Single T-cell Research Articles

Clinicopathological Presentation, Prognosis, and Overall Survival of Primary Extra Nodal Lymphomas

ABSTRACT Introduction: Primary extranodal lymphoma (PENL) originates from extranodal organs or tissues. Approximately 25–40% of NHL arise primarily from sites other than lymph nodes. Material and Methods: A retrospective study where survival outcome was calculated using the Kaplan–Meier method. Prognostic factors were estimated using the Cox proportional hazard regression model. Results: Out of 228 non-Hodgkin lymphoma (NHL) cases, 97 (42.54%) cases were presented as PENL. Head and neck (31.95%) were the most common site of PENL, followed by the gastrointestinal tract (24.7%) and soft tissue and bone (10.3%). The overall survival (OS) in PENL was 72.1%, while in nodal NHL, the overall survival was 47% at three years. The median survival for PENL was 1737.21 days, with a standard error of 155.31 days. Survival outcome, from KM method, analysis of PENL had a significant association with anatomical location (P = 0.022), given treatment modality (P = 0.000), and histopathological grading (P = 0.004). Associated factors of mortality from the multivariable analysis were noted as high-grade lymphoma histologically (P = 0.011) (Hazard ratio (HR) =3.112, 95% CI 1.592-6.889), T-cell lymphoma (HR = 1.887, 95% CI 0.893-4.121), single line treatment and no treatment (P = 0.028) (HR = 2.728, 95% CI 1.289-3.184), and age of patients >51 years. (HR = 1.772, 95% CI 0.86-3.297) (P = 0.041). Conclusion: The median survival for PENL was significantly better than nodal NHL. Prognostic factors such as age >51 years, single modality treatment T-cell lymphoma, high-grade lymphoma, and nodal lymphoma are associated with poor survival.

Multiomic Single Cell Profiling Identifies Drivers of Progression in T-Cell Prolymphocytic Leukemia

Treatments for T-cell prolymphocytic leukemia (T-PLL), while transiently effective in the frontline, have failed to achieve any substantial improvement in the outcomes of this ultra-rare chronic leukemia. Patients with relapsed T-PLL have a dismal prognosis, with a median overall survival (OS) of less than 6 months. For this study we used samples collected from: 8 patients with untreated T-PLL, 2 with relapsed/refractory (R/R) T-PLL, and 1 from a PDX model of R/R T-PLL. Whole-exome sequencing (WES) was used to elucidate the mutational landscape and single cell T-cell receptor sequencing (scTCRseq), coupled with single cell RNA sequencing (scRNA-seq), was used to generate gene expression and TCR clonality signatures at the single cell level. The WES revealed a higher frequency of mutations in R/R T-PLL compared to their baseline counterparts. R/R T-PLL samples displayed mutations of CXCR4 (37%; p.S312fs), STAT5B (46%; p.N642H), ATM (82%; p.L2890V), SAMHD1 (92%; p.A565T) and BCOR (p.N1585fs) and deletions in ASXL2 which were absent in treatment naïve T-PLL. In addition, all T-PLL samples harbored recurrent mutations on CXCR4, CD27, JAK1, STAT3, CCND1, CDKN1B, and the epigenetic regulators KMT2C, KMT2B, TET2, and KDM5C. The mutational profiles revealed the genomic complexity of T-PLL. The ubiquitous presence of activating mutations affecting the JAK/STAT axis underscored their importance in driving T-PLL survival. We also studied the diversity of TCR clonality and its relationship with pathogenesis and prognosis in T-PLL. All the samples exhibited dominance of at least one to up to 3 specific CDR3 sequences, revealing the anticipated clonal expansion in T-PLL. Expression of TCRα and TCRβ genes in the dominant clone differed among the samples.Monoclonality was validated as biallelic only when the 2nd largest transcripts of CDR3α or CDR3β clones were at least 5 times larger than its subceeder. The coupled TCR seq and RNA seq analysis displayed varied degrees of clonal expansions at a single cell resolution across all the patient samples. The T cell subsets were characterized by the expression of various T cell-markers across the cell clusters. In particular, 60% of the samples showed more expression of IL7R and lesser expression CD8A and CD8B, indicating their ability in suppressing the antitumor immunity. CD3E and CD3D were highly expressed across the clusters of T-PLL datasets.Out of 11 datasets, NKG7 and GZMA (cytotoxic T cell markers) were identified in more than 40% of the samples. The augmented levels of anti-apoptosis (BCL-2) and Jak/Stat signaling (JAK1, STAT3) markers were seen across the clusters of T-PLL scRNA seq datasets. The higher expression of PPP1R14B was found in 70% of the samples indicating its probable role in poor prognosis in T-PLL. In conclusion, T-PLL is characterized by numerous genomic aberrations, presence of 1-3 dominant clones, and gene expression signatures that facilitate survival, and resistance to apoptosis.

P3.06D.14 Liquid Biopsy-Based Single T-Cell Dynamic Polyfunctionality Profiling as a NSCLC Immunotherapy Biomarker

P3.06D.14 Liquid Biopsy-Based Single T-Cell Dynamic Polyfunctionality Profiling as a NSCLC Immunotherapy Biomarker

The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

PoxiPred: An Artificial-Intelligence-Based Method for the Prediction of Potential Antigens and Epitopes to Accelerate Vaccine Development Efforts against Poxviruses.

Poxviridae is a family of large, complex, enveloped, and double-stranded DNA viruses. The members of this family are ubiquitous and well known to cause contagious diseases in humans and other types of animals as well. Taxonomically, the poxviridae family is classified into two subfamilies, namely Chordopoxvirinae (affecting vertebrates) and Entomopoxvirinae (affecting insects). The members of the Chordopoxvirinae subfamily are further divided into 18 genera based on the genome architecture and evolutionary relationship. Of these 18 genera, four genera, namely Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, and Yatapoxvirus, are known for infecting humans. Some of the popular members of poxviridae are variola virus, vaccine virus, Mpox (formerly known as monkeypox), cowpox, etc. There is still a pressing demand for the development of effective vaccines against poxviruses. Integrated immunoinformatics and artificial-intelligence (AI)-based methods have emerged as important approaches to design multi-epitope vaccines against contagious emerging infectious diseases. Despite significant progress in immunoinformatics and AI-based techniques, limited methods are available to predict the epitopes. In this study, we have proposed a unique method to predict the potential antigens and T-cell epitopes for multiple poxviruses. With PoxiPred, we developed an AI-based tool that was trained and tested with the antigens and epitopes of poxviruses. Our tool was able to locate 3191 antigen proteins from 25 distinct poxviruses. From these antigenic proteins, PoxiPred redundantly located up to five epitopes per protein, resulting in 16,817 potential T-cell epitopes which were mostly (i.e., 92%) predicted as being reactive to CD8+ T-cells. PoxiPred is able to, on a single run, identify antigens and T-cell epitopes for poxviruses with one single input, i.e., the proteome file of any poxvirus.

OP04 Endoscopic healing in paediatric IBD perpetuates a persistent signature characterized by pathogenic Th17 cells and tissue-associated molecular and microbial drivers of disease

Abstract Background Endoscopic healing (EH) is the major long-term treatment target for inflammatory bowel diseases (IBD) in adults and paediatric patients. However, EH may not represent disease clearance. Hence, risk of relapse remains high and treatment discontinuation after reaching EH often results in disease exacerbation. We aimed to identify persistent cellular, molecular and microbial drivers of IBD under EH and longitudinally collected endoscopic biopsies from paediatric patients for analysis of single T-cell and bulk transcriptomics as well as mucosa-associated microbiota. Methods We followed disease trajectories of paediatric IBD patients treated according to international guidelines and collected mucosal biopsies from the terminal ileum (TI) and sigmoid colon (SC) at baseline (T1) and after approximately 3-12 months (T2) to assess EH. Non-IBD controls showed no macroscopic or histologic signs of inflammation. Host RNA and bacterial DNA were simultaneously extracted from single biopsies for bulk RNA-seq, and 16S rRNA seq. A second biopsy was used for isolation of lamina propria mononuclear cells (LPMCs), followed by CD45+, CD3+ cell sorting and single T-cell analysis (10X Genomics). Data were quality controlled and integrated. Results 217 biopsies (135 simultaneous extraction of host RNA and bacterial DNA; 82 single T-cell analysis) from 32 paediatric IBD patients (mean age 13 ± 3.5 years, 21 Crohn’s disease [CD], 11 ulcerative colitis [UC]; T1 = 32, T2 = 32, T > 2 = 6) and 5 non-IBD controls were analysed. Time between T1 and T2 averages 40 ± 22 weeks. At baseline, 15 patients (14 CD, 1 UC) were newly diagnosed and 31/32 patients had active mucosal inflammation. Twenty-two patients (71%) achieved EH at T2 (SES-CED ≤2 and absence of ulcerations), mostly on anti-TNF therapy. One patient (EH at T1) experienced relapsing disease within 10 weeks after discontinuing medication. In the bulk RNA-seq, we identified 299 differential expressed genes (DEGs; corrected p-value <0.05, FC >2.0), such as DUOX2, SAA2-SAA4, FCGR3B and NOS2, that are associated with active IBD and remained upregulated in EH in contrast to non-IBD controls. In addition single cell analysis revealed an IBD-specific pathogenic Th17 cluster that continued to exist in EH and showed high correlation with top DEGs after data integration. 16S rRNA gene seq highlighted highly individual mucosa-associated bacterial profiles and a reduced α-diversity in active and EH compared to non-IBD controls. Conclusion A persisting IBD signature in EH reflects ongoing cellular, molecular and microbial activity in comparison to non-IBD controls despite EH and mucosal regeneration. These markers may provide targets for future or sequential therapies.

Measurement of Forces Acting on Single T-Cell Receptors.

Molecular forces are increasingly recognized as an important parameter to understand cellular signaling processes. In the recent years, evidence accumulated that also T-cells exert tensile forces via their T-cell receptor during the antigen recognition process. To measure such intercellular pulling forces, one can make use of the elastic properties of spider silk peptides, which act similar to Hookean springs: increased strain corresponds to increased stress applied to the peptide. Combined with Förster resonance energy transfer (FRET) to read out the strain, such peptides represent powerful and versatile nanoscopic force sensing tools. In this paper, we provide a detailed protocol how to synthesize a molecular force sensor for application in T-cell antigen recognition and hands-on guidelines on experiments and analysis of obtained single molecule FRET data.

Single Cell Multi-Omic Dissection of Response and Resistance to Chimeric Antigen Receptor T Cells Against BCMA in Relapsed Multiple Myeloma

Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment of relapsed/refractory multiple myeloma (RRMM). Robust variables that predict long-term response are currently missing. Limited data are especially available on the impact of bridging therapies on manufacturing and outcome. We conducted a longitudinal single-cell multi-omics study to identify factors that predict response to BCMA-directed CAR T cells. Changes in the immune microenvironment associated with response were analyzed as well as the impact of prior bridging therapy with bispecific antibodies on subsequent CAR T cell manufacturing and outcome. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 29 consecutive MM patients treated with commercially available anti-BCMA CAR T cells on the day of leukapheresis as well as days 30 and 100 after CAR T cell infusion. PBMCs were subjected to single cell RNA, T-cell receptor (TCR) and B-cell receptor (BCR) sequencing. A custom panel of 57 oligonucleotide-coupled antibodies was used to study surface proteomics. Downstream analyses were performed with Seurat. Differences in cellular compositions at all three time points were analyzed with scCODA. To analyze CAR T cell functionality, CAR T cells from peripheral blood were isolated 7 days after infusion and subjected to an in vitro cytotoxicity assay after expansion and stimulation. Patients were grouped based on their best response following CAR T cell infusion (CR: n=12, non CR: n=17). Results: In total, 375,338 cells were sequenced (median 7246 cells/sample, range 1,569-10,972 cells) and 354,878 cells (94.5%) passed quality assessment. Quantitative and qualitative differences in the cellular composition of peripheral blood between CR and non CR patients were detected at the time of leukapheresis as well as on days 30 and 100 following infusion. CR patients harbored significantly more CD8+ effector memory T cells (TEM) at leukapheresis and less NK cells on day 30 after therapy compared to non CR patients. Regulatory T cells isolated at the time of leukapheresis from non CR patients exhibited significantly higher surface protein levels of CXCR3, CD40, CD95 and KLRG1 (p<0.015, respectively) that have been associated with T cell senescence and impaired tumor immunity. No significant differences in cell numbers between CR and non CR were detected on day 100 after CAR T cell infusion. However, single cell TCR analysis revealed an increasing diversity in the TCR repertoire over time in patients with CR, while Shannon diversity decreased from leukapheresis over day 30 to day 100 in non CR patients (p=0.004). The prior administration of the bispecific antibody teclistamab had no significant impact on the quantitative cellular composition at the time of leukapheresis. However, termination of manufacturing in the first attempt occurred in all patients with a close proximity of teclistimab administration and apheresis. Differential gene expression analysis showed that the application of teclistamab was associated with impaired T cell activation and exhaustion indicated by upregulation of e.g. CTLA4, TIGIT, LAG3 and GZMK. After discontinuation of teclistamab (median 4 weeks) and successful manufacturing of CAR T cells, we found no significant differences for in vitro cytotoxicity and in vivo expansion of CAR T cells. CAR T cells isolated at day 7 post-infusion from patients in CR, non CR or with prior teclistamab exposure, effectively eliminated MM cells (U-266). Tracking of single CAR T cells over time showed that the majority of CAR+ cells were CD8+ TEMs regardless of remission achievement or prior teclistamab exposure. Conclusion: We demonstrate that differences between MM patients achieving a CR and patients with suboptimal response upon anti-BCMA CAR T cell therapy can already be identified at the time of leukapheresis. Long-term changes associated with CR include a diversification of the TCR repertoire. Successful CAR T cell manufacturing is hampered by exposure to bispecific antibodies but can be successfully achieved by allowing for a wash-out phase of ca. 4 weeks.

Abstract 16577: Unraveling T-cell and Macrophage Chemokine Crosstalk in Immunotherapy-Induced Myocarditis

Introduction: Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that activate immunity against tumor cells but can cause immune related adverse events such as myocarditis, a rare but serious side effect with up to 40% mortality. Hypothesis: Macrophages contribute to recruitment of cytotoxic T-cell subsets to the heart in the setting of ICI, causing myocarditis. Methods: To study the role of macrophage depletion on ICI myocarditis, we treated MRL/Pdcd1-/- mice with liposomal clodronate. To identify timing of immune infiltration, we performed serial single cell T-cell receptor and RNA sequencing of CD45+ cells from the hearts/blood of MRL/Pdcd1-/- myocarditis mice at 1, 2, and 4 weeks of age. We treated MRL mice with anti-PD1/CTLA4 to induce myocarditis and assessed if anti-CXCR3 could decrease effector CD8+ T-cell cardiac infiltration and mitigate myocarditis. We studied the effects of in vitro CXCR3 blockade on CXCR3+ T-cell migration in a transwell assay. Results: Serial single-cell TCR sequencing/scRNA-seq of immune cells from MRL/ Pdcd1-/- vs. MRL control hearts showed infiltration of CXCL9/10+ macrophages ( Fig. 1, A ) along with progressive clonal expansion of CXCR3+ effector CD8+ T-cells in the heart starting at 2 weeks of age, prior to clinical onset of myocarditis. Macrophage depletion with liposomal clodronate reduced myocarditis and improved survival in MRL/ Pdcd1-/- mice ( Fig. 1, B-C) . CXCR3 blockade with anti-CXCR3 antibody reduced myocarditis, decreased CXCR3+ CD8+ T-cell infiltration, and improved survival in mice treated with anti-CTLA4/anti-PD1 antibodies (Fig. 1, D-E) . CXCR3 blockade in transwell assay also reduced migration of CD8+CXCR3+ T-cells towards the macrophage compartment (Fig. 1, F) . Conclusions: CXCL9/10+ macrophages likely play a critical role in development of ICI myocarditis through the recruitment of CXCR3+ effector T-cells into the heart, making this pathway an attractive therapeutic target.

New Tocilizumab (TCZ) Dosing Guidance for T-Cell Engaging Bispecific Antibody-Related Cytokine Release Syndrome (CRS) in Patients (pts) with Relapsed/Refractory (R/R) B-Cell Non-Hodgkin Lymphoma (B-NHL): Insights from Pooled Clinical Trial Safety Experience and Quantitative Clinical Pharmacology (qCP) Analyses

Background: Mosunetuzumab (Mosun), approved for treatment of pts with R/R follicular lymphoma (FL) and ≥2 prior therapies, and glofitamab (Glofit), approved for treatment of pts with R/R diffuse large B-cell lymphoma, or large B-cell lymphoma arising from FL, and ≥2 prior therapies, are CD20xCD3 T-cell engaging bispecific antibodies that redirect T cells to eliminate B cells. CRS is a common on-target adverse event observed with bispecific antibodies and associated with exposure. TCZ, a humanized anti-interleukin-6 (IL-6) receptor, is often used for CRS management. TCZ dosing recommendations derived from CAR T-cell studies (Le et al. Oncologist 2018) may not be optimal for bispecific antibody-treated pts. Using TCZ clinical usage patterns in Mosun- or Glofit-treated pts with R/R B-NHL, qCP, and cytokine analyses, we explored a new TCZ dosing regimen for CRS management (for pts >30 kg body weight). Methods: Pooled safety data from seven Phase I/II studies evaluating Mosun or Glofit in pts with R/R B-NHL were analyzed. CRS was graded using ASTCT criteria (Lee et al. Biol Blood Marrow Transplant 2019). Modeling was used to predict TCZ concentrations (conc) and soluble IL-6 receptor (s-IL6R) occupancy over time, following administration of different TCZ dosing regimens in a subset of pts. Downstream cytokine pharmacodynamic responses (IL-6 and C-reactive protein [CRP]) were evaluated graphically post-TCZ dosing. Results: Data from 733 Mosun- and 772 Glofit-treated pts across different Mosun/Glofit dose levels were analyzed; of these, 232 (31.7%) and 427 (55.3%) pts, respectively, had ≥1 CRS event, and 36/232 (15.5%) and 140/427 (32.8%) pts received TCZ for CRS management. CRS events were mainly Grade 1/2 and occurred during the first treatment cycle. Most pts who received TCZ for CRS received ≤2 doses of TCZ overall (Mosun: 94.4%; Glofit: 84.2%) ( Table), and 1 dose of TCZ for the management of a single CRS event (Mosun: 91.7%; Glofit: 84.9%). CRS resolution was achieved within 3 days of TCZ administration in 75.0% and 66.9% of Mosun- and Glofit-treated pts, respectively. By then, a reduction in IL-6 and CRP levels was also observed. In a prior pharmacokinetic (PK) analysis of TCZ in pts with CAR T-cell-associated CRS, predicted TCZ conc after administration of 4 intravenous (IV) TCZ doses of 8 mg/kg every 8 hours (q8h) was generally below 649 μg/mL, the observed maximum conc in healthy volunteers and “safety threshold” used by the FDA (Le et al. Oncologist 2018). qCP analyses of TCZ PK and s-IL6R data from 67 pts who received TCZ for CRS management following treatment with Mosun or Glofit indicated that this TCZ regimen may exceed the safety threshold, in part because maximum TCZ conc is one-third lower in pts with CAR T-cell-induced CRS vs bispecific antibody-induced CRS. In addition, typical bispecific antibody regimens involve multiple doses compared with a single CAR T-cell infusion, with the potential for multiple CRS events requiring TCZ treatment; therefore, TCZ accumulation must be considered. qCP simulations predicted that for 1 x 8 mg/kg dose, median duration of >90% s-IL6R saturation was 28 (range: 14-28) days ( Table). Furthermore, simulations indicated that ≤2 consecutive TCZ doses q8h per CRS event and a maximum of 3 TCZ doses within a 6-week time period maintain the TCZ conc below the FDA safety threshold, while ensuring sustained s-IL6R occupancy ( Table). Conclusions: qCP results and clinical experience in Mosun and Glofit studies support a TCZ dosing regimen of ≤2 IV doses of 8 mg/kg per CRS event administered q8h, not exceeding a total of 3 doses within a 6-week period. Further improvement in CRS management and symptom resolution with additional TCZ doses is unlikely given the predicted duration of s-IL6R occupancy and observed rapid decline in systemic inflammatory markers post dosing. This proposed TCZ dosing regimen reflects TCZ usage patterns in clinical trials evaluating Mosun and Glofit, and is included in their Summaries of Product Characteristics. In patients with R/R B-NHL receiving bispecific antibody treatment, TCZ is generally suitable for management of acute CRS, and balances risk-benefit for potential scenarios involving multiple TCZ administrations for pts with CRS recurrence. This recommendation considers TCZ safety factors, the unique PK properties of TCZ in this setting and the expected duration of activity based on pharmacological modeling of target binding (s-IL6R occupancy).

Differentiation of BCMA-specific induced pluripotent stem cells into rejuvenated CD8αβ+ T cells targeting multiple myeloma

AbstractA major hurdle in adoptive T-cell therapy is cell exhaustion and failure to maintain antitumor responses. Here, we introduce an induced pluripotent stem cell (iPSC) strategy for reprogramming and revitalizing precursor exhausted B-cell maturation antigen (BCMA)-specific T cells to effectively target multiple myeloma (MM). Heteroclitic BCMA72-80 (YLMFLLRKI)–specific CD8+ memory cytotoxic T lymphocytes (CTL) were epigenetically reprogrammed to a pluripotent state, developed into hematopoietic progenitor cells (CD34+ CD43+/CD14− CD235a−), differentiated into the T-cell lineage and evaluated for their polyfunctional activities against MM. The final T-cell products demonstrated (1) mature CD8αβ+ memory phenotype, (2) high expression of activation or costimulatory molecules (CD38, CD28, and 41BB), (3) no expression of immune checkpoint and senescence markers (CTLA4, PD1, LAG3, and TIM3; CD57), and (4) robust proliferation and polyfunctional immune responses to MM. The BCMA-specific iPSC–T cells possessed a single T-cell receptor clonotype with cognate BCMA peptide recognition and specificity for targeting MM. RNA sequencing analyses revealed distinct genome-wide shifts and a distinctive transcriptional profile in selected iPSC clones, which can develop CD8αβ+ memory T cells. This includes a repertoire of gene regulators promoting T-cell lineage development, memory CTL activation, and immune response regulation (LCK, IL7R, 4-1BB, TRAIL, GZMB, FOXF1, and ITGA1). This study highlights the potential application of iPSC technology to an adaptive T-cell therapy protocol and identifies specific transcriptional patterns that could serve as a biomarker for selection of suitable iPSC clones for the successful development of antigen-specific CD8αβ+ memory T cells to improve the outcome in patients with MM.

CAMEL NANOBODY-BASED B7-H3 CAR-T CELLS WITH HIGH EFFICACY AGAINST SOLID TUMORS

Abstract Background and significance Chimeric antigen receptor (CAR)-T cell therapy shows promising potency for treating patients with hematological malignancies. However, follow-up data indicate that only 30% to 50% of these patients experience long-term disease control. In solid tumors, the B7-H3 transmembrane protein is an emerging target that harbors in its ectodomain two distinct epitope motifs - IgC and IgV. Here, we developed nanobody-based CAR-T cell strategy targeting B7-H3 and investigated its anti-tumor efficacy in xenograft mouse models. Methods We isolated anti-B7-H3 VHHs from our large dromedary camel VHH nanobody libraries with great diversity (> 1012 total) by phage display technology. The binding of isolated VHHs was validated by ELISA, flow cytometry, and Octet. A B7-H3 peptide library was synthesized to predict the epitope of select VHHs. Anti-tumor effect of B7-H3 CAR-T cells was determined via cell luciferase-based cell killing assay as well as xenograft mouse models. Two tumor models, human neuroblastoma and pancreatic adenocarcinoma, were used in the present study. Single-cell transcriptome RNA sequencing coupled with single T-cell functional proteomics analysis was used to analyze the functionality of nanobody-based B7-H3 CAR-T cells. Results We analyzed the isoforms of B7-H3 at the RNA and protein levels and validated that only 4IgB7-H3 is a therapeutic target as the dominant isoform in tumors. Targeting 4Ig isoform, we obtained a panel of high-affinity nanobodies cross-reactive to human, mouse, rat, and monkey. Furthermore, we demonstrated that CAR-T cells based on the nanobodies had potent antitumor activity against tumors with rigorous T cell signaling and significant tumor infiltration. Mechanistically, we uncovered the top-upregulated genes that might be critical for the persistence of polyfunctional CAR-T cells in the tumor microenvironment. Conclusions Our results provide a novel nanobody-based B7-H3 CAR-T product for use in solid tumor therapy.

Mpox vaccine and infection-driven human immune signatures: an immunological analysis of an observational study

Monkeypox virus has recently infected more than 88 000 people, raising concerns about our preparedness against this emerging viral pathogen. Licensed and approved for mpox, the JYNNEOS vaccine has fewer side-effects than previous smallpox vaccines and has shown immunogenicity against monkeypox in animal models. This study aims to elucidate human immune responses to JYNNEOS vaccination compared with mpox-induced immunity. Peripheral blood mononuclear cells and sera were obtained from ten individuals vaccinated with one or two doses of JYNNEOS and six individuals diagnosed with monkeypox virus infection. Samples were obtained from seven individuals before vaccination to serve as a baseline. We examined the polyclonal serum (ELISA) and single B-cell (heavy chain gene and transcriptome data) antibody repertoires and T-cell responses (activation-induced marker and intracellular cytokine staining assays) induced by the JYNNEOS vaccine versus monkeypox virus infection. All participants were men between the ages of 21 and 60 years, except for one woman in the group of mpox-convalescent individuals, and none had previous orthopoxvirus exposure. All mpox cases were mild. Vaccinee samples were collected 6-33 days after the first dose and 5-40 days after the second dose. Mpox-convalescent samples were collected 20-102 days after infection. In vaccine recipients, gene-level plasmablast and antibody responses were negligible and sera displayed moderate binding to recombinant orthopoxviral proteins (A29L, A35R, E8L, A30L, A27L, A33R, B18R, and L1R) and native proteins from the 2022 monkeypox outbreak strain. By contrast, recent monkeypox virus infection (within 20-102 days) induced robust serum antibody responses to monkeypox virus proteins and to native monkeypox virus proteins from a viral isolate obtained during the 2022 outbreak. JYNNEOS vaccine recipients presented robust orthopoxviral CD4+ and CD8+ T-cell responses. Infection with monkeypox virus resulted in robust B-cell and T-cell responses, whereas immunisation with JYNNEOS elicited more robust T-cell responses. These data can help to inform vaccine design and policies for preventing mpox in humans. National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), and Icahn School of Medicine.

Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study

BackgroundLocally advanced/recurrent head and neck squamous cell carcinoma (HNSCC) is associated with significant morbidity and mortality. To target upregulated ErbB dimer expression in this cancer, we developed an autologous CD28-based...

Abstract 6421: Molecular profiles and single cell analysis identify immunogenic pancreatic ductal adenocarcinoma (iPDAC)

Abstract Most pancreatic ductal adenocarcinomas (PDAC) are lethal and resistant to immunotherapy. Thus, identifying the immunogenic subgroup (iPDAC) and therapeutic targets can save lives. Herein, we present molecular features of iPDAC. 3 cohorts (A, B, C) from 288 patients whose sequenced tumors (MSK-IMPACT) were classified by homologous recombination deficiency groups. MSI-H were excluded. Survival, tumor mutation burden, genomic instability score, and enriched pathways for each cohort are included in Table 1. Patients in A (BRCA1/2/PALB2) had longer survivals vs B/C. 61 samples were selected for bulk RNAseq analysis for A vs C. Gene Ontology was enriched for upregulated humoral, T cell, and neutrophil immunity. CIBERSORT suggested higher infiltration of gamma delta T (Tgd) cells (p=0.039) and neutrophils (p=0.012), but lower Treg (p=0.001). Multidimensional insights in cellular components of cancer, immune, stroma, and neural genes were obtained by single nuclear RNA (snRNAseq) analysis from 30 biopsies for A vs C. 10x Genomics Chromium platform for library and Scanpy for computational analysis after Cell Ranger pipelines were used. 61,868 nuclei were profiled from 18 (13 baseline and 5 matched longitudinal) samples after quality evaluation. UMAP accurately clustered cells from each patient. Long-term survivors (LTS) had heterogenous baseline immune cell infiltrates of plasma cells, neutrophils, and CD8 (+) cytotoxic T cells. In matched samples of LTS, evolution of more prominent CD8 (+) T cells, macrophage, plasma cell, and neutrophil were observed. Single nucleus T-Cell Receptor sequencing for clonal trajectory inference will be done to determine the associated single cell molecular features contributing to iPDAC and identify novel targets for future intervention. Table 1. Cohort (Total: N=288) A: core HRD (BRCA1/2/PALB2) B: non-core HRD (ATM, BARD1, BLM, CHEK2, RAD50, RAD51C, RTEL1, MUTYH) C: others without HR-gene alterations Number (%) 48 (16.6) 19 (6.5) 221 (76) Median overall survival (95% confidence Interval) 33 months (3.6-64) 16 (11- not reached) 16 (14-18) Tumor Mutation Burden (TMB) 4.4 3.5 3.9 Genomic Instability Score (GIS, HRD score) 26 12 13 Gene Ongology term, enrichment score, adjusted p-value Adaptive immune response, GO:0002250, 0.49, 1.69e-10 Not included Reference to cohort A Humoral immune response, GO:0006959, 0.58, 1.67e-9 T cell activation, GO:0042110, 0.44, 2.75e-8 Neutrophil chemotaxis, GO:0030593, 0.73, 4.3e-10 Citation Format: Wungki Park, Catherine O'Connor, Shigeaki Umeda, Roshan Sharma, Yingjie Zhu, Elias-Ramzey Karnoub, Anna Varghese, Kevin C. Soares, Alejandro Jimemez, Asli Yavas, Kenneth H. Yu, Balachandran P. Vinod, Joanne F. Chou, Danny N. Khalil, Kelsen David, Hulya Sahin Ozkan, Olca Basturk, Marinela Capanu, Tal Nawy, Michael F. Berger, Ghassan K. Abou-Alfa, Jorge S. Reis-Filho, Ronan Chaligne, Nadeem Riaz, Dana Pe'er, Christine Iacobuzio-Donahue, Eileen M. O'Reilly. Molecular profiles and single cell analysis identify immunogenic pancreatic ductal adenocarcinoma (iPDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6421.

Associations of granulocyte colony-stimulating factor with toxicities and efficacy of chimeric antigen receptor T-cell therapy in relapsed or refractory multiple myeloma

Background aimsFew studies have reported the associations of granulocyte colony-stimulating factor (G-CSF) with cytokine release syndrome (CRS), neurotoxic events (NEs) and efficacy after chimeric antigen receptor (CAR) T-cell therapy for relapsed or refractory (R/R) multiple myeloma (MM). We present a retrospective study performed on 113 patients with R/R MM who received single anti-BCMA CAR T-cell, combined with anti-CD19 CAR T-cell or anti-CD138 CAR T-cell therapy. MethodsEight patients were given G-CSF after successful management of CRS, and no CRS re-occurred thereafter. Of the remaining 105 patients that were finally analyzed, 72 (68.6%) received G-CSF (G-CSF group), and 33 (31.4%) did not (non G-CSF group). We mainly analyzed the incidence and severity of CRS or NEs in two groups of patients, as well as the associations of G-CSF timing, cumulative dose and cumulative time with CRS, NEs and efficacy of CAR T-cell therapy. ResultsBoth groups of patients had similar duration of grade 3–4 neutropenia, and the incidence and severity of CRS or NEs.There were also no differences in the incidence and severity of CRS or NEs between patients with the timing of G-CSF administration ≤3 days and those >3 days after CAR T-cell infusion. The incidence of CRS was greater in patients receiving cumulative doses of G-CSF >1500 μg or cumulative time of G-CSF administration >5 days. Among patients with CRS, there was no difference in the severity of CRS between patients who used G-CSF and those who did not. The duration of CRS in anti-BCMA and anti-CD19 CAR T-cell–treated patients was prolonged after G-CSF administration. There were no significant differences in the overall response rate at 1 and 3 months between the G-CSF group and the non–G-CSF group. ConclusionsOur results showed that low-dose or short-time use of G-CSF was not associated with the incidence or severity of CRS or NEs, and G-CSF administration did not influence the antitumor activity of CAR T-cell therapy.

Phenotypic plasticity of malignant T cells in blood and skin of a Sézary syndrome patient revealed by single cell transcriptomics.

Sézary Syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL). In SS patients, malignant T cells are circulating through the blood and cause erythroderma. To compare the transcriptome of single cells in blood and skin samples from a patient with advanced SS. We utilized combined single cell RNA and T-cell receptor (TCR) sequencing (scRNA-seq). We scrutinized the malignant T cells in blood and skin in an unbiased manner without pre-sorting of cells. We observed different phenotypes of the same monoclonal malignant T-cell population, confirmed by TCR sequencing and inferred copy number variation analysis. Malignant T cells present in the circulating blood expressed genes resembling central memory T cells such as CCR7, IL7R and CD27. In the skin, we detected two major malignant T-cell populations: One subpopulation was closely related to the malignant T cells from the blood, while the other subpopulation expressed genes reminiscent of skin resident effector memory T cells including GZMB and NKG7. Pseudotime analysis indicated crucial transcriptomic changes in the transition of malignant T cells between blood and skin. These changes included the differential regulation of TXNIP, a putative tumor suppressor in CTCL, and the adaptation to the hypoxic conditions in the skin. Tumor cell proliferation in the skin was supported by stimulating interactions between myeloid cells and malignant T cells. Using scRNA-seq we detected a high degree of functional heterogeneity within the malignant T-cell population in SS and highlighted crucial differences between SS cells in blood and skin.

Quantitative peripheral live single T-cell dynamic polyfunctionality profiling predicts lung cancer checkpoint immunotherapy treatment response and clinical outcomes.

Predictive biomarkers for immune checkpoint inhibitors (ICIs), e.g., programmed death ligand-1 (PD-L1) tumor proportional score (TPS), remain limited in clinical applications. Predictive biomarkers that require invasive tumor biopsy procedures are practically challenging especially when longitudinal follow-up is required. Clinical utility of tissue-based PD-L1 TPS also becomes diluted when ICI is combined with chemotherapies. Peripheral single T-cell dynamic polyfunctionality profiling offers the opportunity to reveal rare T-cell subpopulations that are polyfunctional and responsible for the underlying ICI treatment molecular response that bulk biological assays cannot achieve. Here, we evaluated a novel live single-cell functional liquid biopsy cytokine profiling platform, IsoLight, as a potential predictive biomarker to track ICI treatment response and clinical outcomes in non-small cell lung cancer (NSCLC). Peripheral blood mononuclear cell samples of 10 healthy donors and 10 NSCLC patients undergoing ICI-based therapies were collected longitudinally pre-/post-ICI treatment after ≥2 cycles under institutional review board (IRB)-approved protocols. Cancer blood samples were collected from unresectable advanced stage (III-IV) NSCLC patients. Clinical course and treatment response and survival outcomes were extracted from electronic health records, with treatment response assessed by treating oncologists based on RECIST. CD4+ and CD8+ T-cells were enriched magnetically and analyzed on the IsoLight platform. Single T-cells were captured in microchambers on IsoCode chips for proteomic immune cytokines profiling. Functional polyfunctionality data from 55,775 single cells were analyzed with IsoSpeak software, 2D- and 3D-t-distributed stochastic neighbor embedding (t-SNE) analysis, kappa coefficient, and Kaplan-Meier survival plots. P values ≤0.05 is considered statistically significant. Pre-treatment baseline polyfunctionality profiles could not differentiate NSCLC patients from healthy subjects, and could not differentiate ICI responders from non-responders. We found a statistically significant difference between responders and non-responders in CD8+ T-cells' changes in overall polyfunctionality (ΔPolyFx) (P=0.01) and polyfunctional strength index (ΔPSI) (P=0.006) in our dynamic pre-/post-treatment single cell measurements, both performing better than PD-L1 TPS alone (P=0.08). In the 3D-t-SNE analysis, subpopulations of post-treatment CD8+ T-cells in ICI responders displayed distinct immune cytokine profiles from those in pre-treatment cells. CD8+ T-cells ΔPolyFx and ΔPSI scores performed better than PD-L1 TPS in ICI response correlation. Moreover, combined PD-L1 strong TPS and ΔPSI >15 scores strongly correlated with early ICI response with a robust kappa coefficient of 1.0 (P=0.003), which was previously statistically established to indicate a perfect agreement between the prediction and actual response status. Interestingly, high CD4+ T-cells ΔPSI >5 was found to correlate with a strong trend of improved progression-free survival (3.9-fold) (10.8 vs. 2.8 months; P=0.07) and overall survival (3-fold) (34.5 vs. 11.5 months; P=0.09) in ICI-treated patients. Our study nominates single peripheral T-cell polyfunctionality dynamics analysis to be a promising liquid biopsy platform to determine potential ICI predictive biomarker in NSCLC. It warrants further studies in larger prospective cohorts to validate the clinical utilities and to further optimize cancer immunotherapy.

Single-Cell Profiling of CD8 Landscape in Treatment Naïve and Relapsed/Refractory AML Patients Reveals Distinct Effector Cellular States Predictive of Outcomes

Background: The success of allogeneic stem cell transplantation mediated by the graft versus leukemia effect of engrafted T-cells strongly supports the premise to investigate T cell biology in acute myeloid leukemia (AML). Of particular interest are CD8 cells, which are key players in eliciting anti-cancer cytotoxic activities but are understudied in AML. In this study, we conducted single cell RNA profiling in bone marrows from healthy donors, treatment naïve (RxNaive) and relapsed/refractory (R/R) AML patients to investigate the CD8 landscape in AML. Methods: We conducted paired single cell gene expression (scRNA) and T-cell repertoire (scTCR) profiling of bone marrow mononuclear cells from 3 healthy donors and 23 RxNaive adult AML patients (median age 71.81 years; range 51-86 years) and leveraged our previously published data of 8 relapsed/refractory AML patients (median age 73 years; range 64-88 years). After doublet removal and quality assessment, a total of 51,219 T cells passed the filtering criteria, of which 23,320 (45.5%) were CD8 cells. A total of 17,782/23,320 (74.1%) CD8 cells had paired scTCR. We applied principal component analysis to identify sources of variability across CD8 cells, then used Harmony-based linear correction integrated with K-nearest neighbors for cluster identification. We then applied non-linear dimensionality reduction (UMAP) to project cells into 2 dimensions. Results: In total, 6 CD8 T cell subtypes were defined including naïve, memory, effector memory, cytotoxic and exhausted T cell subtypes. Panel 1 shows the UMAP projection of CD8 cells overlaid with neighborhoods demonstrating differential abundance of cells by patient type (healthy, RxNaive or R/R). Naïve, memory, MAIT and exhausted clusters were shared across healthy, RxNaive and R/R patients (Panel 1, left). Remarkably, the effector clusters (effector memory and cytotoxic) demonstrated distinct patterns of disease states. Specifically, the Top effector clusters (Panel 1, top right) were largely contributed by R/R AML patients. However, the Bottom effector clusters (Panel 1, bottom right) were enriched by cells from healthy and RxNaive. Only 2.5% of R/R effector cells were part of the bottom effector clusters. This indicated that naïve and memory cells of RxNaive and R/R AML patients are biologically similar, however, effector cells took diverging paths in RxNaive versus R/R AML patients. We next explored the biological programs that govern the Top clusters and thus associate with R/R state as compared to the Bottom clusters, associated with RxNaive AML patients. We conducted consensus non-negative matrix factorization coupled with gene expression profiling and pathway analysis. This revealed positive enrichment for IFNa and IFNg signaling and significant negative enrichment for TNFa and TGFb signaling in the Top clusters, suggesting a cytokine dependent remodeling of CD8 effector function in R/R cells. We next explored whether the top 25 differential genes (herein referred to as CD8 Dysfunctional Signature) in the Top clusters (enriched for R/R CD8 cells) can predict outcomes in two cohorts of newly diagnosed AML patients (Abbas et al 2021 and TCGA AML cohorts). Interestingly, the CD8 Dysfunctional Signature was indeed predictive of outcomes, with patients harboring higher CD8 Dysfunctional Signature scores having worse outcomes (p=0.0054, Panel 2). We next explored whether the TCR repertoires are also different in AML of RxNaive and R/R patients. Indeed, TCR profiling revealed lower clonotype diversity and significant clonotype expansion in R/R patients, with 34% of R/R cells with TCR data exhibiting hyperexpansion (clone size >100). However, these hyperexpanded clonotypes were largely derived from dysfunctional CD8 cells. Conclusion: We characterized the CD8 landscape in RxNaive and R/R AML patients and defined distinct states of effector CD8 cells predictive of outcomes. CD8+ T-cells in R/R patients are markedly different from healthy/RxNaive patients, and characterized by a dysfunctional state, skewed distribution, lower clonal diversity, and significant clonal hyperexpansion. This dysfunctional state may be due to the AML microenvironment actively shaping the T-cell repertoires or the chemotherapy received by the patient, which may narrow the repertoire by eliminating active T-cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Resolution Single Cell T-Cell Phenotyping of B-Cell Lineage Lymphomas Reveals Unique Immune Signatures and Potential Targetable Vulnerabilities

High-Resolution Single Cell T-Cell Phenotyping of B-Cell Lineage Lymphomas Reveals Unique Immune Signatures and Potential Targetable Vulnerabilities