Activated Natural Killer Cells Research Articles

Preclinical Characterization of the Anti-Leukemia Activity of the CD33/CD16/NKG2D Immune Modulating TriNKET ® BMS-986357 (CC-96191)

CD33 is widely expressed by myeloid cells and is a validated drug target in acute myeloid leukemia (AML), as shown by the benefit of some patients from the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO). However, GO is often ineffective and toxic, prompting interest in more potent CD33-directed therapies. Success of T cell-engaging bispecific antibodies in lymphoid malignancies has raised enthusiasm for similar agents targeting CD33, but toxicities from activated T cells cause significant challenges for their use and provide a strong rationale to explore drugs engaging natural killer (NK) cells. Here, we investigated BMS-986357, a TriNKET ® that activates NK cells via CD16 and NKG2D receptors while targeting CD33. This format was chosen because simultaneous engagement of multiple activating NK cell receptors yields greater cytotoxic activity against neoplastic cells than engagement via a single activating receptor. Surface plasmon resonance analyses showed BMS-986357 simultaneously binds CD33, NKG2D, and CD16a, and co-engagement of NKG2D and CD16a increases the avidity. In the presence of primary human NK cells from healthy donors or a human NK cell line, the TriNKET ® demonstrated broad cytolytic anti-leukemia activity in a strictly CD33-dependent manner using a panel of CD33-expressing or CD33-deficient/deleted acute leukemia cells. Consistent with this, removing the CD33 binding domain rendered BMS-986357 ineffective against CD33+ AML cells. Drug-induced cytotoxicity was greater with BMS-986357 than with monoclonal CD33 antibodies, including lintuzumab and an afucosylated antibody with enhanced NK function, demonstrating the benefit of the TriNKET ® format. Compared to BMS-986357, substantially less cytotoxicity was observed with an Fc-mutant variant of BMS-986357 that abrogates binding to CD16a or an NKG2D dead-arm TriNKET ®, and cytolytic effects were reduced with a CD16-deficient NK cell line vs. a CD16-expressing subline, indicating maximal efficacy of BMS-986357 required co-engagement of CD16a and NKG2D. Using sublines of OCI-AML3 and KG-1 cells (both cell lines with low endogenous CD33 expression) to overexpress wild-type CD33 at increasing levels, we found low abundance of CD33 curtailed BMS-986357-induced cytotoxicity. Flow cytometric binding studies identified BMS-986357 to recognize the membrane-distal V-set domain of CD33. Unlike GO, BMS-986357-mediated cytolysis was not affected by over-expression of ABC transporter proteins by AML cells. The potency but not maximal activity of BMS-986357 was reduced by super-physiologic concentrations of soluble CD33; in contrast, the soluble form of the NKG2D ligand MICA did not impact TriNKET ® activity. In the presence of CD33+ AML cells, BMS-986357 activated NK cells but not T cells from healthy donor peripheral blood mononuclear cells and produced similar levels of maximum cytolysis as a CD33/CD3 bispecific antibody. Despite this, BMS-986357 elicited 10 to >100-fold lower soluble cytokine levels including IFN-γ, TNF, IL-6, and GM-CSF than a CD33/CD3 bispecific antibody, suggesting BMS-986357 has the potential for CD33/CD3 bispecific-like efficacy without the associated risk of cytokine release syndrome. Finally, in bone marrow specimens from patients with AML, BMS-986357 killed AML cells but not normal monocytes, suggesting selectivity of drug-induced cytotoxicity toward neoplastic cells, consistent with the notion NK cells are capable to differentiate between healthy and leukemic CD33+ cells. This selectivity suggests BMS-986357 may produce less myelosuppressive toxicity than what has been observed with CD33 targeting antibody-drug conjugates. In summary, our findings demonstrate that BMS-986357 TriNKET ® has potent CD33-dependent cytolytic activity in vitro against human AML cells, supporting the drug's exploration in early phase clinical trials. One such trial (NCT04789655) has opened accrual for adults with relapsed/refractory AML.

Engineered Natural Killer Cells Expressing Chimeric Ilt Receptors (CIR) Effectively Target HLA-G Positive AML Tumor Cells

Background Human leukocyte antigen-G (HLA-G), the immune checkpoint of pregnancy, directs immunosuppression by binding to Ig-like transcript (ILT) receptors (ILT2 in lymphoid cells and ILT4 in myeloid cells) via immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling domains. HLA-G is expressed in more than 50% of solid tumors and leukemias as a mechanism to evade immune attack. As a result, HLA-G is an excellent tumor-specific antigen since its expression in normal tissues is low except for cytotrophoblasts in the placenta where it protects the fetus from the mother's immune system. We present here a novel approach to target HLA-G-expressing Acute Myeloid Leukemia (AML) cells by utilizing the natural receptors of ILT2 and ILT4 coupled to positive intracellular signaling domains. Such chimeric ILT receptors, or CIRs drive activation of Natural Killer (NK) cells. Further, we have identified unique intracellular activation modules that function to robustly enhance cytotoxicity and expansion of NK cells upon engagement with tumor cells. Methods CD56 + NK cells were isolated from PBMCs, cultured with IL-15 and activated with a feeder-free cocktail of an immobilized cytokine and activating ligand. Activated NK cells were transduced with γ-retroviruses directing expression of CIR proteins (CIR.4-1BB.CD3ζ or CIR.X.Y where X and Y are candidate activation and coactivation moieties), soluble IL-15 and a ΔCD19 marker or CAR control constructs directed to HLA-G or CD33. After 8 days of expansion, CIR- or CAR-NK cells were cocultured with GFP ffluc-expressing AML target cell lines with or without transgenic HLA-G (G1, G2 and G5) isoforms. Cytotoxicity of CIR-NK cells was determined by reduction of GFP fluorescence in Incucyte or Celligo instruments or luciferase activity and supernatants were analyzed for IFN-γ and TNF-α release by ELISA. Transduced NK cells were labeled with nuclear-RFP for measurement of expansion in coculture. In comparative experiments, T cells from PBMCs were transduced to produce CIR-T cells by standard methods. Results CIR (ILT2 or ILT4.4-1BB.ζ) constructs were efficiently and stably expressed in T and NK cells (>80% ΔCD19 + 8- and 14-days post activation). Mock-transduced NK cells displayed innate killing activity against HLA-G negative AML cells (THP1 and KG1) that was not augmented by CIR constructs (mock = 6.16E6 ± 0.77E6 vs. CIR = 6.43E6 ± 0.37E6 in coculture against KG1 cells). In cocultures against THP1 and KG1 target cells expressing exogenous G1, G2 or G5 isoforms, CIR-NK cells or CIR-T cells displayed enhanced target cell killing in 2 and 7-day assays relative to mock-transduced effector cells (mock = 1.47E6 ± 0.49E6 vs. CIR = 0.57E6 ± 0.6 in coculture against KG1-HLA-G1). Further, while mock NK cells transduced with RFP alone could kill one-third of AML cells expressing endogenous HLA-G1 and G5 (Molm13 and Kasumi1), CIR-NK cells eliminated more than 90% of these AMLs and their activivation resulted in 4-fold higher IFN-γ secretion. Notably, robust cytotoxicity of CIR-NK cells was maintained even at 28 days post activation (mock = 36.95E6 ± 21.85E6 vs. CIR = 7.38E6 ± 1.92 in coculture against Kasumi1). To further enhance the functionality of the CIR constructs, a screen of novel costimulatory molecules to replace 4-1BB and/or CD3ζ resulted in several potent candidates ultilizing MyD88 signaling that greatly improved the cytotoxicity (> 80% tumor elimination), proliferation (> 2-fold expansion) and proinflammatory cytokine production (IFN-γ and TNF-α) of NK cells in cocultures. A subset is displayed in Figure 1. Conclusions The use of natural receptors ILT2 and ILT4 to target HLA-G takes advantage of the physiological pairing of the ILT receptors with the multiple immunosuppressive HLA-G isoforms thereby circumventing the possibility of antigen escape often observed in CAR-directed therapy. We demonstrated that engineered CIR-NK cells can convert HLA-G expression in AML from a potent inhibitor of leukocyte activity into a target for anti-tumor control.

Chimeric Antigen Receptor-Engineered NK Cells Overcome CD226 Downregulation As an Immune Escape Mechanism in Acute Myeloid Leukemia

Background: Natural killer (NK) cells are part of the innate immune system, and their killing capacity depends on the inputs from activating and inhibitory receptors. CD226 is an activating receptor that plays a role in the immune surveillance of acute myeloid leukemia (AML). AML cells have developed mechanisms to escape NK cell cytotoxicity including downregulating activating receptors such as CD226 on the surface of NK cells. Chimeric antigen receptors (CARs) are synthetic receptors integrated into NK cells to direct their anti-leukemia activity, and CD38 is a potential target for NK CAR-based cellular therapy against AML. Understanding the dynamics between the endogenous activating and inhibitory NK cell receptors (such as CD226) and the introduced CAR can determine the development of more effective NK CAR-based therapy for AML. We hypothesize that CD38-directed CAR NK cells may overcome CD226 downregulation as a mechanism of immune escape in AML. Purpose: we aimed to investigate (1) the mechanisms developed by AML cells to escape recognition by the activating receptor CD226, (2) whether and how anti-CD38 CAR-armed NK cells can overcome AML immune escape mechanisms. Methods: we performed multiparametric mass spectrometry to characterize changes in CD226 expression in NK cells of individuals with AML. Then, we investigated CD226 expression in NK cell-AML cocultures to characterize CD226-related mechanisms by imaging flow cytometry. Finally, to explore the impact of CD226 knockout (KO) together with the role of CD38 CAR in NK cell functionality, we retrovirally transduced CD38 CAR combined with CRISPR-Cas9 CD226 KO followed by short- and long-term cytotoxicity assays, cytokine production techniques and immune synapse assays by imaging flow cytometry. Results: NK cells from AML patients expressed low levels of CD226 with impaired cytotoxicity compared to heathy donor NK cells. Co-culture of healthy donor NK cells with AML blasts resulted in CD226 downregulation secondary to its internalization and was dependent on expression of the CD226 ligand CD155 in AML cells. CD226 KO diminished NK cell cytokine production and decreased their killing capacity against AML cells. In contrast, CD38-targeting NK cells displayed enhanced and specific cytotoxic activity against primary AML cells. When we combined the CD38 CAR with a CD226 KO in NK cells, these cells maintained their activating potential, cytokine secretion levels, and anti-leukemic cytotoxicity (Figure 1). CD38 CAR / CD226 KO NK cells could build strong immune synapses (Figure 2) even in the absence of LFA-1, an integrin activated by CD226 signaling and essential for NK cell immune synapse formation, suggesting that the CAR molecule compensates for the absence of CD266-mediated LFA signaling. Furthermore, CD38 CAR NK cells were capable of forming immune synapses, that were at least partially independent from LFA-1. Conclusion: CAR-directed NK cells can surpass resistance mechanisms in AML, such as CD226 downregulation and dampening of LFA-1 recruitment into the immunological synapse. Thus, NK cell activation by artificially introduced and germline-encoded receptors can overcome distinct tumor-resistance mechanisms, CAR-driven NK cells being a potent option for the treatment of AML.

The KIR-HLA-CD16a Immunogenetic Profile Influences NK Cell-Mediated ADCC and Response to Rituximab Therapy in B-Cell Lymphomas

Rituximab-based chemoimmunotherapy (R-CHOP) is the standard of care in most B-cell lymphomas, however there is marked interpatient heterogeneity in treatment response. An important mechanism of Rituximab (anti-CD20) action is through natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but the factors influencing this are poorly understood. Here, we present findings from a preclinical study investigating the role of NK cell receptor polymorphisms and HLA allelic variation on ADCC against aggressive B-cell lymphoma cell lines. Genotyping was conducted using Luminex-based SSO and Sanger sequencing. Multicolor flow cytometry was used to assess the response of activated NK cells from healthy donors against 8 lymphoma cell lines. Individuals carrying the GG or GT allele in CD16a were found to have a 3-8 fold greater NK cell response compared to individuals with a TT allele and rituximab-mediated ADCC was significantly greater in individuals with a KIR-3DL1 allele when tested against B-cell lymphomas lacking the HLA-Bw4 allele. An additive effect was observed when considering both CD16a and KIR-HLA together. As expected, CD20 expression was a strong correlate of NK cell response. In summary, NK cell-mediated ADCC in response to ADCC appears to be influenced by the KIR-HLA-CD16a genotype. These findings suggest a potential role for immunogenotyping in B-cell lymphomas treated with Rituximab. Additionally, the presence of the shared F c receptor domain opens the possibility of applying this framework towards predicting NK cell responses to antibody therapies in other diseases.

Potentiation of natural killer cells to overcome cancer resistance to NK cell-based therapy and to enhance antibody-based immunotherapy.

Natural killer (NK) cells are cellular components of the innate immune system that can recognize and suppress the proliferation of cancer cells. NK cells can eliminate cancer cells through direct lysis, by secreting perforin and granzymes, or through antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC involves the binding of the Fc gamma receptor IIIa (CD16), present on NK cells, to the constant region of an antibody already bound to cancer cells. Cancer cells use several mechanisms to evade antitumor activity of NK cells, including the accumulation of inhibitory cytokines, recruitment and expansion of immune suppressor cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), modulation of ligands for NK cells receptors. Several strategies have been developed to enhance the antitumor activity of NK cells with the goal of overcoming cancer cells resistance to NK cells. The three main strategies to engineer and boost NK cells cytotoxicity include boosting NK cells with modulatory cytokines, adoptive NK cell therapy, and the employment of engineered NK cells to enhance antibody-based immunotherapy. Although the first two strategies improved the efficacy of NK cell-based therapy, there are still some limitations, including immune-related adverse events, induction of immune-suppressive cells and further cancer resistance to NK cell killing. One strategy to overcome these issues is the combination of monoclonal antibodies (mAbs) that mediate ADCC and engineered NK cells with potentiated anti-cancer activity. The advantage of using mAbs with ADCC activity is that they can activate NK cells, but also favor the accumulation of immune effector cells to the tumor microenvironment (TME). Several clinical trials reported that combining engineered NK cells with mAbs with ADCC activity can result in a superior clinical response compared to mAbs alone. Next generation of clinical trials, employing engineered NK cells with mAbs with higher affinity for CD16 expressed on NK cells, will provide more effective and higher-quality treatments to cancer patients.

Association of recurrent implantation failure and recurrent pregnancy loss with peripheral blood natural killer cells and interferon-gamma level.

Fetal uterine survival depends on maintaining an immune balance between the mother and fetus. This study aimed to investigate the correlation of blood peripheral natural killer (NK) cells and interferon-gamma (IFN-γ) with recurrent recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL). In this case-control study, peripheral blood samples were obtained from three groups of RPL, RIF, and parous women without a history of abortion or infertility problems and analyzed by lymphocyte-based flow cytometry. Afterward, the levels of NK cells and IFN-γ were determined. All data were analyzed using one-way analysis of variance and nonparametric Kruskal-Wallis tests. The level of IFN-γ in the RPL group was significantly higher than that in parous women and the RIF group (P<0.05), whereas its level in the RIF group was not significantly different from the control group (P>0.05). A significant correlation was found between the levels of IFN-γ and NK cells in the RPL group (r=0.481; P=0.02). However, no significant correlation was found between the levels of IFN-γ and the active NK cells in the RPL group (P=0.08). Moreover, no significant correlation was found between the levels of NK cells (whether activated or not) and IFN-γ in the RIF patients (P>0.05). Immune dysfunction may not be involved in implantation failure during IVF but may be involved in recurrent miscarriage, probably by increasing IFN-γ levels.

The role of Natural Killer cells in pregnancy and their potential impact on cancer prevention

Immune cells such as Natural Killer (NK) cells play a central role in immunosurveillance, preserving the physiological integrity of tissues by fighting infection and combating tumor cells, as it has the potential to release specific cytotoxic substances. Some studies suggest that a high number and/or inadequate presence/frequency of activated NK cells is involved in the Recurrent Pregnancy Loss (RPL) condition once it might trigger an immunological imbalance in women’s bodies. Our hypothesis states that a higher number of activated NK cells could be associated to lower cancer rates in women with RPL. To test this idea, we propose to evaluate abortion material, peripheral blood and uterine samples from both RPL and non-RPL women, comparing histological, cellular, and molecular features in order to identify and differentiate the immunological profile of both groups. Once established the immunological profile of both RPL and non-RPL women, an investigation of the epidemiological history of women with/without RPL can approach the possible correlation between different NK patterns and cancer reports. Scientific literature concerning this potential correlation is scarce, and we believe that a better understanding of the NK cell behavior associated to different gestational outcomes could increase our comprehension on the mechanisms underlying both cancer development and immunological aspects of pregnancy.

Charting a killer course to the solid tumor: strategies to recruit and activate NK cells in the tumor microenvironment.

The ability to expand and activate natural Killer (NK) cells ex vivo has dramatically changed the landscape in the development of novel adoptive cell therapies for treating cancer over the last decade. NK cells have become a key player for cancer immunotherapy due to their innate ability to kill malignant cells while not harming healthy cells, allowing their potential use as an "off-the-shelf" product. Furthermore, recent advancements in NK cell genetic engineering methods have enabled the efficient generation of chimeric antigen receptor (CAR)-expressing NK cells that can exert both CAR-dependent and antigen-independent killing. Clinically, CAR-NK cells have shown promising efficacy and safety for treating CD19-expressing hematologic malignancies. While the number of pre-clinical studies using CAR-NK cells continues to expand, it is evident that solid tumors pose a unique challenge to NK cell-based adoptive cell therapies. Major barriers for efficacy include low NK cell trafficking and infiltration into solid tumor sites, low persistence, and immunosuppression by the harsh solid tumor microenvironment (TME). In this review we discuss the barriers posed by the solid tumor that prevent immune cell trafficking and NK cell effector functions. We then discuss promising strategies to enhance NK cell infiltration into solid tumor sites and activation within the TME. This includes NK cell-intrinsic and -extrinsic mechanisms such as NK cell engineering to resist TME-mediated inhibition and use of tumor-targeted agents such as oncolytic viruses expressing chemoattracting and activating payloads. We then discuss opportunities and challenges for using combination therapies to extend NK cell therapies for the treatment of solid tumors.

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

Innate Cell Engager (ICE®) AFM13 Combined with Preactivated and Expanded (P+E) Cord Blood (CB)-Derived Natural Killer (NK) Cells for Patients with Refractory CD30-Positive Lymphomas: Final Results

CONCLUSIONS Pts with refractory Hodgkin (HL) and other CD30+ lymphomas have few effective therapies available and targeted NK cell immunotherapy is an active area of research. Transfer of non-targeted NK cells has shown limited clinical benefit as target recognition of cancer cells by NK cells constitutes a barrier. The innate cell engager AFM13 is a first-in-class CD30/CD16A bispecific antibody construct that induces selective killing of CD30+ tumor cells by engaging and activating NK cells. As a single agent, AFM13 has limited clinical activity against HL, likely due to the reduced effector function of autologous NK cells in these pts, which provides the rationale for combining AFM13 with allogeneic NK cells. Our prior preclinical work identified a promising combination of CB-derived NK cells that were first IL-12/IL-15/IL-18-preactivated followed by ex vivo expansion (P+E) with engineered K562 feeder cells expressing membrane-bound IL-21, 4-1BBL and CD48 and exogenous IL-2, and subsequently complexed with AFM13 just before infusion; these cytokine-induced memory-like NK cells presented chimeric antigen receptor (CAR)-NK-like features and increased in vitro and in vivo antitumor activity compared to either non-AFM13-precomplexed P+E NK cells or to AFM13 alone (Kerbauy et al, Clin Cancer Res 2021). Building on this work we wished to study this CB NK-cell therapy in pts with refractory CD30+ lymphomas. This investigator-initiated single-center phase I-II trial (NCT04074746) evaluated the safety and activity of P+E AFM13-precomplexed CB NK cells followed by systemic AFM13. Pts ages 15-75 with CD30+ lymphomas refractory to brentuximab vedotin were enrolled. The goals were to identify the recommended phase 2 dose (RP2D) of NK cells, estimate the overall (ORR) and complete response (CR), event-free (EFS) and overall survival (OS) rates, and study NK cell activation and persistence. Each treatment cycle consisted of fludarabine/cyclophosphamide (days −5 to −3) followed by infusion (day 0) of the AFM13-precomplexed CB NK cells, cultured for 14 days as described above, and three weekly IV infusions of AFM13 (200 mg, days 7, 14 and 21). Two cycles were planned for pts 1-19 and up to 4 cycles from patient 20 on. Response was evaluated on day 28 of each cycle. Enrollment proceeded at 3 dose levels (DL) of 10 6, 10 7 and 10 8 NK/Kg, respectively. Each patient received the same NK dose in all cycles. Between 09/20-12/22 we enrolled 42 pts (37 HL, 5 NHL), pretreated with a median 7 lines, at DL1 (N=3), DL2 (N=3) and DL3 (N=36), with 1 (N=2), 2 (N=21), 3 (N=3) or 4 cycles (N=16). All had active progressive disease at enrollment and no bridging therapy was given. The cords used for each of the 117 cycles were selected without consideration for HLA match, which was 0/6 (N=53), 1/6 (N=46), 2/6 (N=14), 3/6 (N=3) and 4/6 (N=1). The product infused consisted of &amp;gt;94% NK cells (expanded &amp;gt;2,700 fold), with only 0.02% T cells. The NK cells were 96% viable, 91% bound to AFM13, and 97.6% CD16+. The products were infused fresh. There was 1 grade 2 infusion-related reaction (IRR) in 115 infusions of AFM13-NK (0.8%) and 27 IRR infusion related reactions in 350 infusions of AFM13 (7.7%) (1 G3, 26 G2). We saw no cases of CRS, ICANS or GVHD of any grade. DL3 (10 8 NK/Kg) was established as the RP2D. The ORR and CR were 92.8% and 66.7%, respectively (94.4% and 72.2%, respectively, in 36 pts treated at the RP2D). Nine pts had a response consolidated with SCT (5 allo, 4 auto). At median follow-up of 14 (6-34) months, the EFS/OS rates are 31%/76%; median EFS/OS are 8 months/not reached. No relapses were associated with antigen loss. In the subset of pts planned for 4 cycles at RP2D, 15 of 23 pts remained event free at 6 mo (4 pts with and 11 without SCT consolidation), for 6-month EFS/OS rates of 65%/83%. Overall, 8 of the 9 pts (89%) receiving a consolidation SCT are event free at 10 to 34 months. AFM-bound CB NK cells were detected in blood from day 1 post infusion for up to 3 weeks. Donor NK levels followed similar patterns after each cycle, which argues against a sensitization after the first NK infusion. CyTOF studies showed increased expression of AFM13 and activation markers in donor NK cells. We confirmed trafficking of AFM13-bound donor NK cells to tumor sites shortly after infusion. CB-derived cytokine-induced memory-like NK cells precomplexed with AFM13 have excellent tolerability and activity for pts with heavily pretreated and refractory CD30+ lymphoma.

T cell-Dependent Bispecific Therapy Enhances Innate Immune Activation and Antibody-Mediated Killing.

T cell-retargeting therapies have transformed the therapeutic landscape for hematologic diseases. T cell-dependent bispecific antibodies (TDB) function as conditional agonists that induce a polyclonal T-cell response, resulting in target cell destruction and cytokine release. The relationship between this response and its effects on surrounding innate immune populations has not been fully explored. Here we show that treatment with mosunetuzumab in patients results in natural killer (NK) cell activation in the peripheral blood. We modeled this phenomenon in vitro and found that TDB-mediated killing activated NK cells, increasing NK function and antibody-dependent cellular cytotoxicity (ADCC), and enhanced the capability of macrophages to perform antibody-dependent cellular phagocytosis (ADCP). This enhancement was triggered by cytokines released through TDB treatment, with IL2 and IFNγ being major drivers for increased ADCC and ADCP, respectively. Surprisingly, cytolytic ability could be further augmented through neutralization of IL10 for NK cells and TNFα for macrophages. Finally, we showed that TDB treatment enhanced the efficacy of Fc-driven killing to an orthogonal solid tumor target in vivo. These results provide rationale for novel antibody therapy combinations that take advantage of both adaptive and innate immune responses.

Antitumor Immune Responses in B2M-Deficient Cancers.

β2-microglobulin (B2M) is a critical component of the MHC class I molecule and is required to present tumor antigens to T cells. Its loss results in acquired resistance to immune checkpoint blockade (ICB) therapies. However, there have been well-documented cases of B2M-inactivated tumors responding to ICB, justifying investigation of how an antitumor immune response can be generated to tumors without surface MHC class I. We knocked out B2M in three murine models with varying baseline MHC class I expression and sensitivity to anti-programmed death receptor (PD-1) therapy and analyzed the immune responses. MC38 and YUMMER2.1 without B2M responded to anti-PD-1 alone or with an IL2 agonist, and this was mediated by CD4+ T cells and natural killer (NK) cells. The more aggressive B16 without B2M expression only partially responded to the IL2 agonist, and this was dependent on NK cells. When analyzing nearly 300 pretreatment biopsies from patients with melanoma receiving PD-1 blockade-based therapies, we found infrequent B2M mutations or homozygous loss but more frequent LOH or copy-number gains. B2M LOH was enriched in biopsies from patients without response to therapy, and these biopsies were more frequently infiltrated by activated NK cells. We conclude that in the absence of B2M, activation of CD4+ T cells and NK cells can mediate responses to murine models of PD-1 blockade therapy. In addition, in human melanoma, the intratumoral presence of activated NK cells upon partial B2M loss likely selects against tumor escape through low surface MHC class I expression.

Intensified NK cell therapy in combination with low-dose chemoradiotherapy against human colorectal cancer.

The therapeutic potential of adoptive natural Killer (NK) cells immunotherapy in combination with chemoradiotherapy, the main treatment modality for colorectal cancer (CRC), has not yet been explored. Here, we aimed to investigate the efficacy of NK cells to potentiate primary tumor control and improve survival outcomes, especially in combination with low-dose chemoradiotherapy. Ex vivo activated NK cells (> 90% purity) from healthy donors were obtained. NK cells were administered intravenously to the CRC-bearing mice and intensified in vivo in combination with low-dose 5-fluorouracil (0.5mg/kg or 1mg/Kg) and irradiated tumors with low doses (2Gy or 4Gy). Real-time NK cell cytotoxicity demonstrated a synergistic killing effect of a combination of low-dose chemoradiotherapy, mainly through NKp30 and NKG2D, showing a decrease in NK cell degranulation after blocking NKG2D and NKp30. In vivo tumor characteristics after combination treatment showed decreased CD112, CD155, MICA, and MICB expression. Under the combination strategy, 70% of the mice had free lung metastasis and 90% without secondary gross tumors, indicating suppressed distant metastasis to lung and axillary regions. This combination therapy resulted in significantly synergistic antitumor activity against primary solid tumors compared to chemoradiotherapy only. Furthermore, the intensified NK cell administration showed significantly better primary tumor control and survival outcomes than the non-intensified NK cell administration in a human colorectal HT-29 model treated with low-dose chemoradiotherapy. Optimized NK cell therapy combined with low-dose chemoradiotherapy can provide effective therapeutic potential for intractable cold human colorectal cancer.

Tumour-associated macrophages as a potential target to improve natural killer cell-based immunotherapies.

Adoptive transfer of natural killer (NK) cells has been proposed as a novel immunotherapy for malignant tumours resistant to current therapeutic modalities. Several clinical studies have demonstrated that the NK cell-infusion is well tolerated without severe side effects and shows promising results in haematological malignancies. However, patients with malignant solid tumours do not show significant responses to this therapy. Such disappointing results largely arise from the inefficient delivery of infused NK cells and the impairment of their functions in the tumour microenvironment (TME). Tumour-associated macrophages (TAMs) are the most abundant stromal cells in the TME of most solid tumours, and a high TAM density correlates with poor prognosis of cancer patients. Although our knowledge of the interactions between TAMs and NK cells is limited, many studies have indicated that TAMs suppress NK cell cytotoxicity against cancer cells. Therefore, blockade of TAM functions can be an attractive strategy to improve NK cell-based immunotherapies. On the other hand, macrophages are reported to activate NK cells under certain circumstances. This essay presents our current knowledge about mechanisms by which macrophages regulate NK cell functions and discusses possible therapeutic approaches to block macrophage-mediated NK cell suppression.

Therapeutic Anti-KIR Antibody of 1-7F9 Attenuates the Antitumor Effects of Expanded and Activated Human Primary Natural Killer Cells on In Vitro Glioblastoma-like Cells and Orthotopic Tumors Derived Therefrom.

Glioblastoma (GBM) is the leading malignant intracranial tumor, where prognosis for which has remained extremely poor for two decades. Immunotherapy has recently drawn attention as a cancer treatment, including for GBM. Natural killer (NK) cells are immune cells that attack cancer cells directly and produce antitumor immunity-related cytokines. The adoptive transfer of expanded and activated NK cells is expected to be a promising GBM immunotherapy. We previously established an efficient expansion method that produced highly purified, activated primary human NK cells, which we designated genuine induced NK cells (GiNKs). The GiNKs demonstrated antitumor effects in vitro and in vivo, which were less affected by blockade of the inhibitory checkpoint receptor programmed death 1 (PD-1). In the present study, we assessed the antitumor effects of GiNKs, both alone and combined with an antibody targeting killer Ig-like receptor 2DLs (KIR2DL1 and DL2/3, both inhibitory checkpoint receptors of NK cells) in vitro and in vivo with U87MG GBM-like cells and the T98G GBM cell line. Impedance-based real-time cell growth assays and apoptosis detection assays revealed that the GiNKs exhibited growth inhibitory effects on U87MG and T98G cells by inducing apoptosis. KIR2DL1 blockade attenuated the growth inhibition of the cell lines in vitro. The intracranial administration of GiNKs prolonged the overall survival of the U87MG-derived orthotopic xenograft brain tumor model. The KIR2DL1 blockade did not enhance the antitumor effects; rather, it attenuated it in the same manner as in the in vitro experiment. GiNK immunotherapy directly administered to the brain could be a promising immunotherapeutic alternative for patients with GBM. Furthermore, KIR2DL1 blockade appeared to require caution when used concomitantly with GiNKs.

Abstract PO-071: Pseudonormal morphology of adenoid cystic carcinoma cells subverts the antitumor reactivity of immune cells

Abstract Objectives: Adenoid cystic carcinoma (ACC) is a rare salivary gland cancer most frequently arising in the head and neck region. The ACC microenvironment exhibits low immunogenicity (low tumor infiltrating lymphocytes and dendritic cells). This scenario favours immune evasion and partially explains poor prognosis because of the activation of immune inhibitory proteins. Gipie is a hook-related protein, that is involved in protein trafficking. Gipie (HkRP3, encoded by CCDC88B) is associated with T cell maturation and inflammatory functions and target cell killing in natural killer (NK) cells. Gipie is present in ACC patient samples and cells. Therefore, we investigated proliferation, apoptosis, and morphologic changes caused by Gipie in oral and salivary gland cancer cells in presence of immune cells. Methods: We assayed UM-HACC-2A (adenoid cystic carcinoma), A-253 (mucoepidermoid carcinoma, MEC), SCC4, SCC9, SCC25, CAL27 (oral squamous cell carcinoma, OSCC), OKF6 (normal oral) cells cocultured with, NK-92 (natural killer), and Jurkat, Clone E6-1 (acute T cell leukemia) cells, to construct multiple salivary gland and oral cancer-immune cell co-culture models. Gipie silenced cancer cells (silencing maintained for 48 hours) in the cancer immune coculture models were compared with their unaltered subsets. Cancer-immune cells interactions were maintained for 16 hours. Overall three hundred thirty-six (336) co-culture models were used to determine apoptosis, immune activation, morphology, transmigration, and protein expression. Additionally, we performed targeted mass spectrometry analysis to identify changes in protein profiles. Results: ACC cells exhibited Pseudonormal morphology after interacting with immune cells in the 3D coculture model. Gipie-silenced ACC cells in coculture model transformed to “lymphoblast-like” morphology. Gipie-silenced ACC cells in co-culture model (n = 24) showed significant increase of apoptotic cells compared to unaltered ACC cells, lowering of T regulatory cells (FoxP3+/IL-2Rα+/CD25+) (n = 24), and increase of activated NK cells (NKp30+/IFN-γ+) (n = 24) with significantly higher release of granzyme (n = 12) and perforin (n = 12). Increased activation of immune cells (transmigration and fluorescence intensity) via 3D-z-stack images were observed in Gipie-silenced subsets (n = 24). MEC cells responses were significantly less compared to ACC in co-culture models followed by silencing of Gipie, while OSCC and OKF6 cell responses were negligible, under identical conditions. Conclusions: The presence of Gipie confers pseudonormal morphology to ACC cells thereby reducing immune attack. Thus, trafficking proteins may impact ACC cancer-immune cell interaction during conventional chemotherapy or monoclonal immunotherapy. Citation Format: Rajdeep Chakraborty, Charbel Darido, Giuseppe Palmisano, Arthur Chien, Aidan Tay, Thiri Zaw, Shoba Ranganathan. Pseudonormal morphology of adenoid cystic carcinoma cells subverts the antitumor reactivity of immune cells [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-071.

Identifying lupus Patient Subsets Through Immune Cell Deconvolution of Gene Expression Data in Two Atacicept Phase II Studies.

To use cell-based gene signatures to identify patients with systemic lupus erythematous (SLE) in the phase II/III APRIL-SLE and phase IIb ADDRESS II trials most likely to respond to atacicept. A published immune cell deconvolution algorithm based on Affymetrix gene array data was applied to whole blood gene expression from patients entering APRIL-SLE. Five distinct patient clusters were identified. Patient characteristics, biomarkers, and clinical response to atacicept were assessed per cluster. A modified immune cell deconvolution algorithm was developed based on RNA sequencing data and applied to ADDRESS II data to identify similar patient clusters and their responses. Patients in APRIL-SLE (N=105) were segregated into the following five clusters (P1-5) characterized by dominant cell subset signatures: high neutrophils, T helper cells and natural killer (NK) cells (P1), high plasma cells and activated NK cells (P2), high B cells and neutrophils (P3), high B cells and low neutrophils (P4), or high activated dendritic cells, activated NK cells, and neutrophils (P5). Placebo- and atacicept-treated patients in clusters P2,4,5 had markedly higher British Isles Lupus Assessment Group (BILAG) A/B flare rates than those in clusters P1,3, with a greater treatment effect of atacicept on lowering flares in clusters P2,4,5. In ADDRESS II, placebo-treated patients from P2,4,5 were less likely to be SLE Responder Index (SRI)-4, SRI-6, and BILAG-Based Combined Lupus Assessment responders than those in P1,3; the response proportions again suggested lower placebo effect and a greater treatment differential for atacicept in P2,4,5. This exploratory analysis indicates larger differences between placebo- and atacicept-treated patients with SLE in a molecularly defined patient subset.

Dynamic Changes in miRNA Expression during the Generation of Expanded and Activated NK Cells.

Therapies based on allogenic Natural Killer (NK) cells are becoming increasingly relevant, and our laboratory has produced expanded and activated NK (eNK) cells that are highly cytotoxic against several hematological cancers when used alone or in combination with currently approved therapeutic monoclonal antibodies. In order to produce eNK cells, healthy human donor NK cells undergo a 20-day expansion protocol with IL-2, IL-15 and Epstein-Barr virus (EBV)-transformed lymphoblastoid feeder cells. In order to produce an even more potent eNK-based therapy, we must elucidate the changes our protocol produces within healthy NK cells. To understand the post-transcriptional changes responsible for the increased cytolytic abilities of eNK cells, we performed microRNA (miRNA) expression analysis on purified NK cells from day 0 and day 20 of the protocol using quantitative reverse transcription PCR (RT-qPCR). Of the 384 miRNAs profiled, we observed changes in the expression of 64 miRNAs, with especially significant changes in 7 of them. The up-regulated miRNAs of note were miRs-146a, -124, -34a, and -10a, which are key in the regulation of cell survival through the modulation of pro-apoptotic genes such as PUMA. The down-regulation of miRs-199a, -223, and -340 was also detected and is associated with the promotion of NK cell cytotoxicity. We validated our analysis using immunoblot and flow cytometry studies on specific downstream targets of both up- and down-regulated miRNAs such as PUMA and Granzyme B. These results corroborate the functional importance of the described miRNA expression patterns and show the wide variety of changes that occur in eNK cells at day 20.

Early expansion of activated adaptive but also exhausted NK cells during acute severe SARS-CoV-2 infection.

The analysis of immunological parameters during the course of a SARS-CoV-2 infection is of great importance, both to identify diagnostic markers for the risk of a severe course of COVID-19, and to better understand the role of the immune system during the infection. By using multicolor flow cytometry we compared the phenotype of Natural Killer (NK) cells from hospitalized COVID-19 patients during early SARS-CoV-2 infection with samples from recovered and SARS-CoV-2 naïve subjects. Unsupervised high-dimensional analysis of 28-color flow cytometric data revealed a strong enrichment of NKG2C expressing NK cells in response to the acute viral infection. In addition, we found an overrepresentation of highly activated NK cell subsets with an exhausted phenotype. Moreover, our data show long-lasting phenotypic changes within the NK cell compartment that did not completely reverse up to 2 months after recovery. This demonstrates that NK cells are involved in the early innate immune response against SARS-CoV-2.

Perforin and granzyme A release as novel tool to measure NK cell activation in chickens

Natural killer (NK) cells are cytotoxic lymphocytes that are present in the circulation but also in many organs including spleen and gut, where they play an important role in the defense against infections. Interaction of NK cells with target cells leads to degranulation, which results in the release of perforin and granzymes in the direct vicinity of the target cell. Chicken NK cells have many characteristics similar to their mammalian counterparts and based on similarities with studies on human NK cells, surface expression of CD107 was always presumed to correlate with granule release. However, proof of this degranulation or in fact the actual presence of perforin (PFN) and granzyme A (GrA) in chicken NK cells and their release upon activation is lacking. Therefore, the purpose of the present study was to determine the presence of perforin and granzyme A in primary chicken NK cells and to measure their release upon degranulation, as an additional tool to study the function of chicken NK cells. Using human specific antibodies against PFN and GrA in fluorescent and confocal microscopy resulted in staining in chicken NK cells. The presence of PFN and GrA was also confirmed by Western blot analyses and its gene expression by PCR. Stimulation of NK cells with the pectin SPE6 followed by flow cytometry resulted in reduced levels of intracellular PFN and GrA, suggesting release of PFN and GrA. Expression of PFN and GrA reversely correlated with increased surface expression of the lysosomal marker CD107. Finally it was shown that the supernatant of activated NK cells, containing the NK cell granule content including PFN and GrA, was able to kill Escherichia coli. This study correlates PFN and GrA release to activation of chicken NK cells and establishes an additional tool to study activity of cytotoxic lymphocytes in chickens.