Primary Human T-cells Research Articles

Cell therapy biomanufacturing: integrating biomaterial and flow-based membrane technologies for production of engineered T-cells.

Adoptive T-cell therapies (ATCTs) are increasingly important for the treatment of cancer, where patient immune cells are engineered to target and eradicate diseased cells. The biomanufacturing of ATCTs involves a series of time-intensive, lab-scale steps, including isolation, activation, genetic modification, and expansion of a patient's T-cells prior to achieving a final product. Innovative modular technologies are needed to produce cell therapies at improved scale and enhanced efficacy. In this work, well-defined, bioinspired soft materials were integrated within flow-based membrane devices for improving the activation and transduction of T cells. Hydrogel coated membranes (HCM) functionalized with cell-activating antibodies were produced as a tunable biomaterial for the activation of primary human T-cells. T-cell activation utilizing HCMs led to highly proliferative T-cells that expressed a memory phenotype. Further, transduction efficiency was improved by several fold over static conditions by using a tangential flow filtration (TFF) flow-cell, commonly used in the production of protein therapeutics, to transduce T-cells under flow. The combination of HCMs and TFF technology led to increased cell activation, proliferation, and transduction compared to current industrial biomanufacturing processes. The combined power of biomaterials with scalable flow-through transduction techniques provides future opportunities for improving the biomanufacturing of ATCTs.

Abstract 4168: Tumor cell stimulator of interferon genes (STING) activation primes NK cell therapy in mesothelioma

Abstract Background: Activation of the anti-viral STING pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Understanding their mechanism of action in human tumors is key to developing STING-based therapies and combinations. Higher tumor STING expression correlates with better response to treatment and multiple cancers have been shown to silence STING and the downstream interferon response to avoid immune detection, demonstrating an important role for tumor cell STING in antitumor immunity. Recent mouse studies have explored the complex interplay of STING activation in the tumor immune microenvironment (TIME), identifying unexpected T-cell toxicity and novel effector mechanisms including NK cells. Yet how STING agonists impact the human TIME has not been carefully examined and could inform development of novel therapeutic combinations. Here we address this question using human tumors cultured ex vivo to maintain the TIME, dissecting STING agonist response in malignant pleural mesothelioma (MPM), an inflamed cancer with high STING expression. Methods: We performed STING immunohistochemistry on 300 thoracic tumor specimens, followed by functional ex vivo studies of STING agonist response in human mesothelioma explants, measuring cell death and cytokine production. Dynamic single cell RNA sequencing was performed after treatment with a STING agonist. Primary human T-cells and NK cells were used to study STING agonist toxicity in the context of STING cycling and patient derived organotypic tumor spheroids (PDOTS) were treated for six days with STING agonists +/- NK and anti-mesothelin chimeric antigen receptor (CAR)-NK cell therapies. Results: Amongst thoracic cancers, MPM robustly expressed tumor cell STING and was responsive to STING agonist treatment ex vivo. We observed tumor cell death >20%, p<0.05 in 12/35 MPM PDOTS after STING agonist treatment. Cell death was prevented by CD8 neutralizing antibody co-treatment. Dynamic single-cell RNA sequencing of MPM explants treated with a STING agonist unveiled CXCR3 chemokine activation primarily in tumor cells and cancer associated fibroblasts, coupled with an interferon stimulated gene program. However, STING agonism was also toxic to T-cells. In contrast, primary NK cells manifested rapid STING turnover and resisted STING agonist-induced toxicity. STING agonists enhanced NK and especially anti-mesothelin CAR-NK cell migration and killing, improving therapeutic activity in MPM PDOTS. Conclusions: MPM, an inflamed cancer type with marginal response to immune checkpoint blockade, demonstrated high tumor cell STING expression and response to STING agonists in combination with NK cell therapies ex vivo. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies, identifying STING agonism as an effective strategy to prime NK cell therapy. Citation Format: Erik H. Knelson, Elena Ivanova, Mubin Tarannum, Marco Campisi, Patrick H. Lizotte, Matthew A. Booker, Brittany Meisenheimer, Minyue Chen, Nathaniel Spicer, Bonje Obua, Ha V. Vo, Thomas P. Albertson, Marina Vivero, Michael Y. Tolstorukov, Rizwan Romee, Cloud P. Paweletz, Raphael Bueno, David A. Barbie. Tumor cell stimulator of interferon genes (STING) activation primes NK cell therapy in mesothelioma [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 4168.

Engineering a single-chain variable fragment of cetuximab for CAR T-cell therapy against head and neck squamous cell carcinomas

The monoclonal antibody cetuximab recognizes domain III of the epithelial growth factor receptor (EGFR) with high-affinity and is an important element in the treatment of several malignancies that overexpress non-mutated wild-type EGFR. In order to create an EGFR recognizing chimeric antigen receptor (CAR) for cellular immunotherapy of head and neck squamous cell carcinoma (HNSCC), we rationally designed single chain fragments of different lengths based on the cetuximab variable heavy and light chains. We then cloned the different cetuximab fragments into our second generation CAR construct, expressed CARs on primary human T-cells from healthy donors using mono- or biscistronic lentiviral vectors and tested the stability, functionality and specificity of the CARs. Our smallest CAR construct was most efficient with greatly improved vector production and T-cell transduction efficacy. Finally, we demonstrated that the new cetuximab CAR construct expressed on T-cells is highly reactive against EGFR-positive HNSCCs and also malignant cells from other solid cancer entities. In conclusion, we generated an optimized high-affinity EGFR CAR construct for the next steps in cancer immunotherapy, which need to focus on the development of armored CAR T-cells that will be more resistant and effective in the hostile microenvironment present in solid cancers.

Modular design of synthetic receptors for programmed gene regulation in cell therapies

Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors that we call synthetic intramembrane proteolysis receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the therapeutic potential of the receptor platform by engineering human primary Tcells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.

Disruption of c-MYC Binding and Chromosomal Looping Involving Genetic Variants Associated With Ankylosing Spondylitis Upstream of the RUNX3 Promoter.

Background: Ankylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 (p = 2.6 × 10−15) lies ∼13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C). Results: Interrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14+ monocytes, in contrast to the Jurkat T cell line or primary human T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 we consistently observed reduced binding to the AS-risk “C” allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences (n = 3; p < 0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889. Conclusion: The lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3. In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS.

Discovery of Spiro-azaindoline Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1).

Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1. Using a structure-based drug design approach, the kinase selectivity of the molecules was significantly improved by inducing and stabilizing an unusual P-loop folded binding mode. The metabolic liabilities of the initial 7-azaindole high-throughput screening hit were mitigated by addressing a key metabolic soft spot along with physicochemical property-based optimization. The resulting spiro-azaindoline HPK1 inhibitors demonstrated improved in vitro ADME properties and the ability to induce cytokine production in primary human T-cells.

Regulation of Heterochromatin Landscape in T-Cell Acute Lymphoblastic Leukemia

Ikzf1 encodes a zinc finger, DNA-binding protein that functions as a tumor suppressor in acute lymphoblastic leukemia (ALL). Deletion and/or loss of Ikaros function results in the development of high-risk leukemia. In the nucleus, Ikaros forms complexes with histone deacetylase complex, NuRD, and it participates in the formation of heterochromatin. The role of Ikaros-mediated formation of heterochromatin in tumor suppression in leukemia is unknown. We determined global genomic occupancy of Ikaros, global heterochromatin distribution, chromatin accessibility, DNA methylation landscape, and gene expression in primary human T-cell ALL (T-ALL), as well as in mouse T-ALL to analyze how Ikaros regulates heterochromatin landscape and gene expression in T-ALL. Results showed that Ikaros DNA occupancy is essential for the recruitment of histone deacetylase 1 (HDAC1), Polycomb repressive complex 2 (PRC2) and formation of facultative heterochromatin, as well as the formation of constitutive heterochromatin (characterized by H3K9me3 occupancy). T-ALL cells with deletion of both Ikzf1 alleles have severely impaired HDAC1 DNA occupancy and reduced H3K27me3. Re-introduction of Ikzf1 via retroviral transduction resulted in the restoration of H3K27me3 facultative heterochromatin, along with HDAC1 DNA occupancy. The H3K27me3 genomic distribution following Ikzf1 re-introduction showed high homology to the H3K27me3 genomic distribution in normal thymocytes. Analysis of H3K9me3 genomic distribution showed that Ikzf1 deletion results in dramatic redistribution of H3K9me3 global occupancy, with reduced H3K9me3 occupancy at pericentromeric loci. Reintroduction of Ikzf1 enhances H3K9me3 enrichment in pericentromeric loci, as well as at the promoters of genes that are involves in cellular proliferation. Analysis of DNA methylation distribution showed that Ikzf1 expression regulates global DNA methylation landscape. The presence of facultative heterochromatin, with enrichment of H3K27me3, inversely correlated with DNA methylation. Global analysis of chromatin accessibility revealed that Ikaros binding resulted in the loss of chromatin accessibility at over 3400 previously-accessible chromatin sites. Dynamic analyses demonstrate the long-lasting effects of Ikaros's DNA binding on heterochromatin distribution and chromatin accessibility. Analysis of gene expression in T-ALL with both Ikzf1 alleles and in Ikzf1-defficient cells (from Ikzf1-defficient T-ALL, and from Ikzf1-wild-type T-ALL following Ikzf1 deletion by CRISPR) showed that Ikaros-induced redistribution of facultative and constitutive heterochromatin results in the repression of several genes that are critical for cell cycle progression, PI3K-AKT-mTOR, and WNT signaling pathway. In conclusion, results suggest that Ikaros' tumor suppressor function in T-ALL occurs via global regulation of the heterochromatin, DNA methylation landscape, and chromatin accessibility, as well as via epigenetic regulation of transcription of the genes that play essential roles in signaling pathways that promote cellular proliferation. DisclosuresNo relevant conflicts of interest to declare.

Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells

The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human Tcells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.

The Targeting Effect of Cetuximab Combined with PD-L1 Blockade against EGFR-Expressing Tumors in a Tailored CD16-CAR T-Cell Reporter System

The switchable chimeric antigen receptors (CARs) have shown many advantages in CAR T-cell therapy. However, human primary T-cells are required to evaluate antigen-specific adaptors by IFN-γ assay or FACS analysis, which limits the throughput of adaptor screening. A sensitive and robust CD16-CAR Jurkat NFAT-eGFP reporter system has been developed to assess the therapeutic efficacy of antibody-targeted CAR-T-cell by effectively evaluating the T-cell activation by various tumor cells and the impact of immune checkpoint inhibitor antibodies. This reporter system facilitates the screening of targeted antibodies in a high throughput manner for the development of improved T-cell immunotherapy.

Synthetic promoters to induce immune-effectors into the tumor microenvironment

Harnessing the immune-system to eradicate cancer is becoming a reality in recent years. Engineered immune cells, such as chimeric antigen receptor (CAR) T cells, are facing the danger of an overt life-threatening immune response due to the ON-target OFF-tumor cytotoxicity and Cytokine Release Syndrome. We therefore developed synthetic promoters for regulation of gene expression under the control of inflammation and Hypoxia-induced signals that are associated with the tumor microenvironment (TME). We termed this methodology as chimeric-antigen-receptor-tumor-induced-vector (CARTIV). For proof of concept, we studied synthetic promoters based on promoter-responsive elements (PREs) of IFNγ, TNFα and hypoxia; triple PRE-based CARTIV promoter manifested a synergistic activity in cell-lines and potent activation in human primary T-cells. CARTIV platform can improve safety of CAR T-cells or other engineered immune-cells, providing TME-focused activity and opening a therapeutic window for many tumor-associated antigens that are also expressed by non-tumor healthy tissues.

A primary human T-cell spectral library to facilitate large scale quantitative T-cell proteomics

Data independent analysis (DIA) exemplified by sequential window acquisition of all theoretical mass spectra (SWATH-MS) provides robust quantitative proteomics data, but the lack of a public primary human T-cell spectral library is a current resource gap. Here, we report the generation of a high-quality spectral library containing data for 4,833 distinct proteins from human T-cells across genetically unrelated donors, covering ~24% proteins of the UniProt/SwissProt reviewed human proteome. SWATH-MS analysis of 18 primary T-cell samples using the new human T-cell spectral library reliably identified and quantified 2,850 proteins at 1% false discovery rate (FDR). In comparison, the larger Pan-human spectral library identified and quantified 2,794 T-cell proteins in the same dataset. As the libraries identified an overlapping set of proteins, combining the two libraries resulted in quantification of 4,078 human T-cell proteins. Collectively, this large data archive will be a useful public resource for human T-cell proteomic studies. The human T-cell library is available at SWATHAtlas and the data are available via ProteomeXchange (PXD019446 and PXD019542) and PeptideAtlas (PASS01587).

Obesity Attenuates T-Cell Function Which Impacts the Efficacy of T-Cell Based Immunotherapies in Acute Lymphoblastic Leukemia

The incidence of obesity continues to rise with over 50% of the world's population predicted to be overweight/obese by the year 2030. The global health impact of this trend is concerning given that obesity is a risk factor for developing cancers of varying etiologies. Alarmingly, the survival outcomes for obese patients with cancer are lower than those observed in lean patients. Obesity is characterized by the accumulation of adipocytes which alters drug dynamics and impacts the function of cancer and immune cells in the tumor microenvironment. Obesity-induced immune defects are troubling given the increasing use of immunotherapy in the treatment of malignancies. Here we show that adipocyte-secreted factors upregulate immunosuppressive mechanisms on human B-cell acute lymphoblastic leukemia (B-ALL) cells, attenuate the function of endogenous T-cells, and compromise the efficacy of T-cell based immunotherapies. To study the impact of adiposity on T-cell function, CD4+ and CD8+ T-cells were purified from the spleens of C57BL/6 mice and activated with PMA/Ionomycin for 72 hours in unconditioned media (UCM), bone marrow stromal-cell conditioned media (SCM), and adipocyte-conditioned media (ACM) followed by flow cytometry analysis for surface marker expression, cytokine production, and the induction of cytolytic mediators. Interestingly, T-cells activated in ACM, but not UCM or SCM, showed an attenuated phenotype highlighted by decreased CD44 and PD-1 expression, diminished cytokine production (IFN-γ/TNF-α) and reduced induction of cytolytic mediators (granzyme B/perforin). These observations were also true in obese, relative to lean, patients with B-ALL where we found that T-cells purified from the peripheral blood mononuclear cells (PBMCs) failed to produce significant levels of TNF-α when stimulated with PMA/Ionomycin. In all, these results demonstrate that adipocyte-secreted factors directly compromise the function of endogenous T-cells, which phenocopies T-cell defects observed in obese relative to lean pediatric patients with B-ALL. We next assessed the impact of adiposity on malignant cells by culturing human B-ALL cell lines in the conditioned mediums described above and performed flow cytometric analysis to assess their surface expression of the B-cell lineage antigen CD19 and proteins that modulate immunity. In addition to being a marker for B-cells, CD19 is the primary target of the T-cell based immunotherapies Blinatumomab and CAR T-cells directed against B-ALL cells. Surprisingly, when human B-ALL cells were co-cultured with adipocytes, every cell line tested (n=6) exhibited lower surface CD19 expression with 5 out 6 reaching statistical significance. Furthermore, adipocyte-secreted factors alone were sufficient to reduce CD19 surface levels on B-ALL cells in 2 of the 6 cell lines tested. Human B-ALL cells cultured in ACM, but not UCM or ACM, also upregulated their surface expression of the immunoinhibitory proteins HVEM, PD-L1, and PD-L2. These results demonstrate that adipocytes directly induce the downregulation of CD19 on human B-ALLs and increase their immune evasive capacity. Given these observations, we hypothesized that adipocyte-secreted factors would compromise T-cell-based immunotherapies targeting CD19-expressing B-ALL cells. To this end, primary human T-cells were engineered to express a CD19-directed chimeric antigen receptor (CAR). CAR T-cells and human B-ALL cells were separately pre-treated for 24 hours in UCM, SCM or ACM followed by co-culture for cytolytic analysis using Annexin-V/PI staining. Adipocyte-secreted factors significantly inhibited CAR T-cell mediated killing of CD19-expressing B-ALL cells at 4 hours. In addition to CAR T-cells, we tested the leukemia killing efficacy of the bispecific T-cell engager, Blinatumomab. After 3 days of culture, we found that Blinatumomab significantly increased the killing capacity of endogenous T-cells with 60-80% of B-ALL cells being killed after 3 days of culture in UCM and SCM. In contrast, we found that ACM significantly compromised the efficacy Blinatumomab with only 30% of B-ALL cells being killed over 3 days when co-cultured with human T-cells. Our pre-clinical data highlights the negative impact of an adipose-rich microenvironment on T-cell function and B-ALL immunogenicity, which subsequently compromises the efficacy of multiple classes of immunotherapies targeting CD19. Disclosures No relevant conflicts of interest to declare.

A Protease-Mediated Regulatory Chimeric Antigen Receptor (CAR): “Double Arm” CAR System Improves Tumor-Cell-Specificity of CAR-T Cell Therapy

【 Introduction 】 CAR-T cell therapy has shown excellent therapeutic effects against some malignancies including refractory B cell lymphoma or leukemia. This adoptive T cell transfer therapy is an attractive methodology; however, there are several disadvantages to be overcome. Current CAR-T cell therapy targets single cell-surface molecule, which leads to damage of normal cells expressing the target protein. Such "On-target / off-tumor" effect is one of the major adverse effects. Improvement of target-cell-specificity is needed to avoid this serious adverse effect and to expand target diseases of this therapy. Here, we show the protease-mediated "Double-Arm" CAR-T cell system, which improved the specificity of CAR-T cell therapy by recognizing two distinct cell-surface proteins. We designed two types of CARs: "Effector CAR" and "Scissors CAR". The "Effector CAR" is constituted of a single chain Fv fragment (scFv) targeting a cell-surface protein (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition polypeptide sequence, and a functional domain of CD3-zeta. The "scissors CAR" is constituted of a recognition portion targeting another protein (protein Y) and HIVPR. The HIVPR induces cleavage of the recognition polypeptide sequence in the effector CAR leading to inactivation of the effector CAR when the CAR-T cells contact with cells expressing both proteins X and Y. 【 Material and Methods 】 For proof of principle, we first constructed "anti-CD19 mCherry CAR" harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition polypeptide sequence. Also, we constructed "anti-CD19 scissors CAR", low affinity "anti-HER2 (4D5-3) scissors CAR" and high affinity "anti-HER2 (4D5-8) scissors CAR". To analyze the target-cell-dependent cleavage of mCherry CAR, 293T cells expressing these CARs were co-cultured with target cells, including K562 (CD19-, HER2-), Raji (CD19+, HER2+), or SK-BR-3 (CD19-, HER2+). To obtain target cells expressing both CD19 and HER2, Raji and SK-BR-3 cells were molecularly manipulated. (1) To evaluate efficiency of this system, after co-cultivation of CAR-transduced 293T and the target cells (K562, Raji, SK-BR-3), the localization of mCherry was examined under the microscopy and Western blotting. (2) To assess the T cell activation, we constructed "anti-CD19 effector CAR" and established Jurkat cells expressing both the "effector CAR" and the "anti-HER2 scissors CAR". These cells were co-cultured with wild type or engineered Raji or SK-BR-3 cells. T cell activation was analyzed with flowcytometric analysis. (3) To assess the CAR activity of this system, we transduced these CARs to primary T cells from healthy donors. After co-cultivation with the target cells, we measured target-specific cytotoxic activity. 【 Results 】 (1) Transduced "anti-CD19 mCherry CAR" was detected as a membrane-bound protein in 293T cells. Co-cultivation of 293T cells expressing both the "anti-CD19 mCherry CAR" and "anti-HER2 scissors CAR" with engineered Raji cells expressing both CD19 and HER2 induced cleavage of the recognition site and translocation of the mCherry from the membrane to the cytoplasm. These results suggested that this system would regulate activities of CAR-T cell through HIVPR-mediated cleavage of the "effector CAR" in vitro. (2) Jurkat cells expressing "anti-CD19 effector CAR" were activated through a target-cell-dependent manner. Furthermore, "anti-HER2 scissors CAR" attenuated T cell activation driven by "anti-CD19 effector CAR" when Jurkat cells expressing both the "anti-CD19 effector CAR" and the "anti-HER2 scissors CAR" contacted with target-cells expressing both CD19 and HER2. In addition, high affinity 4D5-8 scissors CAR showed more potent attenuation than the low affinity 4D5-3 scissors CAR. (3) Primary human T cells expressing "anti-CD19 effector CAR" showed CD19-expressing target-cell-specific cytotoxic activity. We also demonstrated that "Double Arm" primary human CAR-T cells showed tumor-cell-specificity as seen in cell-line models described above. 【Discussion】 Our "Double-Arm" CAR-T cell system has improved tumor-cell-specificity even in primary human T-cells as we expected. This system would attenuate the adverse effects of clinical CAR-T cell therapies. Disclosures No relevant conflicts of interest to declare.

Engineering Tolerance toward Allogeneic CAR-T Cells by Regulation of MHC Surface Expression with Human Herpes Virus-8 Proteins

Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human Tcells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for Tcells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic Tcells, K3-transduced Tcells selectively expanded invivo. Ectopic K5 expression in MHC class I-, MIC-A+/B+ K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, Tcell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

Abstract 6528: Anti-PD-1 induces the endocytosis of the co-receptor from the surface of T-cells: Nivolumab is more effective than Pembrolizumab

Abstract Immune checkpoint blockade (ICB) has revolutionized the treatment of cancer. Present models show that anti-PD-1 operates simply by blocking co-receptor binding to its ligand PD-L1. Surprisingly, no previous studies have assessed whether ICB also induces the internalization or endocytosis of PD-1 from the cell surface. Many, but not all receptors, undergo endocytosis upon antibody or ligand binding. In this study, PD-1 internalization was assessed on activated human T cells by flow cytometry and fluorescence microscopy. Immunoprecipitation and western blotting were performed to assess the destiny of PD-1 after internalization. Our results report that anti-PD-1 induces the rapid internalization of PD-1 from the surface of T-cells in a manner comparable to CD28. A substantial minority of receptors were also resistant to antibody-induced endocytosis. Further, Nivolumab was considerably more effective than Pembrolizumab in inducing the endocytosis of PD-1 in primary human T-cells. This difference was the result of several factors where Nivolumab bound to a greater number of PD-1 molecules on the surface of T-cells than Pembrolizumab, indicating that the co-receptor might exist with different conformations on activated cells. Moreover, Nivolumab induced more rapid endocytosis (5.5 versus 3.6% per minute) and a greater percentage of PD-1 molecules than Pembrolizumab (70% versus 50%). The relative effect of anti-PD-1 molecules on internalization was seen equally on activated CD4 helper and CD8+ effector/memory T-cells. The internalization and the degradation of PD-1 were partially blocked by the inhibition of clathrin-mediated endocytosis and proteasome activity. Our studies show for the first time that ICB involves a second level of modulation involving the internalization of PD-1 from the surface of T-cells, an event which will contribute to the efficacy of ICB. Citation Format: Elham Ben Saad, Andres Oroya, Christopher E. Rudd. Anti-PD-1 induces the endocytosis of the co-receptor from the surface of T-cells: Nivolumab is more effective than Pembrolizumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6528.

Thermal Control of Engineered T-cells.

Genetically engineered T-cells are being developed to perform a variety of therapeutic functions. However, no robust mechanisms exist to externally control the activity of T-cells at specific locations within the body. Such spatiotemporal control could help mitigate potential off-target toxicity due to incomplete molecular specificity in applications such as T-cell immunotherapy against solid tumors. Temperature is a versatile external control signal that can be delivered to target tissues in vivo using techniques such as focused ultrasound and magnetic hyperthermia. Here, we test the ability of heat shock promoters to mediate thermal actuation of genetic circuits in primary human T-cells in the well-tolerated temperature range of 37-42 °C, and introduce genetic architectures enabling the tuning of the amplitude and duration of thermal activation. We demonstrate the use of these circuits to control the expression of chimeric antigen receptors and cytokines, and the killing of target tumor cells. This technology provides a critical tool to direct the activity of T-cells after they are deployed inside the body.

An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human Tcells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair Tcell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human Tcells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells

Regulatory Tcells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory Tcells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the Tcell-specific kinase, protein kinase C theta, which promotes Tcell effector functions and represses regulatory Tcell differentiation, augments regulatory Tcell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 Tcells. When differentiated exvivo into induced regulatory Tcells, treated cells expressed elevated levels of the regulatory Tcell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory Tcells. The invitro suppressive capacity of these induced regulatory Tcells was 10-fold greater than that of Tcells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory Tcells were superior to non-treated Tcells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory Tcells as a cell-based therapy for immune modulation.

Efficient Nuclease-Directed Integration of Lentivirus Vectors into the Human Ribosomal DNA Locus

Lentivirus vectors (LVs) are efficient tools for gene transfer, but the non-specific nature of transgene integration by the viral integration machinery carries an inherent risk for genotoxicity. We modified the integration machinery of LVs and harnessed the cellular DNA double-strand break repair machinery to integrate transgenes into ribosomal DNA, a promising genomic safe-harbor site for transgenes. LVs carrying modified I-PpoI-derived homing endonuclease proteins were characterized in detail, and we found that at least 21% of all integration sites localized to ribosomal DNA when LV transduction was coupled to target DNA cleavage. In addition to the primary sequence recognized by the endonuclease, integration was also enriched in chromatin domains topologically associated with nucleoli, which contain the targeted ribosome RNA genes. Targeting of this highly repetitive region for integration was not associated with detectable DNA deletions or negative impacts on cell health in transduced primary human Tcells. The modified LVs characterized here have an overall lower risk for insertional mutagenesis than regular LVs and can thus improve the safety of gene and cellular therapy.

A CD3 x FRα T-cell engaging bispecific antibody for efficient killing of ovarian cancer cells with minimal cytokine release.

e18050 Background: Ovarian Cancer (OvCa) is the leading cause of gynecologic cancer mortality in women. Since the introduction of platinum-based chemotherapy there has been little change in the prognosis of OvCa patients, with &lt; 30% overall survival in advanced disease, creating an urgent medical need for novel therapies. Few ovarian epithelium-specific surface proteins are suited for Ab targeting. However, studies have shown folate receptor α (FRα) to be highly over-expressed in OvCa; expression level and stage correlate, and FRα is absent or minimally expressed in normal tissues. However, naked Ab therapy has shown limited efficacy while CAR-T therapy has been plagued by toxicity and limited efficacy. ADCs have demonstrated some activity but present the risk of toxin-mediated side effects. Using Teneobio’s unique antibody discovery platform, we have developed a CD3 x FRα T-BsAb that retains the potent cytotoxicity of other T-cell redirecting therapies but with significantly reduced cytokine release. Methods: Antibodies targeting CD3 and FRα were generated via immunization of our proprietary transgenic animals. Candidate antibodies were selected by repertoire deep sequencing of B-cells from draining lymph nodes, high-throughput gene assembly, recombinant expression, and functional screening. Bispecific antibodies targeting CD3 and FRα were assembled and evaluated for their ability to selectively activate primary human T-cells and mediate killing of FRα+ tumor cells in vitro and in vivo. T-cell activation surface markers, cytokine production and tumor cell cytotoxicity were measured. Results: Primary human T-cells were activated only in the presence of both the CD3 x FRα T-BsAb and FRα (either recombinant or cell-surface protein). Potent and selective cytotoxicity against FRα+ tumor cells was observed in co-cultures of primary human T-cells and OvCa tumor cell lines. Strikingly, our T-BsAb mediated comparable tumor cell cytotoxicity to CD3 x FRα T-BsAbs containing a high affinity anti-CD3 domain but with significantly reduced cytokine production. Our Ab showed preliminary evidence of tumor growth inhibition in xenograft models of OvCa in vivo. Conclusions: We have created a novel CD3 x FRα T-BsAb that mediates T-cell killing of FRα+ tumor cells with minimal production of cytokines. This molecule may improve safety, efficacy, and offer opportunities for combination therapy to treat OvCa.