Inhibitory Signals Research Articles

CD80-Fc fusion protein as a potential cancer immunotherapy strategy.

The activation of T lymphocytes is a crucial component of the immune response, and the presence of CD80, a membrane antigen, is necessary for T-cell activation. CD80 is usually expressed on antigen-presenting cells (APCs), which can interact with cluster of differentiation 28 (CD28) or programmed cell death ligand 1 (PD-L1) to promote T-cell proliferation, differentiation and function by activating costimulatory signal or blocking inhibitory signal. Simultaneously, CD80 on the APCs also interacts with cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on the surface of T cells to suppress the response of specific effector T cells, particularly in the context of persistent antigenic stimulation. Due to the pivotal role of CD80 in the immune response, the CD80-Fc fusion protein has emerged as a promising approach for cancer immunotherapy. This review primarily focused on the crucial role of CD80 in the cancer immunotherapy. We also reviewed the current advancements in the research of CD80-Fc fusion proteins. Finally, we deliberated on the challenges encountered by CD80-Fc fusion proteins and proposed the potential strategies that could yield the benefits for patients.

CS585 Demonstrates Favorable Selectivity and Sustained In Vivo Action in Preventing Platelet Activation and Thrombosis Compared to Existing IP Receptor Agonists

Anti-platelet therapeutics are common tools in the treatment of thrombotic diseases, decreasing morbidity and mortality resulting from cardiovascular complications. However, many patients remain at risk for a cardiovascular event while others experience bleeding due to hemostatic dysregulation. The need for target specificity and lasting stability presents a major challenge in the development of novel antithrombotic therapeutics. While increasing platelet cAMP levels through activation of the prostacyclin (IP) receptor is a viable therapeutic approach, the IP receptor is currently considered a challenging target due to the non-selectivity and instability of existing IP agonists. Our lab has developed a novel IV and orally available IP receptor agonist, CS585, with selectivity towards the IP receptor and sustained in vivo protection from injury-induced thrombosis without observed bleeding. CS585 demonstrates IP receptor-dependent activity, leading to activation of protein kinase A (PKA), a key player in inhibitory signaling. While indications of current IP receptor agonists are limited to pulmonary arterial hypertension and temporary relief of peripheral artery disease, CS585 presents a new approach to selectively regulate platelet activation and thrombosis. CS585 and other IP receptor agonists, iloprost and selexipag, were assessed by direct comparison to elucidate potential differences in selectivity and sustained action. Selectivity was determined using aggregation and VASP phosphorylation. The effects of CS585 are inhibited by pharmacological inhibition of the IP receptor, but not by inhibition of the DP1, EP2, or EP4 prostaglandin receptors. Meanwhile, the activities of iloprost and selexipag were affected by other prostaglandin inhibitors, supporting the improved selectivity toward activation of the IP receptor with CS585. Arterial thrombosis was measured and quantified in a laser-induced cremaster thrombosis assay in IP agonist-treated WT mice to assess platelet activation and thrombus formation in vivo in mouse models of thrombosis. In WT mice treated with CS585, we observed sustained effects of CS585 following both IV and oral administration. Platelets and fibrin were labeled and accumulation at the site of injury was measured using intravital microscopy. In contrast to the FDA-approved IP receptor agonists (iloprost and selexipag) which showed short-term protection from thrombosis, treatment with CS585 resulted in sustained inhibition of clot and fibrin formation at the site of injury in the same model between 24 and 48 hours post-administration. To determine if the anti-thrombotic effects of CS585 impact coagulation, coagulation parameters were assessed in whole blood using thromboelastography (TEG). Treatment with CS585 did not impact coagulation parameters in whole blood, while a significant increase in lysis was observed with iloprost. Here, we show for the first time a head-to-head comparison of CS585, a novel IP receptor agonist, with selexipag and iloprost. Our preclinical results with CS585 indicate a favorable profile for inhibiting platelet activation and clot formation and demonstrate a sustained duration of action in mice in the ability of CS585 to inhibit platelet activation through multiple routes of administration. Importantly, CS585 does not affect coagulation parameters and previous studies demonstrated no potential for increased bleeding in mouse models. Overall, CS585 provides a new option of activating the IP receptor to decrease platelet reactivity and could represent the first viable option for targeting the IP receptor on platelets for primary inhibition of thrombosis with a reduced risk of bleeding.

G-CSFR Is a Checkpoint of Natural Killer Cells Against Tumor

G-CSFR is a cell surface receptor that can activate cell proliferation and differentiation after binding to granulocyte colony stimulating factor(G-CSF). However, in natural killer (NK) cells, the biological role of G-CSFR has not been fully elucidated. Here we show that granulocyte colony-stimulating factor receptor, that is encoded by the CSF3R gene, is a novel and potent checkpoint in both human and mouse NK cells fighting against tumors. Previous studies have shown that NK cells under resting state with low expression of G-CSFR, but highly expressed in peripheral blood and bone marrow NK cells after G-CSF mobilization. We therefore used G-CSFR blocking agent in vitro to evaluate the effect of G-CSFR on human NK cells proliferation and cytotoxicity capacity. The results showed that G-CSFR agonist significantly down-regulated the proliferation and cytotoxicity of NK cells in vitro and this is independent of G-CSF stimulation. Since NK92 and YT cell lines highly express G-CSFR, we then knocked down G-CSFR in NK92 and YT cells. The results showed that the low expression of G-CSFR enhanced the expression of CD107a in vitro, indicating G-CSFR may be an inhibitory signaling in NK cells. To the further evaluate the specific role of G-CSFR in NK cells and we therefore constructed NK-specific CSF3R deficiency mice (CSF3R △NK/NK; CSF3Rfl/flNCR1cre, thecre group) and the control group ( CSF3Rfl/fl ( CSF3RWT/WT mice, the flox group). We then evaluated the phenotype, differentiation and functional state of NK cells between the two group. NK-specific CSF3R deficiency increased mature NK subsets in mice, and these CSF3R-deficient NK cells exhibited enhanced activation, cytotoxicity and proliferation, which is in accordance with the role in human NK cells. After constructing four tumor-bearing models, namely MC38 (colon tumor), LLC (lung tumor), MLL-AF8(acute myeloid leukemia), B16-F10(melanoma), we found that CSF3R specific depletion in NK cells reduced the tumor burden, prolonged the survival and reduced tumor metastasis in CSF3R△NK/NK mice (Figure 1A-D), indicating CSF3R depletion in NK cells promotes increased ability against tumors. To further evaluate the molecular mechanism of G-CSFR regulation of NK cell function, single cell RNA sequencing on bone marrow cells from both CSF3R△NK/NK mice and CSF3RWT/WT mice and NK cells were extracted for further subpopulation analysis. UMAP analysis revealed the clustering of bone marrow NK cells from both genotypes into ten subsets and on the basis of the key gene expression of NK cell effector molecules, transcriptional factors, maturation markers, and proliferation marker, we identified NK cell maturation program as mBM5+mBM8, mBM2+mBM3+ mBM6 and mBM0, by which immature NK cells should go through to gain optimal effector function, according to this model, CSF3R△NK/NK mice contained more NK cells belonging to the terminally mature mBM0 than CSF3RWT/WT mice. “Cell Killing” and “leukocyte mediated cytotoxicity” pathway was enriched in CSF3R△NK/NK mice, indicating enhanced activation state of NK cells. In conclusion, our results uncover a potent checkpoint in NK cells protecting against tumors, suggesting a promising approach of targeting G-CSFR for NK cell-based immunotherapies.

Expression of a Novel Chimeric Inhibitory Receptor Attenuates CD19 CAR T Cells: A Promising Strategy for Toxicity Mitigation

Introduction Chimeric antigen receptor (CAR) T cells targeting CD19 are highly effective in patients with refractory/relapsed acute lymphoblastic leukemia and lymphoma but often trigger severe cytokine release syndrome (sCRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). For r/rALL patients, as many as 77% develop Grade 3-5 CRS and 40% Grade 3-5 ICANS. sCRS occurs when hyperactivated CAR T cells undergo exponential expansion accompanied by the release of excessive amounts of inflammatory cytokines. This leads to a systemic pro-inflammatory state that can cause hypotension, multi-organ failure and even death. Tocilizumab is the only FDA-approved drug for CRS and despite concurrent corticosteroids, often fails to control CRS. Attempts to improve safety have been proposed, but have significant limitations, such as permanent removal of CARs. New and innovative methods to mitigate toxicities are needed. The purpose of this study was to design and test the efficacy of a novel autoregulatory element in the form of a chimeric inhibitory receptor (CIR), that when engaged by a specific CRS-associated pro-inflammatory molecule would trigger inhibitory signaling within CAR T cells. We hypothesize that as cytokine levels increase following activation, these signals can be redirected through responsiveness of the CIR, leading to an attenuation of CAR activity. In this study, we have tested a CIR construct that couples the recognition of IFNγ to the signaling domain of the TIGIT inhibitory receptor. We co-expressed this CIR with a CD19.28ζ CAR to evaluate its ability to attenuate activation and cytokine production and to assess its cytotoxic activity. Methods An anti-IFNγ scFv followed by the transmembrane and intracellular domains of TIGIT were arranged in sequence with the truncated EGFR receptor followed by the CD19.28ζ FMC63 CAR with intervening T2A cleavage domains and cloned into a retroviral backbone. Transduction efficiency was assessed by staining with biotinylated CD19-Fc protein or biotinylated EGFR. Following expansion, memory and effector cell differentiation was evaluated by staining for CD45RO, CD45RA, CCR7, CD127, and CD95. Activation following stimulation with NALM-6 tumor cells was measured by staining for CD69, CD25, CD38, PD-1, and CD107a. Cytotoxicity was evaluated using bioluminescence. To mimic cell types present during CRS, we utilized a coculture assay that consists of CAR T cells, NALM-6 tumor cells, and either macrophages or dendritic cells (DC) differentiated from autologous CD14 + monocytes. Cocultures were incubated for 48 hr. and supernatants were collected for analyses by Luminex. Results CD19 CARs co-expressing a CIR composed of an anti-IFNγ scFv coupled to TIGIT inhibitory signaling expanded during the manufacturing process as well as T cells expressing only CD19 CAR and displayed normal levels of CAR expression. The presence of the CIR did not affect the differentiation profile of memory and effector subsets. CIR-expressing CD19 CARs exhibited robust tumor cell killing of NALM-6 targets equivalent to CD19 CARs. Next, CD19 CAR/CIR cells were pretreated with IFNγ for 24 hr. and then cultured with NALM-6 cells. Exposure to IFNγ resulted in a significant abrogation in cytotoxicity, which was eventually restored with time. Activation profile analysis of CARs equipped with CIR signaling after target stimulation revealed an overall diminution of CARs expressing multiple (4 or 5) activation markers relative to CD19 CARs. Importantly, CIR signaling resulted in significant reductions in the levels of effector, stimulatory, and inflammatory mediators including IFNγ, TNFα, GM-CSF, and IL-17. CARs expressing the CIR exhibited slower killing kinetics when challenged with sequential stimulations by fresh tumor cells. However, in vivo anti-tumor cell activity was not adversely affected as CAR/CIR T cells robustly eliminated disease in leukemia-bearing NSG mice. Lastly, using an in vitro CRS assay, we sought to determine if attenuated CAR T cells would be less potent in activating myeloid cells. We observed that monocyte-derived macrophage and DC activation by CD19 CAR/CIR T cells was reduced as evidenced by decreased secretion of proinflammatory cytokines and chemokines. Conclusion These results provide a compelling proof of concept of a novel self-regulating CAR T cell design with the potential to mitigate severe toxicities in patients.

Immunological Footprints of Pediatric Acute Myeloid Leukemia in Local Bone Marrow T Cells

Introduction: Despite notable advancements, childhood acute myeloid leukemia (AML) remains associated with one of the poorest prognoses among pediatric malignancies. Conventional chemotherapies carry significant toxicity, underscoring the urgent need for novel e.g. immunotherapy-based approaches. Despite AML being previously considered non-immunogenic due to its low mutational burden, the complex interplay between leukemic blasts and T cells within the bone marrow in pediatric patients remains largely unexplored. This study investigates the AML-induced footprint on local bone marrow T cells (bmT cells) representing tumor-infiltrating lymphocytes, with the aim to reveal functional points of action for future immunotherapy. Methods: Cryopreserved bone marrow samples from both pediatric AML patients (n=29) and age-matched healthy bone marrow donors (HD, n=9) were analyzed. Multicolor flow cytometry quantified surface expression of inhibitory signaling and activation receptors, as well as T cell differentiation stages. RNA-Seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) were performed of sorted CD8 + bmT cells. Bulk RNA and ATAC-Seq data was visualized by principal component analysis (PCA). Another PCA was performed on the combined dataset of RNA- and ATAC-Seq. Differential analysis using DESeq2 (fitType “parametric”, testType “Wald”) identified 135 differentially expressed genes in CD8 + bmT cells between HD and AML patients based on fold change (≥1/≤-1) and statistical criteria (s-value<0.05). Results: Based on CD45RO, CD62L and CD95 surface expression, late differentiation stages of bmT cells were enriched in AML. Namely, the frequency of naïve bmT cells was reduced (33.9% vs. 57.5%, mean values, p=0.0018), while the frequency of effector memory bmT cells was increased in AML patients (21.5% vs. 9.4%, p=0.0010). This could be validated by RNA-Seq of sorted CD8 + bmT cells which documented higher expression of cytotoxic effector T cell genes such as Perforin, Granulysin and Granzyme B. In addition, our analysis also revealed higher expression of functional relevant genes associated with T cell cytotoxity (ADGRG1), effector function (BATF/TPX2) or exhaustion (SLAMF7) in AML patients. Next, surface expression of receptors for inhibitory signaling and activation was analyzed. Several of these receptors have also been described as immune checkpoints (ICP). Analyzing differential expression between HD and AML patients showed pronounced upregulation of inhibitory markers (TIM-3: 7.1% vs. 1.7%, p<0.0001, CD39: 7.7% vs. 2.0%, p=0.0103, LAG-3: 8.0% vs. 2.9%; CTLA-4: 3.9% vs. 1.6%, p=0.0024; PD-1: 34.8% vs. 21.3%, p=0.0016). Since CD8 + bmT cells showed the most promising effects in the flow cytometry analysis, this subpopulation of bmT cells was further characterized by RNA- and ATAC-Seq. Here, clustering of HD and AML patients' CD8 + bmT cells was observed in PCA. Interestingly, PCA also revealed differential clustering of CD8 + bmT cells from AML primary and relapse samples. Confirming the observed differences in phenotype of bmT cells in AML patients, we identified distinct bmT cell populations specifically induced in AML patients compared to HD, based on phenotype and differential ICP expression patterns. Through tSNE analysis, we discovered a CD8 + effector memory T cell population with a TIM-3 +PD-1 +2B4 + phenotype, comprising 2.3% of all bmT cells in AML samples compared to only 0.03% in HD. Conclusion: This study reveals significant alterations in the activation and inhibition potential of bmT cells in pediatric AML compared to HD, as confirmed by comprehensive analysis of the epigenome, transcriptome and surfaceome. We confirm the observed differences through the identification of a T cell population with a typically exhausted phenotype specific for AML patients when compared to HD, implying persistent antigen exposure and potential direct or indirect interactions with leukemic blasts. Although prospective clinical trials will have to confirm functional relevance in patient cohorts, this study provides crucial insights into the immune landscape of pediatric AML, underscoring the potential for targeted immunotherapeutic interventions.

Chimeric Antigen-Receptor (CAR)-Engineered Natural Killer (NK) Cells Targeting Chronic Myeloid Leukemia (CML) Blast Crisis

Background The use of tyrosine kinase inhibitors (TKIs) has dramatically modified the therapy of chronic myeloid leukemia (CML), generating durable remissions and prolonging survival in TKI-responders. However, progression to blast crisis (BC) still occurs especially in TKI-resistant patients and represents a clinical challenge. We and others have identified IL2RA/CD25 as a typical cell surface marker of BC-CML and reported that its overexpression is correlated with the progression of CML from CP-CML to BC-CML (Imeri et al, Cells 2023). Here we show the experimental development of a third-generation CAR-NK therapy strategy against the CD25 based on the scFV of the clinically approved monoclonal humanized antibody, Basiliximab. Methods: As NK cell model, we have used the NK92 cell line which has a well-established and clinically demonstrated NK cell activity. We have lentivirally transduced NK92 cells with the CAR construct containing a selectable gene (GFP). After FACS-sorting of GFP-positive cells, phenotypical characterization was performed by FACS. The expression of the CAR-CD25 at the surface of the cells was demonstrated an anti-Fab antibody and double-positive (Fab/GFP) cells were further purified. The functionality of the cells was evaluated using CD107a degranulation assay after 3h co-culture and ELISA for IFN-gamma release. We have in parallel engineered K562 cells expressing CD25 by lentiviral transduction (K562-CD25) as well as also a second target cell line (RAJI) using the same strategy. Annexin V staining of target K562 cells was used for in vitro cytotoxicity assessments. For in vivo assays, NSG mice were intraperitoneally (IP) injected with K562-CD25 cells expressing Luciferase at Day-3 (3.10 6 cells/mouse, n = 13). At Days 0, 3, and 7, mice were treated by IP injection of either irradiated CD25 CAR-NK92 cells (10 .10 6/mouse n=6) or irradiated Wild-type (WT)-NK92 cells (n=5). The clinical evolution of mice transplanted mice was followed weekly by luminescence (IVIS 200). Results: After cell sorting, we obtained more than 90% of double-positive NK92 CAR+/GFP+ cells. Lentiviral transduction did not affect the activatory or inhibitory signals of NK92 cells. No statistical differences were observed between CD25 CAR-NK92 and WT- NK92 cells for the expression of NKp30, NKp46, KirDl2-3, TIGIT, and DNAM. However, we observed a strong increase in the Granzyme B and Perforin in CD25 CAR-NK92 cells after co-culture with K562-CD25 as compared to WT NK92 cells (p<0.001). Importantly, we have found increased levels of degranulation after co-culture of target K562-CD25 with CD25-CAR-NK92 cells (40%) as compared to cells co-cultured with WT NK92 alone (20% ) suggesting strongly the occurrence of an additional specific effect due to CAR-CD25. IFN-gamma levels after co-culture of CAR CD25 NK92 cells were also found to be significantly increased ( 400 pg/ml) in as compared to co-cultures of target cells with WT-NK92 (200 pg/ml)(p<0.0001). Similarly,in vitro cytotoxicity assays showed induction of higher levels of apoptosis in target cells (K562-CD25 and Raji-CD25) when co-cultured with CD25 CAR-NK92 as compared to NK92 WT (p<0.0001). In in vivo experiments, we have analyzed K562-CD25 leukemia-bearing mice treated with CAR-NK92 cells (n=6) or WT-NK92 cells (n=5). These experiments analyzed at D+30 post-transplant showed stronger anti-leukemia effect of CAR-NK therapy by IVIS imaging with a survival rate of 84% for mice treated with CD25 CAR-NK92 versus 40% for those treated with WT-NK92. All control mice transplanted with K562-CD25 cells and left untreated died by D+20. Conclusion: We show here for the first time the potential use of an NK cell-mediated CAR therapy strategy targeting CD25 which has been shown to be upregulated in CML blast crisis. The experimental data show a significantly increased and selective in vitro and in vivo cytotoxicity of CD25 CAR-NK92 cells against CD25-expressing leukemia cells as compared to WT-NK92 cells. These results suggest that targeting CD25 by a CD25 CAR based on Basilixiamb's scFV might be an interesting tool in BC-CML and in all acute leukemias overexpressing CD25. In order to translate these findings to NK cells derived from induced pluripotent stem cells (iPSCs), we have produced iPSCs expressing CAR-CD25 constructs and experiments are underway to evaluate the therapeutic potential of iPSC-derived CD25 CAR-NK cells in CML blast crisis or AML models.

The HLA-B Leader MM Genotype Has a Negative Impact on Outcomes in HLA-B One Antigen Mismatched and HLA-B Matched Cord Blood Transplantation without Anti-Thymocyte Globulin: Analysis of the Japanese Society for Transplantation and Cellular Therapy

Background: The dimorphism at position -21 of exon 1 in HLA-B gives rise to leader peptides featuring either methionine (M) or threonine (T) as the second residue in the processed leader peptide (HLA-B leader). The cell surface abundance of HLA-E is positively correlated with the copy number of −21M HLA-B. In hematopoietic cell transplantation (HCT), the MM genotype in the recipients is associated with worse outcomes after HLA-matched and mismatched unrelated bone marrow and peripheral blood stem cell transplantation compared with TT (Kanaya M, et al. ASH Annual Meeting 2021, Petersdorf EW et al. Lancet Haematol. 2020 and Blood. 2020). Eurocord/EBMT study revealed HLA-B leader mismatch in HLA-B mismatched combination is associated with relapse and non-relapse mortality (NRM) risks after cord blood transplantation (CBT) (Petersdorf EW et al. Haematologica 2021). Notably, approximately 70% of patients in the literature administrated anti-thymocyte globulin (ATG) for graft-versus-host disease (GVHD) prophylaxis. The impacts of HLA-B leader dimorphism on the outcomes of CBT still needed to be elucidated more. Particularly, we were interested in the significance of HLA-B leaders in CBT without an ATG, then we conducted a retrospective study to elucidate it. Methods: Clinical data of 10,569 patients who underwent single-unit CBT for acute myeloid leukemia (AML, n=5,518), myelodysplastic syndrome (MDS, n=1,483), acute lymphoblastic leukemia (ALL, n=2,020), and lymphoma (n=1,548) between 2008 - 2021 were provided by the Japanese Data Center for Hematopoietic Cell Transplantation. HLA-A, -B, -C, and -DRB1 typing was performed at least at the antigen level. The leader genotype is defined based on the two HLA-B allotypes. All patients were not used ATG for GVHD prophylaxis. Study outcomes were overall survival (OS), cumulative incidences of relapse, NRM, and grade II-IV acute GVHD (aGVHD). Multivariable Cox regression analysis including covariables; disease risk index (DRI), myeloablative/reduced-intensity conditioning, presence of total body irradiation as a conditioning regimen, short-term methotrexate or mycophenolate mofetil for GVHD prophylaxis, recipient cytomegalovirus serostatus. All statistical analyses were performed with EZR. Results: The median patient's age was 53 (range 0-88) years. The numbers of patients with HLA-B leader TT, MT, and MM genotype were 7,639 (72.3 %), 2,707 (25.6 %), and 223 (2.1 %), respectively. To classify HLA-B match/mismatch status, we stratify “HLA-B one locus mismatched (GVH direction)” and HLA-B matched (no GVH direction mismatch)“. First, we focused on HLA-B one antigen mismatched (HLA 5/6 antigen matched, n = 1172) setting as shown in the previous literature. Recipient MM genotype showed significantly worse OS and relapse in the multivariate analysis compared to MT or TT genotype (Tx) (Figure) (OS: hazard ratio [HR] 0.543 [95% CI, 0.311 - 0.947]; p = 0.032, relapse: [HR] 2.863 [95% CI, 1.330 - 6.165]; p = 0.007). Recipient MM didn't relate to relapse and grade II-IV aGVHD. Next, we explored the impacts of HLA-B leaders in HLA-B antigen-matched CBT (3/6 - 6/6 matched, n = 3995). Donor MM genotype demonstrated a negative impact for OS and NRM, not relapse and aGVHD, compared to Tx donor (Figure) (OS: [HR] 0.738 [95% CI, 0.542 - 1.000]; p 0.050, NRM: [HR] 1.695 [95% CI, 1.097 - 2.621]; p = 0.018). Especially, OS and NRM of donor MM genotype were significantly inferior to Tx in HLA 6/6 matched (n = 914) CBT (OS: [HR] 0.311 [95% CI, 0.187 - 0.518]; p < 0.001, NRM: [HR] 3.216 [95% CI, 1.931 - 5.356]; p = 0.034). Conclusions: ATG would affect immune dynamics strongly in CBT via in-vivo T cell depletion, yet the current study demonstrated the HLA-B leader was still a significant risk factor in CBT without ATG for the first time. Recipient MM genotype in HLA-B one locus mismatched (HLA 5/6 matched) and donor MM genotype in HLA-B matched were the risk factors for worse CBT outcomes. We concluded MM genotype donors would be avoided to decrease NRM in HLA-B matched CBT. The precise mechanism of negative impact in CBT outcomes in patients or donor MM genotype remains to be clarified, however, we assume higher level HLA-E expression may be crucial in terms of NKG2A inhibitory signaling to T cells/NK cells and T-cell direct recognition for peptide/HLA-E complex in CBT.

A Multicenter, Open-Label, Phase 1b/2 Study to Evaluate the Effects of Maplirpacept in Combination with Tafasitamab and Lenalidomide in People with Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Background Maplirpacept (PF-07901801) is an anti-CD47 molecule designed to enhance phagocytosis and antitumor activity by preventing CD47-mediated inhibitory signaling. Maplirpacept currently is being evaluated in an ongoing, multi-center, two-part, Phase 1a/1b study in participants with advanced hematologic malignancies (NCT03530683). Early safety and efficacy results for maplirpacept monotherapy have previously been reported in patients with refractory or relapsed lymphomas (Patel et al. Blood 2021;138:3560). Study Design This trial is a multicenter, open-label, Phase 1b/2 study (NCT05626322), to evaluate the effects of maplirpacept, in combination with tafasitamab and lenalidomide in participants with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). The estimated enrollment is 70 patients, and the study will be split into two parts (Figure 1). Phase 1b will determine the maximum tolerated dose or recommended Phase 2 dose (2 dose levels) of maplirpacept, in combination with approved doses of tafasitamab and lenalidomide. In Phase 2, participants will be randomized to high or low doses of maplirpacept, in combination with approved doses of tafasitamab and lenalidomide, as per the Oncology Center of Excellence ‘Project Optimus’ initiative for dose optimization and selection in oncology drug development. Study interventions will be administered in 28-day cycles. Maplirpacept will be given weekly, by intravenous (IV) infusion, for the first three cycles and subsequently every two weeks. Tafasitamab will be administered, by IV infusion, on Days 1, 4, 8, 15 and 22 in cycle 1, weekly in cycles 2 and 3, then every 2 weeks in cycle 4 and beyond. Lenalidomide will be taken orally, daily for Days 1 to 21 of each 28-day cycle, for the first 12 cycles. In Phase 1b, approximately 20 participants will be allocated to sequential doses of maplirpacept, co-administered with approved doses of tafasitamab and lenalidomide, to select doses for further assessment in Phase 2. The primary endpoint for Phase 1b is to assess dose limiting toxicities up to 28 days following the first dose. In Phase 2, approximately 50 participants will be randomized to 1 of 2 different doses of maplirpacept, co-administered with tafasitamab and lenalidomide. The primary endpoint for Phase 2 is objective response as per the Lugano Response Classification Criteria 2014. Secondary endpoints are frequency of adverse events, frequency of clinical laboratory abnormalities, objective response (Phase 1b only), complete response, duration of response, duration of complete response, progression free survival, maplirpacept, tafasitamab and lenalidomide PKs, and immunogenicity of maplirpacept and tafasitamab. Inclusion criteria (Table 1) are adults with histologically confirmed, measurable relapsed and/or refractory DLBCL not otherwise specified, T cell/histiocyte rich large B-cell lymphoma, or Epstein-Barr virus positive DLBCL according to the 5th edition of the WHO classification of lymphoid neoplasms. Additionally, participants with follicular lymphoma grade 3b, or composite lymphoma with a DLBCL component with a subsequent DLBCL relapse are also eligible. Participants with evidence of histological transformation to DLBCL from an earlier diagnosis of low-grade lymphoma are eligible if relapsed or refractory to at least one line of therapy for DLBCL. Further inclusion criteria are participants who are not a candidates for, or are unwilling to receive, high-dose chemotherapy and subsequent stem cell transplant and/or are unable to undergo chimeric antigen receptor T-cell (CAR-T) therapy (e.g. due to logistical limitations), treatment with at ≥1 prior line of systemic therapy (for Phase 2, at least 1, and no more than 2, prior lines of systemic therapy) and prior therapy must include an anti-CD20 antibody. Participants must have adequate bone marrow, renal and hepatic function and an Eastern Cooperative Oncology Group performance status ≤2. Participants must provide a baseline tumor tissue sample (fresh or archival), for biomarker analysis. Exclusion criteria include previous treatment with anti-CD47, anti-CD19 (other than CAR-T) or immunomodulatory agents, prior allogeneic stem cell transplantation or autologous stem cell transplantation within 12 weeks prior to enrolment, and participants with active, uncontrolled viral, bacterial or fungal infections.

H2-K1 on MLL-AF9 Leukemia Cells Facilitates the Escape of NK Cell-Mediated Immune Surveillance

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, and prognosis is poor; 5-year survival approximately 30%. Recently, there is an emerging recognition of the innate immune system, in particular Natural killer (NK) cells and macrophages, for immune surveillance against AML, but the mechanistic basis for this is mostly unknow. Identifying how AML cells escape immune surveillance by NK cells may translate into new treatment opportunities for AML patients. To identify new therapeutic opportunities, we recently performed an in vivo CRISPR/Cas9 screen targeting 961 cell surface genes using the MLL-AF9 AML mouse model. One of the top leukemia stem cell dependencies in the bone marrow niche was H2-K1, an ortholog of human HLA-A, a classical MHC class-I molecule. In validation experiments, we observed a seven-fold depletion (p<0.001) of H2-K1 sgRNA-expressing c-Kit +Cas9 + leukemia cells in the bone marrow. In contrast, genetic disruption of H2-K1 did not impact the growth and survival of MLL-AF9 leukemia cells in culture. Given the known suppressive role of MHC class-I molecules for immune cells, we speculated that H2-K1 mayprovide inhibitory signals that counteract immune-surveillance mechanisms in the bone marrow niche. To test this hypothesis, we depleted NK cells and macrophages prior to transplantation of the c-Kit + MLL-AF9 leukemia cells. Macrophage depletion by clodronate liposomes did not affect the in vivo expansion of H2-K1 sgRNA-expressing leukemia cells demonstrating that macrophages were not suppressed by H2-K1 on the leukemia cells. In contrast, NK1.1 antibody-mediated depletion of NK cells fully rescued the depletion of H2-K1 sgRNA-expressing leukemia cells in vivo. These findings suggest that H2-K1 expression on MLL-AF9 leukemia cells inhibits NK cells in this model. Consistent with these findings, H2-K1 knockdown in leukemia cells triggered a two-fold increase of INFγ production (p<0.01) in the NK cells, accompanied by augmented apoptosis of the leukemia cells (p<0.01). Given that NK cells have been shown to be dysfunctional in AML patients, we next explored whether leukemia development affects NK cell maturation. The expansion of leukemia cells in the mice skewed NK cells towards a M1 (CD27 +CD11b -) state and decreased the level of the more cytotoxic M2 (CD27 +CD11b +) and M3 (CD27 -CD11b +) populations. H2-K1 disruption in the leukemic cells restored the level of M2 and M3 NK cell population in the bone marrow. Notably, restoration of matured NK cell populations was accompanied by an increased expression of NKG2D, an activating receptor, indicating a more cytotoxic state of the NK cells. In line with these findings, ablation of H2-K1 in leukemia cells induced JAK/STAT and NF-κβ signaling in the NK cells. Taken together, our study identifies that H2-K1 on MLL-AF9 leukemia stem cells facilitates immune evasion by suppressing NK cells. H2-K1 alters the maturation and activation of NK cells in the bone marrow niche. These findings increase our understanding of how leukemia cells escape immune surveillance and suggest that the identification of corresponding mechanisms in human AML could pave the way for new therapies that boost the endogenous NK cells by restoring immune surveillance mechanisms.

Phase I Study of Response-Adapted Treatment with Venetoclax, Obinutuzumab, and Magrolimab (VENOM) in Relapsed and Refractory Indolent B-Cell Malignancies

Background and Significance Targeted agents and immunotherapy have changed the treatment landscape for follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and chronic lymphocytic leukemia (CLL), but indefinite therapy leads to cumulative toxicity and excessive cost. CD47 is a checkpoint for the innate immune system providing an inhibitory signal to phagocytic macrophages and a rational therapeutic target in B-cell lymphoma. Magrolimab is a fully humanized IgG4 anti-human CD47 antibody, with high activity in FL when combined with rituximab (Advani NEJM 2018). Our objective is to test the safety of adding escalating doses of venetoclax to magrolimab and obinutuzumab in a response-adapted study design with planned dose expansion cohorts (www.clinicaltrials.govidentifier NCT04599634). Study Design and Methods Patients with grade 1-3A FL, MCL, MZL, or CLL relapsed after and/or refractory to ≥2 prior lines of therapy are eligible. Pts with FL are eligible after 1 prior therapy if FLIPI ≥2 or disease progression within 24 months of the end of the last therapy. Pts with MCL are eligible after 1 prior therapy if blastoid histology, 17p deletion, TP53 mutation, Ki67 ≥30%, or received Bruton's tyrosine kinase inhibitor (BTKi) as first-line therapy. Pts with CLL are eligible after 1 prior therapy if 17p deletion, TP53 mutation, or received both a BTKi and a BCL2 inhibitor. Eligibility includes age ≥18 and adequate organ function unless dysfunction secondary to lymphoma. A positive direct antiglobulin test without hemolytic anemia is allowed. Patients with a history of hemolytic anemia or autoimmune thrombocytopenia within 3 months of enrollment are excluded. No washout period is required for prior anti-lymphoma treatment. Active HIV, CMV, and hepatitis B or C are excluded. The safety of venetoclax, obinutuzumab, and magrolimab will first be determined in 2 dose-finding cohorts of up to 24 pts (Cohort 1: 6-12 pts with FL and Cohort 2: 6-12 pts with MZL, MCL, or CLL). During dose-finding, all pts start triplet therapy with venetoclax, obinutuzumab, and magrolimab (Figure 1). Magrolimab IV is administered at a 1mg/kg priming dose on D1 of the first cycle, followed by 30mg/kg x 3 weekly loading doses, then 30 mg/kg on D1 of each 4-wk cycle. Obinutuzumab IV is administered at 100mg on D1, 900mg on D2, and 1000mg on D8 and D15 of the first cycle, followed by 1000mg on D1 of each 4-wk cycle. Pts with FL receive venetoclax 800mg (DL1) daily throughout therapy duration, whereas pts with MZL, MCL, and CLL start venetoclax at 20mg with weekly escalation to 400mg (DL1) over 5 weeks. Dose-limiting toxicity is assessed during the first 4 wks for FL, and the first 5 wks for MZL, MCL, and CLL. If ≥2 of 6 pts in either arm experience DLT, 6 additional pts will be enrolled to the respective arm at DL(-1) of venetoclax (600mg for FL and 200mg for MZL, MCL, and CLL). After dose-finding is completed, expansion cohorts of each histology will first receive magrolimab and obinutuzumab for two 4-wk cycles in a window for translational research. On-treatment tumor biopsies are optional during the window. After the window, venetoclax will be added at the dose determined during dose-finding. FDG-PET and CT scans are performed at baseline, after window, and after cycles 3 and 6 of triplet therapy. During both dose-finding and dose-escalation, pts with complete response (CR) after 6 cycles of triplet therapy will stop therapy. If these pts relapse, they can be retreated with an additional 6 cycles of triplet therapy. Pts with partial response (PR) after 6 cycles of triplet therapy receive an additional 6 cycles of triplet therapy. All pts stop treatment after 12 cycles. The primary objective is safety, with secondary endpoints of overall response rate, duration of response, and PFS. Exploratory objectives include the identification of a molecular signature that predicts response to magrolimab and obinutuzumab, and correlation of response with circulating tumor DNA levels.

Radiation and Immune Checkpoint Inhibitors: Combination Therapy for Treatment of Hepatocellular Carcinoma.

The liver tumor immune microenvironment has been thought to possess a critical role in the development and progression of hepatocellular carcinoma (HCC). Despite the approval of immune checkpoint inhibitors (ICIs), such as programmed cell death receptor 1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte associated protein 4 (CTLA-4) inhibitors, for several types of cancers, including HCC, liver metastases have shown evidence of resistance or poor response to immunotherapies. Radiation therapy (RT) has displayed evidence of immunosuppressive effects through the upregulation of immune checkpoint molecules post-treatment. However, it was revealed that the limitations of ICIs can be overcome through the use of RT, as it can reshape the liver immune microenvironment. Moreover, ICIs are able to overcome the RT-induced inhibitory signals, effectively restoring anti-tumor activity. Owing to the synergetic effect believed to arise from the combination of ICIs with RT, several clinical trials are currently ongoing to assess the efficacy and safety of this treatment for patients with HCC.

A synaptic corollary discharge signal suppresses midbrain visual processing during saccade-like locomotion

In motor control, the brain not only sends motor commands to the periphery, but also generates concurrent internal signals known as corollary discharge (CD) that influence sensory information processing around the time of movement. CD signals are important for identifying sensory input arising from self-motion and to compensate for it, but the underlying mechanisms remain unclear. Using whole-cell patch clamp recordings from neurons in the zebrafish optic tectum, we discovered an inhibitory synaptic signal, temporally locked to spontaneous and visually driven locomotion. This motor-related inhibition was appropriately timed to counteract visually driven excitatory input arising from the fish’s own motion, and transiently suppressed tectal spiking activity. High-resolution calcium imaging revealed localized motor-related signals in the tectal neuropil and the upstream torus longitudinalis, suggesting that CD enters the tectum via this pathway. Together, our results show how visual processing is suppressed during self-motion by motor-related phasic inhibition. This may help explain perceptual saccadic suppression observed in many species.

Dual-inhibitory domain iCARs improve the efficiency of the AND-NOT gate CAR T strategy.

CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

Trial in Progress: Phase 1 Trial Evaluating the Safety and Tolerability of Odronextamab in Combination with Cemiplimab in Relapsed/Refractory Aggressive B-Cell Non-Hodgkin Lymphoma

Background and Significance CD20×CD3 bispecific antibodies (bsAbs) have emerged as an important immunotherapeutic approach for relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (B-NHL). These agents have shown compelling efficacy in indolent B-NHL, with the CD20×CD3 bsAb odronextamab demonstrating 75% complete response (CR) rates and encouraging durability of responses (median duration of response [DoR], 20.5 months) in heavily pretreated patients with R/R follicular lymphoma in the ELM-2 study (Kim TM, et al. ASH. 2022). In comparison, patients with R/R diffuse large B-cell lymphoma who were treated with odronextamab in the same study demonstrated a CR rate of 31%, and median DoR was 10.2 months (Kim WS, et al. ASH. 2022). These data indicate an unmet need for treatment strategies that can improve the depth and durability of responses in patients with aggressive R/R B-NHL. Combination therapy with drugs that complement the mechanism of action of CD20×CD3 bsAbs may provide an opportunity to address this unmet need. Cemiplimab is a human immunoglobulin G4 (IgG4) monoclonal antibody directed against programmed cell death protein 1 (PD-1), blocking PD-1- and programmed cell death ligand 1 (PD-L1)-driven inhibitory T-cell signaling. Combining odronextamab and cemiplimab provides an attractive therapeutic opportunity to improve anti-tumor efficacy while restricting T-cell exhaustion, and preliminary results for this combination provided proof of concept, with evidence of clinical activity and manageable tolerability in patients with B-lymphoid malignancies (n=12; Topp MS, et al. ASH. 2017). Here, we investigate whether the addition of cemiplimab to odronextamab can improve the depth and durability of response in patients with previously treated aggressive B-NHL. Study Design and Methods This is a Phase 1, open-label, multicenter, dose-escalation and -expansion study that will take place at up to 28 sites globally (NCT02651662). Intravenous (IV) odronextamab is administered in 21-day cycles, initially as monotherapy and with step-up dosing during Cycle (C) 1 to mitigate the risk of cytokine release syndrome (Figure). Full-dose odronextamab is administered on Day (D) 1, D8 and D15 of C2-C4, and then on D15 of each cycle during maintenance (C5 onwards) until disease progression or treatment discontinuation. IV cemiplimab starts on C2D15 at the specified dose level (DL1-DL7) and is administered on D15 of subsequent cycles. The dose-limiting toxicity (DLT) period is 28 days from the start of cemiplimab administration. At the end of dose escalation, a regimen will be selected for further investigation in dose expansion. Approximately 60 patients will be enrolled. Patients will be aged ≥18 years with aggressive CD20+ B-NHL that is R/R after ≥2 lines of systemic therapy containing an anti-CD20 antibody and an alkylating agent. Patients must have measurable disease, Eastern Cooperative Oncology Group performance score (ECOG PS) ≤1, and adequate bone marrow and organ function. Exclusion criteria include primary central nervous system lymphoma, prior allogeneic stem cell transplantation, autoimmune disease, prior adverse events from anti-PD-1/PD-L1 therapy, and uncontrolled infections. Primary endpoints are safety, tolerability, DLTs, and to determine the recommended Phase 2 dose of the combination regimen of odronextamab and cemiplimab. Secondary endpoints include pharmacokinetics, immunogenicity, and preliminary anti-tumor activity (overall response rate, CR rate, and DoR, according to the Lugano classification). There are no formal hypotheses for this study and analyses will be descriptive in nature.

Modakafusp Alfa Demonstrates Type I Interferon-Mediated Innate and Adaptive Immune Enhancement in a Phase 1/2 Study in Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Modakafusp alfa (moda), a novel immunocytokine, is an innate immunity enhancer comprising two attenuated interferon (IFN) α2b molecules fused to an anti-CD38 IgG4 monoclonal antibody backbone, driving preferential IFNα signalling in CD38-expressing innate and adaptive immune cells as well as myeloma cells. Here, we present pharmacodynamic (PD) data from the first-in-human Phase 1/2 study of moda as a monotherapy in patients with RRMM (iinnovate-1; NCT03215030). A total of 37 patients treated with moda at 1.5 or 3 mg/kg every 4 weeks were included in the analysis. Peripheral blood (PB) and bone marrow (BM) samples were collected during screening or pre-dose on cycle 1 day 1 (C1D1) and at multiple timepoints after treatment. BM and PB samples were evaluated by cytometry time of flight (CyTOF) and bulk RNA sequencing (RNAseq) to assess pharmacodynamic changes in immune cell populations, cell activation, and gene expression. Administration of moda led to enhanced innate immune cell activation and cytotoxic function as demonstrated by increased proportions of CD69+ and granzyme B+ in peripheral NK cells analyzed by CyTOF. This was accompanied by a decrease in the proportion of TIGIT+ NK cells, indicating reduced inhibitory signaling. Furthermore, enhanced proliferation (%Ki67+) of NK cells was observed in both BM and PB. Moda also impacted myeloid cell populations in BM and PB. Dendritic cells (DCs) and monocytes in PB showed increased surface expression of the co-stimulatory molecule CD86, suggesting a potential for enhanced antigen presentation and co-stimulation ability. Increased proliferation (%Ki67) of DCs and monocytes was detected in both PB and BM, accompanied by a decrease in numbers of peripheral DCs which may indicate recruitment or homing to secondary lymphoid organs. Additionally, RNAseq analysis revealed that treatment with moda led to upregulation of CD68 expression in both PB and BM, indicating a proinflammatory M1 macrophage type response. Finally, CyTOF analysis also indicated that moda significantly impacted adaptive immunity. We observed enhanced activation (%CD69, %PD1 and %CD40L) and cytotoxic function (%granzyme B) of peripheral CD8 T cells, and increased CD8 T cell proliferation (%Ki67 CD8) in both PB and BM samples. Enhanced activation of peripheral CD4 T cells (%CD69, %CD40L) was also detected. Consistent with activation of the adaptive immune system, there was a reduction in naïve CD8 T cells accompanied by an increase in CD8 T effector memory cells in PB. Analysis of exhaustion markers (TIGIT, PD-1) in cycle 1 (C1D8, C1D15, and C2D1 predose) showed that moda did not induce CD8 T cell exhaustion in either PB or BM, and there was no change in FoxP3+ Treg cells. Moda-induced innate and adaptive immune cell phenotypic changes were evaluated for correlation with clinical response at timepoints with sufficient number of evaluable samples. While none of the associations were statistically significant after correcting for multiplicity testing (using the false discovery rate method), we observed trends warranting further investigation. There was a greater increase of CD40L+ NK cells and PD1+ CD8 cells in responders vs. non-responders at C1D2. Additionally, at C1D15 responders showed a lower increase in proliferation (%Ki67+) of classical monocytes, NK cells, and myeloid DCs in PBas compared to non-responders. A similar trend was also observed in the BM classical monocytes. Even though the peak of Ki67 expression was observed at C1D8, diminished increases of proliferating cells at C1D15 in responders was a surprising observation that needs further consideration. The PD biomarker data from this first-in-human clinical trial demonstrated that treatment with moda enhances both innate and adaptive immune cell activation in PB and BM. Importantly, moda-mediated immune activation did not result in T cell exhaustion during cycle 1, as evaluated in both PB and BM. We have observed interesting trends of immune phenotypic changes that may correlate with response. Follow-up analyses of the correlation of biomarkers with clinical response are in progress and will be presented. Further clinical trials are underway (iinnovate-2, NCT05556616; iinnovate-3, NCT05590377) to evaluate moda's novel immune activating mechanism in combination with standard of care anti-myeloma therapies.

CD58 Genetic Alterations and Its Contribution to Upregulation of PD-L1 and IDO Via LYN/CD22/SHP1 Axis in DLBCL

Abstract Background: Recurrent abnormalities of immune surveillance-related genes play a crucial role in DLBCL progression. Prior studies have shown that CD58, a key adhesion molecule that acts as a ligand for the T-cell costimulatory molecule CD2, is frequently mutated or deleted in certain hematological malignancies. Aberrant expression of CD58 may result in tumor cells evading immune surveillance mechanisms mediated by T/NK cells. Downregulation or loss of CD58 is linked to resistance to ICB therapy in melanoma and CAR-T therapy in B-cell malignancies. Nevertheless, the role of CD58 in cancer is not yet well understood. Thus, the aim of this study was to comprehensively characterize CD58 genetic alterations in DLBCL, unveil the multiple roles of CD58 in anti-tumor immunity, and provide insights for potential therapeutic strategies. Methods: Comprehensive analysis of the genetic characteristics of CD58 were performed through targeted deep sequencing (n=176), whole exome sequencing (n=38), and RNA-sequencing (n=162) in patients with de novo DLBCL. To investigate the mechanistic impacts of CD58 alterations on co-inhibitory molecules expression and immune cell function, we performed bulk and single-cell RNA-sequencing analysis of tumor samples and conducted co-IP, flow cytometry and co-culture assays in vitro. Results: We identified that CD58 mutagenesis rate was 9.1%, with 88.9% of the identified mutations resulting in truncated protein or impairment of its normal function. 61.1% mutations occurred in exon 2, which may disrupt CD58-CD2 binding. Co-mutation analysis revealed higher frequencies of KMT2D, CD79B and MYD88 mutations in patients with CD58 mutations(p < 0.05) (Fig 1A). The CD58 copy number loss rate was 44.7% (Fig 1B). Both mutations and copy number loss led to a mild decrease in mRNA expression of CD58. Patients with CD58 mutation, copy number loss or low expression exhibited lower complete response rates following first-line R-CHOP therapy, as well as significantly poorer PFS and OS. To reveal the profound impact of CD58 on the immune microenvironment of DLBCL, we performed bulk and single-cell RNA sequencing and revealed that increased NK cells, activated CD4 +/CD8 + T cells, as well as the weaker exhaustion status of CD8 + T cells, correlated with CD58 high expression. By co-culturing assays, we identified that CD58 downregulation or mutation in DLBCL cells suppressed T cell proliferation and CAR-T cell-mediated killing. Interestingly, blocking CD58-CD2 signaling with a CD2 antibody did not fully eliminate the inhibitory effect, indicating that CD58 alterations could trigger additional immunosuppressive mechanisms. We further revealed that CD58 downregulation or mutation in DLBCL cells resulted in elevated expression of PD-L1 and IDO. This suggested that CD58 may have a dual effect on T cells, whereby its downregulation or mutation may lead to a lack of CD2 co-stimulatory signaling, as well as an increase in PD-L1 and IDO inhibitory signaling. We found that CD58 downregulation or mutation caused JAK2/STAT1 phosphorylation, which in turn promoted PD-L1 and IDO expression. Co-IP identified that CD58 can interact with Lyn, which phosphorylated ITIM of the inhibitory receptor CD22, leading to recruitment and activation of the inhibitory phosphatase SHP1. CD58 downregulation suppressed the negative regulation of the LYN/CD22/SHP1 axis, thereby promoting the activation of JAK2/STAT1 and upregulating PD-L1 and IDO expression. To overcome the dual effects induced by CD58 alterations, anti-PD-L1 antibody or IDO inhibitor and recombinant CD58-Fc chimera protein were added alone or in combination to the co-culture cells. We showed that the combination regimens reversed the inhibitory effect of CD58-downregulated or mutated DLBCL cells on T cell proliferation and restored sensitivity to CAR-T cell-mediated killing (Fig 1C, D). Conclusions: Our study comprehensively characterized CD58 genetic alterations in DLBCL. We demonstrated that CD58 downregulation or mutation led to upregulation of PD-L1 and IDO expression mainly by regulating the LYN/CD22/SHP1 axis. Moreover, we explored strategies to directly activate CD2 co-stimulatory signaling or in combination with immune checkpoint inhibitors to address the diverse effects of CD58 alterations. Our findings provide novel insights for individualized therapy for DLBCL patients with CD58 mutation or deletion.

The Relationship Between PD-1(rs2227981) and PD-L1(rs2890658) Polymorphisms and Urothelial Cell Carcinoma.

Background Urothelial cell carcinoma, which is believed to develop from the urothelium (transitional epithelium), is the most common aggressive tumor and accounts for the ten most prevalent cancers in the world. The risk factors for urothelial cell carcinoma are aging, smoking, gender, and genetic alternations. Programmed cell death1 (PD-1) has been widely described as a negative regulator of T-cells by sending inhibitory signals to the T-cell. Through PD-1 binding with PD-L1 (ligand for PD-1), an inhibitory signal is propagated to the T cell. The polymorphisms of PD-1 and PD-L1 lead to an efficient T-cell response and affect an anti-tumor reaction. The polymorphisms of PD-1 and PD-L1 could also affect the carcinogenesis of human cancer, including urothelial cell carcinoma. Therefore, in this study, we evaluated the relation between PD-1(rs2227981) and PD-L1(rs2890658) polymorphisms and the carcinogenesis of urothelial cell carcinoma. Materials and methods This study was conducted using 211 healthy controls and 256 cases of urothelial cell carcinoma among the Japanese population. The DNA samples were extracted from the peripheral white blood cells of each subject. The genotype was detected by using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. Results C/T (Adjusted OR 1.55, 95% CI:1.02-2.35) and C/T+T/T (OR 1.46, 95% CI:1.01-2.12) genotypes of PD-1 rs2227981 were significant and risk factors for urothelial cancer.Male with A/A genotype in PD-L1 and CT genotype in PD-1 has a significant higher risk factor compared with other genotypes (Adjusted OR 1.83, 95% CI:1.05-3.21). Conclusions and discussion We found that C/T(PD-1) and "A/A (PD-L1) and C/T(PD-1)" were predominant in urothelial cell carcinoma cases. This indicates that C/T(PD-1) and "A/A (PD-L1) and C/T(PD-1)" genotypes could increase susceptibility to urothelial cell carcinoma. However, since our findings indicated that the effects of PD-1 and PD-L1 polymorphisms included discrepancies, additional research will be needed to evaluate the relationship between human cancer and PD-1 and PD-L1 polymorphisms. This is the first study that seeks to find the relation between PD-1(rs2227981) and PD-L1(rs2890658) polymorphisms concerning urothelial cell carcinoma among the Japanese population.

Constitutive Interleukin-7 Cytokine Signaling Enhances the Persistence of Epstein-Barr Virus-Specific T-Cells.

The efficacy of therapeutic T-cells is limited by a lack of positive signals and excess inhibitory signaling in tumor microenvironments. We previously showed that a constitutively active IL7 receptor (C7R) enhanced the persistence, expansion, and anti-tumor activity of T-cells expressing chimeric antigen receptors (CARs), and C7R-modified GD2.CAR T-cells are currently undergoing clinical trials. To determine if the C7R could also enhance the activity of T-cells recognizing tumors via their native T-cell receptors (TCRs), we evaluated its effects in Epstein-Barr virus (EBV)-specific T-cells (EBVSTs) that have produced clinical benefits in patients with EBV-associated malignancies. EBVSTs were generated by stimulation of peripheral blood T-cells with overlapping peptide libraries spanning the EBV lymphoma antigens, LMP1, LMP2, and EBNA 1, followed by retroviral vector transduction to express the C7R. The C7R increased STAT5 signaling in EBVSTs and enhanced their expansion over 30 days of culture in the presence or absence of exogenous cytokines. C7R-EBVSTs maintained EBV antigen specificity but were dependent on TCR stimulation for continued expansion. C7R-EBVSTs produced more rapid lymphoma control in a murine xenograft model than unmodified EBVSTs and persisted for longer. The findings have led to a clinical trial, evaluating C7R-EBVSTs for the treatment of refractory or relapsed EBV-positive lymphoma (NCT04664179).

TIGIT contributes to the regulation of 4-1BB and does not define NK cell dysfunction in glioblastoma

TIGIT is a receptor on human natural killer (NK) cells. Here, we report that TIGIT does not spontaneously induce inhibition of NK cells in glioblastoma (GBM), but rather acts as a decoy-like receptor, by usurping binding partners and regulating expression of NK activating ligands and receptors. Our data show that in GBM patients, one of the underpinnings of unresponsiveness to TIGIT blockade is that by targeting TIGIT, NK cells do not lose an inhibitory signal, but gains the potential for new interactions with other, shared, TIGIT ligands. Therefore, TIGIT does not define NK cell dysfunction in GBM. Further, in GBM, TIGIT+ NK cells are hyperfunctional. In addition, we discovered that 4-1BB correlates with TIGIT expression, the agonism of which contributes to TIGIT immunotherapy. Overall, our data suggest that in GBM, TIGIT acts as a regulator of a complex network, and provide new clues about its use as an immunotherapeutic target.