Natural Killer Cell-mediated Antibody-dependent Cellular Cytotoxicity Research Articles

Abstract 2722: CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms

Abstract Many therapeutic monoclonal antibodies mediate the immune effector functions through the engagement of their fragment crystallizable (Fc) domain and Fcγ receptors on the immune cells. However, these functions are limited due to the low affinity of their interactions and the requirement of abundancy of antigens for the avidity effects. Recently, mutation-based Fc engineered antibodies showed their enhanced immune effector functions mostly through the NK cell-mediated ADCC. To overcome the limited improvement of current Fc engineering, we developed an innovated platform, Twin Fc-Immune Cell Engager (Twin Fc-ICE) composed of a single Fab domain and two Fc domain with drug-like properties. Here we demonstrated the superior efficacy of CTN001, a first Twin Fc-ICE, in in vitro and in vivo Her2+ tumor models. CTN001 bound to various types of Fcγ receptor-expressing immune cells with stronger affinity, supported by its better in vitro affinity to all types of Fcγ receptors than wild type and Fc-engineered antibodies. To evaluate the pharmacological efficacies, CTN001 was tested for its functions of in vitro antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) and in vivo efficacy with Her2+ xenograft models. In Her2+ models, both CTN001 and trastuzumab induced ADCC-mediated effects, but CTN001 showed better anti-tumor cytotoxicity through ADCP and CDC than trastuzumab. Importantly, CTN001 also showed cytotoxic effects on Her2-low tumor cells. Consistently, CTN001 showed better anti-tumor efficacy than trastuzumab or its combination with pertuzumab in Her2+ tumor xenograft model and, surprisingly, even in Her2-low or resistant models. These results suggest that CTN001 can provide new therapeutic options for treating Her2-low patients differentiated from antibody-drug conjugates (ADCs), and that the Twin Fc-ICE can be an innovative platform to overcome limitations of existing therapeutics. Citation Format: Deokjae Lee, Sujin Yoon, Jiseon Bae, Minju Kim, Ki-June Lee, Eunmi Kim, Hyunkyu Park, Eun S. Choi, Gi-Hyeok Yang. CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2722.

Elotuzumab Enhances CD16-Independent NK Cell-Mediated Cytotoxicity against Myeloma Cells by Upregulating Several NK Cell-Enhancing Genes.

Multiple myeloma (MM) is an intractable hematological malignancy caused by abnormalities in plasma cells. Combination therapy using antibodies and natural killer (NK) effectors, which are innate immune cells with safe and potent antitumor activity, is a promising approach for cancer immunotherapy and can enhance antitumor effects. Elotuzumab (Elo) is an immune-stimulatory antibody that targets the signaling lymphocytic activation molecule family 7 (SLAMF7) expressed on the surface of MM and NK cells. We confirmed that Elo strongly promoted NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against SLAMF7-positive MM cells in a CD16-dependent NK cell line, and also activated expanded NK cells derived from peripheral blood mononuclear cells of healthy donors and patients with MM in the present study. However, the antitumor effects and genes involved in the direct promotion of NK cell-mediated activation using Elo in CD16-independent NK cells are not clearly known. In this study, we demonstrated that Elo pretreatment significantly enhanced CD16-independent NK cell-mediated cytotoxicity in both SLAMF7-positive MM.1S and SLAMF7-negative K562, U266, and RPMI 8226 tumor cells. Upon direct simulation of CD16-independent NK cells with Elo, increased levels of CD107a degranulation and IFN-γ secretion were observed along with the upregulation of granzyme B, TNF-α, and IL-1α gene expression. The enhanced NK cell function could also be attributed to the increased expression of the transcription factors T-BET and EOMES. Furthermore, the augmentation of the antitumor effects of CD16-independent NK cells upon pretreatment with Elo enhanced the expression of CRTAM, TNFRSF9, EAT-2, and FOXP3 genes and reduced the expression of HSPA6. Our results suggest that Elo directly promotes the cytotoxic function of CD16-independent NK cells against target cells, which is associated with the upregulation of the expression of several NK cell-enhancing genes.

A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

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.

AFM13 in Combination with Allogeneic Natural Killer Cells (AB-101) in Relapsed or Refractory Hodgkin Lymphoma and CD30 + Peripheral T-Cell Lymphoma: A Phase 2 Study (LuminICE)

Background and Significance Limited treatment options are available for patients with relapsed or refractory (R/R) Hodgkin lymphoma (HL) and no standard-of-care therapy is established for R/R peripheral T-cell lymphoma (PTCL); novel therapies to improve outcomes in these patients are required. AFM13 is a tetravalent, bispecific innate cell engager that binds CD16A on natural killer (NK) cells and CD30 expressed (CD30 +) on HL and PTCL cells, enhancing NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). AFM13 monotherapy trials in patients with R/R HL and PTCL have shown promising clinical activity and a tolerable safety profile (Sasse et al. Blood 2020; Kim et al. Cancer Res 2023). Recently, a Phase 1/2 study of AFM13 in combination with cord blood (cb)-derived NK cells in patients with R/R CD30 + lymphomas treated at the recommended phase 2 dose (n=35) achieved an objective response rate (ORR) of 94% and a complete response (CR) rate of 71% (Nieto et al. Blood 2022, oral presentation). AB-101 is a non-genetically modified, allogeneic, cryopreserved, off-the-shelf, cb-derived NK cell product optimized for enhanced ADCC through selection for the KIR-B haplotype and the CD16 F158V polymorphism. AB-101 has demonstrated potent killing of tumor cell lines in vitro and in vivo and preliminary results of a Phase 1/2 trial of AB-101 alone and in combination with rituximab in patients with R/R B cell non-Hodgkin lymphoma demonstrated AB-101 is well tolerated (Khanal et al. J Clin Oncol 2023). Combining AFM13 with AB-101 has the potential to synergistically enhance and redirect antitumor immune responses to target HL and CD30 + PTCL cells. Study Design and Methods This Phase 2, open-label, multi-center, multi-cohort study (NCT05883449) aims to evaluate the efficacy and safety of AFM13 in combination with AB-101 in patients with R/R HL and certain R/R CD30 + PTCL subtypes. PTCL subtypes permitted are PTCL not-otherwise specified, angioimmunoblastic T-cell lymphoma, and ALK-positive and -negative anaplastic large cell lymphoma (ALCL). Patients aged ≥18 years are planned for enrolment and patients with R/R HL must have received at least two prior lines of therapy including prior combination chemotherapy, brentuximab vedotin (BV) and a checkpoint inhibitor. Patients with R/R PTCL must have confirmed CD30 expression of ≥1% by immunohistochemistry and have received at least one prior line of combination chemotherapy; patients with ALCL must have received or been intolerant to BV. Prior autologous or allogeneic hematopoietic stem cell transplant is permitted. Exclusion criteria include treatment with any anti-cancer agent ≤21 days prior to enrolment, continuing toxicity from a prior therapy, central nervous system involvement, or previous treatment with AFM13 or NK cells. The primary objective is to determine the ORR (complete and partial responses) by Independent Radiology Committee (IRC) based on PET-CT per Lugano classification. Secondary objectives include safety and immunogenicity, complete response rate, duration of response and progression free survival. Treatment will be given intravenously (IV) over 48-day cycles for up to 3 cycles. A run-in phase will assess two dose levels of AFM13 and AB-101 in 4 cohorts (Figure). A standard lymphodepletion regimen of fludarabine (30 mg/m 2/day) and cyclophosphamide (300 mg/m 2/day) will be administered IV from Day −5 to Day −3 at the start of each treatment cycle. Following this, AFM13 (200 mg or 300 mg once weekly) will be given, with AB-101 (dose level 1 or 2, see Figure) given 1 hour later per cycle. Patients will also receive 6 ×10 6 IU of IL-2 subcutaneously at least 1 hour after each AB-101 dose. Cohorts 1 and 2 will enrol in parallel; cohorts 3 and 4 will start only if cohorts 1 and 2 are cleared per protocol safety criteria. After cycle 1 has completed for each subject enrolled in all 4 cohorts, an analysis of the safety and clinical responses will be performed to determine the two dose levels to be evaluated in the main study. In addition, an exploratory cohort (cohort 5) will begin enrolment of patients with CD30 + PTCL. Disease and efficacy assessments will be conducted at screening and on Day 43 (± 3 days) of each cycle.

AFM24 in combination with autologous NK cells (SNK01) in patients with advanced/metastatic epidermal growth factor receptor (EGFR) expressing solid tumors: Initial results from the phase 1 dose-escalation study.

26 Background: AFM24 is a novel tetravalent bispecific IgG1-scFv fusion antibody (scFv-IgAb) binding specifically to EGFR on target cells and CD16A on NK cells, mediating specific lysis of EGFR+ tumor cells via NK cell-mediated antibody-dependent cellular cytotoxicity. SNK01 cell therapy is an NK cell-derived adoptive cellular immunotherapy technology that can produce large scale ex vivo expanded and activated autologous NK cells (1,000–10,000-fold expansion of NK cells with >95% cell purity), even in patients with low primary NK cell counts. The antitumor activity of SNK01 may be enhanced when combined with AFM24. Methods: This is a proof-of-concept phase 1, 3+3 dose escalation study to determine the maximum tolerated dose and/or recommended phase 2 dose of AFM24 in combination with SNK01 (NCT05099549). Secondary endpoints are safety/tolerability, preliminary efficacy and pharmacokinetics (PK). Patients with advanced or metastatic, histologically or cytologically confirmed EGFR+ solid tumors (≥1% of positive tumor cells) refractory to standard-of-care therapies were enrolled. AFM24 starting dose was 160 mg followed by 480 mg. AFM24 is administered once weekly, concomitantly with 4.0 x109 SNK01 cells once weekly until disease progression, intolerable toxicity, patient withdrawal. Cycles are 28 days and CTs are at screening, cycle 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of January 27, 2023, 6 patients (5 microsatellite stable colorectal and 1 head and neck cancer) received combination therapy, 3 in each cohort. Median (range) age was 59 (41–64), mean number (range) of prior therapies was 5 (3–7). ECOG performance status was 0 for 3 patients and 1 for the other 3. No dose limiting toxicities or unexpected toxicities were observed. Preliminary results suggest consistent safety events with both drugs in monotherapy. The most frequent treatment-emergent adverse events related to AFM24 was Grade 1/2 infusion related reactions occurring in 5/6 patients. There were no Grade ≥3 events related to AFM24 or SNK01. In the low dose cohort (160 mg), 2 patients had stable disease for 106 days (15 weeks) and 100 days (14 weeks), respectively. At 480 mg, 1 patient with SD had 2% tumor burden reduction and 2 subsequent carcinoembryonic antigen reductions. The patient proceeded to Cycle 3. No difference in PK data was observed in comparison to AFM24 alone. Enrollment continues at 480 mg. Conclusions: In this first study combining a bispecific NK cell engager with autologous NK cell infusions; two dose cohorts have completed, and escalation has proceeded. Early PK data are consistent with AFM24 monotherapy with no new safety signals, suggesting that the combination of AFM24 and SNK01 cells is tolerated and warrants further investigation. Clinical trial information: NCT05099549 .

Antitumor effects of NK cells expanded by activation pre‑processing of autologous feeder cells before irradiation in colorectal cancer.

Natural killer (NK) cells play a crucial role in early immune defenses against transformed cells and are used in the therapeutic management of cancer. However, it is difficult to sufficiently obtain high purity activated NK cells for clinical application. The function of NK cells is dependent on the balance of activating and inhibitory signals. Strong and diverse stimuli are required to increase the function of NK cells. Radiotherapy modulates the expression of various immunomodulatory molecules that recruit and activate NK cells. NK cell-mediated antibody-dependent cellular cytotoxicity is one of the most potent cytotoxic effects of NK cells against target cancer cells. To generate activated and irradiated autologous peripheral blood mononuclear cells (PBMCs), cytokine and monoclonal antibody stimulation followed by ionizing radiation was performed in the present study. The expanded NK cells were cultured for 21 days using activated/irradiated autologous PBMCs. Colorectal cancer cells (SW480 and HT-29) were used to analyze the expression of NK group 2D ligands and EGFR by radiation. The cytotoxicity of radiation plus NK cell-based targeted therapy against colorectal cancer cell lines was analyzed using flow cytometry. Activated and irradiated PBMCs exhibited significantly increased expression of various activating ligands that stimulated NK cells. In total, >10,000-fold high-purity activated NK cells were obtained, with negligible T-cell contamination. To confirm the antitumor activity of the NK cells expanded by this method, the expanded NK cells were treated with cetuximab, radiotherapy, or a combination of cetuximab and radiotherapy in the presence of human colorectal cancer cells. Expanded NK cells were effective at targeting human colorectal cancer cells, particularly when combined with cetuximab and radiotherapy. Thus, in the present study, a novel method for high-purity activated NK cell expansion was developed using activated and irradiated PBMCs. In addition, combined radiotherapy and antibody-based immunotherapy with expanded NK cells may be an effective strategy to enhance the efficiency of treatment against colorectal cancer.

Single-Cell Transcriptomic Analysis Reveals Reduction of Cytotoxic NK Cells in a Subset of Newly Diagnosed Multiple Myeloma Patients Impacting Outcome after Daratumumab Therapy

Introduction Anti-CD38 antibody-based therapies in multiple myeloma (MM) rely to a large degree on natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Classically, NK cells are divided into cytotoxic CD56dim NK cells, important for ADCC, and cytokine-producing CD56bright NK cells. Accumulating evidence in other types of cancer suggests that alterations in the balance between CD56dim and CD56bright NK cells is associated with inferior clinical outcome. We hypothesized that changes in NK cell subset composition in the MM microenvironment would impact therapy-responses during NK cell-driven immunotherapies. Here, we used single-cell RNA sequencing and flow cytometry to investigate the heterogeneity of the NK cell compartment in the bone marrow (BM) of newly diagnosed MM (NDMM) patients, and the effects of altered NK cell subset composition on therapy response in patients undergoing first-line daratumumab-containing therapy. Results We performed single-cell RNA sequencing of the BM CD38+ immune cell compartment from 19 NDMM patients. NK cells were identified in silico resulting in a single-cell transcriptomic dataset of 24,664 NK cells. UMAP dimensionality reduction identified 5 transcriptomic distinct NK cell clusters, present in all NDMM patients. Conventional CD56bright and CD56dim NK cells were identified by increased transcription of GZMK or GZMB, respectively. To study the balance of cytotoxic vs cytokine-producing NK cells in MM we compared the relative abundance of cytokine-producing GZMK+CD56bright NK cells vs cytotoxic GZMB+CD56dim NK cells in individual patients in our cohort. The MM BM was characterized by heterogeneity in the ratio of cytotoxic vs cytokine-producing NK cell cluster composition, in which most patients had a predominance of cytotoxic NK cells, while a subset of patients presented with a relative decrease in cytotoxic NK cell clusters (Figure 1A). The reduction in cytotoxic NK cell subsets in this patient subgroup was reflected in an altered overall transcriptome of the total BM NK cell population, with enrichment for inhibitory receptors such as KLRB1 and KLRC1, and a relative loss of activating receptors such as FCGR3A (CD16), NCR3 (NKp30) and CD226 (DNAM-1), and cytotoxic effector genes including NKG7 and GNLY. To define the relative abundance of a reduced cytotoxic NK cell potential in NDMM patients, we performed flow cytometric analyses of 246 NDMM patients from 2 independent clinical cohorts. This revealed a reduced frequency of cytotoxic CD56dim NK cells, defined as < 90% CD56dim NK cells, in 17.9 % of 162 transplant-eligible treatment-naïve NDMM and in 28.6% of 84 treatment-naïve frail or unfit transplant ineligible MM patients. To test whether such relative decrease in cytotoxic NK cells would impact responses to daratumumab, we tested the association of BM NK cell composition with clinical outcome in the context of first-line daratumumab therapy in a cohort of frail NDMM patients ineligible for transplant from the HO143 trial (n=43). NDMM patients with reduced frequencies of BM cytotoxic NK cells presented with significantly shorter progression-free survival (PFS) (Figure 1B, PFS, p = 0.031, log-rank test), while OS was not impacted (p = 0.18, log-rank test). Univariate Cox regression analyses confirmed the correlation between reduced frequencies of CD56dim NK cells in the BM and significantly shorter PFS (hazard ratio (HR) 1.38; 95% confidence interval: 1.057 - 1.803; P = 0.018). Whether reduced frequencies of cytotoxic CD56dim NK cells affect outcome in transplant-eligible patients warrants additional study. Conclusion Here we show that about 20% of NDMM patients have an altered BM NK cell composition with a relative reduction in cytotoxic NK cells. Such altered NK cell composition correlates with significantly shorter PFS following daratumumab-containing therapy in a cohort of frail NDMM patients. Our data suggest that defining BM NK cell composition could stratify patients and identify a subgroup less likely to benefit from therapies driven by NK cell-mediated ADCC. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The combination of CD16A/EGFR innate cell engager, AFM24, with SNK01 autologous natural killer cells in patients with advanced solid tumors.

TPS2675 Background: Natural killer (NK) cells are a critical component of the innate immune system involved in the eradication of transformed cells via antibody-dependent cellular cytotoxicity (ADCC). For treatment of solid tumors autologous NK cell transfer represents a promising treatment strategy, with ex vivo expansion and activation enhancing the specificity and anti-tumor activity of NK cells. The efficacy of this approach may be enhanced through the addition of tumor-targeting antibodies, augmenting NK cell-mediated ADCC. Innate Cell Engagers (ICE) are bispecific antibodies that target a tumor cell-surface antigen and bind to CD16A expressed on NK cells. AFM24 is a novel ICE that targets epidermal growth factor receptor (EGFR), which is often overexpressed in solid tumors. The Phase 1 study of AFM24 monotherapy showed patients had a manageable safety profile, and SNK01 monotherapy has also shown to be well-tolerated in patients with rapidly progressive solid tumors. This study seeks to investigate AFM24 in combination with SNK01 autologous NK cells in patients with advanced EGFR+ solid tumors. Methods: An ongoing Phase 1/2a open-label, non-randomized, multicenter, dose escalation (Phase 1) and dose expansion (Phase 2a) study was initiated in November 2021 (NCT05099549) to evaluate the safety, tolerability and efficacy of AFM24 in combination with SNK01 NK cells in EGFR+ solid tumors. The primary aim of the Phase 1 study is to determine the maximum tolerated dose and/or recommended Phase 2 dose (RP2D) of AFM24 in combination with SNK01 at a fixed dose NK cells using a standard 3+3 design. Eligible patients must have advanced or metastatic disease with positive immunohistochemical staining for EGFR in &gt;1% of tumor cells and be refractory to standard-of-care treatment. Treatment begins with a safety lead-in phase with a single dose of AFM24 7 days prior to combination therapy. AFM24 will be administered at an escalating dose as weekly intravenous (IV) infusions; the starting dose (160 mg) and dose escalations for each cohort are based on results from the ongoing AFM24 monotherapy trial (NCT04259450). SNK01 NK cells will be given at a fixed dose (4.0 x109 cells) as a weekly IV infusion concomitantly with AFM24. Patients will receive combination therapy until disease progression, intolerable toxicity, patient withdrawal or termination at the investigator’s discretion. Phase 2 will then establish the overall response rate (as per RECIST v1.1) of combination therapy in patients with treatment refractory, advanced or metastatic squamous cell carcinoma of the head and neck, non-small cell lung cancer, or colorectal cancer as the primary endpoint. Efficacy will also be assessed by assessing progression-free and overall survival. Secondary endpoints for both phases include treatment-emergent adverse events, serious adverse events, pharmacokinetics and immunogenicity. Clinical trial information: NCT05099549.

High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

PurposeHost genetic variants in activating natural killer (NK) cell receptors may contribute to differences in severity of COVID-19. NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) responses play, however, a controversial role in SARS-CoV-2 infections. It is unclear whether proinflammatory and cytotoxic SARS-CoV-2-specific ADCC responses limit disease severity or rather contribute to the immunopathogenesis of severe COVID-19. MethodsUsing a genetic association approach and subsequent in vitro antibody-dependent NK cell activation experiments, we investigated whether genetic variants in the FcγRIIIa-encoding FCGR3A gene, resulting in expression of either a low-affinity or high-affinity variant, and individual SARS-CoV-2-specific ADCC response contribute to COVID-19 severity. ResultsIn our study, we showed that the high-affinity variant of the FcγRIIIa receptor, 158-V/V, is significantly over-represented in hospitalized and deceased patients with COVID-19, whereas the low-affinity FcγRIIIa-158-F/F variant occurs more frequently in patients with mild COVID-19 (P < .0001). Furthermore, functional SARS-CoV-2 antibody-specific NK cell-mediated ADCC assays revealed that significantly higher proinflammatory ADCC responses occur in hospitalized patients with COVID-19, and are especially observed in NK cells expressing the FcγRIIIa-158-V/V variant (P < .0001). ConclusionOur study provides evidence that pronounced SARS-CoV-2-specific NK cell-mediated ADCC responses are influenced by NK cell FcγRIIIa genetic variants and are a hallmark of severe COVID-19.

Natural killer cell-mediated ADCC in SARS-CoV-2-infected individuals and vaccine recipients.

COVID‐19, caused by SARS‐CoV‐2, has emerged as a global pandemic. While immune responses of the adaptive immune system have been in the focus of research, the role of NK cells in COVID‐19 remains less well understood. Here, we characterized NK cell‐mediated SARS‐CoV‐2 antibody‐dependent cellular cytotoxicity (ADCC) against SARS‐CoV‐2 spike‐1 (S1) and nucleocapsid (NC) protein. Serum samples from SARS‐CoV‐2 resolvers induced significant CD107a‐expression by NK cells in response to S1 and NC, while serum samples from SARS‐CoV‐2‐negative individuals did not. Furthermore, serum samples from individuals that received the BNT162b2 vaccine induced strong CD107a expression by NK cells that increased with the second vaccination and was significantly higher than observed in infected individuals. As expected, vaccine‐induced responses were only directed against S1 and not against NC protein. S1‐specific CD107a responses by NK cells were significantly correlated to NK cell‐mediated killing of S1‐expressing cells. Interestingly, screening of serum samples collected prior to the COVID‐19 pandemic identified two individuals with cross‐reactive antibodies against SARS‐CoV‐2 S1, which also induced degranulation of NK cells. Taken together, these data demonstrate that antibodies induced by SARS‐CoV‐2 infection and anti‐SARS‐CoV‐2 vaccines can trigger significant NK cell‐mediated ADCC activity, and identify some cross‐reactive ADCC‐activity against SARS‐CoV‐2 by endemic coronavirus‐specific antibodies.

Small extracellular vesicles induce resistance to anti-GD2 immunotherapy unveiling tipifarnib as an adjunct to neuroblastoma immunotherapy

BackgroundAnti-GD2 monoclonal antibody immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to treatment, and the molecular...

Abstract 1856: Donor and antibody diversity in NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) detected using an optimized multiplexed assay and advanced flow cytometry

Abstract Tumor-specific monoclonal antibodies (mAb) which induce NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) have proven to be successful against several types of cancer. Evaluating the potency of a new, tumor-specific mAb requires testing with multiple sources of donor cells since the degree of NK cell-mediated ADCC is known to be variable between patients. Thus, in addition to evaluating whether a new tumor-specific mAb can effectively induce NK cell-mediated ADCC, it is also important to characterize the donor NK cells utilized in order to provide insight into potential treatment efficacy. In the current study, we investigated the application of a novel, high throughput assay in assessing the ADCC potency of different anti-hCD20 mAbs against a Burkitt's lymphoma cell line using effector cells obtained from multiple donors. This multiplexed assay incorporates a cells and beads format to enable the simultaneous measurement of target cell killing, along with enumeration and phenotyping of the NK cells, and quantification of up to 8 secreted effector proteins and cytokines. The assay requires a sample volume of just 10 µl and can be performed in a single well of a 96 or 384 well plate using the iQue® platform for advanced flow cytometry. Data analysis and visualization was done using the integrated iQue Forecyt® software package. The results showed a mAb concentration-dependent increase in tumor cell killing, along with corresponding increases in secretion of Granzyme B and CD178 (Fas Ligand), and production of IFNγ, TNF, and RANTES. The degree of NK cell-mediated ADCC and effector protein secretion levels were dependent on both the anti-hCD20 mAb and donor cells used, with higher levels of ADCC and effector protein secretion observed using a non-fucosylated anti-hCD20 mAb as compared to a conventional, fucosylated anti-hCD20 mAb. This method enabled the rapid assessment of the potency of different mAbs in ADCC mediated by NK cells, and also effectively distinguished potential differences in mAb efficacy between donors. Citation Format: Julie Lovchik, Mark Carter. Donor and antibody diversity in NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) detected using an optimized multiplexed assay and advanced flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1856.

Elotuzumab Plus Lenalidomide/Dexamethasone (ELd) Vs Ld in Patients with Newly Diagnosed Multiple Myeloma: Phase 2, Randomized, Open-Label Study in Japan

Introduction: Elotuzumab, a humanized IgG1 monoclonal antibody that binds to SLAMF7 expressed on myeloma and natural killer (NK) cells, has a dual mode of action; it causes targeted myeloma cell death by directly activating NK cells and enhances NK cell-mediated antibody-dependent cellular cytotoxicity. Elotuzumab combined with lenalidomide and dexamethasone (ELd) has been approved in Japan for the treatment of relapsed/refractory multiple myeloma (RRMM). In a 4-y follow-up of the phase 3 ELOQUENT-2 study (NCT01239797) in patients (pts) with RRMM, ELd demonstrated a sustained 29% reduction in risk of disease progression/death and an overall survival benefit vs Ld (Dimopoulos M et al. Haematologica 2017), consistent with prior reports (Dimopoulos M et al. Br J Haematol 2017). We present the first report on the efficacy/safety of ELd vs Ld in pts with newly diagnosed multiple myeloma (NDMM).Methods: In this phase 2, open-label, multicenter study (NCT02272803) in Japan enrolling pts with NDMM ineligible for high-dose chemotherapy plus hematopoietic cell transplantation, pts were randomized 1:1 to intravenous elotuzumab (10 mg/kg weekly for Cycles 1 and 2; every 2 wk for Cycles 3-18; 20 mg/kg monthly thereafter) plus Ld or Ld alone, in a 28-d cycle until disease progression or unacceptable toxicity. All pts in the ELd arm received premedication prior to elotuzumab to mitigate infusion reactions (IRs). In the absence of IRs, infusion rate was increased from 0.5-2.0 mL/min at dose 1, and up to a maximum of 5 mL/min (per FDA/EMA labels for ELd in RRMM) by dose 3 of Cycle 1. Primary endpoint was investigator-assessed objective response rate (ORR) with ELd, per International Myeloma Working Group criteria (1-sided α=0.15 for a true response rate >71%). Secondary endpoints included difference in ORR between the arms and progression-free survival (PFS). Safety was an exploratory endpoint.Results: In total, 40 and 42 pts received ELd and Ld, respectively. At database lock (March 2017; minimum follow-up of 6 mo), 31 (78%) pts in the ELd arm and 25 (60%) in the Ld arm remained on study. At baseline, median (range) age was 72 y (65-83) in the ELd arm and 73 y (62-89) in the Ld arm. Median duration of therapy was 13.0 cycles with ELd and 11.5 cycles with Ld. The most common reason for discontinuation was: ELd (9 pts)-disease progression (4/40 pts [10%]); Ld (17 pts)-drug toxicity, adverse events (AEs) unrelated to study drug, and pt request (each with 4/42 pts [10%]). The primary endpoint was met: investigator-assessed ORR (Table) was 88% with ELd; lower bound of the 2-sided 70% CI in ORR was 80% (> threshold of 71%). The ORR in the LD arm was 74%. ORR was 13% numerically higher with ELd than Ld. Very good partial response or better was observed for 45% of ELd- and 29% of Ld-treated pts. AEs were reported in 40 (100%) and 41 (98%) pts in the ELd and Ld arms, respectively. The most common (≥20%) AEs for ELd vs Ld included constipation (43% vs 31%), pyrexia (35% vs 7%), rash (28% vs 36%), diarrhea (28% vs 21%), nasopharyngitis (25% vs 26%), dysgeusia (23% vs 19%), malaise (23% vs 2%), peripheral edema (20% vs 14%), neutropenia (20% vs 14%), leukopenia (20% vs 7%), and back pain (18% vs 21%). Grade (G) 3-4 AEs occurred in 31 (78%) and 19 (45%) pts in the ELd and Ld arms, respectively. The most common G3-4 AEs (ELd vs Ld) were neutropenia (18% vs 7%) and leukopenia (15% vs 0%). One IR was reported (ELd): G2 hypersensitivity during the first infusion (2-mL/min infusion rate); elotuzumab infusion was interrupted; the pt did not experience any IRs at subsequent infusions. Thirty-eight pts (95%) received elotuzumab at 5 mL/min (representing 1046/1176 [89%] infusions) and no IRs were reported at this increased infusion rate. Secondary primary malignancies were reported in 1 pt in each arm (of the rectum and lung with ELd and Ld, respectively). ELd had a comparable safety profile when given at 10 mg/kg weekly or every 2 wk up to Cycle 18, or at 20 mg/kg monthly from Cycle 19 onwards. PFS data are not yet mature.Conclusions: In this first report of ELd treatment of pts with NDMM, the primary endpoint of ORR was met. ELd had an acceptable safety profile in this pt population, and an increased infusion rate of 5 mL/min, which reduced infusion time and did not induce additional IRs. These data suggested that ELd with a faster infusion rate of 5 mL/min was effective and well tolerated in Japanese pts with NDMM.Study support: Bristol-Myers Squibb K.K. [Display omitted] DisclosuresOhta:Bristol-Meyer Squibb K.K., Celgene K.K., Jansen Pharmaceutical K.K., Novartis Pharma K.K., Ono Pharmaceutical Co. Ltd, Takeda Pharmaceutical Co. Ltd: Honoraria. Hori:Celgene, Fujifilm RI Pharma, Shire Japan: Other: Post-marketing surveillance study fee. Kinoshita:Bristol-Myers Squibb K.K.: Employment. Shelat:Bristol-Myers Squibb: Employment, Equity Ownership. Miyoshi:Bristol-Myers Squibb K.K.: Employment.

Balancing the CD38 Expression on Effector and Target Cells in Daratumumab-Mediated NK Cell ADCC against Multiple Myeloma.

Simple SummaryWe tracked the cytotoxic potential of NK cells towards multiple myeloma cells in daratumumab-mediated antibody-dependent cellular cytotoxicity assays. These cytotoxicity levels could be directly correlated to the expression of the target antigen (CD38) and to the percentage of fratricide between effector cells. Increasing the expression of CD38 on target cells or neutralizing CD38 on effector cells changed the equilibrium between target and effector cell lysis and promoted multiple myeloma cell death. This study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated cellular toxicity. Multiple myeloma (MM) is an incurable cancer characterized by the proliferation and accumulation of monoclonal plasma cells in the bone marrow. The monoclonal anti-CD38 daratumumab has taken a central place in the different treatment regimens for newly diagnosed and relapsed, refractory myeloma. In this study, we correlated the NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and potential fratricide induced by daratumumab with CD38-expression levels on both effector and target cells. We show that CD38 expression can be modulated by adding all-trans retinoic acid (ATRA) or interferon-α to MM cells to further fine-tune these effects. In addition, we observed that ADCC becomes inefficient when fratricide occurs and both ADCC and fratricide depend on the balance between CD38 expression on effector and target cells. However, the addition of adjuvants (retinoic acid or interferon-α) to myeloma cells or the inhibition of fratricide using a CD38-blocking nanobody on NK-cells can reverse this balance towards ADCC and thus promote lysis of target cells by ADCC. ATRA and interferon-α increased the CD38 expression at the surface of MM cells about three-fold and two-fold, respectively. This increase was of interest for MM cells with low CD38 expression, that became susceptible to daratumumab-mediated ADCC after preincubation. A CD38-blocking nanobody prevented the binding of daratumumab to these NK-cells and blunted the fratricidal effect on effector NK cells. In conclusion, our study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated ADCC.

Development of a Macrophage-Based ADCC Assay.

Fc-dependent effector functions are an important determinant of the in vivo potency of therapeutic antibodies. Effector function is determined by the combination of FcRs bound by the antibody and the cell expressing the relevant FcRs, leading to antibody-dependent cellular cytotoxicity (ADCC). A number of ADCC assays have been developed; however, they suffer from limitations in terms of throughput, reproducibility, and in vivo relevance. Existing assays measure NK cell-mediated ADCC activity; however, studies suggest that macrophages mediate the effector function of many antibodies in vivo. Here, we report the development of a macrophage-based ADCC assay that relies on luciferase expression in target cells as a measure of live cell number. In the presence of primary mouse macrophages and specific antibodies, loss of luciferase signal serves as a surrogate for ADCC-dependent killing. We show that the assay functions for a variety of mouse and human isotypes with a model antigen/antibody complex in agreement with the known effector function of the isotypes. We also use this assay to measure the activity of a number of influenza-specific antibodies and show that the assay correlates well with the known in vivo effector functions of these antibodies.

T cells, particularly activated CD4+ cells, maintain anti-CD20-mediated NK cell viability and antibody dependent cellular cytotoxicity

Anti-CD20 monoclonal antibody (mAb) therapy is a mainstay of therapy for B cell malignancies, however many patients fail to respond or eventually develop resistance. The current understanding of mechanisms responsible for this resistance is limited. When peripheral blood mononuclear cells of healthy donors were cultured with Raji cells for 7 days, rituximab (RTX) induced NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), enhanced NK cell viability and increased or maintained NK expression of CD56, CD16, CD57 and KIR. T cells, mainly CD4+, mediated these changes in a contact-dependent manner, with local T cell production of IL2 playing a central role. Similar findings were found when autologous B cells were used as target cells demonstrating the need for T cell help was not due to allogenic reaction. Results with other anti-CD20 and anti-EGFR antibodies were consistent. Small numbers of T cells activated by anti-CD3/CD28 beads or bispecific antibody enhanced RTX-mediated NK cell ADCC, viability and phenotypical changes. Pathway analysis of bulk NK cell mRNA sequencing after activation by RTX with and without T cells was consistent with T cells maintaining the viability of the activated NK cells. These findings suggest T cell help, mediated in large part by local production of IL2, contributes to NK cell ADCC and viability, and that activating T cells in the tumor microenvironment, such as through the use of anti-CD3 based bispecific antibodies, could enhance the efficacy of anti-CD20 and other mAb therapies where NK-mediated ADCC is a primary mechanism of action.

Novel TLR 7/8 agonists for improving NK cell mediated antibody-dependent cellular cytotoxicity (ADCC)

There is a significant interest in designing therapeutic agents that can enhance ADCC and thereby improve clinical responses with approved antibodies. We recently reported the combination of an imidazoquinoline-based TLR7/8 agonist (522) with a monoclonal antibody improved ADCC in vitro and in vivo. In the present study, we tested several new small molecule TLR7/8 agonists that induce significantly higher cytokines compared to both the FDA-approved TLR7 agonist, imiquimod, and 522. We evaluated these agonists in combination with monoclonal antibody therapy, with the main goal of enhancing ADCC. Our studies show these TLR7/8 agonists induce robust pro-inflammatory cytokine secretion and activate NK cells. Specifically, we found the agonists 574 and 558 significantly enhanced NK cell-mediated ADCC in vitro as well as enhanced the anti-cancer efficacy of monoclonal antibodies in two different in vivo mouse models. Additionally, we found the agonists were able to stimulate CD8 T cells, likely indicative of an early adaptive immune response.

Whole-exome sequencing in cervical adenocarcinoma in mainland Chinese patients.

BackgroundCervical cancer is the most common gynecological malignancy worldwide. Adenocarcinoma is an important pathological type of cervical cancer. In recent years, the incidence of adenocarcinoma is rising in some countries and the prognosis of it remains poor. A precise description of the mutational landscape in cervical adenocarcinoma may provide insights into a better selection of treatments and improve prognosis.MethodsIn this study, we conducted whole-exome sequencing (WES) for cervical adenocarcinomas and matched blood samples from a cohort of 24 mainland Chinese patients. Additionally, the Human-Papilloma virus (HPV) infection statuses of these tumor samples were detected, and the genes that were enriched in both HPV positive and negative samples were also analyzed.ResultsThe results of WES revealed the gene expression profile of cervical adenocarcinoma of women in mainland China and identified multiple genes/pathways, which are frequently mutated in these tumors, including the PI3K-AKT (KRAS, PIK3CA and PTEN), estrogen signaling (KRAS, PIK3CA and GNAS) and NK cell-mediated antibody-dependent cellular cytotoxicity pathways. Besides, seven patients had HPV infection, and the mutated genes in HPV-positive tumor tissues were relatively consistent, while the mutation profiles of HPV-negative tumor tissues were relatively scattered.ConclusionsTaken together, these findings provide novel insights into the pathogenesis of cervical adenocarcinomas. They suggest the potential for individualized treatment of cervical adenocarcinoma according to genomic information.

Improved Detection of in vivo Human NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity Using a Novel NOG-FcγR-Deficient Human IL-15 Transgenic Mouse.

We generated an NOD/Shi-scid-IL2Rγnull (NOG) mouse deficient for the Fcer1g and Fcgr2b genes (NOG-FcγR−/− mice), in which monocytes/macrophages do not express activating (FcγRI, III, and IV) or inhibitory (FcγRIIB) Fcγ receptors. Antibody-dependent cellular cytotoxicity (ADCC) by innate immune cells was strongly reduced in this strain. Thus, while the growth of xenogeneic human tumors engrafted in conventional NOG mice was suppressed by innate cells upon specific antibody treatment, such growth inhibition was abrogated in NOG-FcγR−/− mice. Using this novel strain, we further produced NOG-FcγR−/−-mice expressing human IL-15 (NOG-FcγR−/−-hIL-15 Tg). The mice inherited unique features from each strain, i.e., the long-term sustenance of human natural killer (NK) cells, and the elimination of mouse innate cell-mediated ADCC. As a result, segregation of human NK cell-mediated ADCC from mouse cell-mediated ADCC was possible in the NOG-FcγR−/−-hIL-15 Tg mice. Our results suggest that NOG-FcγR−/−-hIL-15 Tg mice are useful for validating the in vivo function of antibody drug candidates.