Target Antigen Density Research Articles

Natural TCRs targeting KRASG12V display fine specificity and sensitivity to human solid tumors.

BACKGROUNDNeoantigens derived from KRASMUT have been described, but the fine antigen specificity of T cell responses directed against these epitopes is poorly understood. Here, we explore KRASMUT immunogenicity and the properties of 4 T cell receptors (TCRs) specific for KRASG12V restricted to the HLA-A3 superfamily of class I alleles.METHODSA phase 1 clinical vaccine trial targeting KRASMUT was conducted. TCRs targeting KRASG12V restricted to HLA-A*03:01 or HLA-A*11:01 were isolated from vaccinated patients or healthy individuals. A comprehensive analysis of TCR antigen specificity, affinity, crossreactivity, and CD8 coreceptor dependence was performed. TCR lytic activity was evaluated, and target antigen density was determined by quantitative immunopeptidomics.RESULTSVaccination against KRASMUT resulted in the priming of CD8+ and CD4+ T cell responses. KRASG12V -specific natural (not affinity enhanced) TCRs exhibited exquisite specificity to mutated protein with no discernible reactivity against KRASWT. TCR-recognition motifs were determined and used to identify and exclude crossreactivity to noncognate peptides derived from the human proteome. Both HLA-A*03:01 and HLA-A*11:01-restricted TCR-redirected CD8+ T cells exhibited potent lytic activity against KRASG12V cancers, while only HLA-A*11:01-restricted TCR-T CD4+ T cells exhibited antitumor effector functions consistent with partial coreceptor dependence. All KRASG12V-specific TCRs displayed high sensitivity for antigen as demonstrated by their ability to eliminate tumor cell lines expressing low levels of peptide/HLA (4.4 to 242) complexes per cell.CONCLUSIONThis study identifies KRASG12V-specific TCRs with high therapeutic potential for the development of TCR-T cell therapies.TRIAL REGISTRATIONClinicalTrials.gov NCT03592888.FUNDINGAACR SU2C/Lustgarten Foundation, Parker Institute for Cancer Immunotherapy, and NIH.

Analysis of polyfunctionality for enhanced BAFF-R CAR T-cell therapy for hematologic malignancies

AbstractChimeric antigen receptor (CAR) T-cell therapy has emerged as a promising immunotherapeutic strategy for eradicating human cancers. Their therapeutic success and durability of clinical responses hinges, in large part, on their functional capacity, including the ability of these engineered cells to simultaneously expand and persist after infusion into patients. CD19 CAR T-cell polyfunctionality, assessing the simultaneous functions of cytokine production, proliferation, and cytotoxicity has been reported to correlate with clinical outcomes. Assay optimization is potentially limited by the heterogeneous nature of CAR T-cell infusion products and target specificity. We optimized a single-cell platform for polyfunctionality using CAR T-cell products manufactured from healthy donors, engineered against a novel target, B-cell–activating factor receptor (BAFF-R) and validated the protocol using CD19 CAR T cells. We observed distinct qualitative differences between BAFF-R and CD19 CAR T cells relative to the proportions of stimulatory vs effector cytokines, based on target antigen density, and, generally, CD19 CAR T cells exhibited lower indices of polyfunctionality. Finally, we applied our assay to the autologous BAFF-R CAR T-cell product generated from the first patient with non-Hodgkin lymphoma treated in an ongoing clinical trial who had progressed after prior CD19 CAR T-cell therapy. We observed robust indicators of polyfunctionality, which correlated with successful CAR T-cell expansion after infusion and achievement of durable complete remission ongoing after 18 months. The precise identification of factors determining the role of BAFF-R CAR T-cell fitness in toxicity and clinical outcome will require the application of this robust assay in the analysis of additional treated patients. This trial was registered at www.ClinicalTrials.gov as #NCT05370430.

Abstract 1236: Characterization of CBX-250, a first-in-class TCR-mimetic-based T-cell engager targeting a cathepsin G peptide-HLA complex for the treatment of myeloid leukemia

Abstract While T-cell engaging therapies have demonstrated clear clinical benefit in the treatment of B-cell malignancies, their application to myeloid malignancies remains a challenge due to the limited repertoire of suitable tumor specific surface antigens. Targeting HLA-restricted peptides (pHLA) derived from intracellular cancer antigens provides an opportunity to explore the entire cancer proteome. CG1 (FLLPTGAEA) has been validated as an HLA-A*02:01 restricted leader peptide from Cathepsin G (CTSG) and is abundantly presented by leukemic versus normal myeloid cells. TCR-mimetic (TCRm) antibodies are ideally suited to target pHLA with high potency and specificity and can be engineered and manufactured using standard antibody technologies. Here we report the preclinical characterization of CBX-250, a novel CG1/HLA-A2 TCRm-CD3 bispecific T-Cell Engager (TCE). CBX-250 induced potent T-cell activation and T-cell mediated killing of leukemia cell lines with varying levels of target antigen density with sub-nM EC50 in vitro. In vivo, a closely related precursor of CBX-250 demonstrated potent tumor control in various AML and CML CDX models at doses as low as 0.01mpk. Although CTSG is a serine protease stored in neutrophil azurophilic granules, the CG1 peptide is preferentially presented by leukemia cells, as validated by mass spectrometry. Moreover, when co-culturing PBMCs with HLA-A2 neutrophils, we did not observe any CBX-250-mediated T-cell activation or IFNγ production. In silico predictions identified HLA-A2-restricted human peptides that share sequence or structural similarity to CG1. We determined the cross-reactivity risk of CBX-250 to be low, based on T-cell activation and T-cell mediated cytotoxicity assays against cells pulsed with supra-physiological concentrations of these peptides. Moreover, we observed no activity against target-negative cancer cells, including a CTSG-KO cell line. To further assess the specificity of CBX-250, we screened a panel of normal cells and found no meaningful T-cell activation or IFNγ production at CBX-250 concentrations exceeding its EC90. Finally, the CBX-250 precursor molecule demonstrated favorable safety in a double transgenic mouse model expressing human HLA-A2 and CG1. Following sequence optimization, CBX-250 demonstrated robust serum and pH stress stability and a favorable melting temperature, while retaining excellent potency and specificity. A full suite of analytical and biophysical assessments supports CBX-250’s favorable developability profile. In summary, these data provide strong preclinical evidence of the potency, specificity, safety, and developability of CBX-250, a novel, first-in-class, off-the-shelf, TCRm-based TCE for the treatment of myeloid malignancies. Citation Format: Geraldine Paulus, Benjamin Lee, Preethi Sankaran, Tanzila Rahman, Jessica Jimenez, Delainey O'Connor, Simon Yue, Yu Huang, Melissa Bikowitz, Sarah Jaffe, Shawn O'Malley, Bhupal Ban, Galina Gabriely, Tao Wang, Amanda Mak, Michael Princiotta, Chunhua Shi, Helen He, Ningping Feng, Jun Yan, Timothy Heffernan, Gheath Alatrash, Jeffrey Molldrem, Dmitri Wiederschain. Characterization of CBX-250, a first-in-class TCR-mimetic-based T-cell engager targeting a cathepsin G peptide-HLA complex for the treatment of myeloid leukemia [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 1236.

Abstract 4017: CAR T cell-mediated cytotoxicity and cytokine release in response to varying levels of antigen expression on target cells

Abstract The development of immunotherapies relies on the use of in vitro potency assays, which are crucial for understanding the complex interactions between immune cells (effectors) and cancer cells (targets). Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapeutic approach that exhibits promise; however, varying levels of antigen expression on the surface of tumor cells can influence the efficacy of CAR T cell-mediated killing. To fully understand the impact of target antigen density on CAR T cell activity, we utilized a matrix approach to assess both target cell death, using an impedance-based in vitro potency assay to quantify CAR T cell-mediated cytotoxicity, as well as cytokine release with homogeneous LumitTM immunoassays. SKOV3 (high HER2 expression), A549 (low HER2 expression), or MDA-MB-231 (no HER2 expression) target cells were seeded into a 96-well microplate with embedded electrodes in the substrate that detect the attachment and proliferation of target cells. HER2 CAR T cells were added after 24 hours at effector-to-target (E:T) ratios of 1:5 or 1:1 and target cell cytolysis was recorded continuously by the Maestro Z for 72 hours. Cytolysis of the target cells was calculated by comparing the treated wells to no treatment control wells and full lysis wells (1% TritonX). At 72 hours post CAR T cell addition, complete killing was observed in SKOV3 cells at the 1:1 ratio, while A549 cells exhibited only 80% cytolysis. MDA-MB-231 cells showed 20% cytolysis, likely due to nonspecific killing by non-engineered T cells, as approximately only 78% of the CAR T cell population was CAR positive. Because the cytolysis assay was label-free, cytokine analysis was able to be multiplexed with the same plates. To assess release of pro-inflammatory cytokines, TNF-α and IFN-γ, we collected supernatant post-antigen exposure at 24, 48, and 72 h. At the 1:1 E:T ratio, CAR T cells co-cultured with target cells expressing low (A549) or high (SKOV3) levels of HER2 had the highest TNF-α production at 24 hours, with CAR T cells co-cultured with SKOV3 cells releasing 30.6% more TNF-α when compared to A549 cells. The highest levels of IFN-γ were detected in both A549 and SKOV3 groups at the 1:1 E:T ratio, with the highest levels observed from CAR T cells co-cultured with SKOV3 cells at 72 hours, releasing approximately 1459.6 +/- 357.3 pg/mL of IFN-γ, compared to 1093.8 +/- 387.5 pg/mL IFN-γ released by CAR T cells co-cultured with A549 cells. As expected, CAR T cells co-cultured with MDA-MB-231 cells did not release any detectable TNF-α or IFN-γ at either E:T ratios during the duration of the experiment. Future work will continue to explore multiplexed potency assays for the evaluation of CAR T cell therapies. Citation Format: Sierra Barnes, Denise Sullivan, Dan Lazar, Kim Haupt, Danielle Califano, Stacie Chvatal, Daniel Millard. CAR T cell-mediated cytotoxicity and cytokine release in response to varying levels of antigen expression on target cells [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 4017.

Impact of Gamma-Secretase Inhibition on the Multiple Myeloma Immune Microenvironment

Background: Therapy with monoclonal antibodies, chimeric antigen receptor (CAR) T-cells, and bispecific T-cell engagers (BiTE) has significantly changed the treatment landscape for multiple myeloma (MM). B-cell maturation antigen (BCMA), a cell surface protein highly expressed in malignant plasma cells, has been established as an effective MM cell target, however, achieving long-term responses in relapsed or refractory patients remains a challenge. Resistance to BCMA targeting may result from the enzymatic cleavage of BCMA by surface membrane gamma-secretase resulting in reduced target antigen density on MM cells, and from the soluble BCMA ectodomain that may hinder tumor cell recognition through binding to circulating therapeutic antibodies or to CAR T-cells. Methods: We have previously demonstrated that gamma-secretase inhibitors (GSIs) abrogate enzymatic function and increase BCMA surface density on plasma cells with a concomitant decrease in soluble BCMA concentrations [Pont, MJ. et al. Blood, 134(2019)]. In a phase I, first-in-human clinical trial (n=18; relapsed/refractory MM) combining the GSI, crenigacestat, with anti-BCMA CAR T-cell therapy (FCARH143), we recently demonstrated that plasma cell BCMA antibody-binding capacity increased a median of 12-fold among 17/18 (94%) of participants after they received a 5-day GSI “run-in” (25 mg orally administered QOD for 3 doses) [Cowan AJ, et al. Lancet Oncology. 24(7) 2023]. Overall, 78% of participants experienced a very good partial response or better to GSI with FCAR143, but the duration of response was significantly longer in patients without prior anti-BCMA exposure. Here, we report results from an analysis of the bone marrow aspirates collected at enrollment and again within 12 hours of the third “run-in” dose. We leveraged single-cell RNA sequencing (scRNAseq) coupled with the single-cell assay for transposase-accessible chromatin sequencing (scATACseq) to profile the transcriptome and chromatin accessibility of individual cells within the bone marrow microenvironment. Results: Single-cell multi-omic analysis was performed on 32 bone marrow mononuclear cell samples from 16 patients collected before and within 12 hours after the third oral dose of GSI. Six patients had previously been treated with BCMA-directed therapy, including antibody-drug conjugates, CAR T-cells, or BiTEs. After quality filtering, including compensation for ambient RNA, 142,498 cells were analyzed (median 6,373 cells/sample, range 613-17,150 cells/sample). Cells were automatically classified into 92 immune cell types using Celltypist and a reference dataset trained on immune sub-populations from 20 tissues in 18 studies. Comparing samples pre- and post-GSI exposure revealed a significant decrease in the abundance of plasmablasts, non-classical monocytes, germinal center B cells, mid-erythroid cells, and central memory/naïve cytotoxic T cells after GSI treatment. Additionally, gene expression analysis detected increased expression of Notch signaling genes, NEURL1 and HES1, in non-classical monocytes following GSI treatment. Although RNA expression of TNFRSF17 ( BCMA) did not significantly change, chromatin accessibility was considerably higher in patients without a history of prior BCMA-directed therapy following GSI treatment. In non-plasma cells, we observed increased chromatin accessibility after GSI treatment involving genes encoding proteins known to be cleaved by gamma-secretase, such as ERBB4, NOTCH1, NOTCH2, and TNFRSF17. Accessibility of CD38, the target of daratumumab, was significantly increased in B cells, and SLAMF7, the target of elotuzumab, was significantly increased in plasma cells. Conclusions: BCMA cleavage from myeloma cells' surface is a putative resistance mechanism to BCMA-targeting immunotherapy. This study assessed the single-cell transcriptome and chromatin accessibility in the bone marrow environment of 16 patients given GSI monotherapy to ultimately enhance the efficacy of subsequent anti-BCMA CAR T-cell therapy. We found that prior BCMA-targeted therapy resulted in reduced chromatin accessibility within the BCMA epigenome. Surprisingly, GSI administration also modified the epigenome of other gamma-secretase sensitive genes. These data provide insight into the molecular consequences of prior BCMA-targeted therapy and GSI exposure that may inform future studies.

γ-Secretase inhibitor in combination with BCMA chimeric antigen receptor T-cell immunotherapy for individuals with relapsed or refractory multiple myeloma: a phase 1, first-in-human trial

γ-Secretase inhibitors (GSIs) increase B cell maturation antigen (BCMA) density on malignant plasma cells and enhance antitumour activity of BCMA chimeric antigen receptor (CAR) T cells in preclinical models. We aimed to evaluate the safety and identify the recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat (LY3039478) for individuals with relapsed or refractory multiple myeloma. We conducted a phase 1, first-in-human trial combining crenigacestat with BCMA CAR T-cells at a single cancer centre in Seattle, WA, USA. We included individuals aged 21 years or older with relapsed or refractory multiple myeloma, previous autologous stem-cell transplant or persistent disease after more than four cycles of induction therapy, and Eastern Cooperative Oncology Group performance status of 0-2, regardless of previous BCMA-targeted therapy. To assess the effect of the GSI on BCMA surface density on bone marrow plasma cells, participants received GSI during a pretreatment run-in, consisting of three doses administered 48 h apart. BCMA CAR T cells were infused at doses of 50 × 106 CAR T cells, 150 × 106 CAR T cells, 300 × 106 CAR T cells, and 450 × 106 CAR T cells (total cell dose), in combination with the 25 mg crenigacestat dosed three times a week for up to nine doses. The primary endpoints were the safety and recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat, an oral GSI. This study is registered with ClinicalTrials.gov, NCT03502577, and has met accrual goals. 19 participants were enrolled between June 1, 2018, and March 1, 2021, and one participant did not proceed with BCMA CAR T-cell infusion. 18 participants (eight [44%] men and ten [56%] women) with multiple myeloma received treatment between July 11, 2018, and April 14, 2021, with a median follow up of 36 months (95% CI 26 to not reached). The most common non-haematological adverse events of grade 3 or higher were hypophosphataemia in 14 (78%) participants, fatigue in 11 (61%), hypocalcaemia in nine (50%), and hypertension in seven (39%). Two deaths reported outside of the 28-day adverse event collection window were related to treatment. Participants were treated at doses up to 450 × 106 CAR+ cells, and the recommended phase 2 dose was not reached. Combining a GSI with BCMA CAR T cells appears to be well tolerated, and crenigacestat increases target antigen density. Deep responses were observed among heavily pretreated participants with multiple myeloma who had previously received BCMA-targeted therapy and those who were naive to previous BCMA-targeted therapy. Further study of GSIs given with BCMA-targeted therapeutics is warranted in clinical trials. Juno Therapeutics-a Bristol Myers Squibb company and the National Institutes of Health.

Generation of novel anti-tumor chimeric antigen receptors incorporating downstream T-cell signaling proteins

Abstract Chimeric antigen receptors (CAR) are molecules with an antibody-derived extracellular domain combined intracellularly with T cell receptor (TCR) signaling proteins. Although CAR-T cells have been a breakthrough clinically, several challenges persist: adverse effects from cytokine secretion, CAR-T cell exhaustion, and limited sensitivity in tumor microenvironments with low density of target antigen (i.e. tumor relapse and solid tumors). While many efforts have focused on identifying new cell surface targets, we have modified CAR intracellular domains. This idea arose from our super-resolution microscopy study that revealed that adapter molecules form clusters distinct from the TCR complex and that full activation necessitates passing a signaling threshold. From these findings, novel Chimeric Adapter Proteins (CAPs) were designed to trigger signaling downstream of the TCRζ chain. CAPs replace the TCRζ with ZAP70 intracellularly and are fused to an extracellular targeting domain. Thus, the potency problem associated with CARs would be ameliorated as CAPs would bypass kinetic proofreading steps defining the signaling threshold and the inhibitory regulation of upstream molecules. Indeed, CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo persistence of tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19–28ζ CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28ζ-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.

Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response

Abstract Chimeric antigen receptor (CAR) T cells are highly effective in treating hematologic cancers, but patients can relapse due to downregulation of the CAR-targeted antigen, leading to the hypothesis that CAR T cells require high density of target antigen to be effective. Non-engineered CD8+ memory T cells generate more rapid and robust effector T cell responses upon secondary infection, relative to their naïve counterparts, and can display higher antigen sensitivity. Our objective was to characterize the role of prior CD8+ T cell antigen experience (AgEx) in dictating functional CAR-mediated anti-tumor responses. To study the effect of T cell AgEx history on CD8+ CAR T cell (CAR8) function in vivo, we used a vaccine model to control prior T cell AgEx history, and a syngeneic adoptive transfer model with murine anti-CD19 CAR8 targeting murine leukemia engineered to express a low or high level of CD19. We analyzed CAR8 expansion and leukemia burden using flow cytometry and performed ATACseq and RNAseq at timepoints pre- and post-CAR transduction and after reinfusion of CAR8 into leukemia-bearing mice to ask whether CAR8 function was determined by the epigenetic and/or transcriptomic state of the starting cell population. We found that starting cell state dictated in vivo CAR8 effector functions, expansion, and differential chromatin accessibility in regions containing binding motifs for AP-1, Runx, Tcf and other transcription factor (TF) families. Motif chromatin accessibility and gene expression of many TFs showed distinct patterns temporally. We conclude that CAR8 maintain functional traits epigenetically imprinted in the starting cell and predict these analyses could inform methods to transcriptionally modulate CAR T cell function.

Preclinical development and evaluation of nanobody-based CD70-specific CAR T cells for the treatment of acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) treatment remains challenging. CD70 was reported as a promising AML-specific antigen. Preclinically, CAR T-cell with single-chain-variable fragment (scFv) or truncated CD27 targeting CD70 has been reported to treat AML. However, various disadvantages including spontaneous exhaustion, proteinase-mediated loss of functional receptors, and high immunogenicity, limited its further application to clinical settings. Alternatively, the single-variable domain on heavy chain (VHH), also known as nanobodies, with comparable binding ability and specificity, provides an optional solution.MethodWe generated CD70 knocked-out novel nanobody-based anti-CD70-CAR T-cells (nb70CAR-T) with two different VHHs for antigen detection. Next, we detected the CD70 expression on primary AML blasts by flow cytometry and associated the efficacy of nb70CAR-T with the target antigen density. Finally, epigenetic modulators were investigated to regulate the CD70 expression on AML cells to promote the functionality of nb70CAR-T.ResultsOur nb70CAR-T exhibited expected tumoricidal functionality against CD70-expressed cell lines and primary AML blasts. However, CD70 expression in primary AML blasts was not consistently high and nb70CAR-T potently respond to an estimated 40.4% of AML patients when the CD70 expression level was over a threshold of 1.6 (MFI ratio). Epigenetic modulators, Decitabine and Chidamide can up-regulate CD70 expression on AML cells, enhancing the treatment efficacy of nb70CAR-T.ConclusionCD70 expression in AML blasts was not fully supportive of its role in AML targeted therapy as reported. The combinational use of Chidamide and Decitabine with nb70CAR-T could provide a new potential for the treatment of AML.

Target Densities in Malignant and Normal Cells Determine CAR T Cell Efficacy and Off-Target Hematotoxicity

Target antigen density has been established as a key factor for CAR activation and there are ongoing investigations into the number of surface molecules required for target cell lysis. A more precise understanding of differential target expression in malignant vs normal cells is needed to select the optimal CAR design, maintaining efficacy while limiting toxicity on normal cells. In AML, the lack of a leukemia-specific surface target antigen requires fine-tuned combinatorial approaches that enable distinctive target elimination based on differential antigen co-expression patterns in AML vs normal cells. Here, we present our target phenotypic profiling of primary AML and normal hematopoietic cells, and link it with CAR efficacy and changes in normal hematopoietic cell populations. We characterized the AML surface target phenotype across a cytogenetically and molecularly heterogeneous AML patient cohort (n>30) with relapsed/refractory disease. Using spectral flow cytometry on bone marrow and peripheral blood from AML patients and healthy control subjects, we quantified differential target expression of several AML targets, including CD33, CD123, CLEC12A and the novel target ADGRE2. We estimated the numbers of surface molecules per cell on AML bulk, LSC, normal HSPC and differentiated lineages. This allowed us to create two-dimensional maps of combinatorial target densities in AML vs normal. In this analysis, the combination of ADGRE2+CLEC12A offered the best therapeutic window. We developed a novel combinatorial CAR+CCR platform, ADCLEC.syn1, consisting of an ADGRE2-targeting 28z1XX-CAR and a CLEC12A-targeting chimeric costimulatory receptor (CCR) providing additional 4-1BB costimulation. This CAR+CCR configuration triggers killing of cells with high CAR target density alone, whereas cells with low CAR target density are only killed if the CCR target is co-expressed (IF-BETTER gate). Both in vitro and in vivo, we used MOLM13 AML cell line variants that we engineered to match a variety of different ADGRE2 and CLEC12A target density combinations found in AML or normal hematopoietic cells. The observed target density thresholds for ADCLEC.syn1-mediated cell lysis demonstrated potential for sparing vital normal hematopoietic cells. In addition, we evaluated ADCLEC.syn1 in several molecularly distinct AML PDX models with clinically representative target phenotypes, achieving long-term AML remissions in all studied PDX models. Next, we assessed the impact of ADCLEC.syn1 on normal hematopoietic cells. In vitro, we found that soluble factors from antigen-activated CAR T cells led to a reduction of CD34+CD38- HSPC, suggesting a potential role of pro-inflammatory cytokines leading to off-target hematotoxicity. To gain further insight, we established a humanized mouse model that allowed to serially monitor normal hematopoiesis by quantification of HSPC and several differentiated populations in the setting of controlled ADCLEC.syn1 or CD19 CAR activation based on an engineered CD19-positive AML cell line. We found that at time of AML remission, both ADCLEC.syn1 and CD19 CAR-treated mice had detectable CD34+ HSPC, indicating preferential killing of AML cells and relative sparing of HSPC. However, both ADCLEC.syn1 and CD19 CAR led to in vivo reduction of CD34+ HSPC as compared to untreated humanized controls, suggesting an indirect effect of inflammation upon CAR T cell activation. The observed HSPC loss was significantly reduced by blocking pro-inflammatory cytokines, further supporting off-target rather than on-target mechanisms of HSPC reduction. Overall, we profiled antigen densities of several CAR target combinations in AML and normal hematopoietic cells, which informed the development of the ADCLEC.syn1 CAR platform. Pre-clinical in vivo efficacy models based on AML with clinically representative target densities demonstrated high anti-leukemic efficacy of ADCLEC.syn1 and established killing thresholds with potential for sparing vital normal hematopoietic cells. Our in vitro and in vivo hematotoxicity models identified target-independent mechanisms of HSPC reduction in the context of both ADCLEC.syn1 and CD19 CAR activation, a finding that may be related to the off-target hematotoxicity observed clinically in some recipients of CD19 and other CAR T cell products.

Novel 17-Plex Flow Cytometry Assay for Comprehensive Immunophenotyping and Characterization of Plasma Cells (PC) in Relapsed/Refractory (RR) Multiple Myeloma (MM) Patients

Background: Multiple Myeloma (MM) is a hematological malignancy also known as plasma cell myeloma or myeloma, a type of bone marrow cancer characterized by the proliferation of clonal malignant plasma cells (PCs). Characterization of multiple myeloma plasma cells (MMPCs) remains challenging due to their plasticity, especially in response to emerging targeted therapies such as anti-BCMA and anti-CD38. The need for novel options for comprehensive phenotyping remains high due to the evolving target expression profiles and dynamics across MM disease evolution and treatment lines. Recent progress in flow cytometry offers the opportunity to analyze more than 30 surface markers in one panel. This technology advance will help to address the current challenges, answer clinically relevant questions and generate data to enable translational research and identification of new drug targets or combination partners. Methods: We present a novel 17-plex flow cytometry assay for immunophenotyping of PCs in RRMM patients enrolled in a global Phase I multi-center clinical study (NCT04557150). The panel relies on 5 markers for the identification of malignant plasma cells: CD38, CD138, CD45, CD319 and CD229. The inclusion of less commonly used markers (CD229, CD319) allows to overcome some of the challenges in MMPCs identification, such as CD138 instability and CD38 downregulation due to prior therapies. In addition, the assay includes markers commonly used to differentiate normal from malignant PCs (CD19, CD56, CD27, CD81, CD117, CD28). The panel also includes 2 immunotherapy target ligands (PD-L1 and 4-1BBL) and offers the possibility to measure another 3 clinically relevant targets on MMPCs, including BCMA. With this configuration, the assay allows relevant target antigen density quantification via the use of molecules of equivalent soluble fluorochrome (MESF) beads in 3 channels. Results: We present examples of how to apply this strategy for a reliable identification of MMPCs in RRMM patient samples after up to 14 previous lines of therapies. The assay design permits a deep phenotypical characterization of MMPCs and pharmacodynamic changes in markers targeted by immunotherapy drugs, like anti-CD38 or anti-BCMA. As an example, we characterize BCMA and CD38 expression on the surface of MMPCs at the time of enrolment into a Phase I study. Finally, with the goal of discovering new biomarkers that could be overlooked in the traditional manual gating hierarchy applied in flow cytometry, we have visualized the results using dimensionality reduction analysis (Uniform Manifold Approximation and Projection, UMAP). Using this approach, we have observed high inter-patient heterogeneity of MMPC phenotypes. Furthermore, we could compare intra-patient differences in MMPC phenotype in two compartments, whole blood and bone marrow aspirate. Updated data will be presented. Conclusion: The multi parameter flow cytometry assay described here represents a novel tool to reliably identify MMPCs in RRMM patients independently of commonly used unstable surface markers. Furthermore, it represents an important tool in the era of surface antigen targeted therapies (e.g., anti-BCMA) helping to understand drug resistance due to target loss. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

496 Study of Affinity Tuning in AML-Targeted CAR-NK Cells

CD123 is overexpressed on AML blasts compared to healthy myeloid progenitor cells. There is opportunity to design anti-CD123 therapy to discriminate on antigen density. Our goal is to study affinity modification of the single chain variable fragment (scFv) in the context of chimeric antigen receptor (CAR)-modified NK cell activation and target killing. We hypothesize that lower affinity CAR-NK cells will demonstrate improved discrimination between tumor and healthy tissue. We designed NK-cell specific CARs to contain anti-CD123 scFvs binding distinct target epitopes (26292 or 7G3) fused to intracellular portions of NK-activating receptors 4-1BB.CD3Z and 2B4.CD3Z. We synthesized novel 26292 and known 7G3 variants to capture a spectrum of CD123 binding affinities (26292: Kd=15nM; variant Kd range 0.29-160nM; 7G3: Kd=8.75nM; variant Kd range 23.9-64.9nM). These scFv were generated using site-directed mutagenesis of the wild type sequences and subcloned into CAR backbones. Retroviral transduction of primary human NK cells was performed, and CAR expression was measured using FACS (range 23.9%-89.6% CAR+ NK cells). Raji target cells (CD123neg) were engineered and sorted to obtain model cell lines with stable low (Raji.Low) and high (Raji. High) CD123 expression, comparable to healthy cells (CD123.Low) and AML blasts (CD123.High). We tested CAR-NK cell activation and antigen-specific cytotoxicity using in vitro co-culture assays. We established a mouse model of disease using implantation of Raji cells engineered to express firefly Luciferase (Raji.ffLuc). Antitumor cytotoxicity of 26292 and 7G3-based CAR-NK cells with a range of binding affinities showed CAR-specific activation but did not differentiate on target antigen density. Similarly, no differences were seen in CAR-NK activation as measured by TNFa and IFNy secretion following coculture with CD123-positive cells. NSG mice implanted with Raji.ffLuc had median survival of 42 days without treatment. There are no significant differences across a broad range of variable affinity 26292 and 7G3-based anti-CD123 CAR-NK cells in effector cell activation or target killing when assayed short-term. These data support the value of additional long-term investigation of the immune synapse and CAR-NK antitumor activity in vivo. This work is supported by the St. Baldrick's Foundation Fellowship Award and the Johns-Hopkins-Allegheny Health Network Cancer Research Fund.

Abstract 2759: CAR-T engager proteins for the treatment of B cell cancers and acute myeloid leukemia

Abstract Chimeric Antigen Receptor T cells targeting malignancies expressing CD19 (CAR19) have been widely successful, with products approved to treat B cell lymphomas (NHL) and B cell leukemia. A major limitation of CAR19 therapy is the steep relapse rate within 6 months of treatment, often due to the loss or diminution of tumor cell CD19 expression. ALETA-001 is a CAR-T Engager protein that contains the CD19 extracellular domain (ECD), an anti-CD20 VHH, and an anti-albumin VHH for half-life extension. When combined with CAR19 T cells, ALETA-001 triggers cytotoxicity through CD19 bound to CD20, thus increasing total target antigen density and preventing relapse due to loss of CD19 expression. ALETA-001 will enter Cancer Research UK-sponsored Phase 1/2 clinical trials in CAR19-treated NHL patients next year. We made novel variants of ALETA-001 including CAR-T Engagers with alternative anti-CD20 modules and CAR-T Engagers that target both CD20 and a second B cell cancer antigen. Further, we extended our technology to target Acute Myeloid Leukemia (AML), using novel antigen binding domains and immunomodulatory functional domains. Site-directed mutagenesis was used to make ALETA-001 variants. We mutated the complementary determining regions, CDR2 and/or CDR3, within the anti-CD20 VHH. The novel CAR-T Engager Proteins were evaluated for binding to CD19-negative/CD20-positive lymphoma cells and for cytotoxicity against those cells in the presence of CAR19 T cells. Further, CAR-T Engagers were made that contained novel CD20 binding domains and binding domains to other B cell antigens. In addition, we created a novel CAR-T engager that contains the CD19 ECD and binding domains to two AML antigens, plus an immunomodulatory domain. This novel CAR-T engager is designed to treat CD19-positive Mixed Phenotype Leukemia (MPL) and other forms of AML. We evaluated the series of CDR mutations in the ALETA-001 anti-CD20 VHH. A spectrum of activities was observed, with mutations that retained full CD20 binding and cytotoxic activity to those with partial or no activity. A series of constructs with distinct anti-CD20 binding domains were also evaluated, with some found to be equipotent with ALETA-001. Constructs binding both CD20 and a second B cell tumor antigen were prepared and assayed and found to have potent activity against either or both antigens. Finally, CAR-T Engagers directed to AML antigens were able to bind each antigen individually, and mediated potent toxicity with the addition of CAR19 T cells. We conclude that the CAR-T Engager platform offers robust and modular anti-tumor functionality, featuring potent multi-antigen targeting for diverse indications. Finally, we can incorporate other useful immunomodulatory activities into Engager proteins. Citation Format: Paul D. Rennert, Lihe Su, Lan Wu, Roy R. Lobb, Christine Ambrose. CAR-T engager proteins for the treatment of B cell cancers and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2759.

Anti-CCR9 chimeric antigen receptor T cells for T-cell acute lymphoblastic leukemia

AbstractT cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes, associated with higher rates of induction failure compared with those in B cell acute lymphoblastic leukemia. The potent immunotherapeutic approaches applied in B cell acute lymphoblastic leukemia, which have revolutionized the treatment paradigm, have proven more challenging in T-ALL, largely due to a lack of target antigens expressed on malignant but not healthy T cells. Unlike B cell depletion, T-cell aplasia is highly toxic. Here, we show that the chemokine receptor CCR9 is expressed in >70% of cases of T-ALL, including >85% of relapsed/refractory disease, and only on a small fraction (<5%) of normal T cells. Using cell line models and patient-derived xenografts, we found that chimeric antigen receptor (CAR) T-cells targeting CCR9 are resistant to fratricide and have potent antileukemic activity both in vitro and in vivo, even at low target antigen density. We propose that anti-CCR9 CAR-T cells could be a highly effective treatment strategy for T-ALL, avoiding T cell aplasia and the need for genome engineering that complicate other approaches.

Understanding the CD8+ CAR T Cell Response to Antigen-Low Leukemias

Chimeric antigen receptor (CAR) T cell therapy has emerged as a highly efficacious treatment for B-lineage acute lymphoblastic leukemias (B-ALL). However, downregulation of the CAR-targeted antigen on leukemia cells, predicted to reduce cellular avidity, is associated with post-CAR T cell leukemic relapse following CD22 CAR treatment (Fry et al., Nat. Med., 2017). We have observed reduced function of human CAR T cells against low target antigen site density (Ag Lo) human leukemia in immunodeficient mouse models, relative to CARs responding to high-antigen expressing leukemia. Thus, a better understanding of CAR responses to Ag Lo leukemia could help to increase the durability of remissions. We set out to develop a model system in which we could further interrogate the consequences of low-avidity interactions on CAR immunobiology, generating variants of a murine B-ALL driven by the E2A-PBX fusion protein (E2A) with different levels of target antigen to use in an immunocompetent syngeneic mouse model.We observed impaired expansion (p<0.0001) and tumor clearance (p<0.001) of CAR T cells responding to low-antigen variants of E2A (E2A-Ag Lo) as compared to wildtype E2A expressing high levels of antigen (E2A-WT). While CD8+ CAR T cell (CAR8) transcription factor (TF) expression in response to E2A-Ag Lo versus E2A-WT was largely similar early after CAR infusion, by day 9 post-CAR, CAR8s responding to E2A-Ag Lo exhibited decreased expression of multiple TFs, with Eomes (p<0.01), Irf4 (p<0.001) and Blimp1 (p<0.01) showing the largest magnitude change relative to CAR8s responding to E2A-WT. Additionally, CAR8s from mice bearing E2A-Ag Lo became enriched for cells of a “terminally exhausted” phenotype (Eomes+/PD1 Hi/TOX Hi) by day 11 post-CAR, and negatively-enriched for the “progenitor exhausted” (Tcf1+/PD1 Int) phenotype which can be functionally rescued by anti-PD1 therapy (p<0.0001, p<0.01). These data suggest that continual stimulation by low density antigen leads to a gradual reduction in the ability of CAR8s to mount an effector response, and eventually to T cell states with sub-optimal anti-tumor efficacy.Following in vitro stimulation of human CD22 CARs across a range of leukemic antigen densities, we saw that the percentage of CAR+ cells capable of producing IFNγ and IL2 corresponded to target antigen density (p<0.01, p<0.001). As human CARs are commonly manufactured from heterogenous bulk donor T cells, we hypothesized that antigen sensitivity is impacted by the prior antigen-experience of a given T cell. We predicted that T cells which had encountered cognate antigen through their TCR prior to CAR manufacturing (CAR8 AgEx) would have enhanced capacity to respond to low-avidity stimulation compared to CARs manufactured from naïve CD8+ T cells (CAR8 Naïve). We used a well-characterized ovalbumin vaccination model with OT-I TCR-transgenic T cells, allowing defined control of T cell antigen experience, to generate CAR8 AgEx. We found that CAR8 AgEx were highly antigen-sensitive relative to CAR8 Naïve, showing almost no reduction in numbers of cells capable of producing IFNγ and TNFα in vitro against E2A-Ag Lo as compared to E2A-WT. In vivo, CAR8 AgEx showed near complete depletion of E2A-Ag Lo in bone marrow by day 11 post-CAR, while mice treated with CAR8 Naïve maintained a substantial tumor burden (p<0.01). To test our hypothesis in human cells, we manufactured CD22 CAR T cells from naïve (CD45RO-) versus non-naïve (CD45RO+) starting T cell populations, and again found that CAR AgEx outperformed CAR Naïve against Ag Lo leukemia in production of IFNγ and IL2 in vitro (p<0.001, p<0.01) and in early leukemic clearance in vivo (p<0.0001, day 13).In conclusion, we have established a model to study the immunobiology of the CAR T cell response to Ag Lo B-ALL in an intact host. Preliminary findings indicate impaired expansion and tumor clearance of Ag Lo leukemia, associated with altered CAR T cell transcriptional profiles and features of T cell exhaustion. Furthermore, T cell history prior to CAR manufacturing has a drastic impact on the capacity to respond to Ag Lo leukemia. Future studies with this model will expand our characterization of CAR T cells responding to Ag Lo leukemia, with the goal of optimizing antigen sensitivity.We expect that advancing our understanding on the interplay of antigen density and CAR differentiation status will prove useful in developing more effective iterations of this therapy. DisclosuresFry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

IntroductionAcute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs.HypothesisEx vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro.Materials and MethodsT cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n.ResultsEx vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (<16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells.Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated throughE+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown).ConclusionsWe demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients [Display omitted] DisclosuresOelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

Upregulation of CD22 by Chidamide promotes CAR T cells functionality

Treatment failure or relapse due to tumor escape caused by reduction in target antigen expression has become a challenge in the field of CART therapy. Target antigen density is closely related to the effectiveness of CART therapy, and reduced or lost target antigen expression limits the efficacy of CART therapy and hinders the durability of CAR T cells. Epigenetic drugs can regulate histones for molecular modifications to regulate the transcriptional, translational and post-translational modification processes of target agents, and we demonstrated for the first time the role in regulating CD22 expression and its effect on the efficacy of CD22 CART. In this paper, we found that Chidamide promoted the expression of CD22 on the surface of B-cell tumor cells in vitro and in vivo, and enhanced the function of CD22 CART. As for mechanisms, we demonstrated that Chidamide did not affect CD22 mRNA transcription, but significantly increased the expression of total CD22 protein, indicating that Chidamide may upregulate cell surface CD22 expression by affecting the distribution of CD22 protein. In summary, our results suggest that Chidamide may enhance the efficacy of CD22 CART by inhibiting histone deacetylases to regulate post-transcriptional modifications that affect protein distribution to increase the expression of CD22 on the cell surface.

Antibody-Drug Conjugate Efficacy in Neuroblastoma: Role of Payload, Resistance Mechanisms, Target Density, and Antibody Internalization.

Antibody-drug conjugates (ADC) are a targeted cancer therapy that utilize the specificity of antibodies to deliver potent drugs selectively to tumors. Here we define the complex interaction among factors that dictate ADC efficacy in neuroblastoma by testing both a comprehensive panel of ADC payloads in a diverse set of neuroblastoma cell lines and utilizing the glypican 2 (GPC2)-targeting D3-GPC2-PBD ADC to study the role of target antigen density and antibody internalization in ADC efficacy in neuroblastoma. We first find that DNA binding drugs are significantly more cytotoxic to neuroblastomas than payloads that bind tubulin or inhibit DNA topoisomerase 1. We additionally show that neuroblastomas with high expression of the ABCB1 drug transporter or that harbor a TP53 mutation are significantly more resistant to tubulin and DNA/DNA topoisomerase 1 binding payloads, respectively. Next, we utilized the GPC2-specific D3-GPC2-IgG1 antibody to show that neuroblastomas internalize this antibody/GPC2 complex at significantly different rates and that these antibody internalization kinetics correlate significantly with GPC2 cell surface density. However, sensitivity to pyrrolobenzodiazepine (PBD) dimers primarily dictated sensitivity to the corresponding D3-GPC2-PBD ADC, overall having a larger influence on ADC efficacy than GPC2 cell surface density or antibody internalization. Finally, we utilized GPC2 isogenic Kelly neuroblastoma cells with different levels of cell surface GPC2 expression to define the threshold of target density required for ADC efficacy. Taken together, DNA binding ADC payloads should be prioritized for development for neuroblastoma given their superior efficacy and considering that ADC payload sensitivity is a major determinant of ADC efficacy.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

A two-step circuit allows ultrasensitive sensing of antigen density (from Ron Mader via Flickr)Recognition of normal cells expressing a low density of target antigen is a barrier to chimeric antigen receptor (CAR) T–cell therapy for some cancers. Hernandez-Lopez et al. have designed a two-step antigen-recognition circuit enabling CAR T cells to distinguish cells expressing high and low densities of target antigen. They engineer T cells to express a synthetic Notch receptor with low affinity for HER2. HER2 recognition by this receptor drives expression of a CAR with high affinity for HER2. These cells kill tumor cell lines and 3D spheroids expressing high densities of HER2 but spare those with low densities of HER2, showing this approach could limit on-target, off-tumor responses.Hernandez-Lopez RA, …, Lim WA. Science 2021 Mar 12;371:1166–71.Alveolar rhabdomyosarcoma (from Nephron via Wikimedia Commons)Increased understanding of the microenvironment at distant sites prior to metastases becoming detectable at those sites could lead to new strategies for treating metastases. In a syngeneic orthotopic model of rhabdomyosarcoma that spontaneously metastasizes to the lungs, Kaczanowska et al. identify a transcriptional program indicative of myeloid-mediated immune suppression in the premetastatic lung. Intravenous administration of myeloid cells genetically engineered to express IL12 reprograms the premetastatic niche, reversing the immunosuppressive phenotype and inducing T cell–mediated antitumor immunity, such that metastases are reduced and survival improved. These data suggest a new strategy for treating metastatic disease.Kaczanowska S,…, Kaplan RN. Cell 2021 Apr 15;184:2033–52.e21.MRTFs play dual roles in cancer and antitumor immunity (from Fig. 1 of Finch-Edmondson et al., Cell Mol Biol Lett 2016)Tumor cells have multiple ways of evading immune detection. Tello-Lafoz et al. find that myocardin-related transcription factors (MRTF) are “double agents.” On one hand, MRTFs support tumor progression and metastasis, but on the other hand, high expression boosts CTL and natural killer–cell activation, an effect enhanced by anti-CTLA4 therapy. High MRTF expression also alters the rigidity of the tumor cell F-actin cytoskeleton, a mechanical change that renders the tumor cells more susceptible to elimination. The data bring to light “mechanical” changes that can occur that allow for improved immunosurveillance and tumor killing.Tello-Lafoz M, …, Er EE. Immunity 2021 Mar 22. DOI: 10.1016/j.immuni.2021.02.020.CD161 on T cells is inhibitory (T/NK-cell CD161 IHC; from Fig. 2F of Dorfeshan et al., Biomed Res Int 2013)T cells play a vital role in the efficacy of immunotherapies. Using single-cell RNA sequencing, Mathewson et al. analyze tumor-infiltrating T cells in patients with glioma. They identify clonal CD8+ T cells coexpressing cytotoxic programs and natural killer (NK) cell–associated genes, including CD161 (encoded by KLRB1). CD161 is identified as an inhibitory receptor that limits T cell–mediated tumor killing. Inactivation of KLRB1 or blockade of CD161 restores T-cell killing of glioma targets in vitro and in vivo. These data point to CD161 and other NK cell–associated receptors (expressed by T cells) as potential immunotherapeutic targets.Mathewson ND, …, Wucherpfennig KW. Cell 2021 Mar 4;184:1281–98.e26.Bacteria-derived peptides can be presented by tumor cells (by Moustafa Abdou via Ask Hematologist/Understand Hematology)Bacteria are associated with some cancers and have a role in modulating immune function. Kalaora et al. investigate whether bacterial antigens are expressed on melanoma HLA molecules. More than 250 bacteria-derived peptides, originating from 41 bacterial species, are identified. A subset of these peptides are not only presented by HLA-I/II in different tumors within the same patient but also found across samples from different patients. The peptides presented by tumor cells are immunogenic and activate tumor-infiltrating lymphocytes ex vivo, providing further insight into the impact of bacteria on antitumor immunity.Kalaora S, …, Samuels Y. Nature 2021 Apr 1;592:138–43.3-D rendering of a neutrophil (from Blausen.com staff via Wikimedia Commons)Microrobots controlled by rotating magnetic fields have potential to allow precise therapeutic targeting of cancers. However, current microrobots are susceptible to immune clearance and cannot efficiently penetrate biological barriers such as the blood–brain barrier. Zhang et al. show these challenges can be addressed using neutrophil-based microrobots, referred to as “neutrobots.” Neutrobots are made in three steps: first, synthesis of nanogels loaded with hydrophobic Fe3O4 nanoparticles and paclitaxel; second, coating of the nanogels with the outer membrane of Escherichia coli; and third, phagocytosis of the coated nanogels by neutrophils. The neutrobots accurately deliver paclitaxel in a postoperative orthotopic glioma model, increasing survival, thereby highlighting the therapeutic potential of neutrobots.Zhang H, …, He Q. Science Robotics 2021 Mar 24;6:eaaz9519.

Targeting B7-H3 via chimeric antigen receptor T cells and bispecific killer cell engagers augments antitumor response of cytotoxic lymphocytes

BackgroundB7-H3, an immune-checkpoint molecule and a transmembrane protein, is overexpressed in non-small cell lung cancer (NSCLC), making it an attractive therapeutic target. Here, we aimed to systematically evaluate the value of B7-H3 as a target in NSCLC via T cells expressing B7-H3-specific chimeric antigen receptors (CARs) and bispecific killer cell engager (BiKE)-redirected natural killer (NK) cells.MethodsWe generated B7-H3 CAR and B7-H3/CD16 BiKE derived from an anti-B7-H3 antibody omburtamab that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. Antitumor efficacy and induced-immune response of CAR and BiKE were evaluated in vitro and in vivo. The effects of B7-H3 on aerobic glycolysis in NSCLC cells were further investigated.ResultsB7-H3 CAR-T cells effectively inhibited NSCLC tumorigenesis in vitro and in vivo. B7-H3 redirection promoted highly specific T-cell infiltration into tumors. Additionally, NK cell activity could be specially triggered by B7-H3/CD16 BiKE through direct CD16 signaling, resulting in significant increase in NK cell activation and target cell death. BiKE improved antitumor efficacy mediated by NK cells in vitro and in vivo, regardless of the cell surface target antigen density on tumor tissues. Furthermore, we found that anti-B7-H3 blockade might alter tumor glucose metabolism via the reactive oxygen species-mediated pathway.ConclusionsTogether, our results suggest that B7-H3 may serve as a target for NSCLC therapy and support the further development of two therapeutic agents in the preclinical and clinical studies.