Front Immunol Research Articles

α-Mannosylated HLA-II glycopeptide antigens dominate the immunopeptidome of immortalised cells and tumour tissues.

Immunopeptides are cell surface-located protein fragments that aid our immune system to recognise and respond to pathogenic insult and malignant transformation. In this two-part communication, we firstly summarise and reflect on our recent discovery documenting that MHC-II-bound immunopeptides from immortalised cell lines prevalently carry N-glycans that differ from the cellular glycoproteome (Goodson, Front Immunol, 2023). These findings are important as immunopeptide glycosylation remains poorly understood in immunosurveillance. The study also opened up new technical and biological questions that we address in the second part of this communication. Our study highlighted that the performance of the search engines used to detect glycosylated immunopeptides from LC-MS/MS data remains untested and, importantly, that little biochemical in vivo evidence is available to document the nature of glycopeptide antigens in tumour tissues. To this end, we compared the N-glycosylated MHC-II-bound immunopeptides that were reported from tumour tissues of 14 meningioma patients in the MSFragger-HLA-Glyco database (Bedran, Nat Commun, 2023) to those we identified with the commercial Byonic software. Encouragingly, the search engines produced similar outputs supporting that N-glycosylated MHC-II-bound immunopeptides are prevalent in meningioma tumour tissues. Consistent also with in vitro findings, the tissue-derived MHC-II-bound immunopeptides were found to predominantly carry hyper-processed (paucimannosidic- and chitobiose core-type) and hypo-processed (oligomannosidic-type) N-glycans that varied in prevalence and distribution between patients. Taken together, evidence is emerging suggesting that α-mannosidic glycoepitopes abundantly decorate MHC-II-bound immunopeptides presented in both immortalised cells and tumour tissues warranting further research into their functional roles in immunosurveillance.

SB221: A proof-of-concept study to assess the combination of SON-1010 (IL12-FHAB) and atezolizumab in patients with platinum-resistant ovarian cancer.

TPS5629 Background: Recombinant interleukins (rIL) have had limited clinical success due to inefficient tumor targeting and short PK, requiring frequent dosing that leads to aberrant immunostimulation and toxicity. IL-12 potently activates T and NK cells to produce IFNγ and kill tumor cells, yet dosing strategies have failed to provide adequate therapeutic benefit in humans. We developed a novel platform that delivers immunomodulator(s) linked to a fully-human albumin binding (FHAB) domain (Cini, Front Immunol 2023). Single-chain native IL-12 genetically linked to the FHAB provides enhanced tumor targeting and retention through albumin binding to over-expressed FcRn, GP60, and SPARC in the tumor microenvironment (TME), with an improved PK profile, a dose-sparing effect that decreases the toxicity risk, and a broader therapeutic index. Tumor growth inhibition in an immunologically ‘cold’ B16-F10 mouse melanoma model showed the efficacy of IL12-FHAB compared with rIL-12, resulting in significant increases in activated NK, NKT, Th1, and cytotoxic CD8 T cells. SON-1010 is being studied clinically as monotherapy (study SB101) in advanced solid tumors and in healthy volunteers (study SB102) (Chawla, AACR 2023). Atezolizumab (Tecentriq), an anti-PD-L1 immune checkpoint inhibitor (ICI), has shown preliminary clinical activity in Phase 1 studies of patients with platinum-resistant ovarian cancer (PROC) (Liu, Gyn Onc 2019; Moroney, Clin Canc Res 2020). SON-1010 may ‘warm up’ the TME to improve ICI effectiveness in these immunologically-active tumors that have high levels of SPARC. Methods: Study SB221 is a Phase 1b/2a multicenter, dose-escalation and proof-of-concept study to assess the safety, tolerability, PK, PD, and efficacy of SON-1010 administered SC, either alone or in combination with a fixed dose of atezolizumab given IV (NCT05756907). The study is designed in Part 1 to rapidly establish the maximum tolerated dose (MTD) of the combination in patients with advanced solid tumors with up to 5 dose-escalation groups and to expand the dataset using patients with PROC to establish the Recommended Phase 2 Dose (RP2D). Once the likelihood of efficacy is shown in a Simon 2-stage design, this will be followed in Part 2 by an assessment of the potential for improved efficacy of the combination in patients with PROC over SON-1010 alone or the standard of care (SOC). The first dose-escalation cohorts have been enrolled and additional sites are being added to help with recruitment of patients with PROC. Combination of SON-1010 with an ICI offers a unique opportunity to use this extended PK version of IL-12 to augment the potential for tumor control in PROC, which represents a significant unmet medical need. Clinical trial information: NCT05756907 .

Magnetite nanoparticles: an emerging adjunctive tool for the improvement of cancer immunotherapy.

Cancer immunotherapy has emerged as a groundbreaking field, offering promising and transformative tools for oncological research and treatment. However, it faces several limitations, including variations in cancer types, dependence on the tumor microenvironments (TMEs), immune cell exhaustion, and adverse reactions. Magnetic nanoparticles, particularly magnetite nanoparticles (MNPs), with established pharmacodynamics and pharmacokinetics for clinical use, hold great promise in this context and are now being explored for therapeutic aims. Numerous preclinical studies have illustrated their efficacy in enhancing immunotherapy through various strategies, such as modulating leukocyte functions, creating favorable TMEs for cytotoxic T lymphocytes, combining with monoclonal antibodies, and stimulating the immune response via magnetic hyperthermia (MHT) treatment (Front Immunol. 2021;12:701485. doi: 10.3389/fimmu.2021.701485). However, the current clinical trials of MNPs are mostly for diagnostic aims and as a tool for generating hyperthermia for tumor ablation. With concerns about the adverse effects of MNPs in the in vivo systems, clinical translation and clinical study of MNP-boosted immunotherapy remains limited. The lack of extensive clinical investigations poses a current barrier to patient application. Urgent efforts are needed to ascertain both the efficacy of MNP-enhanced immunotherapy and its safety profile in combination therapy. This article reviews the roles, potential, and challenges of using MNPs in advancing cancer immunotherapy. The application of MNPs in boosting immunotherapy, and its perspective role in research and development is also discussed.

Endobronchial Ultrasound Guided Transbronchial Needle Aspiration and PD-L1 Yields

PurposeImmunotherapy is a leading approach for treating advanced non-small cell lung cancer (NSCLC) by targeting the PD-1/PD-L1 checkpoint signaling pathway, particularly in tumors expressing high levels of PD-L1 (Jug et al. in J Am Soc Cytopathol 9:485–493, 2020; Perrotta et al. in Chest 158: 1230–1239, 2020). Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive method to obtain tissue for molecular studies, including PD-L1 analysis, in unresectable tumors (Genova et al. in Front Immunol 12: 799455, 2021; Wang et al. in Ann Oncol 29: 1417–1422, 2018). This study aimed to assess the adequacy of PD-L1 assessment in EBUS-TBNA cytology specimens.MethodsData was collected retrospectively from patients who underwent EBUS-TBNA between 2017 and 2021 for suspected lung cancer biopsy. Samples positive for NSCLC were examined for PD-L1 expression. EBUS was performed by experienced practitioners, following institutional guidelines of a minimum of five aspirations from positively identified lesions. Sample adequacy for molecular testing was determined by the pathology department.ResultsThe analysis involved 387 NSCLC cases (149 squamous cell, 191 adenocarcinoma, 47 unspecified). Of the 263 EBUS-TBNA specimens tested for PD-L1, 237 (90.1%) were deemed adequate. While 84% adhered to the protocol, adherence did not yield better results. Significantly higher PD-L1 adequacy was observed in squamous cell carcinomas (93.2%) compared to adenocarcinoma (87.6%). The number of aspirations and sedation type did not correlate with PD-L1 adequacy in either cancer type, but lesion size and location had a significant impact in adenocarcinomas. Adenocarcinoma exhibited higher PD-L1 expression (68%) compared to squamous cell carcinoma (48%).ConclusionEBUS-TBNA offers high yields for assessing immunotherapy markers like PD-L1, with satisfactory adequacy regardless of NSCLC subtype, lesion size, or location.

Randomized Trial of Prophylactic Donor Lymphocyte Infusion with Positively Selected CD4+ T Cells after HLA-Identical Allogeneic Stem Cell Transplantation

Introduction Allogeneic stem cell transplantation (alloSCT) can cure patients with hematological malignancies by T-cell mediated alloreactivity against patient hematopoiesis, the graft-versus-leukemia (GvL) effect. GvL can coincide with graft-versus-host disease (GvHD) when donor T cells target non-hematopoietic tissues. Increase of donor chimerism in bone marrow (BM) or peripheral blood (PB) after alloSCT indicates GvL response. After T-cell depleted alloSCT, donor lymphocyte infusion (DLI) can restore GvL. Dose and timing of DLI influence the degree of alloreactivity. Low dose of unmodified DLI as low as 0.3x10 6 T cells/kg 3 months after alloSCT appeared not to cause GvHD. (Zouwen et al. Ann Hematol, 2023) We hypothesized that infusion of a higher dose of purified CD4+ donor T cells would not cause GvHD, while inducing GvL by selective targeting of the MHC class II positive hematopoietic cells. Method A randomized controlled phase 2 trial was conducted in patients with hematological malignancies after HLA-identical sibling alloSCT with alemtuzumab added to the graft. Patients without GvHD or relapse were randomized at 2.5 months for prophylactic DLI with 1.0x10 6/kg CD4+ T cells (CD4+ DLI) 3 months after alloSCT or conventional care without DLI (CC). Both groups were eligible for prophylactic unmodified DLI at 6 months. Cumulative incidence (CI) of GvHD requiring immunosuppressive treatment 3 months after randomization was the primary endpoint. Changes in the trajectory of chimerism in BM and subsets in PB (CD4+ T cells, CD8+ T cells, NK cells, B cells and monocytes) were compared by means of mixed effects beta regression as secondary endpoints. Results 66 patients were included (table 1). CI of GvHD did not differ between groups (CD4+DLI: 6.1% vs CC: 9.1%, Gray's test p>0.9). Relapse was comparable (CD4+DLI: 15% vs CC: 12%), although heterogeneity of diseases prevented a formal comparison. In the CD4+ DLI group, BM chimerism was full donor (FD, >95% donor) at baseline in 23/33 patients, while 10/33 had mixed chimerism (MC, ≤95%). In the CC group, 23/33 were FD and 10/33 MC. Some patients with MC at baseline converted to FD in both groups (CD4+ DLI: 1/10 vs CC: 1/10). Changes in BM chimerism did not differ between CD4+ DLI and CC (p = 0.9). Chimerism of B cells, NK cells and monocytes in PB resembled bone marrow chimerism with most patients being FD at baseline. Changes in chimerism did not differ between CD4+ DLI and CC (p = 1.0, 0.5 and 0.9 for B cells, NK cells and monocytes respectively). In the CD4+ DLI group, 25/30 evaluable patients were MC in the CD4+ T-cell subset in PB at baseline. 3 month after randomization 7/25 had converted to FD, 17/25 remained MC and 1/25 was unevaluable. Of the 17 patient who remained MC, CD4+ donor chimerism increased (absolute increase >5%) in 13, decreased (>5%) in 2 and was stable in 2. In the CC group, 26/32 were MC in CD4+ T cells at baseline, of which 6 converted to FD, 18 remained MC and 2 were unevaluable. Of the 18 patients remaining MC, CD4+ chimerism increased in 5, decreased in 9 and remained stable in 4. The CD4+ donor chimerism increased significantly after CD4+ DLI compared to CC (p = 0.01) (Figure 1). In the CD4+ DLI group, 23/29 evaluable patients were MC in the CD8 subset at baseline. 7/23 converted to FD, 15/23 remained MC and 1 was unevaluable. Of these 15 MC patients at 3 months, 9 had increasing, 5 stable and 1 decreasing donor chimerism after 3 months. In CC, 22/31 were MC at baseline. 4/22 converted to FD at 3 months and 17/22 remained in MC. Of the 17 MC patients, 6 had increasing, 3 decreasing and 8 stable chimerism. The CD8+ donor chimerism increased significantly in the CD4+ DLI group compared to CC (p = 0.03). Conclusion CD4+ selected DLI at a dose of 1x10 6 CD4+/kg 3 months after HLA identical T-cell depleted alloSCT did not cause GvHD. Chimerism in BM leukocytes, B cells, NK cells or monocytes did not indicate GvL mediated by CD4+ DLI, possibly because most patients were already FD at baseline. CD4+ and CD8+ T-cell donor chimerism increased significantly after CD4+ DLI, suggesting a GVL-effect - earlier demonstrated in vitro for 4 selected patients. (Van Balen et al. Front Immunol, 2018) However, most patients did not convert to FD in the T-cell compartment, suggesting the GvL-effect of CD4+ DLI was limited. We considered this GvL-effect insufficient to prevent relapse and stress the importance of CD8+ donor T cells as main drivers of alloreactivity after DLI following HLA-identical alloSCT.

Is CAR T a Drug or a Therapeutic Pathway? Systematic Review, (Re-)Estimation of Efficacy Endpoints and Metanalyses in Large B-Cell Lymphoma Real World Reports

In everyday clinical practice, the therapeutic pathway of autologous CAR T-cell starts from leukapheresis, but the drug is infused on average 4-7 weeks later. Between leukapheresis and CAR T infusion patients may die following disease progression or exit the therapeutic pathway due to comorbidities or other reasons, thereby introducing a potential bias in the estimation of survival benefit gained with the drug. Moreover, substantial differences may occur (e.g., timing of leukapheresis and enrollment, use of bridging chemotherapy, lymphodepleting regimens) between trials' designs and every-day clinical practice. All of this precludes a robust comparison among the different available CAR T products and the therapies which share the same indication. Considering analyses limited to treated patients with endpoints estimated from date of infusion should be avoided to prevent misleading treatment choices. We conceived a project involving researchers and cooperative groups worldwide which already published real world effectiveness data on per-protocol (PP, i.e. from infusion) or on intention-to-treat (ITT, i.e. from leukapheresis taking into account also subjects who were scheduled for but finally did not receive CAR T) populations with the aim to re-estimate efficacy endpoints in both modalities. This would offer the clinicians the possibility to draw more meaningful interpretations of the data according to these two different approaches (ALMA IDEA 2022 CUP:J33C22001420001). First a systematic review was conducted searching for real world studies on the effectiveness of anti-CD19 CAR T-cell products administered to large B-cell lymphoma patients in third line and beyond between 2017 (after FDA approval) and October 2022. For each study, we asked researchers about both infused and not infused patients, for overall response rate (ORR), complete response rate (CRR), median follow up, median survivals (event-free [EFS], progression-free [PFS] and overall [OS]) duration of response (DoR). ITT EFS, the new endpoint added for our project, was defined as the time the patients remained in the CAR T pathway from leukapheresis to exit due to any cause. When applicable, meta-regressions, metanalyses and pooled analyses were also performed. The results overview with the 10 selected studies is reported in Table 1 (Bachy et al. Nat Med. 2022; Bastos-Oreiro et al. Front Immunol. 2022; Ghafouri et al. Clin Lymphoma Myeloma Leuk. 2021; Iacoboni et al. Cancer Med. 2021; Kittai et al. Transplant Cell Ther. 2021; Kwon et al. Haematologica. 2023; Mian et al. Leuk Lymphoma. 2021; Nastoupil et al. J Clin Oncol. 2020; Pinnix et al. Blood Adv. 2020; Casadei et al. Cancers (Basel). 2021). Rates of not infused patients ranged from 3% to 29%. Meta-regressions did not indicate a statistically significant influence of the different ratio of infused/not infused patients among studies, nor the different lengths of the median follow up seem to bias project results. Metanalyses for ITT ORR and CRR resulted in 57% and 38% respectively while the PP ORR and CRR were 68% and 45% respectively. Although centers kept track of all patients scheduled for CAR T, around 50% did not record the exit date (decision of not infuse, due to any cause) for not-infused subjects, reflecting the inability to calculate the ITT EFS. Three studies reported a median PP PFS shorter than the ITT PFS, which may indicate a high mean time from leukapheresis to infusion, worthy of further investigation. For all studies but one median PP OS was higher than ITT OS: OS may be over-estimated when using a PP analysis. We should be using an ITT analysis, as reporting data in which we do not include the patients who underwent leukapheresis and were never treated could introduce a significant bias in results interpretation. Hematologists should understand which is the most clinical meaningful endpoint for this new therapeutic approach to reach effective results: have we to consider CAR T-cell therapy as a drug or a therapeutic pathway? The importance of considering this new treatment option as a pathway should translate into implementation of CAR T patient registries that include ITT as an endpoint to establish population-level effectiveness of these drugs. In addition, considering both PP and ITT analyses as well as infused and not infused patients provides a complete overview and leads to more information.

Real-Life Data of Cyclosporine (CsA) Tapering in Patients with Very/ Severe Aplastic Anemia (v/sAA) Treated with Horse Anti-Thymocyte Globulin (hATG/CsA) Suggest Higher Risk of AA Relapse at Cyclosporine Plasma Levels below 100 Nanogram per Milliliter

Introduction: Aplastic anemia (AA) is an acquired bone marrow failure syndrome (BMFS) mediated by autoreactive T-cells. In adults with severe (s) or very severe (vs) AA >40-50 years or ineligible for transplant for other reasons immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG), cyclosporine A (CsA) and Eltrombopag (Epag) has recently become the actual standard of care. With CsA treatment maintained for 6 to 24 months followed by CsA tapering hematologic response is achieved in 60-70 % of AA patients (pts.), while 30-40 % are primary refractory. Approximately 40% of pts. relapse during CsA tapering and most relapsed AA pts. remain CsA dependent. In vitro data indicate a dose dependent inhibition of T cell activity induced by CsA, suggesting the presence of a therapeutic CsA threshold around 100 ng/mL (Flores et al. Front Immunol 2019). Pts. with CsA related side effects such as renal insufficiency would benefit from an established lowest efficient CsA plasma level preventing relapse and reducing CsA related toxicity. As data about correlation of CsA plasma levels and the occurrence of relapse during tapering are sparse, we analyzed data from our registries including CsA plasma levels of 20 s/vsAA pts. with and without relapse during CsA tapering after treatment with horse ATG/CsA. Methods: Descriptive retrospective analysis of 20 AA pts. (17 sAA/ 3 vsAA) from the AA-BMF/Basel AA registry treated with horse ATG/CsA and sufficient clinical data during CsA tapering. Results are given as mean ± standard deviation. Mean age of the 13 female and 7 male pts. was 53 ± 19 years. Follow-up data over 59 ±17 months were compiled between 2010 and 2023. Results: All 20 pts. achieved a hematological response i.e. partial (PR) or complete remission (CR) (as defined in Peffault de Latour et al., 2022) at 3/6/9 months after ATG as follows: PR 90%/ 75%/ 55% and CR 0%/ 20%/ 45%. Relapse of AA occurred in 60 % (12 pts.) at 24 ± 11 months after ATG, while 40 % (8 pts.) remained relapse-free during a follow up period of 59 ±17 months. In the 8 pts. without relapse, dose reduction was initiated 14 ± 6 months after ATG at a hematological response of CR in 4 pts. and PR in 4 pts. In two pts. with PR, early decrease of CsA was required due to renal insufficiency (1 month after ATG) or CsA related adverse drug reaction (4 months after ATG), respectively. The average time of CsA maintenance was 32 ± 13 months. In pts. with relapse (60%, 12 pts.), dose reduction was started after reaching best hematological response i.e. CR (9 pts.) or PR (3 pts.) at 11 ± 7 months after ATG. Relapse i.e. recurrent thrombocytopenia occurred 12 ± 9 months after start of tapering at CsA plasma levels of 44.0 ± 29.2 ng/mL and a daily CsA dosage of 1.01 ± 0.70 mg/kg. In one case, relapse occurred one month after stopping CsA. No patient with CsA levels continuously > 100 ng/mL relapsed, whereas thrombocytopenia was observed with a latency of 2.8 ± 1.6 months after first decrease below 100 ng/mL CsA in pts. with relapse. While 58% (7 pts.) of the relapsed pts. responded to salvage-therapy with CsA+Epag (age 58 ± 16 years), 42 % (5 pts.) required a second course of ATG/CsA, or transplant (age 44 ± 22 years). Conclusion: In s/vsAA pts. with hematological response to ATG/CsA, closer monitoring for relapse is recommended reaching CsA plasma levels <100 ng/mL, as CsA <100 ng/mL was associated with higher risk of relapse during CsA tapering in line with previous reported in vitro data. Pending prospective validation, CsA plasma levels >100 ng/mL might serve as threshold in pts. requiring accelerated tapering due to renal insufficiency or CsA related side effects.

Combination of Ibrutinib with CD19 Chimeric Antigen Receptor (CAR) T Cells Suggests Greater Contribution to CAR T-Cell Efficacy Than Other Approved BTK Inhibitors in the Preclinical Setting

Background: CD19-directed CAR T-cell therapy has demonstrated remarkable clinical benefits for patients with B-cell malignancies, but impaired CAR T-cell fitness can result in lack of response or relapse. Rational combinations that enhance T-cell fitness may overcome primary and secondary resistance. Bruton tyrosine kinase inhibitors (BTKi), such as Ibrutinib (Ibr) have been shown to enhance CAR T-cell efficacy (Fraietta et al, Blood 2016;Liu et al, Cancer Sci 2020), but the precise mechanism remains unclear. In addition to BTK, Ibr potently inhibits IL-2-inducible tyrosine kinase (ITK), an important mediator of T-cell signaling. Ibr has been shown to enhance Th1 polarization through suppression of the Th2 differentiation pathway, thereby potentiating a cytotoxic effector response (Qin et al, J Immunother 2020). Consistent with this finding, the Th1 phenotype was more frequent in peripheral blood mononuclear cells of Ibr-exposed patients than in acalabrutinib (Acala)-exposed patients (Scarfò et al, Blood 2021). Therefore, we aimed to assess the potential impact of ITK inhibition (ITKi) on CD19 CAR T-cell function by comparing Ibr to other approved BTKi that display low or no ITKi activity (Estupiñán et al, Front Cell Dev Biol 2021). Methods: CD19-41bb CAR T cells (CAR19) were generated by lentiviral transduction of healthy donor T cells. BTKi additivity to CAR19 function was evaluated in a Luciferase (Luc)+ Ramos, BTKi resistant systemic tumor model in NSG mice co-treated with covalent BTKis Ibr, Acala, zanubrutinib (Zanu), and CAR T cells. Next, to evaluate the impact of ITKi on CAR19 fitness and cytotoxicity, repeated stimulation in an in vitro cytotoxicity assay was performed at low CAR19 effector-to-tumor ratio (1:20) to mimic higher tumor burden stress conditions in the presence of each BTKi, including non-covalent BTKi pirtobrutinib (Pirto) (500 nM). Tumor cells were replenished every 3 days and cytotoxicity was measured on day 7 by luciferase assay. For phenotypic characterization, CAR T cells were repeatedly stimulated by plate-bound anti-Whitlow linker every 3 days. Viability and proliferation were measured by cell counter. Flow cytometry (FACS) was performed on surface and intracellular T-cell phenotypic, activation, and exhaustion markers. Metabolic fitness (glycolysis and ATP production) was characterized using Seahorse (Van der Windt, 2012). Impact of Ibr on signal strength was evaluated in CD19-expressing Jurkat reporter cells (CAR-J19) with knock-in of mKate in frame with Nur77. Ibr was added to coculture of CAR-J19 and tumor cells and upregulation of Nur77 was measured by FACS. Downstream ITK signaling pathway was measured by Western blot. Results: Preliminary in vivo experiments demonstrated that co-administration of each BTKi with CD19 CAR T cells improved tumor control at day 26 (N=5 per group; P≤0.0004 vs CAR T cells alone). In serial killing experiments, addition of Ibr to the coculture increased proliferation (n=2; P<0.05), viability (n=2; P=0.01), and the cytotoxicity of CAR T cells against Ramos cells ( P=0.02 vs CAR19 alone) to a greater extent than other BTKis tested (Fig. 1A). Prolonged stimulation of CAR19 with Ibr led to downregulation of activation and inhibitory markers on CAR19 and maintenance of a naive T-cell phenotype (Fig. 1B) that was confirmed by increased metabolic fitness (n=5; P<0.05). Addition of Ibr to CAR-J19 resulted in a 72% reduction in CAR activation and reduced phosphorylation of ITK, PLCG, and Zap70. Additional data including the impact of ITK knockout on CAR-T cells will be presented. Conclusions: Our study expands on the hypothesized mechanism of action underlying the clinical benefit observed with Ibr in CD19 CAR T-cell-treated patients with chronic lymphocytic leukemia or mantle cell lymphoma (Liu et al, Front Immunol 2022; Wang et al, NEJM 2020). Our data suggest that Ibr enhances CAR T-cell fitness to a greater extent than both covalent and noncovalent BTKi that display reduced or no ITKi activity. Through ITKi, Ibr tempers CAR T-cell activation induced cell death, maintains a naive state and prevents premature exhaustion, thereby enabling more efficacious tumor cell killing over time. Together, these data support the further exploration of Ibr to enhance CD19 CAR T-cell efficacy.

Mutations in Histone Lysine Methyltransferase Genes Are Associated with Autoimmune Cytopenias

Introduction Epigenetic mechanisms are important in regulation of gene expression by modifying chromatin structure to facilitate DNA accessibility (Lau et al Nat Immunol. 2018). DNA methylation and histone modifications are critical for the pathogenesis of hematologic malignancies and autoimmune diseases (Surace et al Front Immunol. 2019) Alterations in enzymes involved in histone modification are associated with immunodeficiencies, including Kabuki syndrome (KS), a rare genetic disorder associated with autoimmune cytopenias, such as immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) (Margot et al J Am Coll Med Genet 2020) caused by loss-of-function KMT2D and KDM6A mutations. Somatic mutations in the KMT2D gene have also been observed in patients with Cold Agglutinin Disease. (Małecka et al Br J Haematol. 2018). Aims: The aims of this study are: 1) to identify the frequency of mutations in histone methyltransferase genes (MT) KMT2D, KMT2A, KMT2C and KDM6A in patients with autoimmune cytopenias, and 2) to compare clinical, laboratory and immunological characteristics, and response to therapy in patients with or without mutation in MT and autoimmune cytopenias (AIC). Methods Data mining software from the electronic medical record was used to identify patients ≥ 18 years with a diagnosis of AIC, including ITP, warm and cold AIHA, autoimmune neutropenia, and Evans Syndrome (ES), who had Next Generation Sequencing (NGS) performed. Cytopenias due to malignancy, nutritional deficiency, medications, pregnancy, or infection were excluded. Patients were categorized into two groups based on the presence or absence of mutation in the MT genes (MT+, MT-) with variant allelic frequency (VAF) > 2%. Demographic, laboratory, and clinical characteristics, treatment outcomes, and NGS sequencing data were collected and compared between groups. Next, KMT2 mutations seen in patients with AIC were cross-referenced against the 1000 Genome Project (1000GP) population database to evaluate the frequency of polymorphisms in the general population (Auton et al Nature 2015). CADD in silico prediction algorithm was used to score the potential deleterious effect of MT mutations (Kirchner Nat Genet. 2014). Chi-Square test was used for categorical variables. Statistical analysis was completed in R software. Results Among 495 patients who underwent NGS, 79 (16%) had a mutation in MT genes. These mutations were present in 36% of patients with AIC as compared to 13% in those without AIC (p=0.0005). Among 55 patients with AIC, mutations in the MT genes were present in 20 patients (MT+), 35 patients did not have these mutations (MT-). KMT2C mutations were seen in 10 (50%) patients, KMT2D mutations in 9 patients (45%, one patient had two KMT2D mutations), 1 patient (5%) had both KMT2C & KMT2D variants (Figure 1). The median VAF was 39.5% (IQR, 15.45 - 49.80). The MT+ group had a significantly higher proportion of Hispanic (60% vs 31%) and non-Hispanic White patients (25% vs 14%), p=0.038, Table 1. The median age at diagnosis for the MT+ cohort was younger compared to MT- (45.5 vs 58 years old, respectively), though not statistically significant. AIC diagnoses in both groups were ITP (44%) wAIHA (22%) cAIHA (9%) Autoimmune Neutropenia (2%), and ES (24%). Compared to the MT- group, the MT+ group had lower IgG levels (p=0.0041) and absolute lymphocyte count (p=0.041). MT+ patients with AIHA were more likely to have positive Direct Coombs and complement involvement. No significant differences were found in median nadir hemoglobin and platelet counts, need for therapy, or median number of lines of therapy. Best overall response to therapy was similar in the two groups (93%), with more complete responses in MT- group (55% vs 33%) though not statistically significant. Next, we assessed KMT2 polymorphisms in the general population. Most of the polymorphisms were not present in the 1000GP database and none had a frequency > 0.001. The average Phred score was 19.8; 12/20 patients had Phred score > 20, suggesting that most mutations are in the top 1% of the deleterious variants. Conclusion We report for the first time that patients with autoimmune cytopenias have a high frequency of variants in MT genes. Median allelic frequency close to 50% suggests potential germline predisposition to immune dysregulation and autoimmunity however further studies are needed to better understand the impact of these observations.

CXCR4 Loss Reduces the Local Growth of Richter Syndrome Murine and Patient-Derived Xenograft Models and Impairs BCR and VLA-4 Activation

Richter Syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma and is considered the most important unmet clinical need in CLL because of the lack of effective treatments. An important feature of RS cells is the frequent acquisition of genetic lesions in cell cycle regulators, such as CDKN2A/CDKN2B and TP53, resulting in increased proliferative capacity of the transformed cells and reduced dependence on proliferative microenvironmental signals. However, interactions with the tumor microenvironment are still required to promote the expansion of the transformed cells, as evidenced by the delayed growth of murine TCL1-derived or patient-derived xenograft (PDX) RS models following BCR disruption or macrophage depletion (Chakraborty S et al. Blood 2021, Martines C et al. Blood 2022). To further define the relevance of individual microenvironmental signals in supporting the growth of RS cells in vivo, in this study we evaluated the effects of CRISPR/Cas9-mediated knockout of the chemokine receptor CXCR4, which plays a central role in regulating the homing and interaction with the tumor microenvironment of non-transformed CLL cells. CRISPR/Cas9 editing of CXCR4 was done in the murine TCL1-derived RS model TC1-355 TKO and in the RS-PDX model RS9737, which are both characterized by biallelic disruption of TP53, CDKN2A and CDKN2B. The edited cells, comprising a mixed population with more than 80% disrupted CXCR4 alleles, were then transplanted in the peritoneal cavity and subcutaneous tissue of recipient mice (n=3 C57BL/6 mice for TCL1-355 TKO, n=8 NSG mice for RS9737). Analysis of the mutant allele frequency several weeks after injection revealed a significant reduction in the proportion of cells with mutated CXCR4 in the spleens of the injected mice and to a lesser extent in the peritoneal cavity and subcutaneous tumors, suggesting that disruption of CXCR4 signaling affects not only the migration and homing of the malignant B cells but also their local growth. To further evaluate the latter possibility, we transplanted CXCR4 knockout and CXCR4 wild type RS9737 cells in separate flanks of NSG mice (n=8) and after 4 weeks investigated the percentage of proliferating cells in the tumors by in vivo BrdU incorporation assay. A significantly lower percentage of proliferating cells was observed in the CXCR4 knockout compared to CXCR4 wild type tumors (22.3 ± 2.1 vs 37 ± 1.4, respectively, p<0.001), further suggesting that loss of CXCR4 affects the proliferation of the tumor cells. Since CXCL12 stimulation had no effect on the in vitro proliferation rate of the RS cells, we next investigated whether the reduced in vivo proliferation could be an indirect effect. For this purpose, we investigated whether CXCR4 knockout would affect integrin VLA4 or BCR signaling, as both BCR and CXCR4 can mediate inside-out VLA4 activation and there are data suggesting a potential crosstalk between the BCR and CXCR4 pathways (Maity PC et al. Front Immunol. 2018). Analysis of BCR signaling in the TCL1-355 TKO model showed a significant reduction in anti-IgM induced activation of AKT, ERK and reduced calcium mobilization in the CXCR4 knockout cells despite increased surface IgM expression. However, no effect of CXCR4 knockout on BCR signaling was observed in RS9737 cells, whereas CXCL12-induced VLA4 activation and VCAM1 adhesion were impaired in both models by CXCR4 knockout. To investigate whether impaired VLA4 activation could be responsible for the reduced in vivo proliferation rate of CXCR4 knockout RS cells, we performed BrdU incorporation analysis of RS9737 CXCR4 knockout and CXCR4 wild type cells co-cultured with the human stromal cell line HS5 + CXCL12. Co-culture with HS5 + CXCL12 resulted in a significantly increased proliferation of RS9737 CXCR4 wild type cells compared to the unstimulated control condition (58.5% ± 0.9% vs 50.1% ± 4.3%, respectively, p=0.031), whereas no such difference was observed with RS9737 CXCR4 knockout cells (50.0% ± 1.5% vs 49.1% ± 0.5%, respectively, p=n.s.). Together, these data show that CXCR4 disruption results in negative selection and reduced growth of murine and PDX RS models in vivo and affects BCR and VLA-4 signaling, suggesting that the CXCR4 pathway could represent a therapeutic vulnerability in Richter Syndrome.

Engineering Off-the-Shelf Gamma Delta CAR T Cells for the Treatment of Acute Myeloid Leukemia

Chimeric antigen receptor T cell therapy (CART) therapy has shown remarkable success in the treatment of B cell acute lymphoblastic leukemias (B-ALL) and lymphomas. However, CART therapies for acute myeloid leukemia (AML), where 5-year survival rates are significantly lower than for B-ALL, are only in their infancy. CD33-CART have potent activity against AML in preclinical models and a first-in-child/first-in-human phase 1/2 CD33-CART clinical trial for AML is ongoing in the Pediatric Oncology Branch of the National Cancer Institute (NCT03971799). Nonetheless, published outcomes suggest a modest efficacy of approximately 50% (Shahzad et al., Front Immunol 2023), highlighting the critical need to develop new strategies to improve CART accessibility and a more robust anti-AML response. We hypothesized that off-the-shelf gamma delta (γδ) CD33 CART cells could potentially overcome current challenges for the treatment of AML. γδ lineage T cells are unconventional lymphocytes whose functions are not restricted to MHC-mediated antigen presentation; they are primed for immediate responses, including tumor killing. Furthermore, allogeneicγδ T cells have the potential to induce robust anti-tumor cytotoxicity without causing graft versus host disease (GVHD). Here, we generated γδ CAR T cells from healthy donor elutriated lymphocytes by activation with zoledronic acid and IL-2 for 7-14 days. Within 9 days post stimulation, the vast majority of lymphocytes were Vδ2+ and 30-40% were successfully transduced with a lentiviral CD33 CAR construct harboring the 4-1BB costimulatory domain. Importantly, and unlike conventional alpha beta (ab) T lymphocytes, >98% of these γδ CD33CAR T cells expressed IFNγ under basal conditions. This characteristic likely accounted for the efficient in vitro killing of AML cell lines by untransduced γδ T lymphocytes under conditions of high effector/target (E/T) ratios. While untransduced γδ T cells did not exhibit cytotoxicity following repeat AML stimulations, γδ CD33CAR T lymphocytes exhibited proficient in vitro cytotoxicity, with killing rates that were more rapid than those initiated by ab CD33 CART (Figure 1). These characteristics were associated with a prolonged metabolic activity of γδT cells; γδ CD33 CART expressed high levels of the GLUT1 glucose transporter for >14 days post activation whereas GLUT1 levels on ab CD33 CART returned to resting within 10 days. High GLUT1 levels were linked to efficient killing under conditions of basal glucose levels. Most notably, γδ CD33CAR T lymphocytes achieved high in vivo cytotoxicity, assessed using bioluminescent AML cell line xenografts in humanized NSG mice. Together, these data highlight the feasibility of generating allogeneic γδ CD33CART with a strong anti-AML cytotoxic response.

Luspatercept Modulates Inflammation in the Bone Marrow, Restores Effective Erythropoiesis/Hematopoiesis, and Provides Sustained Clinical Benefit Versus Epoetin Alfa (EA): Biomarker Analysis from the Phase 3 COMMANDS Study

Introduction :Anemia is common in patients (pts) with myelodysplastic syndromes (MDS) due to ineffective erythropoiesis, characterized by increased dysplastic erythroid precursors (EPs) and abortive differentiation in the bone marrow (BM). Insufficient red blood cells (RBCs) lead to chronic anemia and heart failure due to oxygen deprivation. Luspatercept is approved to treat anemia after erythropoiesis-stimulating agent (ESA) failure in pts with transfusion-dependent, lower-risk MDS (LR-MDS). In the phase 3 COMMANDS trial (NCT03682536), 58.5% of ESA-naive luspatercept treated pts with LR-MDS experienced RBC transfusion independence for ≥ 12 wk with concurrent mean hemoglobin (Hb) increase ≥ 1.5 g/dL during wk 1-24, vs 31.2% of EA-treated pts. Aims :To differentiate the mechanism of action of luspatercept from EA and determine its correlation with clinical benefit in the COMMANDS study. Methods :Cytomorphology assessments were performed on BM aspirates at baseline, wk 24 and 48. Complete blood count, Hb, and reticulocytes (retics) were measured in peripheral blood. BM mononuclear cells (BMMC) were subjected to bulk RNA-seq. Serum cytokine analysis was performed at screening and wk 24 using Rules-Based Medicine's Human Inflammation MAP ® v.1.1 and a custom Human MAP ELISA panel. Results :In ITT population, EPs, retics, and Hb were increased vs baseline in the luspatercept arm at wk 24 ( P < 0.001) and wk 48 ( P < 0.005). EPs, retics, and Hb were increased in the EA arm at wk 24 ( P < 0.01), but this was not sustained to wk 48. Subgroup analysis showed similar trends for ring sideroblast (RS)+ pts in both arms. While RS− pts in the EA arm showed similar trends to the ITT population, luspatercept RS− pts showed trends of EP expansion post-wk 24 only. Retics increased significantly in RS− luspatercept pts at wk 24 ( P < 0.005). Hb increased more significantly at wk 24 with EA ( P < 0.003) but at wk 48 with luspatercept ( P < 0.005).Gene set enrichment analysis of BMMC revealed enrichment of early, mid, and late EP genes at baseline favored response to luspatercept, but enrichment of mid and late EP genes was unfavorable for EA (Figure A). Significant downregulation of REACTOME IL-1 signaling (normalized enrichment score [NES] = −2.27; adjusted [Adj] P < 0.0001 [vs EA = not significant (ns)]), BIOCARTA IL-6 pathway (NES = −1.99; Adj P = 0.0025 [vs EA = ns]) and IFNα response (NES: −2.26; Adj P < 0.0001 [vs EA = ns]) was observed with luspatercept. BIOCARTA IL-1R pathway was more significantly reduced with luspatercept (NES = −1.78; Adj P = 0.0073) compared with EA (NES = −1.79; Adj P = 0.0172), as was IL-10 signaling (luspatercept NES = −2.39; Adj P < 0.001 vs EA NES = −1.82; Adj P = 0.0125). TGF-β, apoptosis, and spliceosome pathways down regulation was also observed with luspatercept. At wk 24, luspatercept responders had a median 640 pg/mL decrease ( P = 0.0035) of N-terminal pro-brain natriuretic peptide (NT-proBNP), produced by the heart, compared with a median 360 pg/mL decrease in EA arm responders ( P = 0.37). At wk 24, non-responders had a median increase of 775 pg/mL ( P = 0.0032) in the EA arm and 31.5 pg/mL ( P = 0.95) in the luspatercept arm. Conclusions : Multiplatform analysis of primary BM samples from a phase 3 trial of luspatercept revealed novel response-associated biomarkers. Compared with EA, luspatercept led to sustained increase of EPs and retics over 48 wk with a concomitant Hb increase. RS− pts showed Hb accumulation peaked at wk 24 in the EA arm. Conversely, luspatercept in RS− pts significantly increased retics at wk 24 and EPs past wk 24 with concomitant peaking of Hb at wk 48, suggesting gradual and sustained clinical benefit. Unlike EA, which plays a role in early differentiation of hematopoietic stem cells (HSCs), luspatercept acts on different erythroid stages, leading to EP expansion and maturation. Furthermore, downregulation of IL-1, IL-1R, IFNα, IL-6 pro-inflammatory (Figure B), and IL-10 anti-inflammatory signaling with luspatercept suggests modulation of the BM inflammatory environment in MDS to restore HSC function for normal hematopoiesis (Wang J, et al. Front Immunol 2022;13:951937). Additionally, decreased or static levels of NT-proBNP with luspatercept suggest a positive effect in mitigating cardiac damage. These novel insights obtained from analysis of a large controlled clinical trial mechanistically differentiate the superior clinical benefit of luspatercept from EA in treating anemia due to MDS.

Categorization of Hematological Responses to Oral Iptacopan Monotherapy in Anti-C5-Treated Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and Persistent Anemia in the Phase III APPLY-PNH Trial and Complement Inhibitor-Naïve Patients in the Phase III APPOINT-PNH Trial

Background: PNH is an ultrarare disease characterized by complement-mediated hemolysis and consequent anemia. Iptacopan is the first oral, selective complement inhibitor that targets factor B to inhibit the alternative pathway proximally in the complement system. Iptacopan monotherapy led to normal/near normal hemoglobin (Hb) values and transfusion avoidance in the majority of complement inhibitor-naïve patients and anti-C5-treated patients with persistent anemia in the Phase III APPOINT-PNH (NCT04820530) and APPLY-PNH (NCT04558918) trials, respectively, with iptacopan achieving its primary endpoints and demonstrating superiority to anti-C5 treatment in APPLY-PNH. Aim: To apply hematological response categories, adapted from Risitano et al. in Front Immunol 2019, to the data from APPLY-PNH (baseline and Week 24 data) and APPOINT-PNH (Week 24 data). Methods: APPLY-PNH enrolled adult PNH patients with a mean Hb level <10 g/dL who had been receiving eculizumab or ravulizumab for ≥6 months. Patients were randomized 8:5 to receive iptacopan monotherapy 200 mg twice daily or to continue their anti-C5 regimen for 24 weeks. APPOINT-PNH enrolled complement inhibitor-naïve adult PNH patients, with a mean Hb level <10 g/dL and lactate dehydrogenase (LDH) >1.5 × upper limit of normal (ULN); patients received iptacopan monotherapy 200 mg twice daily. Using central laboratory data, hematological responses were categorized primarily based on Hb levels (at baseline and between Days 126 and 168) and the need for packed red blood cell transfusions (in the 6 months prior to baseline and between Days 14 and 168). LDH levels and absolute reticulocyte counts were ancillary indicators to discriminate between complete and major responses. In the original definition of the response categories, absolute reticulocyte count was used to rule out patients with bone marrow failure; however, for this analysis, as patients with laboratory evidence of bone marrow failure were excluded from APPLY-PNH and APPOINT-PNH, absolute reticulocyte count was not used to define the suboptimal categories (ie good, partial, minor and no response categories). Week 24 categories were defined as follows: complete response - median Hb ≥12 g/dL, no transfusions, and both median LDH ≤1.5 × ULN and median absolute reticulocyte count ≤150,000/µL between Days 1 and 168; major response - median Hb ≥12 g/dL, no transfusions, and either LDH >1.5 × ULN or absolute reticulocyte count >150,000/µL between Days 1 and 168; good response - median Hb ≥10 and <12 g/dL and no transfusions; partial response - median Hb ≥8 and <10 g/dL and ≤2 transfusions; minor response - median Hb <8 g/dL and ≤2 transfusions; or median Hb <10 g/dL and 3-6 transfusions; or median Hb <10 g/dL and a reduction in transfusions by ≥50% between Days 14 and 168 compared with the number of transfusions received in the 6 months prior to baseline; no response - median Hb <10 g/dL and >6 transfusions. Results: Sixty-two patients received iptacopan and 35 patients received anti-C5 treatment in the APPLY-PNH trial; 40 patients received iptacopan in the APPOINT-PNH trial. There were no baseline differences between patients in the iptacopan arm and patients in the anti-C5 arm of the APPLY-PNH trial, with most patients having a partial hematological response to complement inhibitor treatment at baseline (62.9% [39/62] of patients in the iptacopan arm and 62.9% [22/35] in the anti-C5 arm; Figure 1). At Week 24, most iptacopan-treated patients in the APPLY-PNH trial achieved a complete response (71% [44/62] of patients versus 0% in the anti-C5 arm; Figure 1). Most patients in the APPOINT-PNH trial also achieved a complete response to iptacopan (62.5% [25/40] of patients; Figure 2). As expected, at Week 24, none of the patients in the anti-C5 arm of APPLY-PNH achieved complete or major hematological responses, with most continuing to maintain a partial hematological response (54.3% [19/35] of patients). Conclusions: This post hoc analysis of the APPLY-PNH and APPOINT-PNH trial data demonstrates that the majority of patients achieved complete or major hematological responses during treatment with iptacopan monotherapy, highlighting the ability of both complement inhibitor-naïve patients and anti-C5-treated patients with persistent anemia to achieve transfusion avoidance and improvement of Hb to normal/near normal levels with iptacopan.

Addressing Resistance to Therapy in Colorectal Cancer

Addressing Resistance to Therapy in Colorectal Cancer

ABPEPserver: a web application for documentation and analysis of substitutants

BackgroundCancer immunotherapy is implemented by identifying antigens that are presented on the cell surface of cancer cells and illicit T-cell response (Schumacher and Schreiber, Science 348:69–74, 2015; Waldman et al., Nat Rev Immunol 20:651–668, 2020; Zhang et al., Front Immunol 12:672,356, 2021b). Classical candidates of such antigens are the peptides resulting from genetic alterations and are named “neoantigen" (Schumacher and Schreiber, Science 348:69–74, 2015). Neoantigens have been widely catalogued across several human cancer types (Tan et al., Database (Oxford) 2020;2020b; Vigneron et al., Cancer Immun 13:15, 2013; Yi et al., iScience 24:103,107, 2021; Zhang et al., BMC Bioinformatics 22:40, 2021a). Recently, a new class of inducible antigens has been identified, namely Substitutants, that are produced as a result of aberrant protein translation (Pataskar et al., Nature 603:721–727, 2022).MainCatalogues of Substitutant expression across human cancer types, their specificity and association to gene expression signatures remain elusive for the scientific community's access. As a solution, we present ABPEPserver, an online database and analytical platform that can visualize a large-scale tumour proteomics analysis of Substitutant expression across eight tumour types sourced from the CPTAC database (Edwards et al., J Proteome Res 14:2707–2713, 2015). Functionally, ABPEPserver offers the analysis of gene-association signatures of Substitutant peptides, a comparison of enrichment between tumour and tumour-adjacent normal tissues, and a list of peptides that serve as candidates for immunotherapy design. ABPEPserver will significantly enhance the exploration of aberrant protein production in human cancer, as exemplified in a case study.ConclusionABPEPserver is designed on an R SHINY platform to catalogue Substitutant peptides in human cancer. The application is available at https://rhpc.nki.nl/sites/shiny/ABPEP/. The code is available under GNU General public license from GitHub (https://github.com/jasminesmn/ABPEPserver).

Immunomodulatory AVM0703 lacks the association between anti-cancer activity and immune-mediated side-effects of the checkpoint Inhibitors.

e14585 Background: Immune checkpoint inhibitors are approved to treat a variety of solid tumors and Hodgkin’s Lymphoma. However, they have not been shown to be effective against Non-Hodgkin’s Lymphoma (NHL). Unfortunately, their use is associated with induction of autoimmune reactions, termed immune mediated side-effects (irAEs) (1-3). AVM0703 is an immunomodulatory drug which mobilizes bispecific gd and invariant TCR double-positive NKT-like cells (AVM_NKT), currently enrolling a Phase 2 clinical trial to treat R/R NHL of all subtypes based on strong activity against the aggressive, immune-resistant mouse A20 lymphoma. Unlike the checkpoint inhibitors which trigger irAEs, gdTCR expression of the AVM0703 mobilized AVM_NKT, suggests that AVM0703 should not trigger irAEs, and in fact might be a treatment for autoimmune diseases such as type 1 diabetes (T1D). As monotherapy in the A20 lymphoma model, AVM0703 completely eradicates ~20% of flank tumors, and used as a neoadjuvant prior to cyclophosphamide/fludarabine (CyFlu) it provided additive/synergistic A20 killing. As a single dosed monotherapy, AVM0703 prevented T1D onset in the gold standard NOD model. Reversal of already established T1D has proven difficult, and monotherapy approaches abandoned for lack of efficacy. Based on its activity in combination with CyFlu, and since teplizumab anti-CD3 has been approved to prevent progression to grade 3 T1D in at risk people (4), we investigated the combination with AVM0703 to reverse recent onset T1D in the NOD mouse. Methods: One hundred twenty hyperglycemic non-obese diabetic (NOD) mice were randomized to receive an insulin pellet followed by 1) Oral Placebo (18 mg/kg HED) 2) 5 µg anti-CD3 (for 5 days) i.p. 2) Oral AVM0703 (18 mg/kg HED) – weekly/biweekly/tri-weekly 3) AVM0703 (18 mg/kg HED) followed by 5 µg anti-CD3 (for 5 days) and then AVM0703 – weekly/bi-weekly/tri-weekly. Mice were monitored for body weight, body condition and hyperglycemia. Results: Timing between 18 mg/kg AVM0703 and 5 ug anti-CD3 dosing was taken from lymphoma studies that demonstrated that chemotherapy 3 and 24 hours after AVM0703 dosing maximally eradicated lymphoma. The combinations of AVM0703 (day 1) and anti-CD3 (day 2-6) were able to reverse diabetes in 26 mice for 0-9 weeks, with six mice in ongoing normoglycemia at the time of this report. The combination group that received weekly AVM0703 dosing was more effective than the bi-weekly or tri-weekly AVM0703 groups. Monotherapy with AVM0703 did not reverse diabetes, in contrast to its profound ability to prevent or delay diabetes onset in the NOD model. Conclusions: We hypothesize that the combination of AVM0703 and anti-CD3 will be more effective when anti-CD3 is added 5 days after AVM0703. 1) Tocut, Autoimmun Rev 2018. 2) Sakowska, Front Immunol 2022. 3) Sherry, Lancet. 2011. 4) Hagopian, Diabetes 2013.

Abstract 685: Engineering semi-allogeneic whole cancer vaccines with enhanced immunogenicity for the treatment of advanced solid tumors

Abstract Background: Therapeutic cancer vaccines are designed to program a patient’s own immune system to recognize and eliminate tumor cells. We sought to harness gene-modified tumor cells as a vaccine platform and developed cancer vaccines composed of breast cancer cells expressing GM-CSF (SV-BR-1-GM). We have recently reported favorable clinical outcomes in patient populations that match SV-BR-1-GM at one or more HLA alleles. Mechanistically, SV-BR-1-GM cells can directly activate CD4+ T-cells in an antigen-specific HLA-restricted manner, as demonstrated by an in vitro antigen presentation assay (1). All these observations led us to hypothesize that semi-allogeneic cell-based cancer vaccines (partially matching host HLA genotype) would mount an enhanced immune response by forcing the host immune response to recognize tumor-associated antigens in the context of allogeneic HLA-I or II molecules or in proximity of strong non-self-antigens. Therefore, steps were taken to generate semi-allogeneic cancer cell lines expressing an extended repertoire of stimulatory molecules to induce optimal immune activation (Bria-OTS). Methods: SV-BR-1 (Breast cancer), PC-3 (prostate cancer), SK-MEL-24 (Melanoma), and NCI-H2228 (Lung cancer) were selected for vaccine development. To generate semi-allogeneic cell lines matching >99% of the population at least at 1 HLA allele, four cell lines (for each parental tumor cell line) were developed, each carrying two (2) HLA-A and two (2) HLA-DRB3/4/5 alleles, for a total of eight HLA-A and seven HLA-DRB3/4/5 alleles. We transduced selected co-stimulatory molecules and immune-modulatory cytokines. Next, the unique combinations of HLA-A and HLA-DRB3/4/5 alleles were transduced into the cells using lentiviral based vectors. Finally, functionality of the engineered cells was validated by using molecular and cellular based assays. Preliminary data: Four cell lines (for each tumor type) that secreted GM-CSF, IFNα, IL12, IL7 and expressed CD80, CD86, 4-1BBL, and different combinations of both Class I and Class II HLA alleles were selected. Using cell-based assays - including mixed lymphocyte reaction assays -, we demonstrated that the generated cells stimulate naïve T-cells. Evaluation of the cells ability to present antigens in an HLA-restricted antigen-specific manner is also planned. Discussion and conclusions: Different types of cancer vaccines have been developed with moderate success, often hampered by lack of strong immunogenicity and complex manufacturing. BriaCell’s Bria OTS provides a solution by increasing vaccine immunogenicity and decreasing manufacturing costs while personalizing the therapy based on matching the patient’s HLA type.1.Lacher MD et al, Front Immunol. 2018 May 15;9:776 Citation Format: Miguel A. Lopez-Lago, Vikas Bhardwaj, Xiaoyi Zheng, Sagarika pachhal, Renee Cortez, Charles Wiseman, William Williams. Engineering semi-allogeneic whole cancer vaccines with enhanced immunogenicity for the treatment of advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 685.

Abstract 4055: CD40L stimulates melanoma infiltrating B cells and enhances ex vivo TIL expansion

Abstract Adoptive transfer of tumor infiltrating lymphocytes (TIL) is a feasible and effective therapy for melanoma and lung cancer[1,2]. Multiple factors may determine the quality of the TIL product including components of the tumor microenvironment. In this work, we analyzed the role of melanoma infiltrating B cells in the context of TIL expansion based on their documented association with response to other types of immunotherapies[3]. We stimulated melanoma infiltrating B cells using human recombinant CD40L on the first day of ex-vivo TIL expansion. Samples were expanded from cryopreserved melanoma tumor single cell suspensions, in high dose IL-2 alone (standard protocol), or in high dose IL-2 plus CD40L. After 48h, analysis of activation markers on the CD40-expressing cells by flow cytometry was performed. For further investigation of the changes induced by CD40L stimulation, TIL expansion cultures (+/- CD40L) were analyzed using scRNA-seq (10X Genomics Chromium NextGEM Single Cell 5’ v2 and V(D)J Reagent kits; Illumina NovaSeq 6000 instrument with S4 sequencing flow cell) at 48h of culture (n=7 patients). The TIL expansion success rate was 68% with the CD40L treatment condition compared to 36% with the standard protocol. TILs cultured in the presence of CD40L expanded to on average three times more than with the standard protocol (P ≤ 0.01). Treatment with CD40L increased the percentage of CD39- CD69- T cells (P ≤ 0.05). Within the tumor digests, a higher percentage of B cells, including switched memory B cells (CD27+ IgD− ), was associated with successful TIL expansion (P=0.04). scRNA-seq analysis demonstrated different clustering patterns within the B cell compartment based on culture conditions. No clear partition was observed for other cell types, including the myeloid compartment. B cells displayed 126 DEGs associated to CD40L addition, CCL22, CD83, EBI3 and CD58 were among the upregulated genes in the CD40L-treated B cells. Other cell types experienced minimal to no change in transcriptomic profiles. B cell clusters were sub-classified based on CD27 and IgD expression[4], showing a predominance of naïve and switched memory B cells. Our results show that higher presence of B cells within tumors is associated with better TIL expansion, suggesting an interplay between T and B cells, and providing rationale for the design of improved TIL expansion protocols based on B cell stimulation with CD40L. This work has been supported in part by the Flow Cytometry, Genomics and Biostatistics and Bioinformatics Core Facilities at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We acknowledge Moffitt’s Melanoma Center of Excellence and the Mark Foundation for the financial support. [1] Sarnaik, A.A. et al. JCO 39, 2656-2666 (2021). [2] Creelan, B. et al. Nat Med 27, 1410-1418 (2021). [3] Cabrita, R. et al. Nature 577, 561-565 (2020). [4] Sanz, I. et al. Front Immunol 10, 2458 (2019). Citation Format: Renata Ariza Marques Rossetti, Leticia Tordesillas, Matthew Beatty, Dongliang Du, Yian Ann Chen, Amod Sarnaik, Shari Pilon-Thomas, Daniel Abate-Daga. CD40L stimulates melanoma infiltrating B cells and enhances ex vivo TIL expansion. [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 4055.

Abstract P3-06-08: Turning tumor cells into antigen-presenting cells for cancer immunotherapy

Abstract Miguel Lopez-Lago1, Sagarika pachhal1, Charles L. Wiseman1, William V. Williams1. 1BriaCell Therapeutics Corporation, Philadelphia, PA Turning tumor cells into antigen-presenting cells for cancer immunotherapy Background: Therapeutic cancer vaccines are based on specific stimulation of the immune system using tumor antigens to elicit an antitumor response. We have been conducting clinical trials using the breast cancer cell line SV-BR-1-GM as a therapeutic vaccine. SV-BR-1-GM is a GM-CSF expressing breast cancer cell line with features of an antigen presenting cell (APC) owning to the expression of several immunomodulatory molecules, including MHC-I (HLA-A, B & C) and MHC-II (HLA-DRB3 & -DRA). Initial results in patients treated with irradiated SV-BR-1-GM cells, low dose cyclophosphamide and local IFNα suggest that patients that match SV-BR-1-GM at least at 1 HLA allele are more likely to derive clinical benefit. This clinical observation, together with the fact that SV-BR-1-GM cells can directly activate CD4+ T-cells in an antigen-specific HLA-restricted manner, as demonstrated by an in vitro antigen presentation assay (1), lead us to hypothesize that SV-BR-1 (the parent cell line) can function as APC. To further enhance direct antigen presentation to T-cells, SV-BR-1 cells have been genetically modified to express co-stimulatory molecules, immunomodulatory cytokines, and an extended repertoire of HLA alleles. Methods: To generate an off-the-shelf semi-allogeneic cell therapy covering most of the population, SV-BR-1 was genetically modified to express an extended repertoire of HLA alleles. Based on population analysis, four cell lines, each carrying two (2) HLA-A and two (2) HLA-DRB3/4/5 alleles, should produce at least a single match in 99% of the population, with a 92% match at Class I HLA-A alleles and a 98% match at Class II HLA-DRB3/4/5 alleles. SV-BR-1 was genetically modified using CRISPR/cas9 deletion of the endogenous HLA-A and HLA-DRB3 alleles and subsequent lentiviral mediated expression of alternative HLA-A and DRB3 alleles. To generate tumor cell lines with enhanced direct antigen presentation to T-cells, SV-BR-1 cells were genetically modified to express co-stimulatory molecules and immunomodulatory cytokines by using a lentiviral mediated expression system. Results: Following sequential CRISPR/Cas9 editing, the SV-BR-1 cells were cloned, and one clone selected (clone 17) for further engineering. Lack of expression of HLA-A and HLA-DRB3 was confirmed using flow cytometry and DNA sequencing. Clone 17 was subsequently transduced with 6 lentiviral vectors each expressing 2 genes under the control of separate promoters:CD86-IL12, CD80-HLA-DRA, 4-1BBL-IL7, GM-CSF-IFN, HLA-allele-1-HLA-allele-2 and HLA-DR-allele-1-HLA-DR-allele-2. Four cell lines were generated with different combinations of HLA alleles. Following selection, cells were evaluated by ELISA, flow cytometry and RT-PCR to confirm gene expression. Cell lines that secreted GM-CSF, IFN IL12, IL7 and expressed CD80, CD86, 4-1BBL, and both Class I and Class II HLA alleles are then transferred to GMP manufacturing. These modified breast cancer cell lines will be used in clinical studies designed to first evaluate the safety of intradermal inoculation with the irradiated cells and later combined with other agents to augment the immune response. Each patient will be treated with the cell line(s) that match them at least at one HLA allele. 1. Lacher M. D. et al, Front Immunol. 2018 May 15;9:776 Citation Format: Miguel Lopez-Lago, William Williams, Charles Wiseman. Turning tumor cells into antigen-presenting cells for cancer immunotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-08.

How COVID-19 and other pathological conditions and medical treatments activate our intravascular innate immune system.

COVID-19 has been shown to have a multifaceted impact on the immune system. In a recently published article in Front Immunol, we show that the intravascular innate immune system (IIIS) is strongly activated in severe COVID-19 with ARDS and appears to be one of the causes leading to severe COVID-19. In this article, we describe the IIIS and its physiological function, but also the strong pro-inflammatory effects that are observed in COVID-19 and in various other pathological conditions and treatments such as during ischemia reperfusion injury and in treatments where biomaterials come in direct contact with blood in, e.g., extracorporeal and intravasal treatments. In the present article, we describe how the IIIS, a complex network of plasma proteins and blood cells, constitute the acute innate immune response of the blood and discuss the effects that the IIIS induces in pathological disorders and treatments in modern medicine.