T-cell Surface Research Articles

Evaluation of inflammatory biomarkers and their association with anti-SARS-CoV-2 antibody titers in healthcare workers vaccinated with BNT162B2.

Vaccine-induced immunity against COVID-19 generates antibody and lymphocyte responses. However, variability in antibody titers has been observed after vaccination, and the determinants of a better response should be studied. The main objective of this investigation was to analyze the inflammatory biomarker response induced in healthcare workers vaccinated with BNT162b2, and its association with anti-Spike (a SARS-CoV-2 antigen) antibodies measured throughout a 1-year follow-up. Anti-spike antibodies and 92 biomarkers were analyzed in serum, along with socio-demographic and clinical variables collected by interview or exploration. In our study, four biomarkers (ADA, IL-17C, CCL25 and CD8α) increased their expression after the first vaccine dose; and 8 others (uPA, IL-18R1, EN-RAGE, CASP-8, MCP-2, TNFβ, CD5 and CXCL10) decreased their expression. Age, body mass index (BMI), smoking, alcohol consumption, and prevalent diseases were associated with some of these biomarkers. Furthermore, higher baseline levels of T-cell surface glycoprotein CD6 and hepatocyte growth factor (HGF) were associated with lower mean antibody titers at follow-up, while levels of monocyte chemotactic protein 2 (MCP-2) had a positive association with antibody levels. Age and BMI were positively related to baseline levels of MCP-2 (β=0.02, 95%CI 0.00-0.04, p=0.036) and HGF (β=0.03, 95%CI 0.00-0.06, p=0.039), respectively. Our findings indicate that primary BNT162b2 vaccination had a positive effect on the levels of several biomarkers related to T cell function, and a negative one on some others related to cancer or inflammatory processes. In addition, a higher level of MCP-2 and lower levels of HGF and CD6 were found to be associated with higher anti-Spike antibody titer following vaccination.

Varnimcabtagene Autoleucel (IMN-003A): Pharmacokinetic Profile with Predominant Naïve and Central Memory Phenotype Demonstrates Sustained In Vivo Persistence and Durable Responses in a First-in-India Industry Phase-2 Study (IMAGINE)

Background: Varnimcabtagene autoleucel (IMN-003A) is an autologous CD19 directed CAR-T cell product with a 4-1BB co-stimulatory domain and a non-FMC63 murine single chain variable fragment (A3B1 binder), manufactured in India, tested in the IMAGINE study (CTRI/2022/03/041162), a phase-2 clinical trial for patients (pts) with relapsed and/or refractory B cell malignancies (RR BCM). A fractionated infusion of 1 x 10 6 CAR+ cells (IMN-003A)/kg for B-ALL cohort and 5 x 10 6 CAR+ cells (IMN-003A)/kg for B-NHL cohort was administered over 3 days as 10%, 30% and 60% fractions after Fludarabine-Cyclophosphamide lymphodepletion regimen (LR). Here we present var-cel pharmacokinetic data and correlation with efficacy. Methods: Varnimcabtagene autoleucel was manufactured using a cGMP compliant closed system (CliniMACS Prodigy ®). The manufacturing data were obtained and analyzed, including % CAR transduction in the final product (FP) by flow cytometry (FC). Peripheral blood (PB) samples were obtained after consent at screening and as per patient schedule after infusion. Persistence of IMN-003A was evaluated in PB after infusion by ddPCR. Safety and efficacy data were collected for analysis. Bone marrow (BM) MRD (B-ALL) and PET-CT (B-NHL) was assessed at screening, D+28 and D+90 for efficacy. Minimal residual disease (MRD) was performed by FC with 10 -4 sensitivity. Apheresis, FP, and PB/BM samples on D+28 were analyzed for T-cell subpopulations and surface markers by FC. Statistical analysis was calculated using appropriate tests. Results: Of 25 patients (pts) enrolled and apheresed, 24 pts received var-cel (1 withdrawal) with majority of infusions on Days 0, +3, +7. Median product doubling time and manufacturing time was 1.04d (range 0.74-2.12) and 14 days (range 10 - 27) with 100% success with T cell purity median 99.4% (range 96.3-99.9). The mean % transduction of var-cel was 33.12% (range 8.50 - 58.35) with median transgene copies / genome of 2.57 (range 0.76 - 4.16). The mean CD4/CD8 ratio at apheresis and final product was 0.47 and 2.58 respectively. There was reversal of CD4/CD8 ratio from apheresis to final product (CAR+ T cells) which was statistically significant (p<0.01). There was statistically significant increase (p<0.01) in the number of naïve (CCR7+ RA+) and central memory (CCR7+ RA-) T cell subsets between apheresis and final product (CAR+ T cells). There was predominance of naïve and central memory CAR+ T cell subsets in the FP (58.19%; Figure 1A). Median Apheresis to Infusion time was 25d (range 16 - 95). There was a statistically significant trend (p<0.01) towards reversal of CD4/CD8 ratio in the PB from D0 to D+90. Var-cel showed maximum PB expansion (T max) at D+10 (range 7 - 28 days) with median C max 125,242 CAR copies / µg gDNA (range 18256 - 413,968). Among pts with 28-day and 90-day and last follow-up, 79.2%, 29.2% and 20.8% respectively had evidence of var-cel in PB, with median persistence of 28 days (range 10 - NR). Post-infusion, peak interleukin-6 level was median 42.3 pg/mL (range 6.71 - 17,481). B cell aplasia was observed in all pts concurrent with CAR-T expansion (Figure 1B). Median loss of B cell aplasia was not reached (range 42 - NR), with no apparent association with response at D+28 and D+90. Hypogammaglobulinemia was noted in 11 pts and 16 pts received IVIg. Of evaluable patients with CD4+ T-cell counts below 200/μL, recovery (>200/μL) was seen at median of 12 days (range 2 - NR) after var-cel infusion. Overall response rate (ORR) was 91.7% (22/24) at D+28 (B-ALL 91.7%, MRD neg 83.3% (n=10/12); B-NHL 91.7%, CR 66.7%) and 80.9% (17/21) at D+90 [B-ALL 80%, MRD neg 80% (n=8/10); B-NHL 81.8% (9/11), CR 63.6%]. One B-NHL pt in PR at D+28 achieved CR at D+90. Three pts relapsed by D+90. Updated results will be presented at the meeting. Conclusion: Varnimcabtagene autoleucel manufacturing process results in a drug product characterized by highly pure T cells, with high proportion of naïve and central memory polyfunctional cells. In this industry-led first-in-India phase-2 study, var-cel resulted in a cytokine response with sustained in vivo proliferation and persistence resulting in robust and durable responses.

CLL Cell-Derived Exosomes Impair Immune Function and Normal Hematopoiesis in CLL

CLL is characterized by immune and hematopoietic abnormalities such as T-cell dysfunction, hypogammaglobulinemia, increased levels of inflammatory cytokines, aberrant monocyte polarity, anemia, neutropenia, and low-grade bone marrow fibrosis. As a result, CLL patients become susceptible to increased rates of infection and/or second neoplasms. Exosomes are nanovesicles that participate in bidirectional communication between neoplastic cells and their microenvironment. Using electron microscopy, we found that CLL cells produce and release CD19 +/CD5 +exosomes, and in agreement with other investigators, we detected high levels of CLL cell-derived exosomes in the plasma of patients with CLL. By using bead-assisted flow cytometry, we found that at least 50% of circulating extracellular vesicles are CLL cell-derived exosomes, identified as CD63 +/CD41a - particles co-expressing CD19 +/CD5 +, whereas only a small proportion of CLL patients' plasma-derived exosomes expressed T-cell or endothelial cell surface markers. Furthermore, we found a positive correlation between levels of CLL cell-derived exosomes in the plasma of patients with CLL and the patients' peripheral white blood cell counts, indicating that CLL cells are the primary source of circulating exosomes. To investigate the effects of CLL cell-derived exosomes, we conducted an analysis of CLL cells and CLL cell-derived exosomes from a cohort of 78 previously untreated patients. To identify the molecular profiles of CLL cell-derived exosomes, we analyzed their RNA transcripts using RNA-seq and qRT-PCR. Our data revealed that CLL cell-derived exosomes expressed significant levels of oncogenic micro-RNAs. Additionally, we identified 1052 differentially expressed genes, and enrichment analysis further unveiled the significant upregulation of gene clusters associated with p53, NF-κB, MAPK, mTOR, and interleukin signal transduction pathways, consistent with their neoplastic cell origin. The distinctive transcriptional profile of CLL cell-derived exosomes led us to explore their effects on normal cell populations. We labeled ultra-centrifuged CLL cell-derived exosomes with fluorescent markers and incubated them with various cell types in culture conditions conducive to their physiological activity. Using confocal microscopy and flow cytometry, we observed significant uptake of CLL cell-derived exosomes by normal monocytes, fibrocytes, B cells, T cells, and CLL cells. Furthermore, incubation with CLL exosomes resulted in notable alterations in normal CD14 + monocyte polarity and subset-specific shifts in the expression of inflammatory cytokines. Remarkably, CLL cell-derived exosomes induced overexpression of the T-cell makers CD5 and CD8 in normal CD19 + cells, suggesting that CLL exosomes may promote immune-evasive reprogramming of normal B cells. Moreover, when normal T cells were incubated with CLL cell-derived exosomes, CyTOF analysis using 46 markers revealed elevated levels of immune checkpoints, including PD1, CTLA4, and LAG3, in both CD4 + and CD8 + subsets, indicating that exosomes derived from CLL cells induce T-cell dysfunction. Given their profound effect on immune cells, we wondered whether CLL exosomes affect hematopoietic cells. We previously found that normal monocyte derived fibrocytes stimulate hematopoietic colony proliferation. In sharp contrast to control fibrocytes, fibrocytes preincubated with CLL exosomes and CLL cell-derived exosomes inhibited normal BFU-E and CFU-GM colony formation, suggesting that CLL exosomes might contribute to CLL patients' anemia or neutropenia. Wondering whether CLL cell-derived exosomes affect the viability of other cell types, we found that they induced a dose-dependent apoptosis of CD19 + B cells, CLL cells but not normal T cells. In summary, CLL cell-derived exosomes exert a profound effect on gene and cell surface protein expression in immune and tissue-remodeling cells, leading to functional modifications, alteration of inflammatory cytokine levels, and suppression of normal hematopoiesis. The impact of CLL exosomes may vary across different cell types; however, as the leukemic cell mass increases, exosome levels escalate, invariably affecting CLL cell environment. The identification of exosomes with distinct molecular signatures holds promising potential for novel therapeutic interventions in future treatment of CLL.

In Vivo Anti-BCMA CAR T-Cell Expansion Kinetics Correlate with Early IMWG Response in RRMM: A Single Institution Study with Comparative Analysis of Idecabtagene Vicleucel and Ciltacabtagene Autoleucel

Introduction: The most recent breakthrough in the treatment of relapsed/refractory MM (RRMM) is chimeric antigen receptor (CAR) T-cell (CAR T) therapy which led to high overall response rates with acceptable toxicity. Here we present our single-institution experience with MM-specific CAR T performing a comparative analysis of clinical data and expansion kinetics after infusion of two commercially available myeloma CAR-T products. Methods: Out of 76 RRMM patients treated with CAR T, 23 patients provided consent, enrolled, apheresed and treated with commercial CAR-T between 6/20/2022 and 4/24/2023. Majority of patients had relapsed and multi-refractory MM. 11 patients received idecabtagene (idecel) and 12 received ciltacabtagene (ciltacel). Manufacturing slot availability was the primary deterministic factor for CAR T-type assignment. Study was IRB approved and informed consent was obtained to collect blood and clinical data. Blood samples were obtained at apheresis, pre-lymphodepleting (LD) chemotherapy, and on days 0, +7, +14, +21, +28 and +3 months post CAR T-cell infusion. PBMCs were generated from whole blood using density gradient centrifugation and were stained using BCMA or control CAR detection reagents and monoclonal antibodies against T-cell surface antigens. Samples were analyzed using MacsQuant-10 flow cytometer, gating on CD3+ and CAR+ cells. Fisher's Exact Test and Pearson Chi-Square tests were used for assessment of categorical variables whereas Independent-Samples Median Test was employed for non-categorical variables. Mann-Whitney U test was used for CAR T cell expansion analyses at individual timepoints. Results: As of July 10, 2023, 23 commercial patients were enrolled. Baseline clinical characteristics were comparable between the idecel and ciltacel cohorts with regard to age (median 66, p=0.827), prior lines of treatment, ASCT history (Table 1). Two patients in each cohort were penta-refractory. Three patients in each cohort had EMD (27.3%). There was balanced distribution of del 17p and t(4;14) across cohorts (p=1.000, p=0.175). Each cohort had 1 RISS-3 patient (p=0.476). Seven patients (30.4%) were previously treated with belantamab. Pre-apheresis and pre-LD absolute lymphocyte count (ALC) were comparable across cohorts. (Table 1). There were no G3 CRS events whereas G1/2 CRS events were not statistically significant between cohorts (p=0.256). Three G3 ICANS were observed, 2 in idecel and 1 in ciltacel cohorts (18.2% and 8.3%). Distribution of all-grade ICANS across cohorts were not statistically significant (p=0.676). Post-CAR T ferritin max correlated with the baseline b2m (p<0.001) and tocilizumab dose number (p=0.009) but none correlated with peak CAR T expansion (p=0.315). Responses were assessed by IMWG criteria at months 1,3,6,9 for efficacy-evaluable patients at that timepoint (n=19,14,8 and 2 in grand cohort (82.6%, 60.9%, 34.8% and 8.7%)). Global cohort ORR was 78.3% (n=18) and 60.9% (n=14) at days 30 and 90 respectively. +1 month IMWG responses were comparable with 72.7% (8/11) in idecel and 83.3% (10/12) in ciltacel cohort (p=0.625). Similarly, there was no statistically significant ORR difference between cohorts at 3, 6, and 9 months. Two-way ANOVA revealed a trend towards an effect of treatment type on CAR-T expansion (p=0.0595) (Figure 1A). Time-to-peak analysis reached statistical significance (p=0.0127) between two products (Figure 1B). Despite its delayed peak, ciltacel had significantly higher magnitude of expansion (p=0.043) (Figure 1C). Peak CAR-T expansion was associated with clinical responses across cohorts at +1 (p=0.034) and +3 months (p=0.050) but not at +6 months (p=0.449). Day +30 responses were indicative of Day +90 and Day +180 responses (p<0.001, p=0.011). Conclusions: In our single-center study of comparative analysis of commercial BCMA targeting CAR T-cell treatments, peak CAR T-cell expansion showed a positive correlation with +1 and +3 month post-CAR-T clinical responses. Despite the delayed peak expansion with ciltacel, the expansion was more robust with a higher level of circulating CAR T-cells. Baseline ALC or fludarabine dose reduction did not have an impact on overall CAR T expansion in combined cohort. Studies are underway at our institution investigating anti-BCMA CAR T cell responses in more detail and larger numbers of patients and functional studies will be further presented.

99mTc-Labeled Cyclic Peptide Targeting PD-L1 as a Novel Nuclear Imaging Probe.

Recent cancer therapies have focused on reducing immune suppression in the tumor microenvironment to prevent cancer progression and metastasis. PD-1 is a checkpoint protein that stops the immune response and is expressed on immune T cells. Cancer cells express a PD-1 ligand (PD-L1) to bind to the T-cell surface and activate immunosuppressive pathways. This study aimed to design, synthesize, and evaluate a 99mTc-labeled PD-L1-targeting cyclic peptide inhibitor (99mTc-iPD-L1) as a novel SPECT radiopharmaceutical for PD-L1 expression imaging. AutoDock software (version 1.5) was used to perform molecular docking for affinity calculations. The chemical synthesis was based on the coupling reaction of 6-hydrazinylpyridine-3-carboxylic acid with a 14-amino-acid cyclic peptide. iPD-L1 was prepared for 99mTc labeling. Radio-HPLC was used to verify radiochemical purity. The stability of the radiopeptide in human serum was evaluated by HPLC. iPD-L1 specificity was assessed by SDS-PAGE. [99mTc]Tc-iPD-L1 cellular uptake in PD-L1-positive cancer cells (HCC827 and HCT116) and biodistribution in mice with induced tumors were also performed. One patient with advanced plantar malignant melanoma received [99mTc]Tc-iPD-L1. The iPD-L1 ligand (AutoDock affinity: -6.7 kcal/mol), characterized by UPLC mass, FT-IR, and UV-Vis spectroscopy, was obtained with a chemical purity of 97%. The [99mTc]Tc-iPD-L1 was prepared with a radiochemical purity of >90%. In vitro and in vivo analyses demonstrated [99mTc]Tc-iPD-L1 stability (>90% at 24 h) in human serum, specific recognition for PD-L1, high uptake by the tumor (6.98 ± 0.89% ID/g at 1 h), and rapid hepatobiliary and kidney elimination. [99mTc]Tc-iPD-L1 successfully detected PD-L1-positive lesions in a patient with plantar malignant melanoma. The results obtained in this study warrant further dosimetric and clinical studies to determine the sensitivity and specificity of [99mTc]Tc-iPD-L1/SPECT for PD-L1 expression imaging.

Cellaca® PLX image cytometer as an alternative for immunophenotyping, GFP/RFP transfection efficiencies, and apoptosis analysis

Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.

Nivolumab in Lymphomatoid Granulomatosis (LYG) and Other Epstein-Barr Virus (EBV)-Positive Lymphoproliferative Disorders (LPDs) and Non-Hodgkin Lymphomas (NHLs) with Systemic and/or CNS Involvement: Preliminary Analysis of Efficacy and Safety from an Ongoing Phase II Study

Background: Programmed cell death protein-1 (PD-1) is a signaling molecule on the surface of T-cells that suppresses the cytotoxic effects of T-cells on tumor cells. We previously demonstrated increased PD-1 expression on CD8+ T-cells compared to healthy controls in pts with LYG, which could be reversed with immunotherapy using interferon alpha-2b (Melani et al. Lancet Haematol. 2023). Additionally, elevated expression of programmed death-ligand 1 (PD-L1) has been observed in other EBV-LPDs and NHLs as well as in chronic viral infections, such as EBV. Given this likely shared disease pathobiology, we hypothesized that treatment with the anti-PD-1 antibody, nivolumab, may reverse T-cell exhaustion and result in anti-tumor responses in pts with LYG and other EBV-LPDs and NHLs, including those with systemic and/or CNS involvement. Methods: Relapsed/refractory (R/R) EBV-LPD and NHL pts age ≥ 12y with adequate organ function were eligible. Untreated pts were eligible if EBV-LPD. Exclusions included prior use of PD-1/PD-L1/PD-L2/CD137/CTLA-4 antibodies, solid organ transplant, and HIV infection. Pts with a known immunodeficiency or autoimmune illness were eligible if not requiring steroids or immunosuppression within 14d of study. CNS involvement was permitted if no seizure activity within 4w of study. Nivolumab 480 mg IV was given q4w for up to 2y. Pts who achieved CR discontinued nivolumab after 1y of treatment. Responding patients with relapse or progression within 1y of discontinuing treatment were eligible for retreatment. Baseline evaluation included CT, PET, MRI brain, flow cytometry of peripheral blood and CSF, BM biopsy, and optional tumor biopsy. Restaging CT scans were performed after cycles 3, 6, 13, and 19, and end of treatment (EoT). PET was performed after cycles 1, 3, and EoT. Surveillance CT was performed q3m for 1y, q6m for yrs 2-5, and annually thereafter. Results: 11 pts were enrolled and treated; 6 (55%) with EBV-LPD and 5 (45%) with EBV-NHL (all DLBCL). EBV-LPD subtypes included 4 pts with G1-2 LYG and 1 pt each with EBV B-LPD of the CNS and CAEBV. Median age was 48y (range 20-67), and all pts had stage III/IV disease with extranodal involvement of the lung most common in 64% (7/11) and CNS disease in 18% (2/11). Median baseline absolute CD8 count (cells/mcL) was 86 (range 9-1237; normal: 178-853), CD4 count (cells/mcL) was 255 (range 99-1304; normal: 359-1565), and EBV VL in plasma (Log10 IU/mL) was undetected (range undetected-6.78; normal: <2.3). Median prior therapies were 1 (range 0-6) with 3 previously untreated LPD pts and 50% of previously tx pts were refractory to last tx. In 10 pts evaluable for response(1 came off tx prior to restaging), ORR and CR rate were both 60% (6/10). CR rate was 67% (4/6) in EBV-LPD, including 75% (3/4) with LYG, and 50% (2/4) in EBV-NHL (Fig 1A). One (50%) of 2 EBV-LPD pts with CNS involvement achieved CR after 3C of therapy and remains in ongoing response. Median TTR was 3.0m with 50% (3/6) responses ongoing from 3-56m after first response (Fig 1B). With a median follow up of 32m, 2y PFS and OS was 33% and 72% overall. By cohort, median PFS was 36m in EBV-LPD and 1m in EBV-NHL (p=0.003), with a 2y OS of 100% in EBV-LPD and 40% in EBV-NHL (p=0.04). Three EBV-NHL pts died, all of disease progression. One EBV-NHL pt stopped therapy after 2C due to radiographic disease progression in the kidney, but later developed CR without further therapy and remains in remission 56m after stopping therapy consistent with initial pseudoprogression. Adverse events (AEs) were as expected for nivolumab therapy with the most common AEs (% pts) including maculopapular rash (27%), diarrhea (18%), hypothyroidism (18%), and fatigue (18%). One pt developed an immune-mediated G2 myositis and G3 hepatitis following 3C of therapy which resolved after drug discontinuation and corticosteroids. Conclusion: Nivolumab is well tolerated in pts with LYG and other EBV-LPDs and NHLs without unexpected toxicities. Preliminary activity, including durable CRs, is noted across all pt cohorts, including 1 EBV-LPD pt with CNS involvement. With small numbers, statistically longer PFS and OS was observed with nivolumab in LYG and EBV-LPD compared to EBV-NHL, indicating that monotherapy may be sufficient for LYG and other EBV-LPDs, but combination therapy is likely needed for pts with EBV-NHL. Study enrollment continues to better assess the activity and durability of nivolumab in LYG and other EBV-LPDs and NHLs.

Prophylactic Tocilizumab to Prevent Cytokine Release Syndrome (CRS) with Teclistamab Administration

Introduction: Teclistamab is a T-cell redirecting bispecific antibody that targets both CD3 on the T-cell surface and B-cell maturation antigen (BCMA) expressed on myeloma cells. Teclistamab gained accelerated approval for RRMM patients that had previously been treated with 4 prior lines of therapy including a PI, IMID and a CD38 monoclonal antibody. This approval was based on the results of the MajesTEC-1 study demonstrating an ORR of 63%. Cytokine release syndrome (CRS), a known complication of T-cell engaging therapies, occurred in 72% patients which required a REMS program to monitor the patients in hospital for 48 hours post both priming doses and the target dosing. Several approaches are in place to minimize the risk of CRS to allow for the administration of teclistamab safely as an outpatient. We have evaluated the role of administering tocilizumab prophylactically to mitigate the incidence and the severity of CRS. Methods: A total of 48 patients were admitted to the Emory University hospital from December 2022 until July 2023 as mandated by REMS program for teclistamab step-up dosing followed by the first full dose at least 48 hours apart (0.06, 0.3, and 1.5mg/kg) with premeds per institutional guidelines (Benadryl 50 PO, Tylenol 650 PO, and Dexamethasone 16mg PO 30 minutes prior to each dose). Upon evaluation of our first 15 patients, the median time to CRS from the administration of the first-priming dose was 48 hours. We subsequently administered tocilizumab 8mg/kg IV over an hour (max dose of 800 mg) prophylactically at 44 hours (4 hours prior to the second step-up dose level) for the next 33 patients. CRS was graded per the American Society for Transplantation and Cellular Therapy criteria and managed according to institutional guidelines. Results: The median age of the patients was 66 years (range, 44-82). All patients were IMID, PI and CD38 mab refractory. The rate of all grade CRS amongst the entire cohort was 21 of 48 patients (43.8%). CRS occurred in 10 of the 33 patients (30.3%) who received prophylactic tocilizumab, compared to 11 of 15 (73.3%) of patients that did not. The majority the events in the prophylactic tocilizumab group are grade 1 [8 (24.2%)]. 1 patient had grade 2 and another had grade 3 CRS (patient with an aggressive relapse at time of teclistamab administration, requiring 3 doses of tocilizumab every 8 hours). The median number of tocilizumab doses was 1 (range, 1-3). The median duration of CRS was 1 day (range, 1-3). 1 dose of steroids (outside of premeds) was administered in the 1 patient who experienced grade 3 CRS but in no additional patients treated with prophylactic tocilizumab. Concurrent immune effector cell neurotoxicity syndrome (ICANS) was also decreased with the incorporation of prophylactic tocilizumab (20% vs 6.1%, respectively). All ICANS in the prophylactic cohort were grade 1 and managed symptomatically. The median duration of ICANS was 1 day. The non-prophylactic cohort experienced 3 of 15 (20%) patients with readmissions, with 1 additional patient going to hospice. None of the patients in the prophylactic cohort were readmitted to the hospital within 14 days of discharge. Conclusion: Prophylactic tocilizumab prior to the second priming dose has decreased the incidence and severity of CRS in heavily refractory RRMM patients receiving teclistamab. These results are lower in absolute incidence and severity when compared to the MajesTEC-1 trial where CRS was seen in 72% of patients. Prophylactic tocilizumab prevented usage of steroids, prevented dose delays, prevented readmission to the hospital, and possibly reduced the occurrence of ICANS. These data make a case for early incorporation of prophylactic tocilizumab and support outpatient administration of teclistamab in most patients.

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced > 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.

Cutaneous adverse effects associated with LAG-3 inhibitor use in cancer treatment: A systematic review.

Immunotherapy has become a mainstay of treatment for many cancers. Multiple immune checkpoint inhibitors have been used to treat malignancies, including anti-programed death-1 (PD1) and anti-cytotoxic T-lymphocyte-associated protein (anti-CTLA4). However, a significant percentage of patients develop resistance to these immunotherapy drugs. Therefore, novel strategies were developed to target other aspects of the immune response. Lymphocyte activation gene-3 (LAG-3) is a cell-surface molecule found on natural killer cells and activated T-cells which negatively regulates T-cell proliferation and function. LAG-3 inhibitors interact with LAG-3 ligands on the surface of T-cells to block T-regulatory (Treg) cell activity, suppress cytokine secretion and restore dysfunctional effector T-cells which subsequently attack and destroy cancer cells. This review reports the dermatologic side effects associated with LAG-3 inhibitors used in the treatment of melanomas. Using PRISMA 2022 guidelines, a comprehensive literature review of PubMed, Google Scholar, Embase, Cochrane, and Web of Science databases was conducted. Three studies were identified that demonstrated that the use of LAG-3 inhibitors, whether as a single agent or in combination with other immune checkpoint inhibitors, resulted in stomatitis, pruritus, rash, dry skin, erythema, and vitiligo. Further research is warranted to assess the cutaneous adverse events observed with LAG-3 inhibitors in treating melanoma and to identify populations most vulnerable to such side effects.

Characterization of CD3+/CD20+ canine large-cell lymphoma.

Immunophenotyping of canine large-cell lymphoma (LCL) for B-cell and T-cell surface antigens is commonly performed to better predict the clinical outcome. Expression of surface antigen CD3 is associated with T-cell malignancies; surface antigen CD20 is expressed on B cells. However, a small subset of canine LCLs expresses both CD3 and CD20 (CD3+/CD20+); this form of lymphoma remains poorly defined at the molecular level. In a retrospective study, we aimed to better characterize immunophenotypic properties and antigen receptor clonality of CD3+/CD20+ LCL. We selected formalin-fixed, paraffin-embedded tissues from 10 cases of CD3+/CD20+ LCL and breed-matched controls of peripheral large T-cell lymphoma (PTCL) and diffuse large B-cell lymphoma (DLBCL). Using PCR for antigen receptor rearrangement (PARR), we identified monoclonal T-cell receptor gamma (TCRγ) rearrangements in all CD3+/CD20+ cases. Three of 10 cases had monoclonal rearrangements in the immunoglobulin heavy chain (IgH), supportive of cross-lineage rearrangement. There was no significant difference in the frequency of antigen receptor rearrangement between CD3+/CD20+ and PTCL cases. In comparison with DLBCL, CD3+/CD20+ LCL had TCRγ rearrangement more frequently and IgH rearrangement less frequently, respectively. Immunolabeling of the B-cell marker PAX5 occurred less frequently in all CD3+/CD20+ LCL cases compared to the DLBCL controls. Immunolabeling for BCL-2 was robust, regardless of immunophenotype. Nuclear Ki67 positivity was variable in CD3+/CD20+ cases, indicating a heterogeneity in proliferation. Overall, cases of canine CD3+/CD20+ LCL had properties similar to PTCL, suggesting a similar histogenesis of these 2 subsets.

Deletion of the inhibitory co-receptor CTLA-4 enhances and invigorates chimeric antigen receptor T cells

Chimeric antigen receptor (CAR) Tcell therapy targeting CD19 has achieved tremendous success treating Bcell malignancies; however, some patients fail to respond due to poor autologous Tcell fitness. To improve response rates, we investigated whether disruption of the co-inhibitory receptors CTLA4 or PD-1 could restore CART function. CRISPR-Cas9-mediated deletion of CTLA4 in preclinical models of leukemia and myeloma improved CAR Tcell proliferation and anti-tumor efficacy. Importantly, this effect was specific to CTLA4 and not seen upon deletion of CTLA4 and/or PDCD1 in CAR Tcells. Mechanistically, CTLA4 deficiency permitted unopposed CD28 signaling and maintenance of CAR expression on the Tcell surface under conditions of high antigen load. In clinical studies, deletion of CTLA4 rescued the function of Tcells from patients with leukemia that previously failed CAR Tcell treatment. Thus, selective deletion of CTLA4 reinvigorates dysfunctional chronic lymphocytic leukemia (CLL) patient Tcells, providing a strategy for increasing patient responses to CAR Tcell therapy.

Update on Immunotherapy Cardiotoxicity: Checkpoint Inhibitors, CAR T, and Beyond.

Immunotherapy is an innovative approach to cancer treatment that involves using the body's immune system to fight cancer. The landscape of immunotherapy is constantly evolving, as new therapies are developed and refined. Some of the most promising approaches in immunotherapy include immune checkpoint inhibitors (ICIs): these drugs target proteins on the surface of T-cells that inhibit their ability to attack cancer cells. By blocking these proteins, checkpoint inhibitors allow T-cells to recognize and destroy cancer cells more effectively. CAR T-cell therapy: this therapy involves genetically modifying a patient's own T-cells to recognize and attack cancer cells. CAR T-cell therapy exhibits favorable response in many patients with refractory hematological cancers with growing clinical trials in solid tumors. Immune system modulators: these drugs enhance the immune system's ability to fight cancer by stimulating the production of immune cells or inhibiting the activity of immune-suppressing cells. While immunotherapy has shown great promise in the treatment of cancer, it can also pose significant cardiac side effects. Some immunotherapy drugs like ICIs can cause myocarditis, which can lead to chest pain, shortness of breath, and heart failure. Other cardiac side effects of ICIs include arrhythmias, pericarditis, vasculitis, and accelerated atherosclerosis. It is important for patients receiving immunotherapy to be monitored closely for these side effects, as prompt treatment can help prevent serious complications. Patients should also report any symptoms to their healthcare providers right away, so that appropriate action can be taken. CAR T-cell therapy can also illicit an exaggerated immune response creating cytokine release syndrome (CRS) that may precipitate cardiovascular events: arrhythmias, myocardial infarction, and heart failure. Overall, while immune modulating therapy is a promising and expanding approach to cancer treatment, it is important to weigh the potential benefits against the risks and side effects, especially in patients with high risk for cardiovascular complications.

CD1a and bound lipids drive T-cell responses in human skin disease.

In addition to serving as the main physical barrier with the outside world, human skin is abundantly infiltrated with resident αβ T cells that respond differently to self, infectious, microbiome, and noxious stimuli. To study skin T cells during infection and inflammation, experimental biologists track T-cell surface phenotypes and effector functions, which are often interpreted with the untested assumption that MHC proteins and peptide antigens drive measured responses. However, a broader perspective is that CD1 proteins also activate human T cells, and in skin, Langerhans cells (LCs) are abundant antigen presenting cells that express extremely high levels of CD1a. The emergence of new experimental tools, including CD1a tetramers carrying endogenous lipids, now show that CD1a-reactive T cells comprise a large population of resident T cells in human skin. Here, we review studies showing that skin-derived αβ T cells directly recognize CD1a proteins, and certain bound lipids, such as contact dermatitis allergens, trigger T-cell responses. Other natural skin lipids inhibit CD1a-mediated T-cell responses, providing an entry point for the development of therapeutic lipids that block T-cell responses. Increasing evidence points to a distinct role of CD1a in type 2 and 22 T-cell responses, providing new insights into psoriasis, contact dermatitis, and other T-cell-mediated skin diseases.

Immune сheckpoint inhibitors in ovarian cancer

The review considers modern immunotherapy for ovarian cancer with immune checkpoint inhibitors that interfere with the ability of the tumor to activate control proteins on the surface of T-cells, preventing cancer from evading the immune response and allowing the immune system to generate an antitumor response. The presence of spontaneous tumor-specific T-cells in cancer patients is a factor in creating ways to overcome the blockade of the immune system's ability to inactivate tumor cells. The use of immunotherapy with monoclonal antibodies makes it possible to influence the checkpoints of immunity, leads to the activation of the immune response, blocking the interaction of the PD-1 protein with the corresponding PD-L1/PD-L2 ligands and the attack reaction on tumor cells. The antitumor effect is achieved by releasing effector T-cells, reducing the function, number and suppressor activity of intratumoral Tregs. For antitumor drug therapy, anti-PD-1 and anti-PD-L1 monoclonal antibodies are used. This therapy is accompanied by various adverse events that are associated with the ability of PD-1 to interact with CD80 and the second PD-L2 ligand. Encouraging results of anti-PD-1/PD-L therapy in ovarian cancer resistant to platinum-containing chemotherapy may become an additional option in the treatment against the progression of ovarian cancer.

Serum sPD-L1 levels are elevated in patients with viral diseases, bacterial sepsis or in patients with impaired renal function compared to healthy blood donors.

Immune checkpoints play an important role in maintaining the balance of the immune system and in the development of autoimmune diseases. A central checkpoint molecule is the programmed cell death protein 1 (PD-1, CD279) which is typically located on the surface of Tcells. Its primary ligand PD-L1 is expressed on antigen presenting cells and on cancer cells. Several variants of PD-L1 exist, among these soluble molecules (sPD-L1) present in serum atlow concentrations. sPD-L1 was found elevated in cancer andseveral other diseases. sPD-L1 in infectious diseases has received relatively little attention so far and is therefore subject of this study. sPD-L1 serum levels were determined in 170 patients with viral infections (influenza, varicella, measles, Dengue fever, SARS-CoV2) or bacterial sepsis by ELISA and compared to the levels obtained in 11 healthy controls. Patients with viral infections and bacterial sepsis generally show significantly higher sPD-L1 serum levels compared to healthy donors, except for varicella samples where results do not reach significance. sPD-L1 is increased in patients with impaired renal function compared to those with normal renal function, and sPD-L1 correlates significantly with serum creatinine. Among sepsis patients with normal renal function, sPD-L1 serum levels are significantly higher in Gram-negative sepsis compared to Gram-positive sepsis. In addition, in sepsis patients with impaired renal function, sPD-L1 correlates positively with ferritin and negatively with transferrin. sPD-L1 serum levels are significantly elevated in patients with sepsis, influenza, mesasles, Dengue fever or SARS-CoV2. Highest levels are detectable in patients with measles and Dengue fever. Also impaired renal function causes an increase in levels of sPD-L1. As a consequence, renal function has to be taken into account in the interpretation of sPD-L1 levels in patients.

Circulating proteins and peripheral artery disease risk: observational and Mendelian randomization analyses.

We conducted observational and Mendelian randomization (MR) analyses to explore the associations between blood proteins and risk of peripheral artery disease (PAD). The observational cohort analyses included data on 257 proteins estimated in fasting blood samples from 12 136 Swedish adults aged 55-94 years who were followed up for incident PAD via the Swedish Patient Register. Mendelian randomization analyses were undertaken using cis-genetic variants strongly associated with the proteins as instrumental variables and genetic association summary statistic data for PAD from the FinnGen study (11 924 cases and 288 638 controls) and the Million Veteran Program (31 307 cases and 211 753 controls). The observational analysis, including 86 individuals diagnosed with incident PAD during a median follow-up of 6.6-year, identified 13 proteins [trefoil factor two, matrix metalloproteinase-12 (MMP-12), growth differentiation factor 15, V-set and immunoglobulin domain-containing protein two, N-terminal prohormone brain natriuretic peptide, renin, natriuretic peptides B, phosphoprotein associated with glycosphingolipid-enriched microdomains one, C-C motif chemokine 15, P-selectin, urokinase plasminogen activator surface receptor, angiopoietin-2, and C-type lectin domain family five member A] associated with the risk of PAD after multiple testing correction. Mendelian randomization analysis found associations of T-cell surface glycoprotein CD4, MMP-12, secretoglobin family 3A member 2, and ADM with PAD risk. The observational and MR associations for T-cell surface glycoprotein CD4 and MMP-12 were in opposite directions. This study identified many circulating proteins in relation to the development of incident PAD. Future studies are needed to verify our findings and assess the predictive and therapeutic values of these proteins in PAD.

Machine learning approach to screen new diagnostic features of adamantinomatous craniopharyngioma and explore personalised treatment strategies.

Adamantinoma craniopharyngioma (ACP) is a non-malignant tumour of unknown pathogenesis that frequently occurs in children and has malignant potential. The main treatment options are currently surgical resection and radiotherapy. These treatments can lead to serious complications that greatly affect the overall survival and quality of life of patients. It is therefore important to use bioinformatics to explore the mechanisms of ACP development and progression and to identify new molecules. Sequencing data of ACP was downloaded from the comprehensive gene expression database for differentially expressed gene identification and visualized by Gene Ontology, Kyoto Gene, and gene set enrichment analyses (GSEAs). Weighted correlation network analysis was used to identify the genes most strongly associated with ACP. GSE94349 was used as the training set and five diagnostic markers were screened using machine learning algorithms to assess diagnostic accuracy using receiver operating characteristic (ROC) curves, while GSE68015 was used as the validation set for verification. Type I cytoskeletal 15 (KRT15), Follicular dendritic cell secreted peptide (FDCSP), Rho-related GTP-binding protein RhoC (RHOC), Modulates negatively TGFB1 signaling in keratinocytes (CD109), and type II cytoskeletal 6A (KRT6A) (area under their receiver operating characteristic curves is 1 for both the training and validation sets), Nomograms constructed using these five markers can predict progression of ACP patients. Whereas ACP tissues with activated T-cell surface glycoprotein CD4, Gamma delta T cells, eosinophils and regulatory T cells were expressed at higher levels than in normal tissues, which may contribute to the pathogenesis of ACP. According to the analysis of the CellMiner database (Tumor cell and drug related database tools), high CD109 levels showed significant drug sensitivity to Dexrazoxane, which has the potential to be a therapeutic agent for ACP. Our findings extend understandings of the molecular immune mechanisms of ACP and suggest possible biomarkers for the targeted and precise treatment of ACP.

Abstract 6346: Discovery of HX011, a novel OX40 mAb, for potential cancer immunotherapy

Abstract OX40, tumor necrosis factor receptor superfamily member 4, is an immune co-stimulatory receptor, together with its ligand OX40L, forming OX40-OX40L trimer-trimer complex between the surface of T-cells (OX40) and APCs/NKs (OX40L). OX40 expresses constitutively on Treg as well as on conventional Teff, particularly within TME. OX40/OX40L interactions directly triggers conventional T cell activation via NF-kB1 pathway and inhibits bcl-x and survivin to prevent apoptosis; and it also downregulates FoxP3/CTLA4, thus Treg function. OX40 agonistic antibodies have also been shown to activate tumor immunity and increase anti-tumor activity in preclinical models. Here we report that we identified/constructed a novel OX40-agonistic humanized IgG1 monoclonal antibody (mAb), HX011, with high affinity through hybridoma technology. HX011 was subsequently confirmed of the similar binding properties and functions as the original mouse mAb clone, 9C12: 1) binding to human OX40 (EC50 ~0.012 nM; 2) similar function binding in the reporter assay (EC50 ~6.6 nM); but 3) has little blockade of the OX40L binding to OX40. In another word, HX011 is similar to BMS-986178 for the first two assays, but contrastingly different for the third assay. Non-competing with OX40L may actually maximize T-cell activation, a potential advantage over a competing antibody. In vivo pharmacology modeling of HX011 in the humanized syngeneic CRC model, MC38, where syngeneic C57BL/6 mouse mice were knocked in with huOX40 (“HuGEMM-OX40”), demonstrated strong anti-tumor activity (TGI ~100% at 1 mg/kg, twice weekly). HX011 is being further investigated as a potential new immunotherapy for the treatment of cancers. Citation Format: Hang Ke, Faming Zhang, Jialin Li, Feiyu Peng, Cen Chen, Henry Qixiang Li. Discovery of HX011, a novel OX40 mAb, for potential cancer immunotherapy [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 6346.

Abstract 6560: Identification of distinct patterns in diffuse large B-cell lymphoma through alternative splicing analysis

Abstract Background Aberrant splicing in cancer cells contributes to cellular proliferation, escape from cell death, growth inhibition, invasion and metastasis, and immune escape. Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, but the effect of AS on its pathogenesis has not been fully elucidated, so it is essential to find out DLBCL-specific splicing patterns and their potential as treatment targets. Material and Method RNA-Seq dataset from DLBCL patients was retrieved from The Cancer Genome Atlas (TCGA) DLBCL (N=48) and Pan-cancer Analysis of Whole Genomes (PCAWG) DLBC (N=7) were used as the cancer group. In order to identify spliced genes that are significantly different from normal tissue, RNA-Seq data of normal lymphoid tissue produced in two studies of E-MTAB-513 (N=1) and E-MTAB-1733 (N=13) from the BioStudies were downloaded. Sequence reads were aligned to the GRCh38 reference genome using STAR aligner. Results 5,470 genes identified as differentially spliced compared to normal in both rMATS and MAJIQ tools were studied. After sorting the AS events of these genes in order of highest average PSI value, events with an average PSI value of 0.7 or more in the top 2/3 samples and events with an average PSI value of 0.3 or less in the bottom 2/3 samples are selected. 8 exons of 7 genes were involved in Exon skipping (ES) events, 6 exons of 5 genes were involved in mutually exclusive exon (MXE) events, and an exon of a gene was involved in alternative 5' splice site (A5SS) Among these genes, exons 6 and 7 in CD53 showed high PSI values between exons 4 and 9. The average PSI values for exons 6 and 7 of CD53 in DLBCL were 0.923855 and 0.89096, respectively, which were significantly higher than the Normal group (p<0.05). CD53 mediates IL-7R signaling and plays a role in the regulation of normal B-cell development. As a gene coding for cell surface protein, it is involved in various immune systems in T-cell and B-cell surfaces. In healthy tissue, transcripts that skip exon 5 and have a junction between exon 4 and exon 6 or 7 are not yet known and should be further studied. In addition, SLAMF8, which has an exon with a PSI value specific to DLBCL, is also involved lymphocyte activation and regulates B-cell receptor signaling. Conclusion These DLBCL-specific patterns of selective exon usage are expected to be potential targets for cancer immunotherapy. Citation Format: Sanyeowool An, Youngil Koh, Sung-Soo Yoon. Identification of distinct patterns in diffuse large B-cell lymphoma through alternative splicing analysis [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 6560.