Antibody-dependent Cellular Cytotoxicity Assays Research Articles

The relevance of tumor target expression levels on IgA-mediated cytotoxicity in cancer immunotherapy

Recent advances in cancer immunotherapy, particularly the success of immune checkpoint inhibitors, have reignited interest in targeted monoclonal antibodies for immunotherapy. Antibody therapies aim to minimize on-target, off-tumor toxicity by targeting antigens overexpressed on tumor cells but not on healthy cells. Despite considerable efforts, some therapeutic antibodies have been linked to dose-limiting side effects. Our hypothesis suggests that the efficacy of IgG leads to a lower target expression threshold for tumor cell killing, contributing to these side effects. Earlier, therapeutic IgG antibodies were reformatted into the IgA isotype. Unlike IgG, which primarily engages Fc gamma receptors (FcγR) to induce antibody-dependent cellular cytotoxicity (ADCC) by NK cells and antibody-dependent cellular phagocytosis (ADCP) by monocytes/macrophages, IgA antibodies activate neutrophils through the Fc alpha receptor I (CD89, FcαRI). In previous studies, it appeared that IgA may require a higher target expression threshold for effective killing, and we aimed to investigate this in our current study. Moreover, we investigated how blocking the myeloid checkpoint CD47/SIRPα axis affect the target expression threshold. Using a tetracycline-inducible expression system, we regulated target expression in different cell lines. Our findings from ADCC assays indicate that IgA-mediated PMN ADCC requires a higher antigen expression level than IgG-mediated PBMC ADCC. Furthermore, blocking CD47 enhanced IgA-mediated ADCC, lowering the antigen threshold. Validated in two in vivo models, our results show that IgA significantly reduces tumor growth in high-antigen-expressing tumors without affecting low-antigen-expressing healthy tissues. This suggests IgA-based immunotherapy could potentially minimize on-target, off-tumor side effects, improving treatment efficacy and patient safety.

A Novel Tetravalent Bispecific Immune Cell Engager Activates Natural Killer Cells to Kill Cancer Cells without Mediating Fratricide.

We previously reported the structure, affinity, and anticancer activity of a bivalent bispecific natural killer cell engager (BiKE) composed of one anti-CD16a VHH and one anti-HER2 VHH fused via a linker. In this study, we explored the engineering of a tetravalent BiKE by fusing two anti-CD16a and two anti-HER2 VHHs in tandem, using bivalent BiKE as a template. The tetravalent BiKE was genetically engineered, and its tertiary structure was predicted using in silico modeling. The antigen binding and affinity of the tetravalent BiKE were assessed using ELISA, flow cytometry, and biolayer interferometry. The ability of the BiKEs to kill cancer cells was evaluated through classical and residual antibody-dependent cellular cytotoxicity (ADCC) assays. Additionally, we investigated the potential for NK cell fratricide via CD16a-CD16a crosslinking. Our results revealed that the tetravalent BiKE exhibited at least 100-fold higher affinity toward its target antigens compared to its bivalent counterpart. The residual ADCC assay indicated that the tetravalent BiKE was more effective in killing cancer cells than the bivalent BiKE, attributable to its lower Koff value, which prolonged its binding to NK cell surfaces. Fratricide assays demonstrated that neither the bivalent nor the tetravalent BiKE mediated fratricide. Notably, our findings showed that daratumumab-induced NK fratricide was restricted to CD38-CD38 crosslinking and was not related to ADCC via CD16a-CD38 crosslinking. This study is the first in the literature to show the successful engineering of a tetravalent immune cell engager composed of tandem VHH units, which achieves high affinity and anticancer activity without mediating fratricide.

Sex Differences during Influenza A Virus Infection and Vaccination and Comparison of Cytokine and Antibody Responses between Plasma and Serum Samples.

In this study, we evaluated sex differences during infection with mouse-adapted H1N1 and H3N2 influenza A viruses (IAVs) in the C57BL/6J mouse model and compared the cytokine and antibody responses between plasma and serum samples during IAV infection and vaccination. Lethal doses for both H1N1 and H3N2 IAVs were lower for adult females and they suffered with greater morbidity than adult males when infected with sublethal doses. In influenza virus-infected mice, cytokine responses differed between plasma and serum samples. After inactivated influenza virus vaccination and drift variant challenge, adult female mice had greater antibody responses and were better protected. In influenza-vaccinated and challenged mice, binding antibodies were unaffected between paired plasma or serum samples. However, functional antibody assays, including hemagglutination inhibition, microneutralization, and antibody-dependent cellular cytotoxicity assays, were affected by the use of plasma and serum sample types. Our results indicate that careful consideration is required while selecting plasma versus serum samples to measure cytokine and antibody responses during IAV infection and vaccination.

Potent and specific killing of SLE donor B Cells with AlloNK (AB-101), an allogeneic cord blood-derived NK cell therapy, in combination with anti-CD19 or anti-CD20 monoclonal antibodies

Abstract B cells play a crucial role in the pathogenesis of SLE by producing autoantibodies that target self-antigens, leading to tissue damage and inflammation. Here we demonstrate proof of concept for the use of a NK cell therapy to enhance the depletion of SLE donor B cells in the presence of anti-CD19 or anti-CD20 antibodies by an antibody dependent cellular cytotoxicity (ADCC) mechanism. AlloNK™ is a non-engineered, allogeneic, off-the-shelf, cryopreserved NK cell product, currently being evaluated in a clinical trial in combination with rituximab in subjects with lupus nephritis. AlloNK, which has been optimized for ADCC, was tested in combination with anti-CD20 (rituximab, obinutuzumab) or anti-CD19 (tafasitamab) mAbs in a short-term ADCC assay to show specific killing of SLE donor B cells. PBMC were isolated from SLE donors and co-cultured with AlloNK at various effector to target (E:T) ratios and mAbs concentrations. After 4h, the percentage of caspase 3/7+ B and T cells was determined by flow cytometry. At a 1:1 E:T ratio, AlloNK mediated killing of B cells in the presence of obinutuzumab (range 79-95%), rituximab (range 19-62%), and tafasitamab (range 24-77%) in a dose-dependent manner. Killing was specific as no off-target apoptosis of T cells was observed. Taken together, these data suggest that AlloNK has the potential to be effective in combination with mAbs to induce deeper B cell depletion and improved efficacy, over the mAbs alone, in SLE.

Abstract 83: NK cell ADCC assays: Leveraging flow cytometry and reporter cell lines for enhanced biological relevance and throughput

Abstract Amid the growing arsenal of cancer treatments, antibody-dependent cellular cytotoxicity (ADCC) assays have emerged as a significant contributor to the quest for more effective therapies. The ADCC-based antibody therapy often exploits Natural Killer (NK) cells as effectors. This immunotherapeutic approach harnesses the innate cytotoxic potential of NK cells, offering a promising solution for the precision targeting of malignant cells. In this study, we employed two methods that can be used to measure ADCC potency and specificity during preclinical drug discovery. The first was a flow cytometry-based approach that focused on enhancing biological relevance by utilizing primary human peripheral blood mononuclear cells (PBMCs) as a source for NK effector cells. To this end, target cells were fluorescently labeled with CFSE and cultured with freshly isolated PBMCs at 3 effector to target (E:T) ratios, in the presence of ADCC antibody. After 16h incubation, a viability dye was added, and percent cytotoxicity was measured in CFSE+ targets cells by flow cytometry. The second method offered a high-throughput plate reader-based approach (ADCC Reporter Bioassay; Promega), that utilized an ADCC reporter effector cell line harboring a luciferase cassette driven by an NFAT promoter, which is activated when effector and targets cells are bridged by a therapeutic antibody. Cytotoxicity was read out by measuring light output on a Cytation 3 plate imaging reader (Agilent Technologies) after a 6h cell culture incubation using the recommended 6:1 E:T ratio. Preclinical application for both assays was demonstrated using an anti-HER2 therapeutic antibody (trastuzumab) against BT-474, which is a HER2 positive cell line. To demonstrate specificity, an anti-VEGF antibody (bevacizumab) and Hs 578T cells (HER2 low) were used as controls. Our results revealed trastuzumab induced a strong dose-dependent increase in cytotoxicity in the BT-474 cells but not the Hs 578T cell line (range: 40-70%) while bevacizumab failed to induce any cytotoxicity above background levels at any concentration. Furthermore, the flow cytometry and plate reader methodologies produced similar trends. In summary, both applications can be used to produce reliable ADCC drug screening assessments. Additional considerations include investigational needs surrounding high throughput capability versus biological relevance. Unlike traditional models using immortalized cell lines, primary human PBMCs more faithfully recapitulate the physiological interactions between effector cells and target cells. On the other hand, the more streamlined reporter cell line approach not only accelerates assay throughput but also ensures consistent and reliable results in an attractive option for large-scale screening and drug development efforts. Citation Format: David William Draper, Yewei Xing, Scott Wise. NK cell ADCC assays: Leveraging flow cytometry and reporter cell lines for enhanced biological relevance and throughput [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 83.

Abstract 1863: α-ABN501, a novel antibody targeting claudin-3, demonstrates the potential to be a new therapeutic option for small cell lung cancer

Abstract Small Cell Lung Cancer (SCLC) is the most aggressive and lethal type of lung cancer, characterized by a poor prognosis, with a 5-year survival rate of only 7% for overall SCLC patients. Limited treatment options such as chemotherapy and immune-checkpoint inhibitor therapy are available for SCLC patients, very often followed by rapid recurrence and metastasis. Thus, new effective treatment options for SCLC remain high unmet medical needs. Overexpression of claudin-3 (CLDN3), one of the tight junction proteins, is observed in various types of cancers. The abnormal conformation of tumor cell growth leads to the external exposure of CLDN3, in contrast, the normal cells that maintain high polarity do not expose CLDN3 externally. Therefore, CLDN3 could be a potential therapeutic target and biomarker. Based on the analysis of the expression pattern of CLDN3 using a single-cell RNA sequencing dataset derived from SCLC patients, we found that CLDN3 could be a superior and unique target of SCLC compared to others (e.g., DLL3 and B7H3). Hence, we developed a fully human monoclonal antibody, afucosylated ABN501 (α-ABN501), which has high specificity and strong affinity to CLDN3-expressing cells. The α-ABN501 antibody showed CLDN3-dependent killing effects against the SCLC cells, expressing different levels of CLDN3, as assessed via the antibody-dependent cellular cytotoxicity (ADCC) assay wherein using NK cells expressing CD16a (NK-92MI-CD16a), peripheral blood mononuclear cells (PBMC), and primary NK cells. Further, the efficacy study in the CLDN3-expressing SCLC cell derived xenograft mouse model revealed that α-ABN501 was highly efficacious in inhibition of tumor growth. Collectively, we elucidated that the ADCC activity of α-ABN501 was dependent on the expression level of CLDN3 and especially specific to the SCLC cells, which underlines significant suppression of tumor growth. Present study data sets suggest that the α-ABN501 can be a potential new therapeutic option for SCLC. In addition, the α-ABN501 antibody is currently under way of development into new drug platforms for further applications. Citation Format: Jae Choi, Sunghyun Hong, Ha Yeon Park, Jaemun Kim, Sangin Lee, Myeung-Ryun Seo, Sang-Beom Bang, Hyo-Jin Shin, Kwang-il Jeong, Hobin Yang, Sungyoul Hong, Jun Young Choi, Na Young Kim, Young Kee Shin, Saehyung Lee. α-ABN501, a novel antibody targeting claudin-3, demonstrates the potential to be a new therapeutic option for small cell lung cancer [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 1863.

Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers

BackgroundTargeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. Development of mAbs with improved cytotoxicity, targeting new and known tumor-associated antigens, therefore...

Higher CD56 Expression on Multiple Myeloma Cells Increases CD38 Expression, Reduces Intracellular NAD+ Levels, and Enhances the Efficacy of Daratumumab-Based Treatment Strategies

NCAM1 (Neural Cell Adhesion Molecule 1), also known as CD56, is a member of the immunoglobulin superfamily and is deregulated in many tumors. In multiple myeloma (MM), high levels of CD56 are observed on MM cells in 70% of patients. This molecule has several functions including tumor growth, adhesion and response to therapy, as suggested by several studies. However, an extensive analysis of the specific landscape underlying its presence and its role as a prognostic biomarker is lacking, especially in MM. Here, we evaluated CD56 surface levels on tumor cells collected from 136 newly diagnosed MM patients (Policlinic San Martino Hospital, Genoa, Italy) and found that low CD56 expression significantly correlated with extramedullary disease (EMD) (p = .016) while high CD56 levels were associated with a lower risk of disease progression or death (p = .003). When evaluating the surface expression of MM cells, we surprisingly found a positive correlation between CD56 and CD38 levels (p < .0001), which was replicated in a panel of MM cell lines (p = .02). To gain insight into the biological significance of this, we challenged a panel of MM cell lines with Daratumumab (DARA), a standard-of-care CD38 antibody that kills MM cells through multiple mechanisms including antibody-dependent cellular cytotoxicity (ADCC). Given the correlation between the expression of CD56 and CD38, we hypothesized that higher CD56 expression would lead to enhanced DARA-mediated ADCC. We performed standard 3-hour ADCC assays using NK cells from healthy donors as effector cells and CD56-positive or -negative cell lines. Myeloma cells were stained with calcein-AM and co-cultured with NK cells at indicated effector:target (E:T) ratios. The assay was assessed in the presence or absence of 1μg/mL Daratumumab. We found that MM cell lines with high expression of CD56 were more sensitive to DARA-mediated NK cell killing (p = .004 at ratio 1:1 E:T). Consistently, MM patients with higher levels of CD56 (N = 8) had better overall response rates to DARA-based therapies compared to patients with lower levels (N = 9). CD56 and CD38 are not known to interact, so this correlation may be mediated by the NAD+ biosynthesis pathway. CD38 is a critical enzyme for breaking down NAD + in cells. We found that ectopic expression of CD56 led to lower intracellular NAD + levels (MM1S overexpressing CD56 p = .0875; RPMI 8226 overexpressing CD56 p = .0003) and increased CD38 enzymatic activity (MM1S overexpressing CD56 p = .0035; RPMI 8226 overexpressing CD56 p = .0478), as measured by a cyclase activity assay. As a result, CD56 surface levels affected MM cells' sensitivity to NAD +-lowering agents, including next-generation NAMPT inhibitors. Indeed, MTS assays confirm that CD56-overexpressing MM cells were more vulnerable to these small molecules, whereas CD56 silencing made cells more resistant. In conclusion, our data suggest CD56 levels as an important determinant of sensitivity to DARA-based therapies. Moreover, our results suggest the next-generation NAMPT inhibitors as an additional therapeutic strategy for CD56-expressing MM patients.

Iberdomide Enhances Dara Mediated Cytotoxicity through Upregulation of CDC Activity and Elevated NK Cell Mediated ADCC

Background: Multiple Myeloma (MM) is an incurable hematological malignancy with current treatments comprising combination regimens, typically featuring immunomodulatory agents (IMiDs) due to their potent anti-tumor activity and immunomodulatory effects. Clinical studies utilizing the combination of IMiD agents and daratumumab (DARA), an anti-CD38 monoclonal antibody, have shown improved patient outcomes for MM. However, patients ultimately relapse demonstrating the need for novel therapies. Iberdomide (IBER) is a novel cereblon E3 ligase modulator. It promotes faster and deeper degradation of the substrate targets compared to IMiDs, resulting in enhanced anti-proliferative, pro-apoptotic and immune stimulatory activity. In early clinical studies, IBER has been shown to overcome resistance to IMiDs and in the ongoing Phase 3 EXCALIBER study (NCT04975997) is being explored for treatment of newly diagnosed (ND) and relapsed/refractory (RR) MM in combination with DARA and dexamethasone. Here, we investigate the potential mechanisms of synergy between IBER and DARA in preclinical models. Methods: Complement dependent cytotoxicity (CDC) of DARA was assessed with 15% human serum for human complement components and assays were performed by titrating either single agent or combinations in a tritiated thymidine assay. Antibody dependent cellular cytotoxicity (ADCC) was performed by co-culturing MM cells with varying expression levels of CD38 and effector cells and assessing target cell viability with annexin-v/topro-3 flow. A humanized PBMC adoptive transfer mouse model, utilizing human PBMC engraftment 14 days prior to implantation, was used to assess in vivo activity of IBER, DARA and IBER + DARA. Results: In CDC assays, we observed variable sensitivity of MM cells to complement-specific CDC in the presence of DARA (0.1-1 µg/ml), regardless of cell surface CD38 expression. Notably, IBER treatment resulted in upregulation of CD38 surface expression in 60% of MM cell lines tested. LP1 and MOLP8 displayed susceptibility to DARA-mediated CDC. DARA showed a dose-dependent increase in CDC activity in both cell lines. Notably, IBER+DARA also showed ~35% more synergistic CDC activity in both cell lines, than the single agents. In ADCC assays, we observed pretreatment of PBMC with IBER (vs. DMSO) significantly increased DARA mediated ADCC in H929 and LP-1 cells. To confirm that this effect was directed by NK cells, we cultured NK cells for 24 hr in supernatants from stimulated PBMC in presence of IBER prior to co-culture with MM cells. We noted that IBER-treated PBMC supernatant significantly enhanced the NK ADCC activity of DARA suggesting that IBER enhancement of cytokine secretion from PBMC increases NK cell mediated ADCC. Orthogonally, we pre-treated MM cells with IBER or DMSO prior to co-culture in ADCC assay. Notably, IBER treatment of MM cells, without treating immune cells, also increased DARA-mediated ADCC, indicating that IBER directly increases sensitivity to DARA-directed ADCC. To investigate the MOA of IBER-DARA synergy in ADCC, we analyzed gene expression and cell surface changes of a panel of NK-specific receptor ligands on MM cells following treatment with IBER. We observed IBER-induced upregulation of MICA mRNA levels (~2x) and surface expression (~40%) in MM target cells. We used RNAi to knock down Ikaros, an IBER substrate, which resulted in increased MICA expression (~3x) in MM cells. Furthermore, we observed that RNAi knock down of MICA in MM cells resulted in a significant decrease (60%, p<0.0001) in MM cell killing by NK cells, suggesting IBER-enhanced sensitivity of MM cells to NK-cell killing was in part, due to the upregulation of MICA through degradation of Ikaros. Lastly, we examined the anti-MM effects of IBER in a humanized xenograft model with a lenalidomide resistant cell line. Longitudinal analysis identified persistence of human PBMC engraftment and activation of immune subsets (NK cells and T-cells) that were increased upon IBER treatments, consistent with clinical observations. The combination of IBER/DARA resulted in ~22% tumor growth inhibition (TGI) in comparison to single agents, DARA and IBER and ~37% TGI in comparison to vehicle. Conclusion: Taken together, these data demonstrate that IBER and DARA work synergistically in preclinical models primarily through enhancement of both ADCC and CDC. This combination has the potential to improve outcomes in MM.

An Antibody-Dependent Cellular Cytotoxicity Assay for Detecting Ocrelizumab Neutralizing Antibody.

Ocrelizumab (OCREVUS®) is a humanized anti-CD20 monoclonal antibody approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). Here, we discuss the strategic and technical considerations needed to develop a robust antibody-dependent cellular cytotoxicity (ADCC)-based neutralizing antibody (NAb) assay to detect anti-ocrelizumab NAb in patients enrolled in the ocrelizumab registered clinical trials. The NAb detection assay consisted of a two-tier assay that included a screening assay and a confirmation assay. In the screening assay, patient samples were analyzed in the presence of ocrelizumab. Samples that tested positive in the screening assay were subsequently analyzed in the confirmatory assay where another anti-CD20 mAb, obinutuzumab, was replaced by ocrelizumab, to verify NAb specificity. Both assays utilized MEC-2 cells, a chronic B cell leukemia cell line, pre-labeled with calcein AM as the target cells, and natural killer (NK) cells engineered to stably express Fc gamma receptor IIIa_ F158 as effector cells. Both cell lines were prepared to be thaw-and-use cells. The NAb assay measures fluorescence from the calcein AM released into the assay media upon the lysis of target cells by ADCC in the presence of ocrelizumab or obinutuzumab. Our validated NAb assay showed a relative sensitivity of 743ng/mL and can detect 1500ng/mL of a surrogate positive control antibody in the presence of 1500ng/mL ocrelizumab. This ADCC assay is the first reported NAb assay that directly measures target cell lysis by using thaw-and-use target and effector cells simultaneously.

IgA antibody immunotherapy targeting GD2 is effective in preclinical neuroblastoma models

BackgroundImmunotherapy targeting GD2 is very effective against high-risk neuroblastoma, though administration of anti-GD2 antibodies induces severe and dose-limiting neuropathic pain by binding GD2-expressing sensory neurons. Previously, the IgG1 ch14.18 (dinutuximab)...

Targeting CXCR4 abrogates resistance to trastuzumab by blocking cell cycle progression and synergizes with docetaxel in breast cancer treatment

BackgroundAlthough trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms.MethodsImmunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4 expression. Three-dimensional co-culture (tumor cells/breast cancer-associated fibroblasts/human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effects of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms.ResultsUsing a panel of cell lines and patient breast cancer samples, we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using a panel of trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel.ConclusionsOur findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.

Development of anti-GD2 Antibody-producing Mesenchymal Stem Cells as Cellular Immunotherapy.

Using the tyrosine hydroxylase (TH)-MYCN mouse neuroblastoma (NB) model, we have previously reported the accumulation of mouse mesenchymal stem cells (mMSCs) on tumors in vivo and the antitumor effect of mMSCs transfected with a small molecule (IFN-β) expression gene. In this study, we have developed novel MSCs secreting anti-disialoganglioside GD2 antibody (anti-GD2-MSCs) and evaluated their antitumor effects in vitro. We generated an anti-GD2 antibody construct (14.G2a-Fcx2-GFP) incorporating FLAG-tagged single-chain fragment variable against GD2 fused to a linker sequence, a fragment of the constant portion of human IgG1, and GFP protein. The construct was lentivirally transduced into mMSCs and the transduction efficiency was assessed by GFP expression. The secretion of FLAG-tagged anti-GD2 antibody was detected by Western blotting using anti-FLAG antibody. Antibody binding capacity was confirmed by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using human NB cells and human natural killer (NK) cells to assess whether the antitumor activity was enhanced in the presence of the produced antibodies. The transduction efficiency of anti-GD2-MSCs was more than 90%. anti-GD2-MSCs secreted antibodies extracellularly and these antibodies had high affinity to GD2-expressing human NB cells. ADCC assays showed that the addition of antibodies secreted from anti-GD2-MSCs significantly increased the cytotoxic activity of NK cells against NB cells. Newly developed anti-GD2-MSCs produced functional antibodies that have affinity to the GD2 antigen on NB cells and can induce ADCC-mediated cytotoxicity. Anti-GD2-MSCs based cellular immunotherapy has the potential to be a novel therapeutic option for intractable NB.

Abstract 4531: Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study

Abstract ZL-1211, a humanized monoclonal IgG1 antibody targeting CLDN18.2, induces cancer cell death through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We developed ex vivo ADCC and CDC assays to study the mechanism of actions of ZL-1211. Our preclinical data has demonstrated greater antitumor activities than the clinical leading benchmark via enhanced ADCC by mutation in Fcγ domain and potent CDC activities. We used those ex vivo assays to further profile the clinical specimens from patients collected at baseline (pre-infusion) and the following cycles (post-infusion) in our phase 1 study (NCT05065710). Preliminary results in clinical samples indicated that baseline ADCC and CDC capabilities were correlated with anti-tumor activity of ZL-1211 in patients with CLDN18.2-positive locally advanced or metastatic solid tumors, suggesting probable predictive biomarkers. Results also showed that changes in circulating ZL-1211 mediated ADCC and CDC activities were dose-dependent and in accordance with the pharmacokinetic (PK) profile. In addition, we performed differential gene expression analysis and real-time PCR to compare gene expression in ZL-1211 responder and non-responder PDX models and CCLE cell lines. Our findings reveal that several immune-related genes may serve to predict the responses to ZL-1211 treatment. Other potential predictive biomarkers such as Fcγ receptor CD16A polymorphism and baseline stromal CD8 expression were also evaluated in clinical samples. Taken together, analytical approaches developed and validated from preclinical studies were successfully translated to characterize clinical samples in the current Phase 1 trial. The exploratory analysis indicated the clinical relevance of multiple biomarkers and their potential as predictive biomarkers of ZL-1211. Citation Format: Zhen Luo, Xiao Wang, Xuehuo Zeng, Jiaqing Yi, Tegan Burns, Herman Liu, Xinyu Zhang, Wenyu Liu, Karl Hsu, Hua Gong. Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4531.

Abstract 5654: A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells

Abstract Background: NEO-201 is a humanized IgG1 mAb reactive against multiple human cancers but not against most normal epithelial tissues. NEO-201 binds to core 1 or extended core 1 O-glycans expressed by its target cells, including neutrophils, various carcinomas, and some human hematological malignancies. NEO-201 can mediate antitumor activity through antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and blockade of the CEACAM5/CEACAM1 ICI pathway. A previous study using flow cytometry demonstrated that NEO-201+/CD4+ T cells were also CD25+/CD127−/Foxp3+/CD15s+ using PBMCs from healthy donors (HD). NEO-201 can kill these Treg cells through CDC in vitro. NEO-201 does not bind to the majority of CD4+ T cells and to other immune subsets. Human gMDSCs are increased in cancer patients and are a population of immature MDSCs deriving from immature neutrophils and alternative activation of mature neutrophils. gMDSC are characterized by HLA-DR−, CD11b+, CD33+, CD15+phenotype.We have shown that NEO-201 recognizes and kill human neutrophils through ADCC. This current investigation was designed to evaluate whether NEO-201 can target and mediate ADCC activity against human gMDSCs. Methods: gMDSCs were generated from human neutrophils from 5 HD isolated using EasySepTM direct human neutrophil isolation kit. Isolated neutrophils were cultured in complete RPMI1640 medium supplemented with human GM-CSF and human IL-6 for 7 days. Phenotypic analysis by flow cytometry was performed on the generated gMDSCs using NEO-201 and mAbs against human CD33, HLA-DR, CD15, CD14, CD66b. Flow cytometry based ADCC assay was performed using gMDSCs stained with both CD33 and HLA-DR as target. PBMCs from a separate HD were used as effectors at different E:T ratios. The ADCC activity of NEO-201 was evaluated comparing the percentage of CD33+/HLA-DR− viable cells in gMDSCs incubated with medium alone to the percentage of CD33+/HLA-DR− viable cells incubated with PBMCs alone and with PBMCs plus NEO-201. Results: Flow cytometry analysis revealed that gMDSCs can be generated from human neutrophils after 7 days of culture with GM-CSF and IL-6 and that they express the following phenotype: HLA-DR−/CD33+/CD15+/CD14−/CD66b+. NEO-201 bound to the majority of these gMDSCs. NEO-201 was functional in mediating ADCC to kill these gMDSCs. Conclusion: This study demonstrated that NEO-201 can be used to identify and kill suppressive gMDSCs in addition to Treg cells. Depletion of suppressive Tregs and gMDSCs in the TME could be an effective strategy to prevent hyperprogressive disease when anti-PD-1 is used in cancer immunotherapy. These data support the rationale for the ongoing phase II clinical trial using NEO-201 in combination with pembrolizumab in checkpoint refractory patients with metastatic solid tumors. Citation Format: Massimo Fantini, Christina M. Annunziata, Philip M. Arlen, Kwong Y. Tsang. A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5654.

Development of a reporter gene assay for antibody dependent cellular cytotoxicity activity determination of anti-rabies virus glycoprotein antibodies.

Rabies is a viral disease that is nearly 100% fatal once clinical signs and symptoms develop. Post-exposure prophylaxis can efficiently prevent rabies, and antibody (Ab) induction by vaccination or passive immunization of human rabies immunoglobulin (HRIG) or monoclonal antibodies (mAbs) play an integral role in prevention against rabies. In addition to their capacity to neutralize viruses, antibodies exert their antiviral effects by antibody-dependent cellular cytotoxicity (ADCC), which plays an important role in antiviral immunity and clearance of viral infections. For antibodies against rabies virus (RABV), evaluation of ADCC activity was neglected. Here, we developed a robust cell-based reporter gene assay (RGA) for the determination of the ADCC activity of anti-RABV antibodies using CVS-N2c-293 cells, which stably express the glycoprotein (G) of RABV strain CVS-N2c as target cells, and Jurkat cells, which stably express FcγRⅢa and nuclear factor of activated T cells (NFAT) reporter gene as effector cells (Jurkat/NFAT-luc/FcγRⅢa cells). The experimental parameters were carefully optimized, and the established ADCC assay was systematically validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 guideline. We also evaluated the ADCC activity of anti-RABV antibodies, including mAbs, HRIG, and vaccine induced antisera, and found that all test antibodies exhibited ADCC activity with varied strengths. The established RGA provides a novel method for evaluating the ADCC of anti-RABV antibodies.

Pre-Clinical Efficacy of AB-101, an Allogeneic Cord-Blood Derived Natural Killer (NK) Cell Therapeutic Candidate, in Combination with Anti-CD38 Antibodies in Models of Multiple Myeloma

Introduction: AB-101 is a non-engineered, allogeneic, off-the-shelf, cryopreserved cord blood-derived natural killer (NK)-cell therapy in development as a cancer therapeutic. A highly scaled manufacturing process enables production of 1000s of doses of AB-101 from a single donor cord blood unit (CBU). AB-101 is derived from CBUs selected for KIR-B haplotype and natural high-affinity variant of CD16 (158V/V), which are associated with improved anti-tumor activity and ADCC enhancement, resulting in a highly active NK cell product without the requirement for additional engineering. We are developing AB-101 for combination with monoclonal antibodies (mAbs) to enhance antibody dependent cellular cytotoxicity (ADCC) in patients, initially in an ongoing Phase I clinical trial for NHL. To further develop AB-101 as an ADCC enhancer, we assessed anti-tumor activity in combination with CD38 mAb, against multiple myeloma (MM) cell lines. CD38 is a transmembrane glycoprotein that is uniformly and highly upregulated on MM cells. Anti-CD38 mAbs exert anti-tumor activity against MM through ADCC and other mechanisms. NK cells play a key role in ADCC responses through CD16 receptor signaling. However, the majority of NK cells express CD38 and are depleted by therapeutic CD38 mAbs which could limit their therapeutic efficacy. Combining NK cell therapies with anti-CD38 mAbs, thus necessitates CD38 knockout to prevent fratricide. AB-101 has a naturally low percent and intensity of CD38 expression. Here we present data demonstrating that AB-101 are resistant to anti-CD38 mAb-induced fratricide and can elicit significant ADCC against MM cell lines both in vitro and in vivo. Since anti-CD38 mAbs are combined with glucocorticoids in the clinic, we assessed the impact of dexamethasone and methylprednisolone on ADCC by AB-101. Here we report robust ADCC following treatment of AB-101 with glucocorticoids. Methods: AB-101 was screened for CD38 expression and incubated with anti-CD38 mAb - daratumumab or hIgG1 control (300 - 0.41 µg/ml) for 4 hours to assess fratricide. In vitro ADCC assays were performed at various effector to target ratios against control (Daudi and K562) and MM cell lines (NCI-H929, RPMI 8226, MM.1S and MM.1R) using 2 µg/ml of daratumumab or hIgG1. To study the effect of glucocorticoids, AB-101 was cultured in various concentrations of dexamethasone and methylprednisolone for 48 hours in the presence of 50 or 500 IU/ml of IL-2, prior to ADCC assay. In vivo efficacy of AB-101 in combination with daratumumab was assessed in the intraperitoneal (IP) MM.1S-Luc xenograft model in NSG mice (Figure 1A). Daratumumab and AB-101 were administered by IP injection. Tumor burden was assessed by bioluminescent imaging (BLI). Results: AB-101 was over 98% CD56+/CD3- with 87% CD56+/CD16+ population. Only 22% of GMP drug product were CD38+ with low intensity of expression compared to peripheral blood NK cells (~88%). In the presence of daratumumab AB-101 viability was unaltered compared to hIgG1 control demonstrating resistance to fratricide. Daratumumab enhanced ADCC potential of AB-101 against MM cell lines and Daudi in in vitro. ADCC against MM lines and Daudi did not decrease on exposure to glucocorticoids compared to no treatment group. In vivo anti-tumor efficacy, as evidenced by reduction in BLI signal, was observed in the MM.1S model in AB-101 + daratumumab groups compared to daratumumab and AB-101 alone (Figure 1B). AB-101 + daratumumab (1 and 3mpk) led to 98 and 99% tumor growth inhibition (TGI), respectively, relative to no treatment. Daratumumab monotherapy at 1 and 3mpk showed 21% and 87% TGI respectively, and AB-101 showed 16% TGI relative to no treatment. Conclusions: AB-101 has high and consistent expression of CD16 and low CD38 expression and is resistant to daratumumab induced fratricide and hence can be combined with daratumumab without additional engineering such as CD38 knockout. Daratumumab is combined in the clinic with glucocorticoids, immunomodulatory drugs (IMiDs) and proteosome inhibitors (PI). While IMiDs and PIs are reported to enhance NK cell function, glucocorticoids have been shown to diminish NK cell cytotoxicity. Data presented here suggests that ADCC potential of AB-101 is not negatively impacted by glucocorticoids. Taken together, the in vitro and in vivo data supports further clinical development of AB-101 in combination with daratumumab to treat MM patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EZH2 Targeting Induces CD38 Upregulation and Response to Anti-CD38 Antibodies in Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy characterized by the accumulation of tumor plasma cells within the bone marrow. MM is characterized by high molecular and clinical heterogeneity. During the last 10 years new therapeutic classes including targeted immunotherapies significantly improved the overall survival of MM patients. However, drug resistance and relapse remain major challenges in MM. In MM, marked reduction of CD38 expression via exocytosis, endocytosis, degradation of the antigen-antibody complex and trogocytosis has been associated with resistance to CD38 targeted immunotherapies. Our group previously described a link and a negative correlation between CD38 and EZH2 (Enhancer of Zeste homolog 2) expression during normal B to plasma cell differentiation in association with a transcriptional control of CD38 expression involving the polycomb PRC2 complex (Herviou et al. Leukemia. 2019). Here we identified a significant negative correlation between CD38 expression and EZH2 expression in 2 independent cohorts of MM patients (p<0.05; Montpellier cohort n=198; Compass cohort n=631). Thus, we hypothesized that PRC2 targeting using EZH2 inhibitor could result in CD38 re-expression to overcome daratumumab resistance. We first characterized the membrane CD38 expression by calculating the Antibody Binding Capacity in our unique panel of 40 Human Myeloma Cell lines (HMCL) representative of MM heterogeneity. We observed a heterogeneous expression of the membrane CD38 expression on MM cell lines with cell lines demonstrating no membrane expression and cell lines demonstrating high membrane expression. Next, we selected 3 HMCLs with low (JJN3), intermediate (XG2), and high (XG7) CD38 expression, and treated these cell lines with sublethal concentration of EZH2 inhibitor (EPZ-6438) for 9 days. CD38 membrane expression was characterized at Day 3, Day 6 and Day 9. Interestingly, EZP-6438 treatment induced upregulation of cell surface CD38 expression on all the HMCLs tested with the highest effect measured at day 9. Similar re-expression pattern was observed by western blot at these timing in the JJN3 CD38low cell line. Remarkably, ChIPseq analyses underlined a significant association of H3K27me3 mark at CD38 promoter in CD38neg and CD38low cell lines compared to CD38highcells. In addition, we investigated the efficiency of CD38 monoclonal antibodies (Daratumumab and Isatuximab) lysis through ADCC (Antibody dependent cellular cytotoxicity) assay after 9 days of EPZ-6438 pretreatment. Using JJN3 CD38low HMCL, EPZ-6438 pretreatment significantly improved the ADCC lysis induced by low doses of Daratumumab and Isatuximab treatment, in presence of purified Natural Killer (NK) from healthy donor, together with CD38 re-expression (p<0.05; n=3). Finally, we searched to validate these results using primary MM cells from patients at relapse after Daratumumab treatment. EPZ-6438 pretreatment resulted in a significant CD38 membrane expression upregulation with a 2 fold difference compared to the control condition together with improved ADCC toxicity of anti-CD38 antibodies in vitro. Altogether we demonstrated that treatment of MM cells with EZH2 inhibitor leads to significant upregulation of membrane CD38 expression in cell lines and primary MM cells from patients. CD38 re-expression was linked to an improvement of Daratumumab and Isatuximab ADCC efficiency. Thus, EZH2 targeting may be of therapeutic interest to overcome resistance to anti-CD38 targeted immunotherapies in Multiple Myeloma.

CD25 Targeting Eliminates Regulatory T Cells and CD25+ Blasts in Acute Myeloid Leukemia

Background Acute Myeloid leukemia (AML) is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. CD25 Mab (also referred to as RG6292 and RO7296682) is an afucosylated, IL-2 non-blocking human IgG1 antibody, shown to efficiently deplete immunosuppressive regulatory T cells (Tregs) in humans and solid tumour models (Kolben 2021) whilst allowing binding of IL-2 to effector T cells and the induction of anti-tumour adaptive immune responses (Solomon, Amann et al. 2020). CD25 Mab binds to CD25+ target cells and its crystallisable fragment (Fc) to Fc receptors (FcR) expressed on the surface of effector cells, such as FcgRIIIa on Natural Killer (NK) cells, monocytes and macrophages. It mediates killing of target cells through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). CD25 Mab is currently being investigated in a phase I dose escalation monotherapy study (NCT04158583) or in combination with atezolizumab (NCT04642365). In AML patients, a higher frequency of immunosuppressive bone marrow Tregs was observed as compared to that of healthy donors and their abundance correlated with poorer outcome (Szczepanski, Szajnik et al. 2009, Han, Dong et al. 2018, Dong, Han et al. 2020). In addition, CD25 is also expressed on a subset of AML cells and may be restricted to leukemic stem cells (LSC) or cells with a progenitor phenotype (Angelini, Ottone et al. 2015, Bertolini, Kageyama et al. 2018, Aref, Azmy et al. 2020). Considering the critical role of LSC in the propagation of the disease and the high relapse rate in AML, it would be important to understand if CD25 targeting could deepen responses and help prevent relapse in patients. In this study, we hypothesized that CD25 Mab may have a dual mode of action whereby it depletes suppressive Tregs and has a direct cytotoxic effect on CD25+ AML cells. Methods We conducted an in-depth characterization of bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry (up to 39 cellular markers) and RNA sequencing using computational analysis. Moreover, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab on Tregs and CD25+ AML cells, as compared to isotype control antibody. Results We found that the abundance of Tregs and CD25+ AML cells correlated strongly with that of the blood in 11 patients with time-matched samples, indicating that blood samples could be used for the identification of predictive biomarkers. Moreover, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing internal tandem duplication mutations in the FLT3 gene (FLT3-ITD). Furthermore, all four patients treated with a hypomethylating agent in combination with venetoclax displayed detectable levels of CD25+ AML cells. We adopted a patient-centric approach (n=14 patients) to study AML clusters with CD25 expression, due to the high inter-patient heterogeneity. Interestingly, CD25+ clusters most commonly expressed CD34, an LSC marker indicating a preferential expression in immature AML cells. Finally, ex vivo treatment of primary AML patient samples with CD25 Mab led to the specific killing of CD25+ AML cells and Tregs by allogeneic NK cells, as compared to isotype control treatment. Taken together, these results provide a proof of concept of CD25 Mab's dual mode of action in AML patient samples. Conclusions Using high dimensional flow cytometry and computational analysis, we provide a deep characterization of AML patient samples and highlight the heterogeneity in the pattern of expression of common AML targets currently under clinical investigation. We dissected the expression of CD25 on healthy T cells and malignant cells and demonstrated that the blood offers a window into the bone marrow composition and could be used to ascertain Treg and CD25+ AML cells prevalence. Mode of action studies demonstrated that CD25 Mab depletes suppressive Tregs and has a direct cytotoxic effect on the CD25+ AML cells. CD25 targeting represents an attractive target for the treatment of AML, especially in patients where CD25 is expressed on LSC or immature AML cells. Our study warrants further exploration of CD25 Mab as combinatorial treatment with, for instance, FLT3 inhibitors or venetoclax.

Methods to Evaluate the Potential Clinical Significance of Antibodies to Red Blood Cells.

Immune-mediated red blood cell (RBC) destruction due to antibodies is an ongoing problem in transfusion medicine for the selection of the safest blood. Serological testing often revealed incompatibility with donors' RBCs. When this incompatible blood was transfused, destruction was due mostly to extravascular-mediated phagocytosis of the antibody-opsonized RBCs; however, intravascular hemolysis was sometimes observed without explanation. Based on serology, antibodies with potential for clinical sequalae could not be ascertained; thus, antigen-negative blood was usually selected for transfusion to avoid problems. Antibodies to antigens having very high frequency in the general population (>95%), however, made selection of antigen-negative blood difficult and sometimes impossible. Some patients, who were sensitized by previous transfusions or by pregnancy, developed multiple antibodies, again creating a problem for finding compatible blood for transfusion, without the ability to discern which of the antibodies may be clinically irrelevant and ignored. Transfusion medicine scientists began searching for an in vitro means to determine the in vivo outcome of transfusion of blood that was serologically incompatible. Methods such as chemiluminescence, monocyte-macrophage phagocytosis, and antibody-dependent cellular cytotoxicity (ADCC) were described. Over the years, the monocyte monolayer assay (MMA) has emerged as the most reliable in vitro assay for the prediction of the clinical relevance of a given antibody. ADCC has not been fully studied but has the potential to be useful for predicting which antibodies may result in intravascular hemolysis. This article captures the protocols for the implementation and readout of the MMA and ADCC assays for use in predicting the clinical significance of antibodies in a transfusion setting. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Monocyte monolayer assay (MMA) Basic Protocol 2: Antibody-dependent cellular cytotoxicity assay (ADCC).