Cell-mediated Cytotoxicity Assay Research Articles

Metformin Suppresses Both PD-L1 Expression in Cancer Cells and Cancer-Induced PD-1 Expression in Immune Cells to Promote Antitumor Immunity.

Metformin, a drug prescribed for patients with type 2 diabetes, has potential efficacy in enhancing antitumor immunity; however, the detailed underlying mechanisms remain to be elucidated. Therefore, we aimed to identify the inhibitory molecular mechanisms of metformin on programmed death ligand 1 (PD-L1) expression in cancer cells and programmed death 1 (PD-1) expression in immune cells. We employed a luciferase reporter assay, quantitative real-time PCR, immunoblotting analysis, immunoprecipitation and ubiquitylation assays, and a natural killer (NK) cell-mediated tumor cell cytotoxicity assay. A mouse xenograft tumor model was used to evaluate the effect of metformin on tumor growth, followed by flow-cytometric analysis using tumor-derived single-cell suspensions. Metformin decreased AKT-mediated β-catenin S552 phosphorylation and subsequent β-catenin transactivation in an adenosine monophosphate-activated protein kinase (AMPK) activation-dependent manner, resulting in reduced CD274 (encoding PD-L1) transcription in cancer cells. Tumor-derived soluble factors enhanced PD-1 protein stability in NK and T cells via dissociation of PD-1 from ubiquitin E3 ligases and reducing PD-1 polyubiquitylation. Metformin inhibited the tumor-derived soluble factor-reduced binding of PD-1 to E3 ligases and PD-1 polyubiquitylation, resulting in PD-1 protein downregulation in an AMPK activation-dependent manner. These inhibitory effects of metformin on both PD-L1 and PD-1 expression ameliorated cancer-reduced cytotoxic activity of immune cells in vitro and decreased tumor immune evasion and growth in vivo. Metformin blocks both PD-L1 and PD-1 within the tumor microenvironment. This study provided a mechanistic insight into the efficacy of metformin in improving immunotherapy in human cancer.

Abstract 1890: HMA800067, a novel CD38-targeting antibody-drug conjugate (ADC), demonstrated superior anti-tumor activity to daratumumab in preclinical B-cell malignancies models

Abstract Introduction: Daratumumab (Dara), an anti-CD38 monoclonal antibody, has been widely used in the treatment of multiple myeloma (MM). However, some of MM patients exhibit primary or acquired resistance to Dara therapy. A CD38 targeting antibody-drug conjugate (ADC) HMA800067 is developed, in which Dara was conjugated with cytotoxic payload via a novel linker, aiming to have superior anti-tumor efficacy to Dara, even in the subjects with resistance to Dara treatment. Methods: HMA800067 was characterized by ELISA-based binding assay, cell internalization assay, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) assays. The in vitro anti-tumor potency was assessed against a panel of 24 MM and B cell lymphoma cell lines with various CD38 expressions. Bystander effect of HMA800067 was evaluated in co-culture of CD38− SU-DHL-2 cells with CD38+ Ramos cells. In vivo anti-tumor efficacy was investigated in the multiple MM and Diffuse Large B-cell Lymphoma (DLBCL) subcutaneous xenograft models, including some non-responsive to Dara. Results: HMA800067 displayed comparable binding affinity and internalization capability to Dara in CD38+ Ramos cells. And HMA800067 maintained the ability of Dara to induce the death of CD38+ Daudi cells by ADCC and ADCP, which indicated that conjugation did not change the property of Dara in the ADC upon antigen binding. HMA800067 exhibited a CD38-dependent cytotoxic activity with median IC50 of 0.46 nM against a panel of tumor cell lines, with a trend that the higher CD38 expression, the stronger potency could be seen. Bystander killing effect of HMA800067 was observed in CD38− cells when co-cultured with CD38+ cells in vitro. Furthermore, with the low release of payload in plasma and sustainable high exposure of payload in the tumor, HMA800067 demonstrated dose-dependent and superior anti-tumor efficacy to Dara in all tested models, and 10 mg/kg induced complete tumor regression in MM RPMI-8226, DLBCL SU-DHL-6 and SU-DHL-10 models. For example, in SU-DHL-6 xenograft model, the treatment of intravenous injection of HMA800067 at 10 mg/kg single dose induced complete tumor regression in all the animals within 10 days of administration, which was maintained until the end of study (day 49), while 10 mg/kg Dara twice a week for 2 weeks inhibited tumor growth within 20%-40% without any tumor regression during a four-week observation post initial dosing. Conclusion: HMA800067 demonstrated potent anti-tumor activities in vitro and in vivo in multiple B-cell malignant tumor lines, including those with poor response to Dara. These results supported further development of HMA800067, as a superior therapeutic option for treatment of patients with CD38+ B cell malignancies. Citation Format: Yan Xu, Junqing Liang, Shuwen Jiang, Haibin Yang, Hui Zhang, Fangfang Mao, Jiahuan Zhu, Jianlin He, Na Yang, Jian Wang, WeiHan Zhang, Yongxin Ren, Weiguo Su. HMA800067, a novel CD38-targeting antibody-drug conjugate (ADC), demonstrated superior anti-tumor activity to daratumumab in preclinical B-cell malignancies models [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 1890.

Abstract 550: Identification of isoform switching events linked with esophageal adenocarcinoma patient survival informs novel prognostic and therapeutic targets

Abstract Esophageal adenocarcinoma (EAC) represents a growing health problem characterized by rising incidence and poor prognosis. Although it is a cancer with a high mutational burden, it lacks highly prevalent oncogenic drivers that can be therapeutically targeted. Recently, our group characterized isoform switching events, defined by the changing of dominant gene isoforms between conditions, associated with EAC progression and identified 75 switching events comparing Barrett’s low-grade dysplasia (BE.LGD) and Barrett’s high-grade dysplasia (BE.HGD) patient samples. Herein, we further investigated whether isoform-switching events may offer prognostic or therapeutic value in EAC. Isoform switching analysis was performed using RNA-seq data derived from patient samples between BE.LGD and BE.HGD combined with EAC (BE.HGD + EAC) alone or in combination with TP53 mutations. Patients were further stratified into tertiles based on isoform expression level followed by survival analysis. Thirty isoforms were significantly (P≤0.05) linked with all-cause mortality, comparing BE.LGD with BE.HGD + EAC, whereas 20 isoforms were identified when TP53 mutation status was included. In terms of cancer-specific mortality, 10 isoforms were significantly linked with survival comparing BE.LGD with BE.HGD+EAC. With the inclusion of TP53 mutation, 16 isoforms were also significantly linked with cancer-specific mortality. Isoform-specific genetic knockdown experiments were designed targeting the top isoforms identified in the analysis, including TTLL12 and HM13 isoforms. Knockdown of TTLL12 and HM13 isoforms led to significant inhibition of cell viability and migration in two EAC cell lines (OE19 and OE33). Chemotherapeutic agents (Paclitaxel + Carboplatin) further decreased cell viability with synergistic effects observed in both siRNA-treated cell lines suggesting a role for specific isoform switches in therapeutic sensitization. Protein-interaction prediction using STRING and immunoblot analysis suggest different mechanisms leading to the inhibition of cell viability and migration. TTLL12 is linked with the activation of chaperon-medicated autophagy (LAMP2A and HSC70) and protein translation, whereas HM13 is linked with unfolded protein response (IRE1α, PERK, and ATF6) and may be linked with immunotherapy outcome. An antigen-dependent cell-mediated cytotoxicity assay is currently underway to investigate whether the knockdown of HM13 isoforms can sensitize EAC cells to PD-1/PD-L1 immunotherapy agents. Future direction includes using FDA-approved agents to target the two major pathways identified above. In conclusion, isoform switching may provide fresh insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC treatment or prevention. Citation Format: Yun Zhang, Rachel Israel, Bryce Vandenburg, Shari Barnett, Jean-Jack Riethoven, Jennifer L. Clarke, Michelle P. Lee, Kiran Lagisetty, Jules Lin, Rishindra Reddy, Andrew Chang, Laura A. Kresty. Identification of isoform switching events linked with esophageal adenocarcinoma patient survival informs novel prognostic and therapeutic targets [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 550.

Abstract 2969: Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models

Abstract Clearing senescent cells has been shown to have promising effects against cancer and age-related pathologies in preclinical models as well as early human clinical trials. However, current senescent cell elimination strategies are focused on targets that do not distinguish between hyper-inflamed or SASP senescent cells versus relatively non-inflamed senescent cells. The use of senolytics in the clinic is still limited due to their cytotoxicity to either normal cells or potentially beneficial senescent cells. A more effective senescence cell targeted approach is needed to reduce side effects and increase the efficiency of senescence cell removal or reduction. Conditionally Active Biologics (CAB) technology is a proprietary platform unique in its ability to be selectively active in the context of diseased tissues, but not normal tissues. The aberrant accumulation of senescent cells in aged and cancerous tissue triggers inflammatory signaling through a senescence-associated secretory phenotype (SASP), promoting aging and tumor progression. Since our CAB technology is currently being tested as a cancer therapy by targeting cell surface markers such as AXL, ROR2, and CTLA4 in clinical studies, we explored whether CAB technology allows selective removal of senescent cells in SASP-associated microenvironments. In our previous report, we demonstrated several novel senescence-specific surface antigens upregulated in senescent cells (1). CAB antibodies targeting these senescence markers have no or very low binding to the target antigen on senescence cells in physiological, alkaline conditions, but have strong binding in glycolytic, acidic SASP conditions in in vitro binding assays. In in vitro antibody-dependent cell-mediated cytotoxicity assays, CAB antibodies were more potent against senescent cells compared to proliferating cells and displayed high selectivity for the glycolytic, acidic condition. To study whether CAB antibody reduces senescent cell occurrence in vivo, we developed a unilateral ureteral obstruction (UUO) mouse model to induce senescent cells, fibrosis, and inflammation in mouse kidney. The mice treated with CAB antibodies had a significant reduction of senescent cells and inflammatory cells infiltrated in the renal cortex, compared to the mice treated with benchmark and isotype antibodies in UUO kidneys. Thus, the CAB antibody-based therapeutic is a promising strategy for senescent cell removal in an inflammatory microenvironment. In conclusion, CAB technology provides a new generation of biologics with an increased safety margin and therapeutic index targeting SASP, acidic senescence cells in cancer and age-related diseases. (1). Chen J, Wang J, Liu H, Chang C, Boyle WJ, and Short JM. Abstract 4795: Conditionally active biologics eliminates senescence cells in cancer and aging. Cancer Res (2023) 83. Citation Format: Jian Chen, Jian Wang, Haizheng Liu, Cathy Chang, William J. Boyle, Jay M. Short. Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models [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 2969.

A CRISPR activation screen identifies MUC-21 as critical for resistance to NK and T cell-mediated cytotoxicity

BackgroundImmunotherapy has significantly advanced cancer treatments, but many patients do not respond to it, partly due to immunosuppressive mechanisms used by tumor cells. These cells employ immunosuppressive ligands to evade detection and elimination by the immune system. Therefore, the discovery and characterization of novel immunosuppressive ligands that facilitate immune evasion are crucial for developing more potent anti-cancer therapies.MethodsWe conducted gain-of-function screens using a CRISPRa (CRISPR activation) library that covered the entire human transmembrane sub-genome to identify surface molecules capable of hindering NK-mediated cytotoxicity. The immunosuppressive role and mechanism of MUC21 were validated using NK and T cell mediated cytotoxicity assays. Bioinformatics tools were employed to assess the clinical implications of mucin-21 (MUC21) in cancer cell immunity.ResultsOur genetic screens revealed that MUC21 expression on cancer cell surfaces inhibits both the cytotoxic activity of NK cells and antibody-dependent cellular cytotoxicity, but not affecting complement-dependent cytotoxicity. Additionally, MUC21 expression hinders T cell activation by impeding antigen recognition, thereby diminishing the effectiveness of the immune checkpoint inhibitor, anti-PD-L1. Moreover, MUC21 expression suppress the antitumor function of both CAR-T cells and CAR-NK cells. Mechanistically, MUC21 facilitates immune evasion by creating steric hindrance, preventing interactions between cancer and immune cells. Bioinformatics analysis revealed elevated MUC21 expression in lung cancer, which correlated with reduced infiltration and activation of cytotoxic immune cells. Intriguingly, MUC21 expression was higher in non-small cell lung cancer (NSCLC) tumors that were non-responsive to anti-PD-(L)1 treatment compared to responsive tumors.ConclusionsThese findings indicate that surface MUC21 serves as a potent immunosuppressive ligand, shielding cancer cells from NK and CD8+T cell attacks. This suggests that inhibiting MUC21 could be a promising strategy to improve cancer immunotherapy.

Evaluation of the novel therapeutic anti-CCR7 antibody CAP-100 as an add-on therapy in chronic lymphocytic leukemia patients receiving venetoclax.

The Bruton's tyrosine kinase inhibitor ibrutinib and the B-cell lymphoma 2 anti-apoptotic protein inhibitor venetoclax provide high response rates in chronic lymphocytic leukemia (CLL). However, there is a growing number of patients that relapse after treatment or show refractory disease, thus new targets and agents are still needed. We have previously reported the chemokine receptor CCR7 as a relevant deregulated target in CLL and have developed CAP-100, a novel therapeutic anti-CCR7 antibody that is under evaluation for relapse/refractory CLL (NCT04704323). While CCR7 expression has been shown to be down-modulated in CLL patients treated with ibrutinib, whether venetoclax acts in a similar manner remains unaddressed. Here, we aimed to document the impact of venetoclax on CCR7 expression in CLL cells, as well as on the pre-clinical activity of CAP-100. To this end, we addressed CCR7 expression by flow cytometry and the antibody efficacy by means of in vitro chemotactic and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Our data indicate that venetoclax treatment did not significantly modify CCR7 expression pattern nor CAP-100 mechanisms of action. Together, these findings support CAP-100 as an adjuvant therapy to venetoclax that may introduce additional modes of action in CLL therapy.

Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method

Abstract In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Traditionally, in vitro CAR-T cell-mediated cytotoxicity is assessed using release assays such as 51Cr (radioactivity), calcein (fluorescence), and LDH (enzymatic). Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo high-throughput plate-based Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. Performing time- and E:T ratio-dependent CAR-T cell-mediated cytotoxicity assays, the GFP-based method demonstrated higher sensitivity in detecting the level of cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions. None

Abstract 5326: Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method

Abstract In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Traditionally, in vitro CAR-T cell-mediated cytotoxicity is assessed using release assays such as 51Cr (radioactivity), calcein (fluorescence), and LDH (enzymatic). However, release assays indirectly measure cell death via molecules released in supernatant and typically limited to only endpoint assays. In addition, handling and disposing of 51Cr hazardous materials is less preferred. Luciferase reporter assay, although highly sensitive, has similar drawbacks as the release assays. Finally, flow cytometry method can directly measure cell death and viability, but can be time-consuming and requires a large number of CAR-T cells when the effector-to-target (E:T) ratio is high. Furthermore, additional steps are required for adherent cells that require trypsinization. Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo high-throughput plate-based Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. Performing time- and E:T ratio-dependent CAR-T cell-mediated cytotoxicity assays, the GFP-based method demonstrated higher sensitivity in detecting the level of cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions. Citation Format: Leo Li-Ying Chan, Yu-Jun Sun, Yi-Chun Chen, Wei-Kai Hua, Sariena Chiung-Yuan Wu. Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method. [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 5326.

Abstract B022: Targeting senescence cells in cancer and aging by conditionally active biologic therapeutics

Abstract Cellular senescence is characterized by stable cell cycle arrest and a secretory program that modulates the tissue microenvironment. The aberrant accumulation of senescent cells in aged and cancerous tissue triggers inflammatory signaling through a senescence-associated secretory phenotype (SASP), promoting aging and tumor progression. Pharmacologically, clearing senescent cells has been shown to have promising effects against cancer and age-related pathologies in preclinical models as well as human pilot clinical trials. However, current senescent cell elimination strategies are focused on the rare senescence targets that are under-represented on normal cells. To date, there are no universal senescence cell specific markers. Therefore, the use of senolytics in the clinic is still limited due to their cytotoxicity to normal cells. A more effective senescence cell targeted approach is needed to reduce side effects and increase the efficiency of senescence cell removal. Conditionally Active Biologics (CAB) technology is a proprietary platform that is unique in its ability to selectively activate in the context of diseased tissues, but not normal tissues, taking advantage of the acidic pH conditions in the inflammatory tumor/aged tissue microenvironment. Senescent cells, like tumor cells, are dependent on glycolysis for energy metabolism, and thus generate a glycolytic microenvironment (GME) that resembles that of the tumor microenvironment. Since our CAB technology is already being deployed in cancer therapy by targeting cell surface markers such as AXL, ROR2, and CTLA4 in the clinical stage, we explored whether CAB technology will allow selective removal of senescent cells in SASP-associated microenvironments such as the GME. To identify senescence-specific surface antigens that are broadly and specifically upregulated in senescent cells, we screened our CAB cell surface antigen library in different senescence models including CDK4/6 inhibitor-induced senescent human breast cancer cells, human lung fibroblast cells, as well as in vivo aging mouse tissues and chronic kidney disease mouse model. We have identified several novel senescence markers which show significantly elevated expression in senescent cells. Furthermore, in vitro binding assays demonstrated that CAB antibodies targeting these senescence markers have no or very low binding to the target antigen on senescence cells in alkaline physiological conditions but have strong binding in glycolytic/acidic SASP conditions. Moreover, we performed antibody-dependent cell-mediated cytotoxicity assays for evaluating the functional activities of CAB antibodies against the senescence targets. Our data demonstrated that CAB antibodies were more potent against senescent cells compared to proliferating cells and displayed high selectivity for the glycolytic/acidic microenvironment condition. In conclusion, CAB technology should provide a new generation of biologics with an increased safety margin and therapeutic index in the clinic targeting senescence cells in cancer and age-related diseases. Citation Format: Jian Chen, Jing Wang, Matt Lucas, Haizhen Liu, Christina Wheeler, Kyrie Johnson, Kathryn Woodard, Cathy Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Targeting senescence cells in cancer and aging by conditionally active biologic therapeutics [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B022.

Triggering of lymphocytes by CD28, 4-1BB, and PD-1 checkpoints to enhance the immune response capacities.

Tumor infiltrating lymphocytes (TILs) usually become exhausted and dysfunctional owing to chronic contact with tumor cells and overexpression of multiple inhibitor receptors. Activation of TILs by targeting the inhibitory and stimulatory checkpoints has emerged as one of the most promising immunotherapy prospectively. We investigated whether triggering of CD28, 4-1BB, and PD-1 checkpoints simultaneously or alone could enhance the immune response capacity of lymphocytes. In this regard, anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins were designed and produced in CHO-K1 cells as an expression host. Following confirmation of the Fc fusion proteins' ability to bind to native targets expressed on engineered CHO-K1 cells (CHO-K1/hPD-1, CHO-K1/hCD28, CHO-K1/hCTLA4, and CHO-K1/h4-1BB), the effects of each protein, on its own and in various combinations, were assessed in vitro on T cell proliferation, cytotoxicity, and cytokines secretion using the Mixed lymphocyte reaction (MLR) assay, 7-AAD/CFSE cell-mediated cytotoxicity assay, and a LEGENDplex™ Human Th Cytokine Panel, respectively. MLR results demonstrated that T cell proliferation in the presence of the combinations of anti-PD-1/CD80-Fc, CD80-Fc/4-1BBL-Fc, and anti-PD-1/CD80-Fc/4-1BBL-Fc proteins was significantly higher than in the untreated condition (1.83-, 1.91-, and 2.02-fold respectively). Furthermore, anti-PD-1 (17%), 4-1BBL-Fc (19.2%), anti-PD-1/CD80-Fc (18.6%), anti-PD-1/4-1BBL-Fc (21%), CD80-Fc/4-1BBL-Fc (18.5%), and anti-PD-1/CD80-Fc/4-1BBL-Fc (17.3%) significantly enhanced cytotoxicity activity compared to untreated condition (7.8%). However, concerning the cytokine production, CD80-Fc and 4-1BBL-Fc alone or in combination significantly increased the secretion of IFN-γ, TNF-α, and IL-2 compared with the untreated conditions. In conclusion, this research establishes that the various combinations of produced anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins can noticeably induce the immune response in vitro. Each of these combinations may be effective in killing or destroying cancer cells depending on the type and stage of cancer.

CircRNF10 Regulates Tumorigenic Properties and Natural Killer Cell-Mediated Cytotoxicity against Breast Cancer through the miR-934/PTEN/PI3k-Akt Axis

Circular RNA (circRNA), a type of non-coding RNA, has received a great deal of attention with regard to the initiation and progression of tumors. However, the molecular mechanism and function of circRNAs in breast cancer (BC) remain unclear. In the current study, we discovered that hsa_circ_0028899 (also called circRNF10) was significantly reduced in BC tissues, and a higher level of circRNF10 was markedly related to a favorable prognosis. The results of CCK8, colony formation, Transwell, ELISA, and NK cell-mediated cytotoxicity assays indicated that increased circRNF10 expression could significantly repress the proliferation, invasion, and migration of BC cells and enhance the killing efficiency of NK cells against BC cells. According to these biological functions, the possible role and molecular mechanism of circRNF10 in BC cells were further investigated. We used bioinformatics prediction tools to predict circRNF10-bound miRNAs, which were verified by many experimental studies, including FISH, luciferase reporter assays, RIP, and Western blots. These data suggest that circRNF10 serves as a molecular sponge for miR-934 to further regulate PTEN expression and PI3k/Akt/MICA signaling in vitro and tumor growth in vivo. Altogether, these findings reveal that circRNF10 functions as a novel anti-oncogene in BC via sponging miR-934 and suppressing the PI3K/Akt/MICA pathway.

Novel Sirpα-Fc Fusion Protein IMM01 Exhibits Dual Anti-Tumor Activities By Targeting CD47/Sirpα Signal Pathway

Background: This study aims to evaluate the anti-tumor mechanism of IMM01, a novel recombinant SIRPα-Fc fusion protein targeting CD47-SIRPα pathway. Methods: IMM01 was a recombinant fusion protein of SIRPα (V2) extracellular segment domain 1 and human IgG1 Fc, with the N glycosylation site N80 mutated to A (Alanine) in D1 region. IMM01 fusion proteins were produced using in house developed CHO-K1 cell expression system. IMM01 blocking activities on Jurkat-CSR (Jurkat-CD47KO-CARSIRPα) cells, antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were done by flow cytometry. Detection of cytokines of IL-1β, IL-2, IL-4, IL-5, IL-6, GM-CSF, IFN-γ, IL-10 and TNF production by PBMC was done CBA assay. In vivo mechanism of action study was done in different mouse models including HL-60 CB17-SCID, Daudi CB17-SCID, SNU-1 CB17-SCID, Raji CB17-SCID and CT26-hPDL1(Tg) hCD47(Tg) mPDL1(KO) mCD47(KO). In addition, human red blood cells binding and erythrocyte agglutination of IMM01 were also evaluated. Results:In vitro binding assay revealed that IMM01 has a strong binding affinity to CD47 with lowest EC50 at 0.4967 nM. The removal of N-glycosylation modification improves the consistence and purity of the IMM01 protein expression, and the potential mitigation of immunogenicity. Using a chimeric SIRPα receptor protein expressing cell line, IMM01 not only can block the CD47 at the protein level, but also block the signal transduction at the cellular levels. Compared to negative (hIgG1-Fc) controls, IMM01 stimulated secretion of minimal amount of IL-10 and TNF but inhibited IL-8 production by PBMC. IMM01 can induce strong ADCP and moderate ADCC, but not the CDC activities. The strong phagocytosis against tumor cells is dependent on interaction of Fc with activating FcγRs on the presence of macrophages. Mechanism of action studies demonstrated that IMM01 induced strong phagocytosis against tumor cells was attributed to dual activities of blocking "don't eat me" signal and activating "eat me" signal. IMM01 has strong and robust in vivo anti-tumor activities in different mouse tumor models either as monotherapy on hematological malignancies, or in combination therapy with PD-L1 mAb, PD-1 mAb, CD33 mAb, and HER-2 mAb, respectively. The in vivo therapeutic efficacy of IMM01 was done in HL-60 xenograft model showed that 100% of the mice achieved CR after administration of IMM01 at 5mg/kg for 2 weeks. Also, 100% of mice achieved CR after IMM01 combined with the above mentioned mAbs in different mouse models, respectively. This illustrated that IMM01 has a great therapeutic potential in combination with these commonly used immunotherapy or targeted therapy agents to be used in the treatment of solid tumors and hematological malignancies. Finally, IMM01 demonstrated a favorable safety profile with no human RBC binding, nor hemagglutination induction. Conclusion: Our study results demonstrated that, as a novel bi-functional SIRPα-Fc recombinant protein, IMM01 has very strong anti-tumor activities with a good curative effect on mouse xenograft tumor models with dual anti-tumor activities by blocking CD47 "don't eat me" signal and activating the phagocytosis "eat me" signal. Furthermore, IMM01 demonstrated a well synergistic effect when combined with different immunotherapeutic agents. Importantly, our results revealed that IMM01 has a favorable safety profile with no human RBC binding activity and no hemagglutination induction. Phase I clinical study is ongoing, and the clinical trial results will be reported soon. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison of chimeric antigen receptor-T cell-mediated cytotoxicity assays with suspension tumor cells using plate-based image cytometry method.

In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. The GFP-based method demonstrated higher sensitivity in detecting CAR-T cell-mediated cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions.

An Innovative Site-Specific Anti-HER2 Antibody-Drug Conjugate with High Homogeneity and Improved Therapeutic Index.

PurposeAntibody-drug conjugates (ADCs) have emerged as a potent cancer therapeutic option in recent years. DP303c is a HER2-targeting ADC with a cleavable linker-MMAE payload. The current study aimed to evaluate the therapeutic potentials of DP303c in vitro as well as in vivo.Materials and MethodsSize exclusion chromatography (SEC), reverse-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to analyze the physicochemical characterization of DP303c. An enzyme-linked immunosorbent assay (ELISA), a cell-based assay, and bio-layer interferometry (BLI) were used to evaluate DP303c’s affinity with HER2 and Fc receptors. A confocal laser scanning microscopy was used to observe the internalization of DP303c. Antibody-dependent cell-mediated cytotoxicity (ADCC) and cytotoxicity assays were used to investigate the activity of DP303c in vitro. The antitumor activity of DP303c was assessed in vivo in the HER2-positive cell-derived xenograft model.ResultsDP303c was a site-specific anti-HER2 antibody-drug conjugate with a monomethyl auristatin E (MMAE) with an average drug-to-antibody ratio (DAR) of 2.0. DP303c showed a high affinity with HER2 and could be effectively internalized. In vitro and in vivo, DP303c showed stronger antitumor activity as compared to trastuzumab-DM1 (T-DM1) in a series of HER2-positive cancer cells and cell-derived xenograft (CDX) models, especially in the lower HER2-expressing cells. DP303c also exhibited high serum stability and a good PK profile.ConclusionDP303c was a steady and homogenous DAR 2 ADC that was predicted to deliver MMAE inhibitor to tumor cells. DP303c demonstrated remarkable anticancer efficacy against T-DM1 in xenograft models. DP303c was a strong candidate for the treatment of patients with HER2-positive cancer.

A Novel CD16xCD33 Bispecific Killer Cell Engager (BiKE) Mediates a Double Hit For Natural Killer (NK) Cells To Target CD33+ Myelodysplastic Syndrome (MDS) Cells and Myeloid Derived Suppressor Cells (MDSCs) At All Disease Stages

MDS is a clonal heterogeneous stem cell disorder identified by pancytopenia that frequently progresses to acute myeloid leukemia (AML). The only curative treatment is hematopoietic cell transplantation (HCT), but many patients are ineligible due to advanced age and comorbidities. New therapeutic strategies are urgently needed to reduce disease burden and improve overall survival. Studies of NK cell function from MDS patients have shown diminished natural cytotoxicity against K562 targets. These NK cell defects have been ascribed to abnormalities in expression and function of the NK cell activating receptors NKG2D, NKp30, NKp46 and DNAM-1. However, the properties of the potent activating receptor CD16 (FcgRIII) and its potential for mediating targeted immunotherapy remain unexplored in MDS. Frozen pre-transplant samples from 67 MDS patients (age 19-74 years, Median=47) receiving unrelated HCT were obtained from the research sample repository of the National Marrow Donor Program and were compared to normal volunteers. NK cell degranulation, cytotoxicity and cytokine production were evaluated using published methods. We focused on CD16-induced NK cell function using a bispecific killer cell engager generated to target CD16 along with the myeloid differentiation antigen CD33 (CD16xCD33 BiKE). CD16 expression on total CD56+ MDS-NK cells was significantly lower compared to normal donors (MDS vs. normal: 63 ± 3% (n=67) vs. 89 ± 2% (n=20); p < 0.0001). However, reverse-antibody dependent cell-mediated cytotoxicity (R-ADCC) assays revealed no significant differences in CD107a (46 ± 5% (n=16) vs. 46 ± 3% (n=12)), IFN-g (6 ± 1% vs. 10 ± 2%) or TNF-a (9 ± 2% vs. 6% ± 1%) production when resting MDS-NK cells were triggered with an agonistic CD16 mAb, indicating MDS-NK cell function triggered through CD16 is intact. To evaluate the therapeutic potential of targeting CD16 in MDS patients, resting MDS-mononuclear cells were coated with 10mg/mL of the CD16xCD33 BiKE and co-cultured for 4 hours with the CD33+ target cell line HL-60. MDS-NK cell CD107a (CD16xCD33 BiKE vs. no reagent: 42 ± 2% (n=62) vs. 22 ± 1% (n=20); p < 0.0001), IFN-g (15 ± 1% vs. 3 ± 1%; p < 0.0001) and TNF-a (13 ± 1% vs. 3 ± 1%; p < 0.0001) production was significantly increased compared to the no reagent and scFv CD16 controls. Moreover, in the absence of HL-60 targets, MDS-NK cells treated with the CD16xCD33 BiKE targeted endogenous CD33+ targets, significantly enhancing degranulation (37 ± 2% vs. 17 ± 1%; p < 0.0001), IFN-g (4 ± 1% vs. 2% ± 1%; p < 0.01) and TNF-a (5 ± 1% vs. 2 ± 1%; p < 0.01) production. Recent data have shown an increase in the frequency of MDSCs, phenotypically defined as CD33+/CD11b+/CD14-/HLA-DRlo/-, during oncogenesis and in the presence of inflammatory cytokines. Indeed, in our MDS patient cohort, the frequency of MDSCs of total viable mononuclear cells was significantly higher compared to normal donors (6 ± 1% (n=64) vs. 0.1 ± 0.02% (n=18), respectively; p < 0.0001) (figure). Furthermore, CD16xCD33 BiKE-treated resting NK cells from normal donors co-cultured with cytokine-induced MDSCs generated from normal PBMCs in a 51Cr-release cytotoxicity assay successfully targeted and lysed MDSCs (CD16xCD33 BiKE vs. no reagent (E:T = 20:1): 63 ± 5% vs. 7 ± 2% [p < 0.0001, n=4]; (E:T = 6.6:1): 35 ± 3% vs. 4 ± 1% [p < 0.0001]). CD107a (48 ± 2% (n=6) vs. 4 ± 1% (n=3); p < 0.0001), IFN-g (8 ± 1% vs. 1 ± 1%; p < 0.0001) and TNF-a (8 ± 1% vs. 1 ± 1%; p < 0.0001) production was also significantly increased against MDSC targets in the presence of the CD16xCD33 BiKE, demonstrating its ability to facilitate killing of both the CD33+ MDS clone and an immune suppressor cell population. Lastly, analysis of MDS patient karyotypes in correlation with CD16xCD33 BiKE-induced MDS-NK cell CD107a, IFN-g and TNF-a production did not reveal any significant differences between favorable, intermediate or poor international prognostic scoring system (IPSS) cytogenetic scores, further demonstrating the therapeutic potential of the CD16xCD33 BiKE to induce optimal MDS-NK cell function against CD33+ targets despite disease heterogeneity. In summary, our data suggest that the CD16xCD33 BiKE may function not only against CD33+ MDS targets themselves, but also against MDSCs. This double hit approach may protect against MDS progression/transformation to AML through immune targeting and may improve blood counts by targeting MDS-MDSCs that contribute to ineffective hematopoiesis. [Display omitted] Disclosures:No relesvant conflicts of interest to declare.

Abstract 1889: Dual-specific antibodies that bind to BCMA and TACI (HMBD-009) enable better targeting of multiple myeloma in pre-clinical models

Abstract Existing immunotherapies for multiple myelomas (MM), including bispecific antibodies, antibody-drug conjugates and chimeric antigen receptor-based strategies, have targeted the B-cell maturation antigen (BCMA) that is expressed at the surface of malignant plasma cells (PCs). Although promising, clinical outcomes are hampered by resistance mechanisms that include BCMA extracellular domain shedding and cell-surface downregulation. In addition, BCMA expression is known to be variable among MM patients. Transmembrane activator and CAML interactor (TACI), another member of the tumor necrosis factor receptor (TNFR) super family, represents another viable drug target. TACI expression largely overlaps with BCMA and expression is also increased in PCs of MM patients. Interestingly, TACI may have a role in maintaining an immunosuppressive tumor microenvironment as its expression was observed to be upregulated in Tregs in MM patient's tumor load. Activation of TACI in Tregs triggers their proliferation and survival further supporting a pro-tumoral role for this receptor. Altogether, the dual targeting of both BCMA and TACI should be beneficial to the efficient removal of malignant PCs. Despite low sequence homology, BCMA and TACI share a similar 3D structure, which is likely the reason that APRIL (A proliferation-Inducing ligand) binds to both receptors. We applied our proprietary Rational Antibody Discovery platform to successfully direct the generation of mouse IgG clones able to bind a conserved epitope on BCMA and TACI with sub-nanomolar affinities. In addition, these antibodies were able to inhibit the binding of BCMA to APRIL - an interaction known to favor MM tumor growth and survival. Two IgG clones were successfully humanized and retained their capacity to bind to both BCMA and TACI when heterologously expressed on HEK293, or endogenously expressed in MM cancer cell lines such as H929. No cross-reactivity to other TNFR family members was observed. In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, these clones were able to induce efficient cell killing by binding either BCMA or TACI. Moreover, our lead dual-specific anti-BCMA/anti-TACI antibody (HMBD-009) showed better tumor control compared to other anti-BCMA only antibodies in pre-clinical tumor models of MM. Dual-specific antibodies able to efficiently target both BCMA and TACI represent a promising new avenue to improve the elimination of malignant PCs from MM patients. Citation Format: Fabien M. Decaillot, Dipti Thakkar, Siyu Guan, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kikurp. Dual-specific antibodies that bind to BCMA and TACI (HMBD-009) enable better targeting of multiple myeloma in pre-clinical models [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 1889.

Engineering Precision Medicine to Enhance Graft-Versus-Lymphoma Activity: Hematopoietic Stem Cells Modified with Chimeric Antigen Receptors

Background: Gene-modified human hematopoietic stem cells (HSC) have been used to introduce genes to correct, prevent or treat diseases. Clinical trials using infusion of autologous T cells engineered with anti-CD19 chimeric antigen receptors (CAR) have demonstrated complete remissions even in chemotherapy-resistant malignancies, but the persistence of the effector cells is transient, limiting clinical efficacy and allowing the cancer to recur. Current trials of CAR T cells have recommended patients in remission post-T cell therapy to undergo hematopoietic stem cell transplantation (HSCT) due to the limited persistence of the CAR+ T cells. Seeking to foster persistence of the CAR-modified immune cells, we propose using gene modification of HSC to create persistent generation of multilineage immune effectors to directly target cancer cells. Concerns regarding malignant transformation, abnormal hematopoiesis and autoimmunity exist, making the co-delivery of a suicide gene a necessary safety measure. We evaluated a second-generation anti-CD19 chimeric antigen receptor (CAR) to human HSC in a humanized mouse model, co-delivered with a truncated epidermal growth factor receptor (EGFRt) as a suicide gene system.Significance: CAR+ HSC can be infused in the context of HSCT, when the chemotherapy-conditioning regimen favors engraftment of gene-modified cells, decreases residual tumor burden and decreases the chance of CAR immunogenicity. The prospect of modifying autologous cells to enhance GVL bears the possibility of decreased morbidity and mortality, a concern for specifically vulnerable populations such as children and elderly patients. In addition, this approach will offer alternative therapy for those without cell sources available for allogeneic HSCT, benefiting ethnic minorities.Methods: Third-generation self-inactivating lentiviral vectors were used to co-deliver an anti-CD19 CAR and EGFRt. In vitro, gene-modified HSC were differentiated into myeloid cells to allow transgene expression. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used incubating target cells with leukocytes and monoclonal antibody cetuximab to determine percentage of surviving cells. In vivo, gene-modified HSC were engrafted into immunodeficient NSG mice treated or not with intraperitoneal cetuximab 1mg/mouse/day for 12 days. Persistence of gene-modified cells was assessed by flow cytometry, ddPCR, and PET imaging using 89Zr-Cetuximab.Results: Gene modification of HSC did not affect engraftment, proliferation or differentiation, at efficiency of 20-40%. Antigen specificity of myeloid, NK and T cells were successfully redirected against CD19. Ablation of gene-modified cells was significantly increased (p=0.01) in target cells expressing EGFRt after incubation with leukocytes and cetuximab 1µg/mL, compared to EGFRt+ cells without cetuximab, and non-transduced cells with or without cetuximab. This was seen at all effector-to-target ratios. Mice engrafted with CAR-modified HSC presented prolonged persistence of gene-modified cells in bone marrow, spleen, thymus and peripheral blood, and CAR+ cells elicited protection against CD19+ tumors, with inhibition or elimination of tumor development and survival advantage. Mice humanized with gene-modified HSC for the expression of the suicide gene had significant ablation of gene-modified cells after treatment with cetuximab (p=0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues. PET imaging of humanized mice documented engraftment of EGFRt-expressing cells in bone marrow, spleen and liver, and confirmed ablation with an average decrease of 82.5% after cetuximab treatment.Conclusions: These results give proof of principle for persistence and anti-tumor activity of CAR-modified HSC regulated by suicide gene, and further studies are needed to enable clinical translation. Cetuximab ADCC of EGFRt-modified cells caused effective killing. Different ablation approaches, such as inducible caspase 9 or co-delivery of other inert cell markers should be evaluated. DisclosuresNo relevant conflicts of interest to declare.

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies.

The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals (n = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A “pooled serum” (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1.

KPMW135, a Biosuperior CD3 Bispecific Version of Rituximab Created By a Novel Chemical Conjugation Technology Demonstrates Increased Anti-Tumor Activity By Adding T Cell-Mediated Cytotoxicity Activity to the Existing Mechanisms of Rituximab

Background We describe KPMW135, a novel CD20 x CD3 bispecific molecule, in which a CD3-directed ScFv (SP34) is conjugated to rituximab (RTX). RTX is a chimeric monoclonal antibody (mAb) specific for CD20, used to treat B-cell lymphomas and lymphocytic leukemias. Monoclonal Ab therapy enhancer (MATE™) enables efficient site-directed chemical conjugation to "off-the-shelf" therapeutic mAbs and allows the development of bispecific therapeutic candidates. Our MATE platform allows for the chemical engineering of existing Abs without the need to create new DNA vectors or genetic engineering of master cell lines. This approach is superior to previous conjugation methods that 1) lack site-directed conjugation specificity by indiscriminately binding to available amino acid residues or 2) require genetic engineering to create conjugate tags. Methods SP34 scFv was constructed using published CDRs. The expression was in FreeStyle 293 cells, followed by affinity purification and SEC to obtain monomeric scFv. The binding of CD20 to KPMW135 was measured by Bio-Layer Interferometry using Octet (Fortebio). Equilibrium dissociation constant (Kd) was calculated using the Octet HT software. The binding of CD3εδ, CD16a, and FcRn were determined by ELISA using neutravidin-coated plates. Biotinylated Human CD3ε &amp; CD3δ heterodimer protein (Avi tag), CD16a (Avi Tag) and human CD16a were used. Readout was determined with anti-human F(ab) HRP. For in vitro T cell-mediated cytotoxicity assays, unfractionated and NK-cell depleted PBMCs were prepared from freshly-thawed and PHA + IL-2 prestimulated PBMCs. Daudi (CD20+) B lymphoblast cells were engineered to stably express a beta-gal reporter fragment using KILR retroparticles (Eurofins). Target cells were treated with KPMW135, RTX, and controls. PBMCs were introduced at an effector:target ratio of 15:1 and incubated for 18h. Luminescence signal was obtained with luminometer to reflect target cell death. Cynomolgus monkeys were intravenously injected with RTX or KPMW135 (30 μg/kg). Endpoints included clinical observations, cytokine profile and flow cytometric immunophenotyping of T cells, monocytes, granulocytes, NK cells, and B cells (CD45, CD3, CD16, CD14, NKG2A, HLA DR) and cellular activation (CD44 and CD69). Results Binding affinity of CD20 to KPMW135 was 0.75nM. CD20 binding to RTX and KPMW135 was similar, thus conjugation of the CD3-binding ScFv did not negatively affect affinity to CD20. ELISA results show KPMW135 binds to CD3εδ, FcRn, and CD16a, indicating that the conjugation does not interfere with binding to their various moieties. In vitro T cell-mediated cytotoxicity results show the activity of Fc/FcR and CD20 binding by the RTX moiety versus CD3 binding to effector T cells. T cell enrichment by functionally prestimulated PBMCs increased killing of target cells by 2-fold. Mechanically depleted NK cells verified that target cell death was induced by T cells, rather than by NK cells via ADCC. KPMW135 elicited target cell killing with an EC50 of 0.03-0.07nM or 0.09-021nM for prestimulated or freshly thawed PBMCs, respectively. Studies in Cynomolgus monkeys showed that KPMW135 can activate T cells in vivo as shown by increased CD69 and CD44 expression (3- and 2-fold, respectively) with a peak at 4h post-dose compared to equivalent dose RTX. A pronounced depletion of B cells was observed in KPMW135 treated animals as early as half hour post-dose, with a partial recovery by days 7-14, while an equivalent dose of RTX induced a transient B-cell depletion followed by a quicker return to baseline levels. Conclusions KPMW135 is a CD20 x CD3 bispecific created by chemical conjugation of CD3-specific ScFv to "off-the-shelf" RTX using our proprietary MATE™ technology, which maintains RTX native binding to FcRs via the Fc domain and its native binding to CD20 on target cells. KPMW135 retains RTX native ability to target B lymphoid malignancies via Fc effector mechanisms like ADCC and ADCP, but now adds T cell-mediated cytotoxicity. Our in vitro data shows increased target cell killing by KPMW135 compared to RTX, and our in vivo study confirms KPMW135 ability to induce target dependent T cell activation and recruitment, making it superior to unconjugated RTX. CD3 MATEs generated with other Abs show similar increased T cell-mediated cytotoxicity, confirming that this technology can be applied to generate "off-the-shelf" biosuperior antibodies. Disclosures Vidal: Kleo Pharmaceuticals: Current Employment, Current equity holder in private company. Cukan:Kleo Pharmaceuticals: Current Employment. Vaill:Kleo Pharmaceuticals: Current Employment. Bunin:Kleo Pharmaceuticals: Current Employment. Rossi:Kleo Pharmaceuticals: Current Employment. Iben:Kleo Pharmaceuticals: Current Employment. Trinh:Kleo Pharmaceuticals: Current Employment. McGrath:Kleo Pharmaceuticals: Current Employment. Berbasova:Kleo Pharmaceuticals: Current Employment. Alvarez:Kleo Pharmaceuticals: Current Employment. Rastelli:Kleo Pharmaceuticals: Current Employment. Welsch:Kleo Pharmaceuticals: Ended employment in the past 24 months.

Development and optimization of a Zika virus antibody-dependent cell-mediated cytotoxicity (ADCC) assay

Zika virus (ZIKV) has become a global public health issue due to its teratogenicity and ability to cause Guillain-Barré syndrome in adults. Although anti-ZIKV envelope protein neutralizing antibodies correlate with protection, the non-neutralizing function of ZIKV antibodies including antibody-dependent cell-mediated cytotoxicity (ADCC) is incompletely understood. To study the role of ADCC antibodies during ZIKV infections, we generated a stably transfected, dual-reporter target cell line with inducible expression of a chimeric ZIKV prM-E protein on the cell surface as the target cell for the assay. By using this assay, nine of ten serum samples from ZIKV-infected patients had >20% ADCC killing of target cells, whereas none of the 12 healthy control sera had >10% ADCC killing. We also observed a time-dependent ADCC response in 2 patients with Zika. This demonstrates that this assay can detect ZIKV ADCC with high sensitivity and specificity, which could be useful for measurement of ADCC responses to ZIKV infection or vaccination.