Lymphocyte Activation Gene-3 Research Articles

Analysis and Investigation of Bioinformatics and Epigenetics Reveal the Underlying Mechanisms by which FLOT2 Modulates the Progression of Diffuse Large B-cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) poses a significant threat to the quality of life for people worldwide. Regrettably, effective treatment strategies for this disease remain elusive in clinical practice due to the unclear understanding of its molecular mechanisms. Therefore, this study was devised to address these issues and identify novel diagnostic, therapeutic, and prognostic biomarkers for DLBCL. Gene expression and clinical data for DLBCL patients were retrieved from The Cancer Genome Atlas (TCGA) database, and relevant clinical data, tumor mutational burden (TMB), and gene expression levels were extracted. Bioinformatics analysis was conducted to screen for differentially expressed genes (DEGs). The prognostic significance of flotillin-2 (FLOT2) was assessed using Kaplan-Meier survival analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses were employed to evaluate mRNA and protein levels of the genes. Cell proliferation, apoptosis, and invasion were assessed using cell counting kit-8 (CCK-8) assay, flow cytometry analysis, and Transwell assay, respectively. Our bioinformatics analysis revealed that FLOT2 was significantly overexpressed in DLBCL tissues compared to normal tissues, a finding corroborated by subsequent immunohistochemistry staining, qRT-PCR, and Western blot analyses. To elucidate its biological functions, shRNAs targeting FLOT2 were transfected into DLBCL cell lines (LY-3 and U2932), resulting in suppressed cell proliferation and invasion, while promoting apoptosis. Furthermore, a positive correlation between TMB and FLOT2 expression in DLBCL was observed. Subsequently, quanTIseq was utilized to calculate the immune score and assess FLOT2 gene expression. In DLBCL, FLOT2 gene expression was found to be associated with T cell CD4+ (non-regulatory) (p < 0.01), monocytes (p < 0.05), and uncharacterized cells (p < 0.05). Regarding immune checkpoint markers, including the cluster of differentiation 274 (CD274), cytotoxic T lymphocyte-associated antigen-4 (CTLA4), hepatitis A virus cellular receptor 2 (HAVCR2), lymphocyte activation gene-3 (LAG3), programmed cell death protein 1 (PDCD1), programmed cell death 1 ligand 2 (PDCD1LG2), Siglec-15 (SIGLEC15), and T cell immunoreceptor with Ig and ITIM domains (TIGIT), our analysis indicated that in DLBCL, FLOT2 exhibited a relationship only with TIGIT (p < 0.05). In summary, FLOT2 functions as an oncogene and is linked to DLBCL prognosis and the tumor microenvironment. Targeting FLOT2 deletion emerges as a novel strategy to impede DLBCL aggressiveness by inhibiting cell proliferation and invasion, ultimately inducing apoptotic cell death.

Multiple myeloma and the potential of new checkpoint inhibitors for immunotherapy

Multiple myeloma (MM), a cancer of the bone marrow, is categorized as the second most common hematological malignancy of adults in the Western world. Despite dramatic improvements in immunotherapies in the field of cancers, MM immunotherapy has not been promising until now. Recent clinical studies of immune checkpoint inhibitor therapy, either alone or in combination with anticancer drugs, showed excessive side effects or low efficacy, particularly in advanced MM patients. In this context, lymphocyte levels of exhaustion markers play a pivotal role in the MM tumor microenvironment (TME). Hence in the present review, the mechanisms relevant to MM of five inhibitory molecules including T-cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell immunoglobulin, and mucin domain 3 (Tim-3), lymphocyte activation gene-3 (LAG-3), V-domain Ig Suppressor of T-cell activation and killer immunoglobulin-like receptors along with bispecific T-cell antibodies (BsAbs) will be discussed. Further, we summarized the underlying biology of these checkpoints in cancer and their rapidly emerging role in pathways in MM along with presenting recent clinical trials in context.

Immune checkpoint inhibitors and cancer immunotherapy by aptamers: an overview.

Efforts in cancer immunotherapy aim to counteract evasion mechanisms and stimulate the immune system to recognise and attack cancer cells effectively. Combination therapies that target multiple aspects of immune evasion are being investigated to enhance the overall efficacy of cancer immunotherapy. PD-1 (Programmed Cell Death Protein 1), CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4), LAG-3 (Lymphocyte-Activation Gene 3), and TIM-3 (T Cell Immunoglobulin and Mucin Domain-Containing Protein3) are all immune checkpoint receptors that play crucial roles in regulating the immune response and maintaining self-tolerance often exploited by cancer cells to evade immune surveillance. Antibodies targeted against immune checkpoint inhibitors such as anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab), anti-CTLA-4 antibodies (e.g., Ipilimumab), and experimental drugs targeting LAG-3 and TIM-3, aim to block these interactions and unleash the immune system's ability to recognise and destroy cancer cells. The US FDA has approved different categories of immune checkpoint inhibitors that have been utilised successfully in some patients with metastatic melanoma, renal cell carcinoma, head and neck cancers, and non-small lung cancer. Although several immune checkpoint inhibitor antibodies have been developed, they exhibited immune-related adverse effects, resulting in hypophysitis, diabetes, and neurological issues. These adverse effects of antibodies can be reduced by developing aptamer against the target. Aptamers offer several advantages over traditional antibodies, such as improved specificity, reduced immunogenicity, and flexible design for reduced adverse effects that specifically target and block protein-protein or receptor-ligand interactions involved in immune checkpoint pathways. The current study aims to review the function of particular immune checkpoint inhibitors along with developed aptamer-mediated antitumor cytotoxicity in cancer treatment.

FGL1 in plasma extracellular vesicles is correlated with clinical stage of lung adenocarcinoma and anti-PD-L1 response.

Fibrinogen-like protein-1 (FGL1) is confirmed a major ligand of lymphocyte activation gene-3 which could inhibit antigen-mediated T-cell response and evade immune supervision. Although hepatocytes secrete large amounts of FGL1, its high expression also be detected in solid tumors such as lung cancer, leading to a poor efficacy of immune checkpoint inhibitors therapy. Here we reported that FGL1 was overexpressed in lung adenocarcinoma (LUAD) but not in lung squamous cell carcinoma. However, FGL1 in tissue and plasma can only distinguish LUAD patients from healthy donors and cannot correlate with clinical Tumor Node Metastasis (TNM) stage. Using lung cancer cell lines, we confirmed that FGL1 can be detected on extracellular vesicles (EVs) and we established a method using flow cytometry to detect FGL1 on the surface of EVs, which revealed that FGL1 could be secreted via EVs. Both animal model and clinical samples proved that plasma FGL1 in EVs would increase when the tumor was loaded. The level of FGL1 in plasma EVs was correlated with clinical TNM stage and tumor size, and a higher level indicated non-responsiveness to anti-programmed cell death ligand 1 (anti-PD-L1) immunotherapy. Its effect on tumor progression and immune evasion may be achieved by impairing the killing and proliferating capacities of CD8+ T cells. Our result demonstrates that FGL1 levels in plasma EVs, but not total plasma FGL1, could be a promising biomarker that plays an important role in predicting anti-PD-L1 immune therapy in LUAD and suggests a new strategy in LUAD immunotherapy.

Development of LAG-3/FGL1 blocking peptide and combination with radiotherapy for cancer immunotherapy

Aside from antibodies, peptides show great potential as immune checkpoint inhibitors (ICIs) due to several advantages, such as better tumor penetration and lower cost. Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint which can induce T cell dysfunction through interaction with its soluble ligand fibrinogen like protein-1 (FGL1). Here, we found that LAG-3 expression was higher than programmed cell death protein 1 (PD-1) in multiple human cancers by TCGA databases, and successfully identified a LAG-3 binding peptide LFP-6 by phage display bio-panning, which specifically blocks the interaction of LAG-3/FGL1 but not LAG-3/MHC-II. Subsequently, d-amino acids were introduced to substitute the N- and C-terminus of LFP-6 to obtain the proteolysis-resistant peptide LFP-D1, which restores T cell function in vitro and inhibits tumor growth in vivo. Further, a bispecific peptide LFOP targeting both PD-1/PD-L1 and LAG-3/FGL1 was designed by conjugating LFP-D1 with PD-1/PD-L1 blocking peptide OPBP-1(8–12), which activates T cell with enhanced proliferation and IFN-γ production. More importantly, LFOP combined with radiotherapy significantly improve the T cell infiltration in tumor and elevate systemic antitumor immune response. In conclusion, we developed a novel peptide blocking LAG-3/FGL1 which can restore T cell function, and the bispecific peptide synergizes with radiotherapy to further enhance the antitumor immune response.

A combined analysis of multi-omics data reveals the prognostic values and immunotherapy response of LAG3 in human cancers

Lymphocyte-activation gene 3 (LAG3) is a highly anticipated immune checkpoint in the context of cancer, exerting regulatory control over immune cell proliferation and function to reinforce the advancement of cancers. However, the comprehensive functional analysis of LAG3 across various cancer types remains undisclosed; thus, this study aims to investigate the pan-cancer expression profile of LAG3. We have investigated the expression profile, prognostic significance, and genetic alterations of LAG3 in various cancers while elucidating its characteristic in immune response regulation. Our findings demonstrated that elevated LAG3 expression is significantly associated with favorable prognosis in patients with cutaneous melanoma (SKCM), and it may be a potential biomarker for SKCM. Furthermore, multiple immune algorithms have highlighted the important regulatory role of LAG3 for the tumor-infiltrating immune cells including CD8 + T cells, B cells, dendritic cells (DCs), macrophages, and natural killer (NK) cells. We also examined the distribution of LAG3 at the single-cell level and explored its functional significance. A comprehensive and systematic analysis of LAG3 would facilitate a comprehensive evaluation of LAG3 in cancer biology and provide valuable insights for cancer management.

Immunohistochemical Expression of Immune Checkpoints; CTLA-4, LAG3, and TIM-3 in Cancer Cells and Tumor-infiltrating Lymphocytes (TILs) in Colorectal Carcinoma.

Colorectal cancer is considered the third most prevalent cancer in both sexes. Immune checkpoint receptors that regulate T-cell response, stimulation, and development include lymphocyte activating gene 3 (LAG-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and T-cell immunoglobulin and mucin domain 3 (Tim-3). In addition, they are crucial for the advancement of cancer and tumor immune escape. This work's aim was to assess the immunohistochemistry expression of Tim-3, CTLA-4, and LAG-3 in cancer cells and tumor-infiltrating lymphocytes (TILs) in colorectal cancer (CRC) and the correlation between these markers and clinicopathological variables and survival data. This study involved 206 CRC specimens processed for CTLA-4, LAG3, and TIM-3 immunohistochemistry and correlated with the clinicopathological and survival parameters of the patients. High CTLA-4 epithelial expression was highly related to the old age group, large tumor size, low tumor-stroma ratio (TSR), high grade, advanced stage, the presence of distant metastasis (DM), perineural invasion (PNI), necrosis, lymphovascular invasion (LVI), relapse, mortality, overall survival (OS), and disease-free survival (DFS), while negative CTLA-4 TILs expression was highly linked with the presence of gross perforation, low TSR, high tumor budding (TB) score, high grade, advanced stage, the existence of lymph node (LN) metastasis, DM, necrosis, LVI, PNI, DFS, mortality, and OS. Positive LAG-3 TILs expression was highly correlated with large tumor size, gross perforation, low TSR, high TB score, high grade, advanced phase, the presence of LN, necrosis, LVI, PNI, relapse DFS, mortality, and OS. High Tim-3 epithelial expression was extremely linked with low TSR, advanced phase, the presence of LN, LVI, PNI, relapse, DFS, mortality, and OS, while positive Tim-3 TILs expression was related to gross perforation, low TSR, high TB score, advanced stage, the presence of LN, DM, necrosis, relapse, DFS, mortality, and OS. The patients' poor prognosis may be related to the immunohistochemistry expression of LAG-3, Tim-3, and CTLA-4 in CRC cancer tissue and TILs. Poor patient consequences can result from the CTLA-4, Tim-3, and LAG-3 co-expression, but CTLA-4 TILs' expression of these proteins may inhibit the growth of tumors.

PD-1 and LAG-3 as early prognostic markers in the treatment of patients with chronic lymphocytic leukemia

Background. Recently, studies have been conducted all over the world to study the role of immune checkpoints in the pathogenesis of chronic lymphocytic leukemia (CLL) and the possibility of their use as prognostic markers. Of greatest interest are PD-1 (programmed cell death-1) and LAG-3 protein (lymphocyte-activation gene 3).Aim. To study the features of PD-1 (CD279) and LAG-3 (CD223) expression on blood B-cells of CLL patients and the possibility of their use as early markers for predicting the hematological response to therapy.Materials and methods. The blood of 30 patients with CLL in stage C according to Binet and 20 healthy individuals was studied by 10-color flow cytometry.Results. In patients with CLL, there were significant differences in the initial lymphocytes level, PD-1 and LAG-3 expression on B-lymphocytes, both with persons in the control group and among themselves with different hematological responses to therapy with rituximab according to the results of minimal residual disease monitoring.Conclusion. PD-1 and LAG-3 can be used as early markers for predicting the response of CLL patients to therapy. The combined use of initial lymphocytes level and PD-1 and LAG-3 expression on CD19+ blood cells has a greater prognostic value. New data obtained from the study of immune checkpoints PD-1 and LAG-3 may be useful in the development of targeted therapeutic agents.

Immune checkpoint inhibitors as potential therapy for reverting T-cell exhaustion and reverting HIV latency in people living with HIV.

The immune system of people living with HIV (PLWH) is persistently exposed to antigens leading to systemic inflammation despite combination antiretroviral treatment (cART). This inflammatory milieu promotes T-cell activation and exhaustion. Furthermore, it produces diminished effector functions including loss of cytokine production, cytotoxicity, and proliferation, leading to disease progression. Exhausted T cells show overexpression of immune checkpoint molecules (ICs) on the cell surface, including programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), and lymphocyte activation gene-3 (LAG-3). The ICs also play a crucial role in T-cell exhaustion by reducing the immune response to cancer antigens. Immunotherapy based on immune checkpoint inhibitors (ICIs) has changed the management of a diversity of cancers. Additionally, the interest in exploring this approach in the setting of HIV infection has increased, including AIDS-defining cancers and non-AIDS-defining cancers in PLWH. To date, research on this topic suggests that ICI-based therapies in PLWH could be a safe and effective approach. In this review, we provide an overview of the current literature on the potential role of ICI-based immunotherapy not only in cancer remission in PLWH but also as a therapeutic intervention to restore immune response against HIV, revert HIV latency, and attain a functional cure for HIV infection.

The identification of a PTEN-associated gene signature for the prediction of prognosis and planning of therapeutic strategy in endometrial cancer.

Endometrial cancer (EC) is one of the most common malignancies among women. To improve the prognosis and treatment of EC, finding out a phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-associated prognostic signature would be beneficial. EC clinical data, genetic mutation data, and transcriptome data were downloaded from The Cancer Genome Atlas (TCGA) database. To clarify the specific PTEN-associated signature, cox regression analyses were performed. The clinical value of the selected signature on the overall survival (OS) and the secretoglobin family 2A member 1 (SCGB2A1)-independent analysis, immune and functional analysis were investigated respectively. Five hundred and fourteen EC samples were screened and PTEN mutation occupied 57%. Enrichment analysis indicated that mutant-type PTEN was enriched for pathways related to the upregulated human T-cell leukemia virus-1 (HTLV-1) infection and estrogen signaling pathway. SCGB2A1 was identified by cox regression analysis. Immune analysis exhibited significant immune infiltration with higher expression of T cells, B cells, and macrophage groups. Immune-checkpoint transcripts CD274 molecule (CD274), and cytotoxic T-lymphocyte associated protein 4 (CTLA4), hepatitis A virus cellular receptor 2 (HAVCR2), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PDCD1), PDCD1 ligand 2 (PDCD1LG2), T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT), and sialic acid binding immunoglobulin like lectin 15 (SIGLEC15) were discovered statistically different. In addition, the low-SCGB2A1 group had worse OS than the high-SCGB2A1 group. SCGB2A1 showed significant area under the curve (AUC) values in a time-dependent receiver operating characteristic (ROC) analysis. Prevalence of microsatellite instability (MSI) was detected and SCGB2A1 showed a negative correlation with EC. Immune checkpoint blockade (ICB) response indicated a worse immune response in the low-SCGB2A1 group. The distribution of one-class linear regression (OCLR) scores reflected the negative correlation between messenger RNA expression-based stemness index (mRNAsi) and prognostic gene expression. Furthermore, several SCGB2A1-related signaling pathways in EC were identified. SCGB2A1 is a prognostic immunometabolic signature for patients with EC, which may help improve the prognosis and therapeutic effect.

Soluble LAG3: A Potential Marker for the Development and Activity of Acute Graft-Versus-Host Disease

Acute graft-versus-host disease (aGVHD), usually developing within 100 days after hematopoietic stem cell transplantation (HSCT), is mainly attributed to donor T cell pro-inflammatory responses against mismatched host histocompatibility antigens. The persistence of increased effector T cell responses following HSCT is partially related to an altered function of T regulatory cells (Tregs) and their failure to prevent GVHD. Certain immune checkpoint molecules, such as cytotoxic T-lymphocyte antigen 4, programmed cell death protein 1 (PD1) and lymphocyte activation gene-3 (LAG3), highly expressed on Tregs, are involved in maintaining immune tolerance and preventing immune-mediated inflammation. LAG3 is a newly identified inhibitory receptor, found on most immune cells, including CD4+ and CD8+ T cells, Tregs, and dendritic cells. Previous studies have demonstrated a role of LAG3 expression on T cells in GVHD suppression. Its soluble isoform sLAG3, released from the cell membrane, has been recently reported to also contribute to enhanced immune-mediated responses in solid tumors and autoimmune diseases, such as anti-PD1-resistant melanoma, rheumatoid arthritis (RA), and ANCA-associated vasculitis. Given this evidence, the current study aimed to explore a potential role of sLAG3 in GVHD development. Serum samples of 25 patients (≥18 years old) who underwent allogeneic HSCT at Rambam from 01.2021 to 01.2023 were drawn on days -7, +100, +180 of transplant. Twelve serum samples obtained from the Biobank were used as healthy control (HC). All samples were assessed for the sLAG3 levels using a commercial ELISA kit. The median level measured in HC (56pg/mL) was considered a cutoff to define the “low and high level sLAG3 groups”. Twenty-three patients were included in the analysis [median age at transplant - 59.5 years (range 19-75); 52% males]. Seventeen patients had acute myeloid leukemia, 3 - myelodysplastic syndrome, 2 - acute lymphoblastic leukemia and 1 had primary myelofibrosis. Fourteen patients had a matched unrelated donor, 6- matched related donors, and 3 - haploidentical donors. Nineteen patients received calcineurin inhibitor (CNI) and methotrexate-based GVHD prophylaxis (13 with ATG); 4 patients received post-transplant cytoxan-based prophylaxis, with CNI and mycophenolate-mofetil. Overall, 17/23 (74%) patients developed GVHD: 9 - aGVHD and 8 - chronic GVHD (cGVHD). A low pre-transplant sLAG3 level (&amp;lt;56pg/mL) was found in 8/23 patients, while it was high (&amp;gt;56pg/mL) in 15/23. Notably, 9/15 (60%) patients in the “high sLAG3 group”, but none in the “low sLAG3 group” were diagnosed with aGVHD, suggesting a high sLAG3 level as a possible indicator for patient-pre-transplant susceptibility to aGVHD development, irrespective of transplant factors, such as conditioning regimen, donor type, etc. Four patients in each group developed cGVHD. Furthermore, there was a significant increase in sLAG3 levels between the baseline and GVHD diagnosis in patients who developed aGVHD (mean: from 156.1±172.8, to 323.9±201.3), but not in those who either had cGVHD (mean: from 119.71±90.5 to 150.1±147.1) or did not have GVHD (mean: from 49.7±36 to 52.1±22.2). Overall, 11/23 patients were in complete remission (CR) at the time of HSCT; 9/11 were in the high-sLAG3 group and 6 of them developed aGVHD. In the low-sLAG3 group, most patients (6/8) had some degree of active disease (persistent disease or minimal-residual disease). These findings may reflect high T-effector cell and/or low Treg activity, possibly contributing to both anti-leukemic effect and GVHD development. In conclusion: The current study demonstrates, for the first time, that the pre-transplant sLAG3 level is significantly increased and continues to further increase in patients who develop aGVHD following HSCT compared to those who don't. These findings suggest a potential role of sLAG3 as a predicting factor for aGVHD development as well as a marker of treatment response that could guide therapeutic decisions in this patient setting.

Open-Label, Randomized, Phase 3 Study of Coformulated Favezelimab and Pembrolizumab Versus Chemotherapy in Patients with Relapsed or Refractory Classical Hodgkin Lymphoma Refractory to Anti-PD-1 Therapy: Keyform-008

Background: The importance of PD-1 therapy in relapsed or refractory (R/R) classical Hodgkin Lymphoma (cHL) is well established, with PD-1 inhibitors such as pembrolizumab being a standard of care option for patients. However, most patients eventually develop progressive disease and optimal therapy after anti-PD-1 failure has not been determined. The inhibitory checkpoint receptor, lymphocyte-activation gene 3 (LAG-3) is expressed in cHL tumor microenvironments and upregulation of LAG-3 is considered to play an important role in anti-PD-1 resistance. Favezelimab (MK-4280) is a humanized immunoglobulin G4 antibody that binds to LAG-3 and blocks interaction with major histocompatibility complex Class II ligands. Results from the ongoing phase 1/2 MK-4280-003 study of the combination of favezelimab and pembrolizumab demonstrated manageable safety and promising antitumor activity in patients with anti-PD-1-refractory R/R cHL. The randomized, open-label, parallel group, active-controlled, phase 3 KEYFORM-008 study (NCT05508867) is designed to evaluate efficacy and safety of the coformulation of favezelimab and pembrolizumab versus physician's choice of chemotherapy in patients with anti-PD-1-refractory R/R cHL. Study Design and Methods: Patients ≥18 years old, with histologically confirmed R/R cHL, an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0-2, and adequate organ function are eligible. Patients must have exhausted all available treatment options with known clinical benefit, including having progressed on anti-PD-1-based therapy and being ineligible for or having failed autologous stem cell transplant (ASCT). In addition, patients should also have been ineligible for brentuximab vedotin (BV), relapsed or failed to respond to BV or discontinued BV due to toxicity. Patients with a history of central nervous system (CNS) metastases or active CNS involvement are excluded. Approximately 360 patients will be enrolled and randomly assigned 1:1 to receive coformulated favezelimab 800 mg and pembrolizumab 200 mg intravenously (IV) every 3 weeks (Q3W) or physician's choice of chemotherapy (gemcitabine 800-1200 mg/m 2 IV on days 1 and 8 of a 21-day cycle or bendamustine 90-120 mg/m 2 IV on days 1 and 2 of either a 21- or 28-day cycle). Randomization will be stratified by prior ASCT (yes vs no) and ECOG PS (0 or 1 vs 2). Treatment will continue for ≤35 cycles for the favezelimab/pembrolizumab coformulation or ≤6 cycles for chemotherapy or until disease progression, unacceptable toxicity, or withdrawal. Patients receiving physician's choice of chemotherapy with progressive disease confirmed by blinded independent central review (BICR) per Lugano criteria may be eligible to cross over to the favezelimab/pembrolizumab coformulation. Response assessments by positron emission tomography (PET) and computed tomography (CT) or magnetic resonance imaging (MRI) will be performed every 12 weeks (Q12W) until disease progression or trial discontinuation. Adverse events will be monitored throughout the study and graded per National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. The primary end point is progression-free survival (PFS) per Lugano criteria by BICR. Overall survival (OS) is a key secondary end point. Other secondary end points are objective response rate (ORR), duration of response (DOR) per Lugano criteria by BICR, and safety. Exploratory end points include PFS on subsequent anticancer therapy and health-related quality of life. PFS, OS, and DOR rates in each treatment group will be estimated using the Kaplan-Meier method. The Clopper-Pearson method will be used to estimate ORR with 95% CI. Patient-reported outcomes will be assessed using EORTC Quality Of Life Questionnaire C30 and the EuroQuol 5-dimension, 5-level questionnaire. Enrollment for this study is open in sites in Australia, Belgium, Brazil, Canada, China, Czech Republic, France, Germany, Israel, Poland, Spain, South Korea, Sweden, Switzerland, Turkey, UK, and USA. ©2023 American Society of Clinical Oncology, Inc. Reused with permission. This abstract was accepted and previously presented at the 2023 ASCO Annual Meeting. All rights reserved.

Favezelimab in Combination with Pembrolizumab in Patients with Heavily Pretreated Anti-PD-1-Refractory Classical Hodgkin Lymphoma: Updated Analysis of an Open-Label Phase 1/2 Study

Background: Programmed cell death protein 1 (PD-1) inhibitors are a standard of care for relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL), but better treatment options are needed for patients with anti-PD-1-refractory disease. Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint receptor with a role in regulating T-cell function. Dual blockade of PD-1 and LAG-3 has demonstrated antitumor activity in patients with advanced melanoma. Combination therapy with favezelimab (anti-LAG-3) and pembrolizumab (anti-PD-1) is currently being investigated in a phase 1/2 study (NCT03598608) in patients with R/R hematologic malignancies. Prior analyses showed that the combination had antitumor activity and manageable safety in patients with heavily pretreated anti-PD-1-refractory cHL (cohort 2). Updated results for this cohort are presented. Methods: The study consisted of a safety lead-in (part 1) followed by a dose-expansion phase (part 2). Eligible patients were ≥18 y; had R/R cHL after autologous stem cell transplantation (ASCT), were ineligible for ASCT, or did not respond to salvage chemotherapy; had an ECOG performance status of 0 or 1; and had experienced disease progression after ≥2 doses of anti-PD-1-based therapy and within 12 weeks of the last dose of anti-PD-1 therapy. In part 1, patients received pembrolizumab 200 mg IV Q3W + favezelimab starting at 200 mg and escalating to 800 mg IV Q3W per a modified toxicity probability interval design. In part 2, patients received pembrolizumab + favezelimab at the established RP2D of 800 mg Q3W for ≤35 cycles (~2 y). The primary end point was safety. ORR per IWG 2007 criteria by investigator review was a secondary end point. DOR and PFS per IWG 2007 criteria by investigator review and OS were exploratory end points. Results: 34 patients with anti-PD-1-refractory cHL were enrolled in cohort 2. The median age was 37.5 y (range, 25-79), 16 patients (47%) were male, 21 (62%) had an ECOG performance status of 0, and 94% had received ≥4 prior lines of therapy. Seventeen patients (50%) had received an anti-PD-1-based regimen as their most recent therapy. At database cutoff (March 2, 2023), 8 patients (24%) had completed 35 cycles of treatment and 26 (76%) had discontinued treatment (14 [41%] progressive disease, 7 [21%] AEs, 5 [15%] other reasons). The median time from first dose to data cutoff was 35.3 mo (range, 15.0-49.4). Treatment-related AEs occurred in 28 patients (82%), of which the most common (≥15%) were hypothyroidism (18%), nausea (18%), and fatigue (15%). Grade 3/4 treatment-related AEs occurred in 6 patients (18%). 6 patients (18%) discontinued treatment because of treatment-related AEs. No deaths due to treatment-related AEs were reported. AEs of clinical interest occurred in 17 patients (50%); 2 patients (6%) had grade 3 events (encephalitis, hepatitis) and 1 patient (3%) had a grade 4 event (type 1 diabetes mellitus). Of the 2 patients who underwent allogeneic hematopoietic stem cell transplantation after discontinuation or completion of study treatment, 1 had a grade 3 AE (acute graft versus host disease) unrelated to study treatment that resolved. The ORR in cohort 2 was 29% (10/34; 95% CI, 15-48), with 3 (9%) complete responses (CR) and 7 (21%) partial responses (PR). Of the 10 responders, 7 had received ≥5 prior lines of therapy (3 CR/4 PR). The ORR in patients with anti-PD-1 as their most recent therapy was 35% (6/17; 95% CI, 14-62; 1 CR/5 PR); the ORR in patients with non-anti-PD-1 as their most recent therapy was 24% (4/17; 95% CI, 7-50; 2 CR/2 PR). Of 28 patients with a baseline and postbaseline assessment available, 25 (89%) had any reduction in target lesion size from baseline, and 12 (43%) had ≥50% reduction. Median DOR was 21.9 mo (range, 0.0+ to 26.1+) and an estimated 17% of responders remained in response ≥24 mo. Median PFS was 9.7 mo (95% CI, 5.1-14.7); 24-mo PFS rate was 21%. Median OS was 34.3 mo (95% CI, 25.7-NR); 24-mo OS rate was 76%. Conclusion: After additional follow-up, the combination of favezelimab + pembrolizumab continued to demonstrate manageable safety and antitumor activity in patients with heavily pretreated anti-PD-1-refractory cHL. Analyses are underway to identify biomarkers predictive of response to the combination of favezelimab and pembrolizumab. The phase 3 KEYFORM-008 study (NCT05508867) is being conducted to evaluate a coformulation of favezelimab and pembrolizumab in patients with anti-PD-1-refractory cHL.

Favezelimab (anti-LAG-3) Plus Pembrolizumab in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma (DLBCL): Cohort 3 of a Multicohort Open-Label Phase 1/2 Study

Background: Lymphocyte-activation gene 3 (LAG-3) is involved in the regulation of T-cell function and is commonly coexpressed with PD-1 on anergic T cells. Favezelimab (MK-4280), a humanized IgG4 anti-LAG-3 monoclonal antibody, plus pembrolizumab (anti-PD-1) is being investigated in the multicohort phase 1/2 MK-4280-003 efficacy and safety study (NCT03598608) in patients with relapsed or refractory (R/R) hematologic malignancies. Prior analyses of the combination demonstrated antitumor activity and manageable safety in patients with anti-PD-1-naive R/R classical Hodgkin lymphoma (cHL; cohort 1; ORR, 73%; CR, 30%) (Johnson NA et al. Blood. 2022;140(suppl 1):6540-2) and anti-PD-1-refractory cHL (cohort 2; ORR, 29%; CR, 9%) (Timmerman J et al. Blood. 2022;140(suppl 1):768-70). We present results from analysis of patients with R/R DLBCL enrolled in cohort 3. Methods: In this study, a safety lead-in phase (part 1) to determine the recommended phase 2 dose (RP2D) was followed by a dose-expansion phase (part 2). In cohort 3, eligible patients were ≥18 years old, had histologically confirmed R/R DLBCL that had progressed after ≥2 lines of previous therapy, including progression after autologous stem cell transplant (ASCT), had declined ASCT, or were ineligible for ASCT. Patients with Richter transformation were not permitted. In part 1, patients received favezelimab at a starting dose of 200 mg that was escalated to 800 mg plus pembrolizumab at 200 mg IV every 3 weeks (Q3W) per the modified toxicity probability interval method. In the dose-expansion phase, patients received favezelimab at the established RP2D of 800 mg plus pembrolizumab 200 mg Q3W for ≤35 cycles (~2 years). Response assessments were performed at weeks 12 and 24 (PET) and Q12W (CT). Adverse events (AEs) were graded per the NCI CTCAE v4.0. The primary end point was safety. ORR per IWG 2007 criteria by investigator review was a secondary end point. Exploratory end points included duration of response (DOR) and progression-free survival (PFS) per IWG 2007 criteria by investigator review and overall survival (OS). Results: A total of 25 patients with R/R DLBCL were enrolled. Patients had a median age of 73 years (range, 25-87), 10 (40%) had ECOG PS 0, and 15 (60%) had ≥3 prior lines of therapy. Most common subtypes of DLBCL were unspecified DLBCL (n = 12; 48%) and germinal center B-cell DLBCL (n = 6; 24%). At database cutoff (March 2, 2023), 1 patient (4%) was ongoing on treatment and 24 (96%) had discontinued because of progressive disease (n = 14), AEs (n = 2), clinical progression (n = 5), or patient noncompliance/nonstudy anticancer therapy (n = 3); 1 patient (4%) discontinued because of treatment-related AEs. No treatment-related deaths occurred. Sixteen patients (64%) had a treatment-related AE; the most common (≥5%) were cough (16%), increased blood alkaline phosphatase (12%), and hypothyroidism, constipation, infusion related reaction, increased AST, muscle spasms, headache and pruritis (8% each). Grade 3 or 4 treatment-related AEs occurred in 4 patients (16%; 1 grade 3 lymphocytic hypophysitis, 1 grade 3 infectious enterocolitis, 1 grade 3 increased AST, 1 grade 3 increased ALT, 1 grade 3 increased amylase, and 1 grade 4 decreased neutrophil count). AEs of clinical interest occurred in 5 patients (20%); only one grade ≥3 occurred (grade 3 hypophysitis). The median (range) time from first dose to data cutoff was 25.9 (19.7-52.1) months. The objective response rate was 12% (95% CI, 2.5-31.2; [2 CR, 1 PR]). Among 17 patients with a postdose scan, 7 (41%) had a reduction from baseline in target lesion size, and 6 (35%) had ≥50% reduction from baseline. Median DOR was not reached (range, 3.0+ to 16.9+ months); 1 responder had an observed response duration of ≥12 months. Median PFS was 2.1 months (95% CI, 1.1-2.7); 12-month PFS rate was 12%. As of the data cutoff, 20 patients (80%) had died. Median OS was 6.4 months (95% CI, 2.3-15.8); 12-month OS rate was 43%. Conclusion: Favezelimab 800 mg plus pembrolizumab 200 mg had limited antitumor activity in patients with DLBCL in cohort 3. Analyses are underway to identify biomarkers predictive of response to the combination of favezelimab and pembrolizumab. The safety profile was manageable and consistent with that observed in other cohorts in the study.

Enhancing anti-tumor immune responses through combination therapies: epigenetic drugs and immune checkpoint inhibitors.

Epigenetic mechanisms are processes that affect gene expression and cellular functions without involving changes in the DNA sequence. This abnormal or unstable expression of genes regulated by epigenetics can trigger cancer and other various diseases. The immune cells involved in anti-tumor responses and the immunogenicity of tumors may also be affected by epigenomic changes. This holds significant implications for the development and application of cancer immunotherapy, epigenetic therapy, and their combined treatments in the fight against cancer. We provide an overview of recent research literature focusing on how epigenomic changes in immune cells influence immune cell behavior and function, as well as the immunogenicity of cancer cells. And the combined utilization of epigenetic medications with immune checkpoint inhibitors that focus on immune checkpoint molecules [e.g., Programmed Death 1 (PD-1), Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4), T cell Immunoglobulin and Mucin Domain (TIM-3), Lymphocyte Activation Gene-3 (LAG-3)] present in immune cells and stromal cells associated with tumors. We highlight the potential of small-molecule inhibitors targeting epigenetic regulators to amplify anti-tumor immune responses. Moreover, we discuss how to leverage the intricate relationship between cancer epigenetics and cancer immunology to create treatment regimens that integrate epigenetic therapies with immunotherapies.

Noninvasive Monitoring of Immunotherapy in Lung Cancer by Lymphocyte Activation Gene 3 PET Imaging of Tumor-Infiltrating Lymphocytes.

Although immunotherapy has revolutionized the entire cancer treatment landscape, small fractions of patients respond to immunotherapy. Early identification of responders may improve patient management during immunotherapy. In this study, we evaluated a PET approach for monitoring immunotherapy in lung cancer by imaging the upregulation of lymphocyte activation gene 3 (LAG-3)-expressing (LAG-3+) tumor-infiltrating lymphocytes (TILs). Methods: We synthesized a LAG-3-targeted molecular imaging probe, [68Ga]Ga-NOTA-C25 and performed a series of invitro and invivo assays to test its specificity. Next, [68Ga]Ga-NOTA-C25 PET was used to monitor immunotherapy in murine lung cancer-bearing mice and in humanized mouse models for assessing clinical translational potential, with confirmation by immunostaining and flow cytometry analysis. Results: [68Ga]Ga-NOTA-C25 PET could noninvasively detect intertumoral differences in LAG-3+ TIL levels in different tumor models. Importantly, in Lewis lung carcinoma tumor models treated with an agonist of a stimulator of interferon genes, [68Ga]Ga-NOTA-C25 PET also detected an immunophenotyping transition of the tumor from "cold" to "hot" before changes in tumor size. Meanwhile, animals carrying "hot" tumor showed more significant tumor inhibition and longer survival than those carrying "cold" tumor. [68Ga]Ga-NOTA-C25 PET also showed markedly higher tumor uptake in immune system-humanized mice carrying human non-small cell lung cancer than immunodeficient models. Conclusion: [68Ga]Ga-NOTA-C25 PET could be used to noninvasively monitor the early response to immunotherapy by imaging LAG-3+ TILs in lung cancer. [68Ga]Ga-NOTA-C25 PET also exhibited excellent translational potential, with great significance for the precise management of lung cancer patients receiving immunotherapy.

Multi-omic analysis in carcinoma of unknown primary (CUP): therapeutic impact of knowing the unknown.

Carcinoma of unknown primary (CUP) is a difficult-to-manage malignancy. Multi-omic profiles and treatment outcome vs. degree of precision matching were assessed. Tumours underwent next-generation sequencing (NGS) [tissue and/or blood-derived cell-free DNA (cfDNA)]. Selected patients had transcriptome-based immune profiling and/or programmed cell death 1 ligand 1 (PD-L1) immunohistochemistry analysis. Patients could be reviewed by a Molecular Tumor Board, but physicians chose the therapy. Of 6497 patients in the precision database, 97 had CUP. The median number of pathogenic tissue genomic alterations was 4 (range, 0-25), and for cfDNA, was 2 (range, 0-9). Each patient had a distinct molecular landscape. Food and Drug Administration (FDA)-approved biomarkers included the following: PD-L1+ ≥ 1%, 30.9% of CUPs tested; microsatellite instability, 3.6%; tumour mutational burden ≥ 10 mutations·Mb-1, 23%; and neurotrophic receptor tyrosine kinase (NTRK) fusions, 0%. RNA-based immunograms showed theoretically druggable targets: lymphocyte activation gene 3 protein (LAG-3), macrophage colony-stimulating factor 1 receptor (CSF1R), adenosine receptor A2 (ADORA2) and indoleamine 2,3-dioxygenase 1 (IDO1). Overall, 56% of patients had ≥ 1 actionable biomarker (OncoKB database). To quantify the degree of matching (tumours to drugs), a Matching Score (MS; roughly equivalent to number of alterations targeted/total number of deleterious alterations) was calculated post hoc. Comparing evaluable treated patients [MS high, > 50% (N = 15) vs. low ≤ 50% (N = 47)], median progression-free survival was 10.4 vs. 2.8 months (95% CI 0.11-0.64; HR 0.27; P = 0.002); survival, 15.8 vs. 6.9 months (95% CI 0.17-1.16; HR 0.45; P = 0.09); and clinical benefit rate (stable disease ≥ 6 months/partial/complete response), 71% vs. 24% (P = 0.003). Higher MS was the only factor that predicted improvement in outcome variables after multivariate analysis. In conclusion, CUPs are molecularly complex. Treatments with high degrees of matching to molecular alterations (generally achieved by individualized combinations) correlated with improved outcomes.

Development and validation of a combined hypoxia- and metabolism-related prognostic signature to predict clinical prognosis and immunotherapy responses in clear cell renal cell carcinoma.

Hypoxia and metabolism are closely correlated with the progression of cancer. We aimed to construct a combined hypoxia- and metabolism-related genes (HMRGs) prognostic signature to predict survival and immunotherapy responses in patients with clear cell renal cell carcinoma (ccRCC). The RNA-seq profiles and clinical data of ccRCC were acquired from the TCGA and the ArrayExpress (E-MTAB-1980) databases. Least absolute shrinkage and selection operator (LASSO) and univariate and multivariate Cox regression analyses were applied to establish a prognostic signature. The E-MTAB-1980 cohort was selected for validation. The effectiveness and reliability of the signature were further evaluated by Kaplan-Meier (K-M) survival and time-dependent receiver operating characteristic (ROC) curves. Further analyses, including functional enrichment, ssGSEA algorithm, CIBERSORT algorithm, and expression of immune checkpoints, were explored to investigate immune status and immunotherapy responses. We constructed a prognostic eight-gene signature with IRF6, TEK, PLCB2, ABCB1, TGFA, COL4A5, PLOD2, and TUBB6. Patients were divided into high-risk and low-risk groups based on the medium-risk score. The K-M analysis revealed that patients in the high-risk group had an apparently poor prognosis compared to those in the low-risk group in the TCGA (p < 0.001) and E-MTAB-1980 (p < 0.005). The area under ROC curve (AUC) of the prognostic signature was 0.8 at 1 year, 0.77 at 3 years, and 0.78 at 5 years in the TCGA, respectively, and was 0.82 at 1 year, 0.74 at 3 years, and 0.75 at 5 years in the E-MTAB-1980, respectively. Independent prognostic analysis confirmed the risk score as a separate prognostic factor in ccRCC patients (p < 0.001). The results of ssGSEA showed not only a high degree of immune cell infiltration but also high scores of immune-related functions in the high-risk group. The CIBERSORT analysis further confirmed that the abundance of immune cells was apparently different between the two risk groups. The risk score was significantly correlated with the expression of cytotoxic T lymphocyte-associated antigen-4 (CTLA4), lymphocyte-activation gene 3 (LAG3), and programmed cell death protein 1 (PD-1). The HMRGs signature could be used to predict clinical prognosis, evaluate the efficacy of immunotherapy, and guide personalized immunotherapy in ccRCC patients.

BIOM-45. GLIOMA CSF PROTEOMICS FOR PHARMACODYNAMIC BIOMARKER AND THERAPEUTIC TARGET DISCOVERY

Abstract Cerebrospinal fluid (CSF) is emerging as an abundant source of candidate biomarkers, including proteins, for gliomas. Longitudinal CSF access may enable insights into treatment response and resistance. To that end, we have established a growing biobank of CSF samples, many of which have been obtained longitudinally via CSF access devices, such as Ommaya reservoirs, through our ongoing biomarker discovery clinical trials (NCT04692337, 04692324). We have previously presented our initial data demonstrating a conserved proteomic signature of HGG that decreased with resection and increased with recurrence. Since then, samples have been acquired throughout individual patients’ treatment course toward our goal of identifying CSF-derived biomarkers of treatment response. CSF samples obtained prior to, during, and throughout treatment underwent global proteomics analysis via the SomaScan® assay by Somalogic. Fold-changes for each protein were calculated between different timepoints and ranked for each comparison of interest. Treatment with pembrolizumab alone results in increased IL-23 (5.01x), IL-12 (4.25x), lymphocyte activation gene 3 protein (LAG3) (3.26x), and granzyme A (2.93x), suggesting potential for combination with multiple agents. Chemoradiation, with or without pembrolizumab, consistently downregulated NAD kinase, acetyl CoA transferase, and Bcl-2 and upregulated proteins related to neuronal signaling, such as neurexins and cerebellins, in addition to LAG-3, suggesting potential for synergy between chemoradiation and anti-LAG-3 antibodies currently in clinical trials. Interestingly, pembrolizumab and chemoradiation decreased MMP-12 and MMP-9 abundance as compared to chemoradiation alone (0.25x vs. 18.5x; 0.54x vs. 4.99x). Finally, bevacizumab treatment consistently increased VEGF-A (7.32x), VEGF-B (2.21x), and MMP-3 (2.99x) abundance, while decreasing the presence of our previously reported plasma-associated protein signature in CSF. In conclusion, the global CSF proteome can be longitudinally accessed to evaluate response to standard-of-care and experimental therapies. Further studies are ongoing to identify signatures of therapeutic resistance and to assess the impact of novel immunotherapeutic combinations on the CSF proteome.

A Comparison of the Antitumor Efficacy of Novel Multi-Specific Tribodies with Combinations of Approved Immunomodulatory Antibodies.

Many advances in antitumor therapies have been achieved with antagonistic antibodies targeting the programmed cell death protein 1 (PD-1) or its ligand (PD-L1); however, many cancer patients still develop resistance to anti-PD-1/PD-L1 treatments often associated with the upregulation of other immune checkpoints such as Lymphocyte Activation Gene-3 (LAG-3). In order to verify whether it is possible to overcome these limits, we analyzed and compared the effects of combinations of the clinically validated anti-LAG-3 mAb (Relatlimab) with anti-PD-1 (Pembrolizumab) or anti-PD-L1 (Atezolizumab) monoclonal antibodies (mAbs) with those of novel bispecific tribodies (TRs), called TR0304 and TR0506, previously generated in our lab by combining the binding moieties of novel human antibodies targeting the same ICs of the mentioned mAbs. In particular, TR0304, made up of a Fab derived from an anti-PD-L1 mAb and two single-chain variable fragments (scFvs) derived from an anti-LAG-3 mAb, was tested in comparison with Relatlimab plus Atezolizumab, and TR0506, made up of an antigen-binding fragment (Fab) derived from the same anti-LAG-3 mAb and two scFvs derived from an anti-PD-1 mAb, was tested in comparison with Relatlimab and Pembrolizumab. We found that the two novel TRs showed similar binding affinity to the targets with respect to validated mAbs, even though they recognized distinct or only partially overlapping epitopes. When tested for their functional properties, they showed an increased ability to induce lymphocyte activation and stronger in vitro cytotoxicity against tumor cells compared to combinatorial treatments of clinically validated mAbs. Considering that tribodies also have other advantages with respect to combinatorial treatments, such as reduced production costs and lower dose requirements, we think that these novel immunomodulatory TRs could be used for therapeutic applications, particularly in monotherapy-resistant cancer patients.