Kill Target Cells Research Articles

Clonally Expanded Cytotoxic CD8 +T cells Target Citrullinated Antigens In ACPA+ Rheumatoid Arthritis

Abstract The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8 +T cells have been described in RA, their role in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8 +T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB +CD8 +subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK +CD8 +memory subpopulation comprises of smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB +CD8 +T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB +CD8 +cells are present in RA synovium. These findings suggest that cytotoxic CD8 +T cells targeting citrullinated antigens have a role in contributing to synovitis and joint tissue destruction in ACPA+ RA. The study was supported by following grants: National Institutes of Health grant R01 AR063676, U19 AI110491, R01 AR078268, and Janssen Biotech, Inc.

Deep phenotypic and functional characterization of NK cells during NK-mediated cytotoxicity

Abstract Natural Killer (NK) cell-mediated cytotoxicity is a critical mechanism of the immune system to respond against harmful stimuli. The development of emerging cell therapies, such as CAR-NK cells, requires a deeper characterization of cultured NK cells. Here we show a phenotyping of ex vivo and in vitro NK cells, and demonstrate how high quality and reliable reagents from BioLegend ensure a more in depth cytotoxic analysis. NK cells were isolated from fresh human PBMCs, using our MojoSort™ Human NK Cell Isolation Kit, and incubated at different ratios with Tag-it Violet™ stained K562 target cells in IMDM + BioLegend’s Cell-Vive™ T-NK Xeno-Free Serum Substitute, in the presence of CD107a-PE. Cells were then harvested for surface phenotyping and the supernatants were collected for cytokine analysis using LEGENDplex™ Human CD8/NK Panel. Tag-it Violet™ and Apotracker™ Green staining of K562 cells demonstrate significant decrease in proliferation and significant increase in apoptosis, respectively. Surface staining of CD107a reveals increased degranulation of both CD56 brightCD16 −and CD56 +CD16 +NK cells that correlates with increased apoptosis of K562 cells. Supernatant cytokine analysis revealed significant increases in Granzyme A and B, Perforin, and Granulysin, among others. Taken together, these results show a more complete picture of the phenotypical and functional state of NK cells by measuring target cell killing as well as characterizing NK surface markers and secreted molecules involved in NK-mediated cytotoxicity. BioLegend reagents are designed to address ever increasing needs for deeper understanding of immunological processes and support the demand to develop sophisticated and consistent cell-based therapies.

The hyperinflammatory spectrum: from defects in cytotoxicity to cytokine control.

Cytotoxic lymphocytes kill target cells through polarized release of the content of cytotoxic granules towards the target cell. The importance of this cytotoxic pathway in immune regulation is evidenced by the severe and often fatal condition, known as hemophagocytic lymphohistiocytosis (HLH) that occurs in mice and humans with inborn errors of lymphocyte cytotoxic function. The clinical and preclinical data indicate that the damage seen in severe, virally triggered HLH is due to an overwhelming immune system reaction and not the direct effects of the virus per se. The main HLH-disease mechanism, which links impaired cytotoxicity to excessive release of pro-inflammatory cytokines is a prolongation of the synapse time between the cytotoxic effector cell and the target cell, which prompts the former to secrete larger amounts of cytokines (including interferon gamma) that activate macrophages. We and others have identified novel genetic HLH spectrum disorders. In the present update, we position these newly reported molecular causes, including CD48-haploinsufficiency and ZNFX1-deficiency, within the pathogenic pathways that lead to HLH. These genetic defects have consequences on the cellular level on a gradient model ranging from impaired lymphocyte cytotoxicity to intrinsic activation of macrophages and virally infected cells. Altogether, it is clear that target cells and macrophages may play an independent role and are not passive bystanders in the pathogenesis of HLH. Understanding these processes which lead to immune dysregulation may pave the way to novel ideas for medical intervention in HLH and virally triggered hypercytokinemia.

Identification of molecular candidates which regulate calcium-dependent CD8+ T-cell cytotoxicity

Cytotoxic CD8+ T lymphocytes (CTL) eliminate infected cells or transformed tumor cells by releasing perforin-containing cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca2+-influx through store operated Ca2+ channels, formed by STIM (stromal interaction molecule)-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca2+-dependent target cell killing. CTL killing efficiency is of high interest considering the number of studies on CD8+ T lymphocytes modified for clinical use. Here, we isolated total RNA from primary human cells: natural killer (NK) cells, non-stimulated CD8+ T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8+ T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca2+-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8+ T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca2+ dependent cytotoxicity, candidates were also analyzed under Ca2+-limiting conditions. Overall, we identified four hits, CCR5 (C-C chemokine receptor type five), KCNN4 (potassium calcium-activated channel subfamily N), RCAN3 (regulator of calcineurin) and BCL (B-cell lymphoma) 2 which clearly affect the efficiency of Ca2+ dependent cytotoxicity in CTL-MART-1 cells, CCR5, BCL2, and KCNN4 in a positive manner, and RCAN3 in a negative way.

Abstract 5135: Standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies

Abstract In the treatment of solid tumors, chimeric antigen receptor(CAR)-engineered NK (CAR-NK) cells have distinct advantages over CAR-T cells, such as a lack graft-versus-host disease in allogenic setting to make “off the shelf” medicine; much safer because they are less likely to cause cytokine storms; and NK cells have their own activated receptors that recognize tumor surface antigens and thus have a natural ability to kill a wide range of tumors. In addition, NK cells are the main performers of antibody-dependent cell-mediated cytotoxicity (ADCC), which binds to the Fc-terminus of antibodies through their surface Fc receptor CD16A, thereby killing antibody-targeted tumor cells. Enhancing the ADCC action of NK cells themselves enhances the therapeutic efficacy of monoclonal antibodies targeting tumor surface antigens and is therefore of great importance in the treatment of tumors, especially solid tumors. We screened two optimal structures among nine different Fc chimeric receptors, and NK cells overexpressing these two receptors killed K562 and Daudi six times more intensely than normal NK cells when combined with rituximab, while the killing ability was comparable to that of normal NK without rituximab, indicating that NK cells expressing chimeric Fc receptors have stronger ADCC effects. Moreover, these NK cells can kill target cells in multiple rounds. In vivo experiments in mice demonstrated that NK cells expressing chimeric Fc receptors in combination with EGFR monoclonal antibodies had a stronger inhibitory effect on tumor growth than monoclonal antibodies or NK cells alone. Here we provide a novel broad “off the shelf” NK cells that can significantly enhance the killing ability of hematological and solid tumors in combination with different monoclonal antibodies. Citation Format: Yanan Lin, Shengbang Zhang, Jie Ran, Can Song, Shengcang Zhu, Hui Shi, Ping Guo, Shengjiang Tan, Yuchun Gu, Lida Wu. Standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies. [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 5135.

Abstract 5329: High throughput screening strategies in the development of logic gated cell therapies

Abstract Chimeric antigen receptor T cell (CAR T) therapy has demonstrated unprecedented therapeutic activity in hematologic malignancies. However, generating potent clinical responses against solid tumors remains a challenge for CAR T therapy. As the field strives to improve the therapeutic efficacy of CAR T cells with novel target antigens and enhanced potency, the risks of on-target toxicity pose a major barrier to progress. To address these challenges, we have developed engineered CAR T cells to target solid tumors through AND logic gates, where CAR expression is conditionally induced by a transcription factor released from a priming receptor (PrimeRTM) upon binding to the PrimeR antigen. The AND gate limits off-tumor toxicity as it requires both CAR and PrimeR antigen expression in the tumor microenvironment. To ensure PrimeR expression and signal transduction upon antigen binding, while minimizing residual ‘‘leaky’’ CAR induction in the absence of PrimeR antigen, we screened hundreds of PrimeR binders using both arrayed and pooled strategies. In an arrayed strategy, we engineered T cells from four donors in multiwell plates using CRISPR-mediated, non-viral, site-specific integration of logic gates bearing a variable PrimeR binder and a fixed MSLN CAR. In addition, we employed a pooled screening strategy, where we engineered T cells from two independent donors with a pool containing a subset of >300 of the same logic gates. Engineered T cells from both strategies were co-cultured with cell lines bearing either both CAR and PrimeR antigens or a single antigen, in order to evaluate fidelity and on-target functionality. In the arrayed setting, on-target functionality was quantified based on the levels of CAR induction, cytokine secretion, T cell activation, and target cell killing in the presence of both antigens, while fidelity was assessed based on the absence of these activity signals in the presence of a single antigen. In the pooled setting, sorting based on functional markers was performed and sequencing was used to quantify the relative abundance of cells with each logic gate in different sorted populations. On-target activity and circuit fidelity were then quantified based on enrichments in different sorted populations. Results from the pooled and arrayed screens were highly concordant. We combined the screen readouts to nominate a small set of PrimeR binders that exhibited both high fidelity and on-target functionality. We confirmed the desired characteristics of these binders with targeted arrayed screens in additional conditions as well as in in-vivo models. We have applied both screen strategies to select a small set of leads from hundreds of candidate PrimeR binders in the context of a logic-gated MSLN CAR. As pooled and arrayed screens come with different sets of limitations and advantages, both serve as important tools for the effective selection of receptors in the development of novel cell therapies. Citation Format: Li Wang, Sofia Kyriazopoulou Panagiotopoulou, Rona Harari-Steinfeld, Dasmanthie De Silva, Michelle Tan, Laura Lim, Angela Boroughs, Cate Sue, Jon Chen, Jamie Thomas, Mary Chua, Ed Yashin, Christine Shieh, Ryan Fong, Sophie Xu, Grace Zheng, Brendan Galvin, Aaron Cooper, Tarjei Mikkelsen, Nicholas Haining. High throughput screening strategies in the development of logic gated cell therapies. [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 5329.

Abstract 3183: Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against solid tumors

Abstract Background: Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense against opsonized target cells wherein the Fc-fragment of an antibody binds to the CD16 Fc receptor (FcγRIIIa) on NK cells, triggering NK cell activation and targeted killing. Cetuximab is a human IgG1 antibody targeting epidermal growth factor receptor (EGFR) that is approved for the treatment of colorectal and head and neck cancers. Binding of cetuximab to EGRF+ cell lines has been shown to trigger ADCC mediated by NK cells. NKX101 is an investigational allogeneic NK cell therapy in development for the treatment of cancer. NKX101 is engineered to express an NKG2D-based chimeric antigen receptor (CAR) and membrane-bound IL-15. To investigate the combination of NKX101 and cetuximab, we developed in vitro assays to (i) demonstrate enhanced activity against cancer cells via CD16-mediated ADCC activity, (ii) compare ADCC activity of NKX101 cells generated from high-affinity CD16 and low-affinity CD16 donors, and (iii) evaluate the synergistic interaction between NKX101 and cetuximab. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. NucRed-labeled EGFR+ NKG2D-L+ human cancer cell lines were pre-incubated with cetuximab or isotype control and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte instrument. For synergy assessments, a dose response matrix of NKX101 cells and cetuximab was used to generate cytotoxicity values. Synergy scores for combinations were determined using the Synergy Finder Plus web application. To determine donor CD16 V/F 158 genotypes, genomic DNA was isolated from each donor and assayed using a TaqMan CD16 genotyping kit. Results: We demonstrate that cetuximab increases the anti-tumor effect of NKX101 in a dose-dependent manner. Assessment of the interaction between NKX101 and cetuximab in vitro revealed that the two agents can combine to kill cancer cells in a synergistic manner. Blocking CD16 on NKX101 cells with a neutralizing antibody significantly decreased potency of the combination, suggesting that the improvement in potency observed is a direct result of CD16-mediated ADCC activity. To determine if common CD16 polymorphisms influence NKX101 ADCC activity, we compared NKX101 cells generated from CD16 158 V/V, V/F, and F/F donors. No significant differences in potency were observed with NKX101 cells expressing the high affinity CD16 158 V/V genotype compared with those derived from CD16 lower affinity 158 V/F or 158 F/F donors. In summary, we show that the combination of an engineered allogeneic NK cell therapy, NKX101, with cetuximab leads to an increase in the specific killing of cancer cells in vitro, supporting further investigation into this combination for the treatment of solid tumors. Citation Format: Cynthia Cho, Kyle Hansen, Max Zhang, Carmel Chan, James Trager. Combination of anti-EGFR antibody cetuximab with NKX101, an allogeneic NKG2D-L targeting NK cell therapy, enhances potency and in vitro cytotoxicity against solid tumors [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 3183.

Abstract 1815: Rapid serial killing of target cells by Vγ9Vδ2 T Cells in cynomolgus macaques and humanized mice treated with a CD20-directed heterodimeric butyrophilin 2A1/3A1 fusion protein

Abstract CD3-directed T cell engagers provide clinical benefit in a variety of hematologic cancers, but it remains unclear whether γδ T cells (GDT) are present at sufficient numbers to be similarly harnessed because they represent only 1-5% of the total T cell pool. The Vγ9Vδ2 T cell receptor is activated by a butyrophilin 2A1 and 3A1 (2A1/3A1) heterodimer as “signal 1”, together with costimulatory signaling through CD28 or NKG2D as “signal 2”. Here we evaluated the ability of a GDT engager (GADLEN) comprising the extracellular domains of 2A1/3A1 adjoined via an Fc to an antibody fragment targeting the CD20 antigen, to mediate target-cell depletion in vivo at physiologically relevant frequencies of GDT. A humanized mouse model was developed wherein human peripheral blood mononuclear cells (PBMC) were engrafted to NSG-IL15 mice and a high degree of human chimerism persisted for over 3 weeks, including a circulating pool of human GDT comprising 0.5 to 3% of total T cells. Across different human donors, the GDT effector to target B cell ratio varied from 1:1 to 1:46. Following treatment with a B cell-targeted GADLEN, rapid and dose-dependent depletion of over 95% of human B cells was achieved in the peripheral blood and spleen, even at E:T ratios of 1:46. Based on these data, a repeat-dose toxicology study was conducted in cynomolgus macaques across a dose range of 0.1 to 25 mg/kg with the CD20-targeted GADLEN. This study also demonstrated dose-dependent B cell depletion within 2 hours of infusion. Durable receptor occupancy was observed on CD20+ cells for over 7 days, whereas transient occupancy was observed on circulating cynomolgus Vγ9Vδ2 T cells. GDT represented approximately 1-3% of total T cells in cynomolgus macaques, and E:T ratios ranged from 1:7 to 1:49. All treatments were well tolerated, and no evidence of cytokine release syndrome nor any other toxicities were observed. Interestingly, in both humanized mice and non-human primates, the proliferation of GDT was not required to achieve B cell depletion. In fact, over several weeks, the circulating frequencies of cynomolgus Vγ9Vδ2 T cells remained stable, and no evidence of either proliferation or migration out of the blood was observed. These observations, together with the speed at which B cell depletion occurred, suggest that the Vγ9Vδ2 T cells eliminated the B cell pool by serial killing following treatment with the GADLEN. Despite a GDT frequency of less than 5% of total T cells in all these studies, Vγ9Vδ2 T cells achieved rapid depletion of target cells following treatment with the GADLEN, without evidence of toxicity, even at high doses. Collectively, these results indicate that low frequencies of GDT can be harnessed to achieve similar target-cell killing potential as the broader CD3+ T cell pool but with potentially less toxicity. Citation Format: Anne Y. Lai, Louis E. Gonzalez, Noah Murr, Karen Lenz, Derek Franklin, Kristen Campbell, Faraha Brewer, Arpita Patel, Kinsley Evans, Mahmud Hussein, Keith Wilson, George Fromm, Taylor H. Schreiber, Suresh de Silva. Rapid serial killing of target cells by Vγ9Vδ2 T Cells in cynomolgus macaques and humanized mice treated with a CD20-directed heterodimeric butyrophilin 2A1/3A1 fusion protein [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 1815.

Abstract 5659: Targeting solid cancers with a cancer-specific monoclonal antibody to surface expressed aberrant O-glycosylated proteins

Abstract The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Most current immunotherapeutic targets are tumor-associated antigens also found in healthy tissues. Such targets often do not display sufficient cancer selectivity to be used for potent antibody based immunotherapeutic treatments, such as chimeric antigen receptor (CAR) T cells. Many solid tumors, however, display aberrant glycosylation that results in the expression of tumor-associated carbohydrate antigens that are not present on healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes within existing or novel glycoprotein targets may provide the cancer selectivity needed for immunotherapy of solid tumours, but only a few such glycopeptide epitopes have been targeted. Here, we used O-glycoproteomics data from multiple cell lines to identify a glycopeptide epitope in CD44v6, a cancer-associated CD44 isoform, and through a glycopeptide immunization strategy developed a cancer-specific monoclonal antibody, 4C8. 4C8 selectively binds to Tn-glycosylated CD44v6 in a site-specific manner with low nanomolar affinity. 4C8 was shown to be highly cancer selective by immunohistochemistry of sections from multiple healthy and cancerous tissues. 4C8 CAR T cells demonstrated target-specific cytotoxicity in vitro and significant tumor regression, and increased survival in vivo. Importantly, 4C8 CAR T cells selectively killed target cells in a mixed organotypic skin cancer model without affecting healthy CD44v6+ keratinocytes, indicating tolerability and safety. Targeting aberrantly glycosylated glycopeptide epitopes may provide the cancer selectivity needed for solid tumour immunotherapy. Citation Format: Mikkel K.M. Aasted, Aaron C. Groen, John T. Keane, Sally Dabelsteen, Edwin Tan, Julia Schnabel, Fang Liu, Hyeon-Gyu S. Lewis, Constantine Theodoropulos, Avery D. Posey, Hans H. Wandall. Targeting solid cancers with a cancer-specific monoclonal antibody to surface expressed aberrant O-glycosylated proteins. [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 5659.

Abstract 693: A novel multi-subset dendritic cell vaccine derived from hematopoietic progenitor cells

Abstract Owing to their molecular features, tissue localization and antigen processing and presentation capacity, dendritic cells (DCs) induce and regulate T cell immunity including licensing T cells to kill target cells. However, tumors often suppress DC differentiation and function leaving cancer patients with DC populations that are quantitatively and qualitatively deficient. This limits both the native immune responses to tumors as well as response to cancer therapy including immunotherapy. Thus, provision of healthy and functionally intact DCs to replace and correct the in vivo function offers an interesting therapeutic opportunity. DCs are composed of multiple subsets which specialize in activation of different immune effectors. Thus, a combination of DC subsets could generate a broad and diverse immunity targeted at cancer. To date, several attempts have been made to generate functional human DCs ex vivo for injection to patients, but the DC subtypes most effective in generation of anti-tumor responses—cDC1 and cDC2s—have proven difficult to make ex vivo using clinically compliant protocols. Here we show that cDC1s and cDC2s can be generated at high yield from hematopoietic progenitor cells (HPCs) from three different HPCs sources (including adult bone marrow) using clinically compliant protocols. We characterize the phenotype and functionality of these cells showing that both cDC1s and cDC2s efficiently process antigens, stimulate, and expand T cells specific for those antigens, and that those T cell populations display targeted killing of tumor cells in vitro. The ability to produce these subtypes using clinically compliant protocols is a major step towards developing effective autologous DC therapy for cancer patients. Citation Format: Chun Yu, Richard L. Barrett, Ananya Zutshi, Karolina Palucka. A novel multi-subset dendritic cell vaccine derived from hematopoietic progenitor 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 693.

Abstract 2925: In vitro characterization of next generation therapeutic antibodies

Abstract Therapeutic antibodies are an established drug class. Checkpoint inhibitors, such as anti-PD-1/PD-L1 and anti-CTLA-4 antibodies for cancer indications, and anti-TNF-α monoclonal antibody against autoimmune disorders represent some of most successful therapeutics in the past decade. Over 100 therapeutic targets suitable for antibody therapy have been identified for various disease indications, with several currently in late-stage clinical trials. In addition, there has been an explosion in the various types of therapeutic antibody modalities due to the increasing number of antibody engineering platforms. The new emerging bi-specific T-cell engaging antibodies (BiTEs) along with antibody-drug conjugates (ADCs) have demonstrated superior therapeutic qualities over the traditional monoclonal antibodies. Here, we describe a pipeline to extensively characterize the various antibody therapeutics in vitro utilizing multiple innovative technologies. Assays to determine target expression and binding potency provide a thorough understanding of the target engagement. Furthermore, we present data from an innovative technology platform, the z-Movi, which measures cell avidity to predict invitro effector functions of T-cell engaging therapies, enabling more robust read-outs and could drive the lead selection of said therapies. Several mechanisms of action of different antibodies were investigated and relevant case studies describing the innate immune effector functions and the signaling pathway modulation are presented. Using a unique label-free approach, the xCELLigence, we report on the kinetics of target cell killing mediated by lymphocytes upon recognition of antigen-antibody complexes. Finally, safety assessment studies focusing on on-target/off-tumor effects of antibodies on primary cells were conducted. The cytokine profiling and cell viability assays to assess potential risk due to exaggerated levels of cytokine, reiterate the specificity and safety of the next generation of antibody therapeutics and support Investigational New Drug (IND) applications. In conclusion, we provide a comprehensive report on the in vitro characterization of the advanced antibody therapeutics, showcasing a pipeline for development and functional characterization of various antibody modalities, including bispecific T-cell engagers. This workflow can be used to progress a wide range of engineered antibodies from pre-clinical to the clinical phase of the drug discovery process. NJ and GM contributed equally to the work. Citation Format: Benita Quist, Hamed Janbazacyabar, David Cobeta Lopez, Namrata Jayanth, Gemma Moiset. In vitro characterization of next generation therapeutic antibodies [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 2925.

Abstract 1787: Design of bispecific adapters for universal CAR T cell therapies of solid tumors

Abstract Recent success in the use of chimeric antigen receptor (CAR) T cell therapies to treat hematologic cancers has encouraged the exploration of CAR T cell technologies to treat solid tumors. For establishment of a productive immunologic synapse between the CAR T cell and cancer cell, multiple CARs on the T cell surface must engage multiple tumor antigens on the cancer cell surface. Whereas these binding interactions are mediated in classical CAR T cells by direct recognition of the tumor antigen by the scFv on the CAR, in the case of universal CAR T cells this recognition must be created by a bispecific adapter that can bridge between the tumor antigen and the CAR. While a diversity of bispecific adapters have been explored in the literature, the bispecific adapter used here is comprised of a low molecular weight ligand that binds avidly to the desired tumor antigen, the yellow dye fluorescein (FL) that binds tightly to an anti-fluorescein scFv on the CAR, and a polyethylene glycol (PEG) spacer that spans between the two. The length of this PEG spacer appears to be critical to ensure that 1) the bispecific adapter is long enough to mediate engagement of the universal CAR T cell with the cancer cell, and 2) the resulting intercellular distance is optimal for activating the necessary co-receptor interactions.In our study, we have utilized protein structure data and molecular modeling to estimate the optimal adapter spacer lengths for bridging between our CAR T cell and two important target cells, namely PSMA positive prostate cancer cells and fibroblast activation protein (FAP) expressing cancer associated fibroblasts. We then synthesized the desired bispecific adapters with a variety lengths of PEG spacer and evaluated their effects in mediating the cytotoxicity of our universal anti-FL CAR T cells both in vitro and in vivo. Our results reveal that the optimal spacer lengths in the bispecific adapters for targeting PSMA (DUPA-FL) and FAP (FAPL-FL) are different and depend on the locations of the adapter binding pockets on their respective protein targets, as predicted by molecular modeling. Specifically, a shorter PEG spacer is required for maximal cytoxicity when employing the PSMA-targeted adapter, whereas a much longer PEG spacer is needed to achieve maximal cell killing with the FAP-targeted adapter. In addition, spacer length can also be adjusted to avoid overactivation-induced T cell exhaustion. Taken together, our studies demonstrate that instead of optimizing the hinge length of a CAR for each antigen epitope, our bispecific adapter/universal CAR platform provides a highly flexible strategy for optimizing the intermembrane distance between the universal CAR T cell and cancer cell to achieve maximal target cell killing with minimal CAR T cell exhaustion. Citation Format: Suilan Zheng, Bo Huang, Yashapal Singh, Haiyan Chu, Suresh K. Bowroju, Md Sazzadul Bari, Madduri Srinivasarao, Sudarsan R. Kasireddy, Ramesh Mukkamala, Laurie Beitz, Byoung Ryu, Andrew M. Scharenberg, Michael Jensen, Philip S. Low. Design of bispecific adapters for universal CAR T cell therapies of solid tumors [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 1787.

Abstract 4421: Targeting myeloid-derived suppressor cells with actinium-225 lintuzumab, a CD33 antibody radioconjugate to enhance antitumor immunity

Abstract Introduction: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid lineage cells with potent immunosuppressive activity found enriched in cancer patients. Crosstalk between MDSCs and the tumor microenvironment can promote tumor immune evasion and thus there is growing interest in developing therapeutic approaches to intervene in the MDSC suppressive function. Both monocytic-MDSC (M-MDSC) and granulocytic-MDSC (G-MDSC) subpopulations express the cell surface CD33 myeloid marker. For well-defined cell surface markers like CD33, there is considerable interest in the use of radionuclides as therapeutic payloads, particularly α-particle emitters such as actinium-225 (225Ac) since they deliver substantially higher decay energies over a much shorter distance than β-emitters, rendering them more suitable for precise, potent, and efficient target cell killing while minimizing toxicity to surrounding bystander cells. Actimab A, the anti-CD33 antibody lintuzumab armed with the 225Ac radioisotope (CD33 ARC), is currently being evaluated in R/R AML and has demonstrated significant anti-leukemic activity in Phase 1/2 clinical trials. We therefore hypothesized that MDSCs can be directly targeted by the CD33 ARC. Hence, we evaluated the therapeutic potential of the CD33 ARC to deplete MDSCs through preclinical studies in vitro and in vivo with humanized mouse model. Methods: CD33 ARC was generated by conjugating lintuzumab with p-SCN-Bn-DOTA and subsequently radiolabeled with 225Ac. Primary MDSCs were isolated from cancer patient peripheral blood or healthy donor PBMCs. The specific binding of CD33 ARC to CD33 positive MDSCs and the decreased viability of MDSCs in response to treatment in vitro was characterized by immunophenotyping using flow cytometry. The therapeutic efficacy of CD33 ARC to deplete MDSC in vivo was evaluated in human CD34 reconstituted humanized NOG-EXL mice. Results: CD33 positive MDSCs (M-MDSCs and G-MDSCs) were identified in human cancer peripheral blood samples, including colorectal and lung. Significantly more MDSCs were found in the peripheral blood of cancer patients in comparison to healthy donors. CD33 ARC treatment of human cancer MDSCs induced a potent dose-dependent reduction in MDSC viability leading to increased depletion of MDSCs. Furthermore, in the humanized NOG-EXL mouse model, CD33 ARC therapy demonstrated in vivo activity of CD33 ARC to deplete human MDSCs. Conclusions: In this study, we demonstrate CD33 ARC alpha targeted radiotherapy depletes human CD33 positive immune suppressing MDSCs present in multiple cancer types, to enhance antitumor immunity. These findings present a translatable strategy that supports further evaluation of 225Ac lintuzumab as a MDSC targeting agent to improve the efficacy of antitumor therapies. Citation Format: Amanda Chin, Mary Chen, Jason Li, Megan McCloskey, Caroline Jennings, Le-Cun Xu, Monideepa Roy, Denis Beckford, Helen Kotanides. Targeting myeloid-derived suppressor cells with actinium-225 lintuzumab, a CD33 antibody radioconjugate to enhance antitumor immunity. [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 4421.

Abstract 3202: NanoBiT technologies for CAR-T characterization and potency testing

Abstract Despite significant clinical advances in CAR-T cell therapies for cancer, the ability to precisely and reproducibly analyze the potency of these drug products remains a challenge. As new CAR-T products and production methodologies are developed, it is becoming increasingly important to be able to rigorously evaluate the relative potencies of each batch. We have developed two MOA-based bioluminescent assays that overcome challenges in quantitative assessment of T cell therapies. Both assays rely on split NanoBiT luciferase technology. In the HiBiT Target Cell Killing Assay, incubation of CAR-T cells with target cells stably expressing HiBiT results in lysis of the target cells and release of HiBiT proteins. These HiBiT proteins bind with high affinity to LgBiT in the detection reagent and form functional NanoBiT luciferase, resulting in production of light in proportion to target cell lysis. Lumit Immunoassays are a simple and homogeneous method for measuring cytokine production from engineered T cells. Monoclonal antibodies are covalently labeled with LgBiT and a low-affinity tag, SmBiT, which have negligible association in solution. When the antibodies are brought together by a common analyte, such as a cytokine, SmBiT and LgBiT can compliment to form NanoBiT luciferase and produce light in proportion to the concentration of analyte in the sample. Lumit Immunoassays can be run directly on cells, removing media transfer, dilution, and wash steps that can be common sources of error. These luminescent assays are homogenous, fast, highly sensitive, and have robust assay windows. They represent a new set of tools for development and characterization of T cell-based immunotherapies. Citation Format: Julia K. Gilden, Pete Stecha, Aileen Paguio, Jim Hartnett, Brock Binkowski, Dan Lazar, Frank Fan, Mei Cong. NanoBiT technologies for CAR-T characterization and potency testing [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 3202.

Abstract 3198: Fully human CD20 and CD19 dual-targeting CAR-T cells mediate long-term remissions and protection from tumor rechallenge in vivo

Abstract Introduction: Despite significant progress in the chimeric antigen receptor (CAR)-T cell therapy of B cell malignancies, the 5-year progression-free survival rate in large B cell lymphoma following CD19-targeting CAR-T cell treatments remains below 50%. While some treatment failures were attributed to CD19 antigen loss, immunogenicity of murine single-chain fragment variable (scFv) elements has been implicated in relapse and resistance to CAR-T cell re-treatment. In this study, we evaluated fully human CD20 and CD19 dual-targeting CAR constructs (h20.19 CARs) combining human scFvs and distinct co-stimulatory domains and architectures, to mitigate tumor antigen escape, reduce immunogenicity, and improve CAR functionality. Methods: The h20.19 CAR constructs were assembled from fully human scFvs against lymphoma antigens CD19 and CD20, with hinge, transmembrane and co-stimulatory domains derived from 4-1BB, CD28, OX40 or ICOS. Tandem second generation (T8/8BBz), tandem third generation (T28/28BBz, T8/28BBz), and bicistronic (D28z/BBz, DBBz/28z, DOXz/ICz) CARs were evaluated. CAR T cells were derived from healthy donors’ CD4+ and CD8+ T cells by lentiviral transduction, and CAR expression was evaluated by flow cytometry. CAR-mediated target cell killing was assessed by co-culture assays, and cytokine elaboration by ELISA. In vivo CAR-T cell function was evaluated in a Raji lymphoma NSG xenograft model using low CAR-T dose (2x106/mouse). Tumor-free survivors were rechallenged with Raji cells to compare CAR-T cell potency and persistence. Results: The hCD20.19 CAR-T cells lysed CD19+CD20+ tumor lines Raji, NALM-6 and REH, and single-antigen lines 293T CD19 and 293T CD20, but not the parental CD19-CD20- 293T cells, indicating antigen-specific efficacy. Cytokines TNFα, IL2 and IFNγ were strongly upregulated in response to Raji cells. During repeat co-culture with Raji cells, the third-generation tandem hCD20.19 CARs and the bicistronic CARs showed greater cytotoxicity than the second-generation CAR T8/8BBz. In vivo, all third-generation tandem and bicistronic CARs induced rapid and durable Raji tumor rejection by study day 7 post CAR-T, with no apparent toxicity. Upon long rest and rechallenge with Raji cells on study day 50, all h20.19 CARs attenuated tumor progression as compared to age-matched untreated mice cohort. CARs T8/28BBz and D28z/BBz afforded the least protection from Raji rechallenge, whereas CAR DOXz/ICz mediated rapid tumor rejection, but mice met euthanasia criteria by study day 71 due to weight loss. The tandem CAR T28/28BBz and bicistronic CAR DBBz/28z, demonstrated tumor rejection, persistence and safety after rechallenge, up to study day 86. Conclusions: In summary, the fully human dual-targeting h20.19 CARs exhibit robust anti-tumor function and persistence in vitro and in vivo, mediate long-term remissions and resist tumor rechallenge. Citation Format: Peirong Hu, Brittany Steimle, Ngoc Tran, Pradyot Dash, Dina Schneider. Fully human CD20 and CD19 dual-targeting CAR-T cells mediate long-term remissions and protection from tumor rechallenge in vivo [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 3198.

Abstract 4120: Depletion of tissue-resident B cells by a CD20xCD3 IgM bispecific T cell engager in cynomolgus monkeys demonstrates effective tissue penetration and potent target cell killing

Abstract Imvotamab (IGM-2323) is an engineered high-affinity, high-avidity bispecific anti-CD20 IgM antibody T cell engager (TCE) that is currently being studied as monotherapy in a Phase 1/2 clinical trial for relapsed/refractory non-Hodgkin’s lymphoma (NHL) (NCT04082936). Imvotamab offers a novel treatment strategy in NHL by depleting CD20-expressing tumor cells through multiple mechanisms, including the recruitment of T cells to kill tumor cells through T cell dependent cellular cytotoxicity, complement-dependent cytotoxicity, and enhanced immune modulation via IFNγ-dominant cytokine stimulation. We evaluated the activity of a surrogate cynomolgus monkey cross-reactive CD20xCD3 IgM bispecific TCE, IGM-2324, in depleting CD20-expressing B cells in peripheral blood and lymphoid tissues of cynomolgus monkeys in vivo. We hypothesized that the high affinity and valency of IGM-2324 would enable potent B cell killing in blood and tissues even when B cells express low levels of CD20. Cynomolgus monkeys were administered vehicle or IGM-2324 at 5 mg/kg or 25 mg/kg through intravenous infusion twice weekly for a total of four doses on days 1, 4, 7, and 10. B cell depletion in peripheral blood was assessed by measuring the frequency of CD19+ B cells through flow cytometry. Administration of IGM-2324 at 5 and 25 mg/kg resulted in a nearly complete depletion in peripheral CD19+ B cells at 8 hours post the 1st dose through 24 hours post the last dose on day 11. Depletion of tissue-resident B cells was evaluated in the spleen, mesenteric lymph node (MLN) and bone marrow (BM) of monkeys at 24 hours post the last dose of vehicle or IGM-2324 on day 11. Immunohistochemistry studies were conducted on the formalin-fixed paraffin-embedded lymphoid tissues by staining for CD19 and CD20 expression. The number and intensity of CD19 or CD20 positive B cells were determined by quantitative imaging analysis. Compared to vehicle-treated animals, significant dose-dependent reductions in both CD19 and CD20-expressing B cells were observed in spleen, MLN and BM following treatment with 5 and 25 mg/kg of IGM-2324. Most importantly, IGM-2324 treatment led to the depletion of not only high and moderate tissue-resident CD20-expressing B cells, but also B cells that expressed low levels of CD20. Our preclinical data indicate that a CD20xCD3 IgM bispecific TCE can penetrate tissues and mediate direct killing of CD20-expressing target cells. B cell depletion in the periphery and tumors of relapsed/refractory NHL patients is currently being evaluated as biomarker of pharmacodynamic activity and/or efficacy for imvotamab in a Phase 1/2 clinical study. Citation Format: Miho Oyasu, Angus M. Sinclair, Haben Ghermazien, Genevive Hernandez, Thomas Manley, Maya K. Leabman, Stephen F. Carroll, Bruce A. Keyt, Maya F. Kotturi. Depletion of tissue-resident B cells by a CD20xCD3 IgM bispecific T cell engager in cynomolgus monkeys demonstrates effective tissue penetration and potent target cell killing. [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 4120.

Emerging Advancements in Piezoelectric Nanomaterials for Dynamic Tumor Therapy

Cancer is one of the deadliest diseases, having spurred researchers to explore effective therapeutic strategies for several centuries. Although efficacious, conventional chemotherapy usually introduces various side effects, such as cytotoxicity or multi-drug resistance. In recent decades, nanomaterials, possessing unique physical and chemical properties, have been used for the treatment of a wide range of cancers. Dynamic therapies, which can kill target cells using reactive oxygen species (ROS), are promising for tumor treatment, as they overcome the drawbacks of chemotherapy methods. Piezoelectric nanomaterials, featuring a unique property to convert ultrasound vibration energy into electrical energy, have also attracted increasing attention in biomedical research, as the piezoelectric effect can drive chemical reactions to generate ROS, leading to the newly emerging technique of ultrasound-driven tumor therapy. Piezoelectric materials are expected to bring a better solution for efficient and safe cancer treatment, as well as patient pain relief. In this review article, we highlight the most recent achievements of piezoelectric biomaterials for tumor therapy, including the mechanism of piezoelectric catalysis, conventional piezoelectric materials, modified piezoelectric materials and multifunctional piezoelectric materials for tumor treatment.

Reinforcement of cell-mediated immunity driven by tumor-associated Epstein-Barr virus (EBV)-specific T cells during targeted B-cell therapy with rituximab.

In immunocompromised patients, Epstein-Barr virus (EBV) infection or reactivation is associated with increased morbidity and mortality, including the development of B-cell lymphomas. The first-line treatment consists of reduction of immunosuppression and administration of rituximab (anti-CD20 antibody). Furthermore, the presence of EBV-specific T cells against latent EBV proteins is crucial for the control of EBV-associated diseases. Therefore, in addition to effective treatment strategies, appropriate monitoring of T cells of high-risk patients is of great importance for improving clinical outcome. In this study, we hypothesized that rituximab-mediated lysis of malignant EBV-infected B cells leads to the release and presentation of EBV-associated antigens and results in an augmentation of EBV-specific effector memory T-cell responses. EBV-infected B lymphoblastoid cell lines (B-LCLs) were used as a model for EBV-associated lymphomas, which are capable of expressing latency stage II and III EBV proteins present in all known EBV-positive malignant cells. Rituximab was administered to obtain cell lysates containing EBV antigens (ACEBV). Efficiency of cross-presentation of EBV-antigen by B-LCLs compared to cross-presentation by professional antigen presenting cells (APCs) such as dendritic cells (DCs) and B cells was investigated by in vitro T-cell immunoassays. Deep T-cell profiling of the tumor-reactive EBV-specific T cells in terms of activation, exhaustion, target cell killing, and cytokine profile was performed, assessing the expression of T-cell differentiation and activation markers as well as regulatory and cytotoxic molecules by interferon-γ (IFN-γ) EliSpot assay, multicolor flow cytometry, and multiplex analyses. By inhibiting parts of the cross-presentation pathway, B-LCLs were shown to cross-present obtained exogenous ACEBV-derived antigens mainly through major histocompatibility complex (MHC) class I molecules. This mechanism is comparable to that for DCs and B cells and resulted in a strong EBV-specific CD8+ cytotoxic T-cell response. Stimulation with ACEBV-loaded APCs also led to the activation of CD4+ T helper cells, suggesting that longer peptide fragments are processed via the classical MHC class II pathway. In addition, B-LCLs were also found to be able to take up exogenous antigens from surrounding cells by endocytosis leading to induction of EBV-specific T-cell responses although to a much lesser extent than cross-presentation of ACEBV-derived antigens. Increased expression of activation markers CD25, CD71 and CD137 were detected on EBV-specific T cells stimulated with ACEBV-loaded APCs, which showed high proliferative and cytotoxic capacity as indicated by enhanced EBV-specific frequencies and increased secretion levels of cytotoxic effector molecules (e.g. IFN-γ, granzyme B, perforin, and granulysin). Expression of the regulatory proteins PD-1 and Tim-3 was induced but had no negative impact on effector T-cell functions. In this study, we showed for the first time that rituximab-mediated lysis of EBV-infected tumor cells can efficiently boost EBV-specific endogenous effector memory T-cell responses through cross-presentation of EBV-derived antigens. This promotes the restoration of antiviral cellular immunity and presents an efficient mechanism to improve the treatment of CD20+ EBV-associated malignancies. This effect is also conceivable for other therapeutic antibodies or even for therapeutically applied unmodified or genetically modified T cells, which lead to the release of tumor antigens after specific cell lysis.

Racing CARs to veterinary immuno-oncology.

Chimeric antigen receptors (CARs) have demonstrated remarkable promise in human oncology over the past two decades, yet similar strategies in veterinary medicine are still in development. CARs are synthetically engineered proteins comprised of a specific antigen-binding single chain variable fragment (ScFv) fused to the signaling domain of a T cell receptor and co-receptors. Patient T cells engineered to express a CAR are directed to recognize and kill target cells, most commonly hematological malignancies. The U.S Food and Drug Administration (FDA) has approved multiple human CAR T therapies, but translation of these therapies into veterinary medicine faces many challenges. In this review, we discuss considerations for veterinary use including CAR design and cell carrier choice, and discuss the future promise of translating CAR therapy into veterinary oncology.

Modification of Cysteine-Substituted Antibodies Using Enzymatic Oxidative Coupling Reactions.

Cysteines are routinely used as site-specific handles to synthesize antibody-drug conjugates for targeted immunotherapy applications. Michael additions between thiols and maleimides are some of the most common methods for modifying cysteines, but these functional groups can be difficult to prepare on scale, and the resulting linkages have been shown to be reversible under some physiological conditions. Here, we show that the enzyme tyrosinase, which oxidizes conveniently accessed phenols to afford reactive ortho-quinone intermediates, can be used to attach phenolic cargo to cysteines engineered on antibody surfaces. The resulting linkages between the thiols and ortho-quinones are shown to be more resistant than maleimides to reversion under physiological conditions. Using this approach, we construct antibody conjugates bearing cytotoxic payloads, which exhibit targeted cell killing, and further demonstrate this method for the attachment of a variety of cargo to antibodies, including fluorophores and oligonucleotides.