxic function, rather stimulation of multiple receptors is required for NK cell effector function.^13,14 The inhibitory receptors prevent NK cell cytotoxicity against normal, healthy cells.¹⁵ Self-tolerance occurs via their recognition of the major histocompatibility complex (MHC) class I molecule, which is expressed on almost all normal nucleated cells. The key inhibitory receptors on human NK cells include inhibitory killer-cell immunoglobulin-like receptors (iKIR) and NKG2A, which contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs).¹⁶ Upon binding of the receptor to their cognate ligands, ITIMs recruit phosphatases that inhibit activating signaling cascades. Healthy cells express MHC-I and thereby avoid NK cell attack. In contrast, virus-infected or malignantly-transformed cells may down regulate MHC-I expression to evade recognition by cytotoxic T cells, while simultaneously becoming targets for NK cell killing.^6,17,18

Cytokine Receptors

Understanding the primary role of the cytokine receptors that NK cells express and how they tune NK responses is critical for the development of optimal memory-like NK cell therapies, particularly in off-the-shelf, allogeneic, settings.^19,20 Cytokine receptor signaling not only regulates the survival, activation, proliferation, and effector function of NK cells, but also modulates the epigenetic and metabolic reprogramming involved in memory-like NK cell development.^21–23 Key cytokines that play the main role in regulating NK cell biology include common gamma chain cytokines like interleukin (IL)-2,15,7, 21.²⁴ IL-12 and IL-18 are also potent NK cell activators that are secreted by dendritic cells and macrophages; they promote NK cell production of cytokines including IFN-γ, and prime NK cells for maximal effector function.²⁵ IL-2 and IL-15 signal via STAT5 and are indispensable for NK cell development, homeostasis and in vivo persistence.²⁶ Additionally, type I interferon (IFN) receptor signaling is important for NK cell maturation, activation, expansion and cytotoxicity.^27,28 Cytokine-induced ML NK cells, a distinct subset of NK cells, have emerged in the field of allogeneic cell therapies and have shown promising therapeutic results via clinical trials.^22,29–32 Brief activation of NK cells with IL-12, IL-15 and IL-18 endow them with memory-like properties including enhanced functionality upon restimulation and persistence in vivo, all underwritten by distinct epigenetic changes.^4,17,21,23

Generating ML NK cells. Allogeneic ML NK cell therapy involves isolating NK cells from healthy donors, followed by brief activation with IL-12/15/18. The activated NK cells are washed of cytokines and infused into recipients. While peripheral blood is the most common source of mature, cytotoxic NK cells suitable for rapid therapeutic use, limited cell numbers may necessitate ex vivo expansion for an off the-shelf-product.

Clinical Applications

Studies in both mice and humans have shown that the enhanced functional properties of ML NK cells are maintained following cell division, indicating a heritable and epigenetically programmed phenotype.^23,33 Moreover, ML NK cells have been detected in vivo for prolonged periods, highlighting their potential for sustained therapeutic activity.^21,30,31 ML NK cells have also shown improved anti-tumor responses across several malignancies, including lymphoma, multiple myeloma, melanoma, colorectal cancer, and head and neck cancers.^34–37 These preclinical insights led to the clinical translation of donor-derived ML NK cells in a first-in-human trial for leukemia, where the approach was well tolerated and resulted in complete remissions (CR) in 87% (13/15) treated patients.³⁰ Although ML NK cell therapies have shown promise in clinical settings for hematologic cancers, their effectiveness against solid tumors requires further investigaton. To fully harness the therapeutic potential of ML NK cells, especially against solid tumors, CAR-engineered ML NK cells have become a potent next-generation cell therapy strategy.³⁸ In this approach, ML NK cells are genetically modified to express CARs which are synthetic receptors designed to target NK cells to tumor-specific antigens.³⁹ Several tumor-associated antigens have been studied as CAR targets, including CD33, CD19, BCMA, and EphA2. Through these studies, CAR engineering has consistently been shown to enhance the responsiveness of ML NK cells against their targets.^34,37,40 Combining the specificity of CAR technology and enhanced functionality of ML NK cells, may be a promising treatment for solid tumors. Currently, significant efforts are directed towards investigating ML NK cell therapies in allogeneic settings through ongoing clinical trials (Table 1).

Table 1.

Overview of current active and enrolling clinical trials using cytokine-induced memory-like NK cells.

NCT Number	Study Title	Conditions	Sponsor	Collaborators
NCT05629546	Memory-Like Natural Killer Cells With Nivolumab and Relatlimab in Advanced or Metastatic Melanoma After Progression on Checkpoint Inhibitors	Melanoma	Washington University School of Medicine	Melanoma Research Alliance|Rising Tide Foundation
NCT06138587	Preemptive CIML NK Cell Therapy After Hematopoietic Stem Cell Transplantation	Acute Myeloid Leukemia, Leukemia, Myeloproliferative Neoplasm MyeloproliferativeDisorders	Dana-Farber Cancer Institute
NCT06321484	Intraperitoneal Cytokine-Induced Memory Like (CIML) Natural Killer (NK) Cells in Recurrent Ovarian Cancer	Ovarian Cancer	Dana-Farber Cancer Institute	ImmunityBio, Inc.
NCT06318871	Memory-like Natural Killer (NK) Cell Therapy in Patients With Renal Cell Carcinoma or Urothelial Carcinoma	Renal Cell Carcinoma, Urothelial Carcinoma	Dana-Farber Cancer Institute	Kidney Cancer Association
NCT06152809	CIML NK Cells With Venetoclax for AML	Acute Myeloid Leukemia	Dana-Farber Cancer Institute
NCT07011004	A Study of Natural Killer Cells in Combination With Atezolizumab in People With Acute Myelogenous Leukemia	Acute Myeloid Leukemia	Memorial Sloan Kettering Cancer Center	Genentech, Inc.
NCT05580601	Cytokine-Induced Memory-Like Natural Killer Cells (CIML-NK) for Relapsed & Refractory Acute Myeloid Leukemia	Acute Myeloid Leukemia	Children's Hospital Medical Center, Cincinnati
NCT06158828	Pilot Study of Memory-like Natural Killer (ML NK) Cells After TCRαβ T Cell Depleted Haploidentical Transplant in AML	Pediatric Acute Myeloid Leukemia	Washington University School of Medicine	The Leukemia and Lymphoma Society|Rising Tide Foundation|St. Louis Children's Hospital Foundation|Children's' Discovery Institute

Challenges and Limitations

Despite the breakthrough and the promising results of allogeneic ML NK cells as off-the-shelf therapy in clinical trials, this strategy requires further optimization. Although ML NK cells showed enhanced longevity and persistence over conventional NK cells, long-term persistence may be limited by host immune rejection of donor cells due to HLA mismatching.⁴¹ Limited in vivo persistence will require either repeated administration or further engineering to enhance NK cell lifespan. Moreover, the immunosuppressive tumor microenvironment (TME) may limit the infiltration and functionality of ML NK cells. Combination therapies that dampen inhibitory signaling in the TME should be investigated.

Strategies for Overcoming Barriers

Strategies for improving ML NK cell-based therapies are directed towards enhancing their targeting, decreasing TME suppression, and increasing functional persistence. Genome editing via CRISPR/Cas9 is being researched as a way to reduce alloreactivity by knocking out HLA molecules or inhibitory molecules.^22,42 Engineered ML NK cells to express CARs designed to target specific tumor antigens and increase ML NK cell homing into solid tumors are also being explored.³⁹ Additionally, combining ML NK cells with immune checkpoint inhibitors such as anti-PD-1/PD-L1 and anti-NKG2A may help overcome the immunosuppressive TME.³⁷ Moreover, optimizing cytokine support to enhance in vivo expansion, persistence and cytotoxic potential, may be critical for improving the clinical efficacy of ML NK cell therapies. A potential method may be to engineer ML NK cells to express membrane-bound IL-15 or IL-2 to improve therapeutic durability and reduce associated toxicities.^43,44

Conclusions and Future Directions

Substantial clinical progress has been made in demonstrating the feasibility and efficacy of cellular immunotherapies for cancer and are starting to be made for viral infections. However, studies have revealed that choices made in the clinic without pre-clinical testing, can negatively impact patient outcomes.⁴⁵ Outstanding questions include 1) How do in vivo dynamics like NK cell expansion correlate with patient responses? 2) Does CAR ML NK cell persistence matter for response? 3) Can factors like chemokine receptors and integrins help direct and retain CAR ML NK cells in the tumor? and 4) What is the capacity for CAR ML NK cells to modulate the endogenous immune system? One major challenge in using CAR-engineered cells in patients is identifying safe, high-density tumor antigens that have the potential for minor on-target-off tumor effects yet maximum on-target-on-tumor activity. However, even with the best scFv, delivering CAR-engineered lymphocytes to solid tumors is a major barrier in the field, underscored by the challenges that have been experienced in moving CAR T cell therapy beyond hematologic malignancies and into solid tumors.⁴⁶ Advances made in correlative immunology associated with clinical trials have revealed a new wave of questions that cannot be experimentally addressed in the clinical setting or using xenograft systems. Indeed, we now require controlled model systems to assess pertinent questions in the field and dissect underlying mechanisms operative in adoptive cell therapy immunomodulation. Basic, mechanistic studies are required to determine factors that impact CAR ML NK cell trafficking, persistence, and effector functions. Murine models will also allow us to simultaneously understand the basic cellular mechanisms that distinguish cytokine-induced ML NK and CAR versus endogenous receptor mediated signals that impact NK cell anti-tumor activity in immune-competent settings.