The Future of Natural Killer Cell Immunotherapy for B Cell Non-Hodgkin Lymphoma (B Cell NHL)

Opinion statement Natural killer (NK) cells have played a critical—if largely unrecognized or ignored—role in the treatment of B cell non-Hodgkin lymphoma (NHL) since the introduction of CD20-directed immunotherapy with rituximab as a cornerstone of therapy over 25 years ago. Engagement with NK cells leading to lysis of NHL targets through antibody-dependent cellular cytotoxicity (ADCC) is a critical component of rituximab’s mechanism of action. Despite this important role, the only aspect of B cell NHL therapy that has been adopted as standard therapy that even indirectly augments or restores NK cell function is the introduction of obinutuzumab, a CD20 antibody with enhanced ability to engage with NK cells. However, over the last 5 years, adoptive immunotherapy with effector lymphocytes of B cell NHL has experienced tremendous growth, with five different CAR T cell products now licensed by the FDA, four of which target CD19 and have approved indications for some subtype of B cell NHL—axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, and tisagenlecleucel. These T cell-based immunotherapies essentially mimic the recognition, activation pathway, and cytotoxic machinery of a CD19 antibody engaging NK cells and lymphoma targets. Despite their efficacy, these T cell-based immunotherapies have been difficult to implement because they require 4–6 weeks of manufacture, are costly, and have significant toxicities. This renewed interest in the potential of cellular immunity—and the manufacturing, supply chain, and administration logistics that have been addressed with these new agents—have ignited a new wave of enthusiasm for NK cell-directed therapies in NHL. With high safety profiles and proven anti-lymphoma efficacy, one or more new NK cell-directed modalities are certain to be introduced into the standard toolbox of NHL therapy within the next few years, be it function-enhancing cytokine muteins, multi-domain NK cell engagers, or adoptive therapy with expanded or genetically modified NK cells.


Introduction
Approximately 90,000 new cases of lymphoma are diagnosed in the USA per year, approximately 90% of which (estimated 81,560 in 2021 per American Cancer Society) are non-Hodgkin lymphoma (NHL), and the majority of these are B cell NHL [1][2][3]. While the survival rate for newly diagnosed children and adolescents with B cell NHL treated with chemotherapy and antibody-based regimens has more than doubled from the 1970s to the early 2000s (45% to 9 90%) [1], the prognosis is dismal in patients with relapsed/refractory B cell NHL [4, 5, 6••, 7, 8, 9•]. Novel approaches with CD19-and BCMAtargeted CAR T cellular immunotherapy have recently been approved by the FDA for patients with relapsed/refractory B cell NHL including CD19 CAR T cells [10, 11••, 12••, 13••]. However, treatment with CAR T cells is complicated by high cost, manufacturing logistics, and toxicity.
In contrast, NK cells have similar cytotoxic effector mechanisms as T cells but appear to have a broader safety profile and are more amenable to generating allogeneic ready-to-infuse (a.k.a. "off-the-shelf") products. Like T cells, NK cells can also be genetically modified for antigen-specific targeting [3]. Liu et al. recently reported the safety and efficacy of cord blood derived CD19 CAR NK cells in patients with relapsed/ refractory CD19 B-cell NHL and chronic lymphocytic leukemia (CLL) [13 •• ]. We now summarize the clinical and preclinical experience with NK cells and CAR NK cells in B-cell NHL.

Endogenous NK cells in B-cell NHL
NK cells are innate lymphocytes that play a key role in the recognition of cells that are cancerous or virus-infected. NK cell activation or inhibition (tolerance) is determined by an integrated balance of signals from NK cell activating and inhibitory receptors binding to their corresponding ligands on target cells [14] (Fig. 1). As part of our first line of defense, NK cells exert their effector function directly via cellular cytotoxicity and indirectly via proinflammatory cytokine secretion. In the last few decades, NK cells have moved to the forefront of immune oncology for several reasons. First, improved understanding of NK cell biology, discovery of new NK cell sources, and advances in NK cell culture techniques have made it possible to expand and activate NK cells for adoptive cell therapy. In addition, in contrast to T cells, NK cells do not depend on antigen presentation but instead utilize a balance of inhibitory and activating cell receptors that recognize self and stress ligands to determine effector function. Without the need for antigen presentation, NK cells can be ubiquitously effective even in cancers where tumor specific antigens remain elusive. Finally, allogeneic NK cell therapy is safe with no reports of dose limiting toxicities including graft versus host disease (GVHD), even with minimal HLA matching [15][16][17].
In patients with lymphoma, decreased number and function of NK cells portends a poor prognosis [18-20, 21•]. Methods of immune evasion in the lymphoma tumor microenvironment (TME) include immune checkpoints, hypoxia-induced immune modulation, and aberrant NK cell receptor/ligand expression [22][23][24][25][26][27]. Similar to the well described graft-versus tumor effect in hematopoietic stem cell transplant (HSCT) for leukemia, early and robust NK cell recovery after autologous and allogeneic HSCT in lymphoma is associated with improved survival [28][29][30][31][32]. To facilitate improved NK cell function after autologous HSCT in lymphoma, several studies administered low dose recombinant IL-2 as maintenance immunotherapy to prevent relapse [33][34][35]. In a study of relapsed/refractory NHL patients undergoing autologous HSCT, 10 patients received low dose recombinant IL-2 (rIL2) as maintenance therapy for 12 months after autologous HSCT. Following rIL2 therapy, peripheral blood (PB) samples from patients had a significant increase in NK cell number, function, and CD16-mediated ADCC compared to their baseline number and function before therapy [33]. None of the 10 patients treated on the protocol had relapse of their disease, with a median follow up of 16 months from the start of rIL2. Interestingly, two patients who still had residual disease after HSCT showed complete resolution of the residual disease while receiving rIL2 therapy. A similar study by Miller et al. using low dose IL-2 after autologous HSCT in patients with lymphoma and breast cancer also demonstrated a more than 10fold increase in PB NK cells with enhanced cytotoxicity against resistant cell lines [35]. To build off of these early studies, clinical trials utilizing adoptive transfer of autologous ex vivo-activated NK cells or lymphokine-activated killer cells in patients with lymphoma emerged with only modest activity [34,[36][37][38][39][40] ( Table 1). In addition, although feasible, the use of autologous NK cells was costly, often required more than one apheresis procedure, and NK cell doses were limited to around 10 7 /kg [44]. To improve clinical efficacy, more recent studies have utilized highly functional expanded NK cells and/or allogeneic NK cell sources to facilitate the graft versus tumor effect. Yang et al. demonstrated the safety of expanded random healthy donor PB NK cells for treatment of malignant lymphoma and other solid tumors [42]. Multiple doses of up to 3 × 10 7 /kg were administered with no dose limiting toxicities and no GVHD. Clinical responses, however, were limited with only 8/17 patients with stable disease as best response overall response and no complete responses. Although the safe use of donor derived NK cells represented progress in the field, alloreactive NK cells alone did not appear to be enough to eliminate bulk disease.

Targeting NK cells to NHL with antibodies
Antibody-based therapy has become a critical part of the treatment landscape in hematologic malignancies in the past few decades and several monoclonal antibodies have been FDA approved that target the lymphoma-specific antigens CD19 (loncastuximab tesirine and tafasitimab-cxix), CD20 (rituximab, obinutuzumab, ofatumumab, ibritumomab tiuxetan), CD30 (brentuximab vedotin), CD52 (alemtuzumab), CD38 (daratumumab, isatuximab), CD79b (polatuzumab vedotin), and CCR4 (mogamulizumab). One of the mechanisms by which antibodies mediate tumor cell lysis is through ADCC by NK cells. NK cells recognize the Fc portion of antibodies bound to the surface of target cells via the Fc-gamma receptor III (CD16). Optimal efficacy of antibody therapy depends on high number and function of NK cells. In patients with DLBCL and follicular lymphoma treated with anti-CD20 monoclonal antibodies, low pre-treatment NK cell count was associated with shorter progression free survival and decreased overall survival compared to patients with higher pre-treatment NK cells [45]. NK cell ADCC has been exploited in antibody therapy by systemic cytokine stimulation of endogenous NK cells [46][47][48][49][50] or in combination with adoptive NK cell therapy [43,51]. Autologous cytokine-expanded NK cells were combined with chemotherapy and rituximab in 9 patients with relapsed CD20positive lymphoma patients to enhance ADCC [43]. A single dose of escalating expanded NK cells (1 × 10 6 /kg, 3 × 10 6 /kg, and 10 × 10 6 /kg) was given on the day after rituximab. Complete responses were observed in 7/9 patients and there was a significant increase in PB NK cells and cytolytic activity in all patients two weeks after infusion. In a phase II trial, patients with relapsed/refractory CD20 + NHL were given IL-2-activated haploidentical PB NK cells after lymphodepleting chemotherapy [21 • ]. A single dose of 0.5-3.27 × 10 7 NK cells/kg was given in combination with IL-2 every other day × 6 doses and weekly rituximab × 4 doses. The NK cells were well tolerated and elicited responses in 4/14 evaluable patients, including 2 complete responses. Importantly, the authors noted improved NK cell persistence and effector function in patients with higher endogenous IL-15 at the time of NK cell infusion highlighting the importance of in vivo cytokine stimulation. Based on this and similar observations, subsequent studies utilizing IL-15 alone or in combination with adoptive NK cell infusion are being investigated. ALT-803 is an IL-15 super agonist complex developed to mimic physiologic trans-presentation of IL-15 and prolong the half-life. A phase I study of ALT-803 in 33 adult patients with hematologic malignancies who relapsed after allogeneic HSCT demonstrated that the cytokine therapy was safe and significantly increased NK and CD8 + T cell number and function [52]. Although only 4 patients had objective responses (1 CR, 1 PR, 2 SD) after 5 doses of ALT-803, the enhanced immune milieu and tolerability has led to further trials of ALT-803 and related compounds combined with rituximab [53] and/or adoptive NK cell therapy, including in lymphoma (NCT02890758) ( Table 2).

Targeting NK cells to NHL with CARs
With the success of CD19 CAR T cell therapy in hematologic malignancies, there has been a push to develop CAR NK cells targeting a wide variety of tumor antigens. Currently, the FDA-approved CAR T cell products are manufactured from autologous T cells due to the risk of GVHD from the native T cell receptor in allogeneic CAR T cells. Manufacturing CAR T cells on a patient-by-patient basis is costly, time consuming, and often fails due to poor T cell function in  heavily pretreated cancer patients. CARs usually contain a single chain variable fragment from a monoclonal antibody, a transmembrane hinge region, a signaling domain such as CD3-zeta, and one or more co-stimulatory domains such as CD28, 4-1BB, or 2B4 (CD244) [54,55] (Fig. 2). Since NK cells utilize many of the same signaling domains and activation pathways as T cells, NK cells can also be redirected to specifically target tumor antigens by the introduction of a CAR, which then permanently recapitulates the targeting function of an antibody and activation pathways for cytotoxicity. Moreover, the addition of a CAR does not abrogate the NK cell's innate recognition of cancer through endogenous receptors. One clear advantage of NK cells is the ability to use donor derived, "off-the-shelf" NK cells that minimize or eliminate the need for HLA matching without the risk of GVHD.  (Fig. 3). The NK cell infusions were safe with no CRS, neurologic toxicity, or GVHD, and the NK cells were detectable (by assaying for the vector transgene) for at least 12 months after infusion. Progress in the field of genetic modification of NK cells has opened the door for true "off-the-shelf" CAR NK cell therapy. Additional CAR NK cell targets for lymphoma currently under clinical investigation include CD19, CD22, and CD7 (Table 3).

Ex vivo NK cell expansion for adoptive NK cell-based immunotherapy for B cell NHL Ex vivo NK expansion with feeder cells for B cell NHL
To overcome the limitation of small number of active NK cells in the donor PB, NK cells can be ex vivo activated and expanded with feeder cells such as irradiated PBMCs [71][72][73], Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL) [74], or gene-modified cell lines such as K562 [75][76][77][78]. Preclinical studies demonstrated that highly cytotoxic NK cells can be expanded with irradiated and activated autologous PBMCs to efficiently kill lymphoma cells in vitro and in mice xenografted with human lymphoma cells [71][72][73]. These NK cells expanded with irradiated autologous PBMC are suitable for allogeneic transfer without the risk of graft-versus-host disease induction [71][72][73]. Our group and others have successfully expanded active PB NK cells or CB NK cells in vitro by coculture with irradiated EBV-LCL, or with K562 cells expressing transfected cell-membrane bound IL-15 and 4-1BBL to kill leukemia and B-NHL [74][75][76][77]. Lee and colleagues have developed a novel feeder cell line what was engineered to express membrane bound IL-21 and 4-1BB ligand (K562-mbIL21-41BBL) to expand PB NK or CB NK ex-vivo [78]. This method resulted in over 35,000-fold increase in NK cells in 3 weeks while avoiding telomere shortening and NK cell senescence and significant increase in NK cell functional activation against lymphoma (Fig. 4) [78].  [79]. Additionally, NK cells can be expanded with a feeder-free, particle-based approach, which uses plasma membrane particles (PM-particles) derived from K562-mbIL15-41BBL or K562-mbIL21-4-1BBL cell lines [80,81], but preclinical studies in B cell NHL have not yet been reported.

Preclinical studies of CAR engineered NK cells for B cell NHL
CAR NK therapy is promising and may provide some advantages over CAR T cells such as low risk of on-target/off-tumor toxicity to normal tissues, reduced risk for GVHD, and reduced frequency and severity of cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity (ICANS) [55]. NK cells can be engineered to express CAR through several technologies such as transposons, messenger ribonucleic acid (mRNA)mediated gene delivery, lentiviruses, and CRISPR/AAV targeted gene insertion [82]. We and others have engineered expanded PB NK (exPBNK) cells with anti-CD19 CAR or anti-CD20 CAR utilizing retroviral integration or CAR mRNA electroporation to target B cell malignancies including B-NHL [83,84]. We previously reported that anti-CD20 CAR exPBNK cells had significantly enhanced in vitro cytotoxicity against BL, limit BL tumor metastasis, and extended the survival of NSG mice xenografted with human BL cells [84]. The combination of anti-CD20 CAR exPBNK cells with a histone deacetylase inhibitor romidepsin, which enhanced expression of NKG2D ligands on the surface of BL, significantly killed BL, reduced tumor burden, and extended the survival in NSG mice xenografted with human BL cells [85]. Similarly, engineering the NKlike lymphoma cell line NK92 with an anti-CD19 CAR significantly increased in vitro cytotoxicity and prominently induced apoptosis in rituximab-and obinutuzumab-resistant cell lines and patient-derived cells, and delayed tumor growth in B-NHL xenografts [86]. Liu et al. genetically modified NK cells expanded from cord blood (CB) cells with a retroviral vector (iC9/CAR.19/IL-15) [56]. Their preclinical studies demonstrated that iC9/CAR.19/IL-15 CB NK cells efficiently killed CD19 + primary leukemia cells and Raji in vitro and significantly prolonged the survival in a xenograft Raji lymphoma murine model [56]. Emerging preclinical evidence shows that IL-12 and IL-18 plus IL-15 can induce murine and human NK cells with long-lasting memory-like functionality [87]. These cytokine-induced memory-like (CIML) NK cells display increased proliferative capacity, prolonged persistence in vivo, and superior functionality in killing of rituximab-coating Raji lymphoma cells compared with control NK cells in vitro, and substantially reduced growth of established lymphoma tumors in mice [88][89][90]. Anti-CD19 CAR-modified CIML NK cells displayed significantly increased interferon-γ (IFNγ) secretion and cytotoxicity against NK-resistant lymphoma lines and primary lymphoma tumor cells, and significantly reduced lymphoma burden and significantly improved the survival in human lymphoma xenograft models [91]. Goodridge et al. developed a CAR NK product FT596, derived from a master induced pleuripotent stem cell (iPSC) line engineered to uniformly express anti-CD19-CAR, an enhanced functioning high-affinity, non-cleavable CD16, and a recombinant fusion of IL-15 and IL-15 receptor alpha (IL-15RF) [92]. FT596 showed significantly enhanced clearance of Raji tumor cells in combination with rituximab in a Raji xenografted mouse model [92]. Furthermore, utilizing an allogenic human CD34 engrafted NSG mouse model, FT596 demonstrated improved survival and safety over primary CAR19 T cells, either as a monotherapy or as a combination therapy with rituximab against Raji tumor cells [92]. These preclinical findings have provided the preliminary results to conduct a Phase I dose-finding study of FT596 as monotherapy and in combination with rituximab or obinutuzumab in subjects with relapsed/refractory B cell lymphoma or CLL (NCT04245722).

Preclinical studies of combinatorial therapies of NK cells for B cell NHL
A large number of preclinical studies of combination therapies incorporating NK cells for B cell NHL have been reported (Table 4), a number of which are summarized below.

Combinatorial therapy of NK cells with a novel type II anti-CD20 antibody Obinutuzumab
Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity and has been approved for the treatment of patients with previously untreated advanced-stage follicular lymphoma [100,101]. Our preclinical studies demonstrated that obinutuzmab has significantly enhanced ADCC compared to rituximab and induced apoptosis in BL in vitro, and the combination of obinutuzumab with exPBNK significantly enhanced overall survival of NSG mice xenografted with Raji tumor cells as compared to the combination of rituximab with exPBNK [93]. Our group is currently conducting a clinical trial to evaluate the safety and response rate of obinutuzumab as a single agent alone and in combination with ifosfamide, carboplatin and etoposide (O-ICE) without exogenous NK cells in children, adolescents and young adults with recurrent refractory CD20 + mature B-NHL including BL (NCT02393157). Combinatorial therapy of NK cells with IL15 superagonist (N-803) and rituximab IL-15 shares similar functions with IL-2 but has a distinct advantage over IL-2 for cancer immunotherapy due to its minimal binding to the low-affinity IL-2 receptor CD25, resulting in a lack of effect on Tregs [103]. N-803 is an IL-15 superagonist (originally named ALT-803) that consists of a high-affinity  interleukin-15 mutein (IL-15N72D) and a dimeric IL-15 receptor alpha (IL-15Rα)/Fc fusion protein [104]. N-803 has at least 25 times the activity of the wild type IL-15 in vivo and a significantly longer serum half-life in vivo than wild-type IL-15 (25 h vs. G 40 min) [104,105]. The study from Rosario et al. showed that the combination of N-803 with rituximab significantly increased the expression of granzyme B and perforin, IFNγ production, and ADCC of human NK cells against BL cell lines or primary follicular lymphoma cells [97].

Combinatorial therapy of NK cells with bispecific or trispecific killer engagers
The study supports the future clinical investigation of N-803 plus NK cells and anti-CD20 mAbs in patients with aggressive B cell NHL.
Combinatorial therapy of NK cells with N-820, a novel antibody-N-803 fusion N-820 was generated by fusing four single-chains of rituximab to the N terminus of N-803 [98]. N-820 activated primary NK cells to enhance ADCC and induced apoptosis of B cell NHL in vitro and in BL xenografted NSG mice [98]. N-820 also significantly enhanced the cytotoxicity of exPBNK against rituximab-sensitive and -resistant BL cells in vitro and in BL xenografted NSG mice in vivo, as compared to controls [99]. Our study and others suggest that N-820 is an attractive novel agent to be combined with NK therapy for CD20 + relapsed/refractory B-NHL [99].

Future directions
Although much is now established regarding the importance of NK cell number and function in the context of cancer survival, the parameters that define the optimal NK cell with respect to phenotype, function, proliferative potential, and long-term persistence are not well defined. In vitro assays that accurately reflect in vivo conditions, tumor microenvironment, trafficking, and cross-talk with other immune cells are much needed, and fully murine models have only partially filled this gap because of significant differences between mouse and human NK cell biology. It is also important to recognize that the definition of "optimal" may vary between different cancers and with different combination therapies. This baseline understanding of optimal will be important in defining differences-pro and con-between the various starting material/sources from which NK cells are generated, expansion methods, genetic modification methods, and cytokine adjuvants. With several new options now enabling engineering of NK cells after decades of difficulty in this area, the wide variety of targeting domains, signaling and cytokine combinations, and cell sources literally provide hundreds of potential CAR NK products that are now feasible and therefor will need to be tested in robust models for each disease.
In addition, the combinations with exogenous cytokines, engagers, immune modulators, and checkpoint inhibition will result in many new options for patients that need careful investigation. To achieve the best outcome quickly, clinical investigation will require cooperation, intelligent trial design, and implementation of multi-cohort, basket, or surrogate endpoints.
Lastly, this review has focused on the relevance of NK cells in the treatment of B cell NHL. Several antibodies have been developed for T cell NHL (e.g., alemtuzumab (anti-CD52), brentuximab (anti-CD30), and mogalizumab (anti-CXCR4)), all of which mediate at least part of their anti-lymphoma efficacy through ADCC. Thus, approaches to enhancing NK cell number and activity may also be highly relevant for T cell NHL.

Summary
NK cell-based immunotherapy holds tremendous promise for patients with B-NHL. A robust, carefully designed, and centrally coordinated systematic investigation of the available modalities should lead to significantly improved outcomes in the near future.

References and Recommended Reading
Papers of particular interest, published recently, have been highlighted as:

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