Lymphocyte Attenuator Research Articles

Small molecules from antibody pharmacophores (SMAbPs) as a hit identification workflow for immune checkpoints.

Small-molecule modulators of immune checkpoints are poised to revolutionize cancer immunotherapy. However, efficient strategies for hit identification are lacking. We introduce small molecules from antibody pharmacophores (SMAbPs), a workflow leveraging cocrystal structures of checkpoints with antibodies to create pharmacophore maps for virtual screening. Applying SMAbPs to five immune checkpoints yielded hits with submicromolar potency in both cell-free and cellular assays. Notably, SMAbPs identified the most potent T cell immunoglobulin and mucin-domain containing-3 and V-domain immunoglobulin suppressor of T cell activation (VISTA) inhibitors reported to date and first-in-class modulators of B and T lymphocyte attenuator, 4-IBB, and CD27. Targeting inhibitory and costimulatory checkpoints with hits identified through SMAbPs demonstrated remarkable in vivo antitumor activity, exemplified by MG-V-53 (VISTA inhibitor) and MG-C-30 (CD27 agonist), which significantly reduced tumor volumes in MC38 and EG7-OVA mouse models, respectively.

Immune Checkpoint Molecule B and T Lymphocyte Attenuator and Induces Anti-inflammatory Dendritic Cells in Experimental GN

Immune Checkpoint Molecule B and T Lymphocyte Attenuator and Induces Anti-inflammatory Dendritic Cells in Experimental GN

Distinct characteristics of BTLA/HVEM axis expression on Tregs and its impact on the expansion and attributes of Tregs in patients with active pulmonary tuberculosis.

Pulmonary tuberculosis (PTB) remains one of the deadliest infectious diseases. Understanding PTB immunity is of potential value for exploring immunotherapy for treating chemotherapy-resistant PTB. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are key players that impair immune responses to Mycobacteria tuberculosis (MTB). Currently, the intrinsic factors governing Treg expansion and influencing the immunosuppressive attributes of Tregs in PTB patients are far from clear. Here, we employed flow cytometry to determine the frequency of Tregs and the expression of B and T lymphocyte attenuator (BTLA) and its ligand, herpesvirus entry mediator (HVEM), on Tregs in patients with active PTB. Furthermore, the expression of conventional T cells and of programmed death-ligand 1 (PD-L1) and programmed death-1 (PD-1) on Tregs in patients with active PTB was determined. We then examined the characteristics of BTLA/HVEM expression and its correlation with Treg frequency and PD-L1 and PD-1 expression on Tregs in PTB patients. The frequency of Tregs was increased in PTB patients and it had a relevance to PTB progression. Intriguingly, the axis of cosignal molecules, BTLA and HVEM, were both downregulated on the Tregs of PTB patients compared with healthy controls (HCs), which was the opposite of their upregulation on conventional T cells. Unexpectedly, their expression levels were positively correlated with the frequency of Tregs, respectively. These seemingly contradictory results may be interpreted as follows: the downregulation of BTLA and HVEM may alleviate BTLA/HVEM cis-interaction-mediated coinhibitory signals pressing on naïve Tregs, helping their activation, while the BTLA/HVEM axis on effector Tregs induces a costimulatory signal, promoting their expansion. Certainly, the mechanism underlying such complex effects remains to be explored. Additionally, PD-L1 and PD-1, regarded as two of the markers characterizing the immunosuppressive attributes and differentiation potential of Tregs, were upregulated on the Tregs of PTB patients. Further analysis revealed that the expression levels of BTLA and HVEM were positively correlated with the frequency of PD-1+Tregs and PD-L1+Tregs, respectively. Our study illuminated distinct characteristics of BTLA/HVEM axis expression on Tregs and uncovered its impact on the expansion and attributes of Tregs in patients with active PTB. Therefore, blockade of the BTLA/HVEM axis may be a promising potential pathway to reduce Treg expansion for the improvement of anti-MTB immune responses.

N-Glycan Branching Regulates BTLA Opposite to PD-1 to Limit T Cell Hyperactivity Induced by Branching Deficiency.

N-glycan branching is a potent and multifaceted negative regulator of proinflammatory T cell and B cell function. By promoting multivalent galectin-glycoprotein lattice formation at the cell surface, branching regulates clustering and/or endocytosis of the TCR complex (TCR+CD4/CD8), CD45, CD25, BCR, TLR2 and TLR4 to inhibit T cell and B cell activation/proliferation and proinflammatory TH1 and TH17 over TH2 and induced T regulatory cell responses. In addition, branching promotes cell surface retention of the growth inhibitory receptor CTLA-4. However, the role of N-glycan branching in regulating cell surface levels of other checkpoint receptors such as BTLA (B and T lymphocyte attenuator) and PD-1 (programmed cell death protein 1) is unknown. In this study, we report that whereas branching significantly enhances PD-1 cell surface expression by reducing loss from endocytosis, the opposite occurs with BTLA in both T cells and B cells. T cell hyperactivity induced by branching deficiency was opposed by BTLA ligation proportional to increased BTLA expression. Other members of the BTLA/HVEM (herpesvirus entry mediator) signaling axis in T cells, including HVEM, LIGHT, and CD160, are largely unaltered by branching. Thus, branching-mediated endocytosis of BTLA is opposite of branching-induced inhibition of PD-1 endocytosis. In this manner, branching deficiency-induced upregulation of BTLA appears to serve as a checkpoint to limit extreme T cell hyperactivity and proinflammatory outcomes in T cells with low branching.

Sepsis-Induced Immunosuppression: The Role of Co-inhibitory Molecules

Sepsis is one of the most common cause of death among hospitalized patients in the intensive care unit (ICU), with current therapeutic options falling short of a comprehensive solution. The condition's pathophysiology is marked by a spectrum of immunological impairments, with a growing consensus that immunosuppression plays a decisive role in the condition's rising morbidity and mortality rates. Extensive preclinical and clinical research has identified the upregulation of several co-inhibitory molecules during sepsis, including Programmed Death-1 (PD-1), Programmed Death Ligand-1 (PD-L1), Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), B and T Lymphocyte Attenuator (BTLA), T Cell Membrane Protein-3 (TIM-3), and Lymphocyte Activation Gene-3 (LAG-3). These molecules, which exert a significant inhibitory effect on T cell function, are believed to contribute to the immunosuppressive state induced by sepsis. The elucidation of the intricate mechanisms by which these molecules induce immunosuppression is essential for devising the most efficacious treatment strategies for sepsis. The burgeoning field of immunotherapy, particularly the blockade of co-inhibitory molecules, represents a significant frontier in sepsis research. This approach holds substantial promise for the future of sepsis therapy, warranting further exploration and clinical investigation to harness its potential fully.

BTLA deficiency promotes HSC activation and protects against hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion injury (IRI) is unavoidable even despite the development of more effective surgical approaches. During hepatic IRI, activated HSC (aHSC) are involved in liver injury and recovery. A proportion of aHSC increased significantly both in the mouse liver tissues with IRI and in the primary mouse HSCs and LX-2 cells during hypoxia-reoxygenation. "Loss-of-function" experiments revealed that depleting aHSC with gliotoxin exacerbated liver damage in IRI mice. Subsequently, we found that the transcription of mRNA and the expression of B and T lymphocyte attenuator (BTLA) protein were lower in aHSC compared with quiescent HSCs. Interestingly, overexpression or knockdown of BTLA resulted in opposite changes in the activation of specific markers for HSCs such as collagen type I alpha 1, α-smooth muscle actin, and Vimentin. Moreover, the upregulation of these markers was also observed in the liver tissues of global BLTA-deficient (BTLA-/-) mice and was higher after hepatic IRI. Compared with wild-type mice, aHSC were higher, and liver injury was lower in BTLA-/- mice following IRI. However, the depletion of aHSC reversed these effects. In addition, the depletion of aHSC significantly exacerbated liver damage in BTLA-/- mice with hepatic IRI. Furthermore, the TGF-β1 signaling pathway was identified as a potential mechanism for BTLA to negatively regulate the activation of HSCs in vivo and in vitro. These novel findings revealed a critical role of BTLA. Particularly, the receptor inhibits HSC-activated signaling in acute IRI, implying that it is a potential immunotherapeutic target for decreasing the IRI risk.

Tifcemalimab combined with toripalimab and chemotherapy as 1st line treatment for extensive-stage small cell lung cancer (ES-SCLC): A phase Ib/II, open-label study.

8089 Background: Tifcemalimab, a humanized IgG4 monoclonal antibody against B and T lymphocyte attenuator (BTLA), has shown preliminary anti-tumor activities in combination with toripalimab (anti-PD-1) as a later line treatment for patients with extensive-stage small cell lung cancer (ES-SCLC). We further conducted a multi-cohort phase Ib/II study (NCT05664971) to evaluate the safety and efficacy of tifcemalimab combined with toripalimab and chemotherapy as a 1st line treatment for patients with advanced lung cancer. Here, we report the preliminary results from the ES-SCLC cohort. Methods: Patients without previous systemic anti-tumor therapy for ES-SCLC were eligible. Patients received tifcemalimab 200mg in combination with toripalimab 240mg and standard chemotherapy (etoposide + carboplatin/cisplatin) intravenously once every three weeks (Q3W) for 4 cycles, then followed by tifcemalimab plus toripalimab maintenance therapy until disease progression, intolerable toxicity, or completion of 2 years treatment. Primary endpoints included safety and objective response rate (ORR) by investigators per RECIST v1.1. Results: From 7/12/2023 to 12/6/2023, a total of 44 ES-SCLC patients were enrolled. As of Dec 28, 2023, the median follow-up duration was 8.3 weeks. The median age of the patients was 65.5 (range 48-73) years, and 84.1% (37/44) were males. Forty-one (93.2%) patients experienced treatment-emergent adverse events (TEAEs), and 26 (59.1%) experienced ≥ grade 3 TEAEs. The most common TEAEs included leukopenia (65.9%), neutropenia (63.6%), anemia (40.9%) and thrombocytopenia (40.9%). No treatment-related adverse events led to discontinuation of tifcemalimab and toripalimab. Four (9.1%) patients experienced immune related AE (irAEs), and 1 experienced ≥ grade 3 irAE. Among 37 evaluable patients, 32 PR and 5 SD were observed. The ORR was 86.5% (32/37) and the DCR was 100% (37/37). By the data cut-off date, 94.6% of the responses were ongoing and the median duration of response was not reached. The preliminary biomarker analysis of tumor tissue suggested 100% ORR was observed among patients with either PD-L1 or HVEM positive expression. Further biomarker analysis will be updated. Conclusions: Tifcemalimab in combination with toripalimab and chemotherapy showed a promising objective response rate with a manageable safety profile as a 1st line treatment for patients with ES-SCLC. Continued follow-up is ongoing for additional safety and efficacy (PFS and OS) evaluation after extended exposure. Clinical trial information: NCT05664971 .

Immune checkpoint inhibitors: breakthroughs in cancer treatment.

Over the past two decades, immunotherapies have increasingly been considered as first-line treatments for most cancers. One such treatment is immune checkpoint blockade (ICB), which has demonstrated promising results against various solid tumors in clinical trials. Monoclonal antibodies (mAbs) are currently available as immune checkpoint inhibitors (ICIs). These ICIs target specific immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1). Clinical trial results strongly support the feasibility of this immunotherapeutic approach. However, a substantial proportion of patients with cancer develop resistance or tolerance to treatment, owing to tumor immune evasion mechanisms that counteract the host immune response. Consequently, substantial research focus has been aimed at identifying additional ICIs or synergistic inhibitory receptors to enhance the effectiveness of anti-PD-1, anti-programmed cell death ligand 1 (anti-PD-L1), and anti-CTLA-4 treatments. Recently, several immune checkpoint molecular targets have been identified, such as T cell immunoreceptor with Ig and ITIM domains (TIGIT), mucin domain containing-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), V-domain immunoglobulin suppressor of T cell activation (VISTA), B and T lymphocyte attenuator (BTLA), and signal-regulatory protein α (SIRPα). Functional mAbs targeting these molecules are under development. CTLA-4, PD-1/PD-L1, and other recently discovered immune checkpoint proteins with distinct structures are at the forefront of research. This review discusses these structures, as well as clinical progress in mAbs targeting these immune checkpoint molecules and their potential applications.

153 Spatial Transcriptomic Profiling of T Cell Exhaustion Markers in Atypical Teratoid Rhabdoid Tumor Subtypes

INTRODUCTION: Atypical teratoid/rhabdoid tumor (ATRT) is one of the most common brain tumors in pediatric populations. Despite advances in treatment, overall survival remains poor and novel effective therapies are needed. METHODS: Tumor purities of 49 atypical ATRT tumors, encompassing 3 epigenetic subtypes (MYC, SHH, TYR) were estimated using known mRNA expression signatures of stromal and immune cells. Using the purity estimates and bulk tumor expression values, non-negative linear regression was implemented to estimate the average expression of each gene in the stromal and tumor compartments for each subtype. RESULTS: Compared to other subtypes, SHH-ATRTs expressed relatively higher levels of T-cell immunoglobulin domain and mucin domain protein 3 (TIM-3) and its ligand GAL9. MYC-ATRTs expressed higher levels of numerous T cell inhibitory receptors including lymphocyte activation gene 3 protein (LAG-3), band T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte antigen-4 (CTLA-4), and programmed cell death protein 1 (PD-1). CONCLUSIONS: The predicted stromal and tumor compartment deconvolution yielded previously unrecognized expression patterns of T cell exhaustion markers which should be evaluated further to develop effective targeted therapies for ATRT.

Abstract 120: A closer Look at colorectal cancer risk: Insights from the complex relationship of HIV, helminths, and immune responses

Abstract The advent of antiretroviral therapy has markedly increased the survival rates of individuals with human immunodeficiency virus (HIV). However, this demographic, particularly in low-middle-income countries (LMICs) where HIV-helminth co-infections are prevalent, remains susceptible to infections. The intricate interplay between helminth infections and HIV susceptibility, often inducing immunological phenotypes that heighten vulnerability, remains poorly understood. Studies have shown the association between HIV-helminth co-infections and elevated susceptibility to colorectal cancer (CRC), introducing an additional layer of health concerns for this already vulnerable population. This retrospective observational study delves into the profound effects of HIV-helminth co-infections on immune responses, with a specific focus on type 2 T helper (Th2) cell immunomodulation and susceptibility to CRC. Utilizing samples from a master project (n=414), 80 systematically randomized individuals were categorized into uninfected control, HIV-infected only, helminth-infected only, and HIV-helminth co-infected groups. Parasite detection employed the Kato-Katz technique, and serum testing was conducted for helminth-specific antibodies. Serological HIV testing, viral load categorization, white blood cell counts, and differential analyses were performed. Human ProcartaPlex Panel 1 was used for cytokine profiling of immune stimulatory and inhibitory soluble proteins. A significant reduction in B and T lymphocyte attenuator (BTLA) (p=0.049) in helminth infections compared to the control. The association of BTLA with its ligand, herpesvirus entry mediator (HVEM), establishes a direct link between the CD28 and tumor necrosis factor receptor (TNFR) families, triggering wide-ranging and robust immune responses. Subsequently, a notable reduction in CD28 was observed in HIV-infected subjects (p=0.003), followed by helminth-infected and HIV-helminth co-infected individuals (p=0.03) compared to the control. Furthermore, programmed death ligand-2 (PD-L2) expression, a major contributor to alternatively activated macrophage polarization during helminth infections, was significantly increased in the HIV-helminth co-infected group (p = 0.05), favoring a shift towards Th2 immune responses. These observed alterations underscore the complexity of the immunological interplay in co-infected individuals, emphasizing an additional risk factor for colorectal cancer development. This necessitates a comprehensive understanding, providing valuable insights for the development of therapeutic strategies in LMICs and similar settings, addressing the heightened risk of CRC within this population. Citation Format: Botle P. Damane, Thanyani V. Mulaudzi, Sayed S. Kader, Ropo E. Ogunsakin, Pragalathan Naidoo, Zodwa Dlamini, Zilungile L. Mkhize-Kwitshana. A closer Look at colorectal cancer risk: Insights from the complex relationship of HIV, helminths, and immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 120.

Graft Protective and Intercellular Immunomodulatory Effects by Adoptive Transfer of an Agonistic Anti-BTLA mAb (3C10) Induced CD4+CD25+ Regulatory T Cells in Murine Cardiac Allograft Transplant Model

We demonstrated that an agonistic anti-B and T lymphocyte attenuator antibody (3C10) prolonged cardiac survival by inducing regulatory T cells (Treg). However, the mechanisms of immune tolerance in the recipients remained unclear. In this study, we investigated the graft-protective and intercellular immunomodulatory effects of adoptive transfer (AT) of 3C10-induced Tregs in a murine cardiac allograft transplant model. Thirty days after transplantation of a C57BL/6 heart into the primary 3C10-treated CBA recipients, splenic CD4+CD25+ cells from these recipients (3C10/AT group) or naïve CBA mice (no-treatment group) were adoptively transferred into secondary CBA recipients with a C57BL/6 heart. To confirm the requirement for 3C10-induced Tregs, we administered an anti-interleukin-2 receptor alpha antibody (PC-61) to secondary CBA recipients. Additionally, histologic and fluorescent staining, cell proliferation analysis, flow cytometry, and donor-specific antibody (DSA) measurements were performed. 3C10/AT-treated CBA recipients resulted in significantly prolonged allograft survival (median survival time [MST], >50 days). Allografts displayed prolonged function with preservation of vessel structure by maintaining high numbers of splenic CD4+CD25+Foxp3+ Treg and intramyocardial CD4+Foxp3+ cells. DSA levels were suppressed in 3C10/AT-treated CBA recipients. Moreover, PC-61 administration resulted in a shorter MSTs of cardiac allograft survivals, a detrimental increase in DSA production, and enhanced expression of programmed cell death (PD)-1. AT of 3C10-induced Tregs may be a promising graft-protective strategy to prolong allograft survival and suppress DSA production, driven by the promotion of splenic and graft-infiltrating Tregs and collaboration with PD-1+ T cells and Treg.

Clinical significance of BTLA gene expression and rs1982809 polymorphism in pan-cancer

The association between the B and T lymphocyte attenuator (BTLA) gene rs1982809 polymorphism and cancer susceptibility has been reported, but these findings are inconsistent. In addition to clarifying the relationship between the rs1982809 polymorphism and cancer susceptibility, the current study also explored the clinical significance of BTLA gene expression. The GSCA tool and Stata software were used to explore the association between BTLA gene expression and tumor stage, immune infiltration, survival prognosis, and drug sensitivity for pan-cancer, and the association of BTLA gene rs1982809 polymorphism with cancer susceptibility, respectively. BTLA gene expression was associated not only with the pathologic stages of thyroid carcinoma, skin cutaneous melanoma, and kidney renal clear cell carcinoma, but also with immune infiltration in 33 types of cancers. In addition, BTLA gene expression was linked to survival prognosis in 8 types of cancers and the sensitivity of 255 drugs such as 5-Fluorouracil, docetaxel, and methotrexate. A meta-analysis of 7 relevant studies with 4002 cancer patients and 5278 healthy controls showed that the BTLA gene rs1982809 polymorphism was unrelated to cancer susceptibility under all genetic models. However, a country-based stratification analysis suggested that the rs1982809 polymorphism could reduce cancer susceptibility in Polish and Tunisian populations. In conclusion, BTLA is expected to serve as a prognostic marker and therapeutic target for certain cancers, and the rs1982809 polymorphism is expected to serve as a cancer susceptibility marker in Polish and Tunisian populations.

Impact of Genetic Mutations on Survival Following Receipt of Commercial CART19 for Patients Diagnosed with Aggressive B Cell Lymphoma

Introduction Anti-CD19 chimeric antigen receptor-modified T cell therapies (CART19) have revolutionized treatment of patients (pts) with relapsed/refractory (R/R) diffuse large B cell lymphoma (DLBCL) and high grade B cell lymphoma (HGBL). However, progression of disease following receipt of CART19 is common with only approximately one-third of pts achieving long-term remission in the standard-of-care setting (Transplant Cell Ther. 2022 Sep;28(9):581.e1-581.e8, Blood (2022) 140 (Supplement 1): 1584-1587). While loss of CD19 antigen expression antigen is one mechanism of CART19 failure, other tumor-intrinsic factors which may result in CART19 failure remain poorly defined. Clinical laboratory mutation analysis (CLMA) performed on tumor specimens can identify recurring genetic mutations which may predict for clinical outcomes; however, the predictive value of CMLA has not been well-established for DLBCL/HGBL pts treated with CART19. Here, we analyze the impact of genetic mutations as detected by CLMA, along with other clinicopathologic characteristics, on survival outcomes of DLBCL/HGBL pts following receipt of CART19. Methods We collected data from 46 pts with DLBCL or HGBL treated at the University of Pennsylvania between 2018 and 2021. Patient tumors were sequenced with one of three sequencing panels comprised of 41-116 genes prior to receiving commercial CART19. Cox proportional hazards regression was used to assess the impact of clinicopathologic characteristics on disease progression and death, with characteristics demonstrating p <0.10 on univariate analysis included in multivariate analysis. Estimated progression free survival (PFS) and overall survival (OS) from the time of CART19 infusion were calculated using Kaplan-Meier estimates. Results Baseline clinicopathologic characteristics are listed in Table 1. A total of 30 of 46 pts had at least one tumor mutation detected prior to receiving CART19, with 22 genes mutated in at least two pts (Figure 1). The most frequent mutations were TP53 (15 of 46, 32.7%), BCL2 (3 of 12, 25%), MYC (4 of 20, 20%), SOCS1 (5 of 38, 13.2%), CD79B (5 of 38, 13.2%), CREBBP (5 of 38, 13.2%), EZH2 (5 of 46, 10.9%), STAT3 (4 of 38, 10.5%), TNFRSF14 (4 of 38, 10.5%) and DNMT3A (2 of 20, 10%). The median length of follow-up from the time of CART19 infusion was 29.6 months. For all pts, the rates of 2 year (y) PFS and OS were 21% (95% confidence interval [CI] 10-34%) and 56% (95% CI 40-69%), respectively. Univariate analysis of mutations present in ≥10% of cases revealed that TNFRSF14 mutations predicted for lower risk of disease progression at 2y (HR 0.16, 95% CI 0.02-1.186, p = 0.07). No other mutation was associated with either disease progression or death at 2y. Univariate analysis of baseline clinicopathologic characteristics also identified International Prognostic Index (IPI) score ≥3 at the time of relapse preceding CART19, HGBL classification, no history of indolent lymphoma (IL), MYC rearrangement (-R) and double hit lymphoma (DHL) as predictive of disease progression at 2y; however, none of these characteristics remained predictive on multivariate analysis. Additionally, IPI score ≥3 at the time of relapse preceding CART19, HGBL classification, no history of IL, MYC-R and DHL were also predictive of death at 2y on univariate analysis; however, none of these characteristics remained predictive on multivariate analysis. Conclusions Tumor characteristics, including recurring genetic mutations as detected by CLMA, may not independently predict for survival outcomes for DLBCL/HGBL pts following receipt of CART19. However, our finding that TNFRSF14 mutations predict for a lower rate of disease progression at 2y on univariate analysis may be explained by loss of the inhibitory effect of HVEM on T helper cells through binding of B and T lymphocyte attenuator (BTLA) (Cell. 2016 Oct 6;167(2):405-418.e13). Additionally, our finding that TP53 mutations did not predict for inferior PFS following receipt of CART19 is in agreement with that of a prior analysis (J Clin Oncol. 2022 Feb 1;40(4):369-381). Performance of CLMA on a larger number of tumor samples from DLBCL/HGBL pts subsequently treated with CART19, which is planned, may be informative.

Vactosertib, a Novel TGFb Type I Receptor Kinase Inhibitor, Improves T-Cell Fitness:a Single-Arm, Phase 1b Trial in Relapsed/Refractory Multiple Myeloma

The prognosis for Multiple Myeloma (MM) patients (pts) has improved substantially over the past two decades with the success of immunomodulatory drugs (IMiDs), monoclonal antibodies, chimeric antigen receptor (CAR) T-cells and bispecific T-cell engagers (BiTEs) that broke the circulus vitiosus of tumor-induced immunosuppression to reengage the immune system. Functional blockade of the transforming growth factor-beta (TGF) signaling pathway improves the efficacy of cytotoxic and immune therapies, therefore we investigator the impact of TGF-inhibition in an steroid free regimn. Methods: We conducted a phase 1b study to determine the safety, efficacy, and maximal tolerated dose (200 mg po bid) of the potent, orally-available TGF type I receptor kinase inhibitor vactosertib in relapsed and/or refractory multiple myeloma (RRMM) pts who had received -2 lines of chemoimmunotherapy in combination with standard dosage of Pomalidomide without any corticosteroids. Results: Twenty one pts were enrolled in the phase 1b dose escalation cohort and received vactosertib plus pomalidomide as indicated (Fig. 1A). Pts' characteristics are described in Table 1The summary of grade 1-4 adverse events is shown (Table 2). A single grade 4 elevated bilirubin (5%) and a single grade 4 elevated lipase (5%) were observed as well as four grade 4 events of decreased neutrophils (19%). Four pts demonstrated a PR, two had a MR, nine had SD and four had disease progression (Fig. 1B). The median time to response for all pts and the responders eight weeks and median duration of response was ~12 weeks. The best overall response for individual pts to treatment is shown (Fig. 1C). Clinical activity was observed across all four dose levels (Table 3). To probe the tumor intrinsic anti-myeloma activity of vactosertib, we first determined the relative effect of vactosertib compared to the IMiDs pomalidomide and lenalidomide (Fig. 2A, B). The effect of vactosertib was greater than that observed with either lenalidomide or pomalidomide. MMCLs and patient CD138 + cells were then treated with vactosertib in combination with pomalidomide, and importantly, vactosertib with pomalidomide synergistically reduced myeloma viability (Fig. 3A). We next probed the systemic, tumor extrinsic effects of vactosertib by analyzing cytokines immune and tumor cells present within patient BM samples. (Fig. 4A). (Fig. 4B). Therefore, we explored the immunophenotype of CD8 + T-cells isolated from patient BM prior to and at the EOT. The relative percentage of T-cells that expressed the immunosuppressive markers programmed cell death-1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), B and T lymphocyte attenuator (BTLA), and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) was reduced following treatment relative to their expression prior to treatment (Fig. 4C). Results indicated that vactosertib and pomalidomide treatment in vivo reduced PD-1, TIM3, BTLA and CTLA-4 expression on patient CD8 + T-cells. Next, CD8 + T-cells were isolated from patient peripheral blood and cultured in the presence of TGF-1, vactosertib, pomalidomide, or vactosertib plus pomalidomide (Fig. 5B). Treatment of patient CD8 + T-cells with vactosertib decreased PD-1 expression up to 40% and decreased TIM3 expression up to 65%. Pomalidomide alone did not significantly reduce PD-1, TIM3, BTLA, or LAG-3 on patient CD8 + T-cells. Vactosertib and pomalidomide also reduced TIM3 levels on CD8 + T-cells from most pts. (Fig. 5C, D). (Fig. 5 G, H). Results indicated that vactosertib or pomalidomide did not increase CD138 + cell death (Fig. 5I). Conclusion: Vactosertib combined with pomalidomide was well-tolerated at all doses, had a manageable adverse event profile and induced durable responses with 80% progression-free survival (PFS-6) at 6 months, Vactosertib reduced TGF- in patient bone marrow and suppressed PD-1 expression on CD8 + T-cells and lead to reduction of PD-L1/PD-L2 expression on CD138 + cells and enhanced autologous T-cell cytotoxicity. Taken together, our results support the safety and efficacy of vactosertib to treat RRMM and revealed that vactosertib modulates the T-cell immunophenotype and reinvigorates T-cell fitness.

The Methylation in B7-H4 and BTLA Genes are Associated with the Risk of Pulmonary Tuberculosis.

The important roles of B7 homologous body 4 (B7-H4), B and T lymphocyte attenuator (BTLA) in patients with pulmonary tuberculosis (PTB) have been reported. This study aims to evaluate the association among B7-H4 and BTLA genes polymorphism, methylation and PTB susceptibility. Here, we assessed the possible relationship of 10 single nucleotide polymorphisms (SNPs) in B7-H4, BTLA genes with PTB susceptibility in a Chinese population (496 PTB patients and 502 controls) by SNPscan technique. Then, the B7-H4, BTLA genes methylation levels among 98 PTB patients and 97 controls were detected using MethylTarget technique. This study found no significant differences in allele and genotype frequencies of B7-H4 gene rs10754339, rs10801935, rs10923223, rs1937956, rs3738414, BTLA gene rs1982809, rs2971205, rs75368388, rs9288953 variants between PTB patients and controls. Haplotype analysis suggested that the lower frequencies of B7-H4 AATTG haplotype, BTLA GATT haplotype and the higher frequency of BTLA AGTC haplotype were found in PTB patients when compared with controls. We also found that the frequency of BTLA gene rs9288953 C allele was significantly increased in PTB patients with drug resistance. Moreover, the methylation levels of B7-H4 and BTLA genes in PTB patients were greater than that in controls, and rs10754339 variant in B7-H4 gene could affect its methylation level in PTB patients. B7-H4, BTLA genes polymorphism might not affect PTB susceptibility, while the abnormal methylation levels of B7-H4, BTLA genes were associated with the genetic background of PTB.

In vitro vascularized immunocompetent patient-derived model to test cancer therapies

This work describes a patient-derived tumoroid model (PDTs) to support precision medicine in lung oncology. The use of human adipose tissue-derived microvasculature and patient-derived peripheral blood mononuclear cells (PBMCs) permits to achieve a physiologically relevant tumor microenvironment. This study involved ten patients at various stages of tumor progression. The vascularized, immune-infiltrated PDT model could be obtained within two weeks, matching the requirements of the therapeutic decision. Histological and transcriptomic analyses confirmed that the main features from the original tumor were reproduced. The 3D tumor model could be used to determine the dynamics of response to antiangiogenic therapy and platinum-based chemotherapy. Antiangiogenic therapy showed a significant decrease in vascular endothelial growth factor (VEGF)-A expression, reflecting its therapeutic effect in the model. In an immune-infiltrated PDT model, chemotherapy showed the ability to decrease the levels of lymphocyte activation gene-3 protein (LAG-3), B and T lymphocyte attenuator (BTLA), and inhibitory receptors of Tcells functions.

Novel PD-L1/BTLA Checkpoint Axis Exploited for Bacterial Immune Escape by Restraining CD8+ T Cell-Initiated Adaptive Immunity in Zebrafish.

Programmed death-ligand 1/programmed cell death 1 (PD-L1/PD-1) is one of the most important immune checkpoints in humans and other mammalian species. However, the occurrence of the PD-L1/PD-1 checkpoint in evolutionarily ancient vertebrates remains elusive because of the absence of a PD-1 homolog before its appearance in tetrapods. In this article, we identified, to our knowledge, a novel PD-L1/B and T lymphocyte attenuator (BTLA) checkpoint in zebrafish by using an Edwardsiella tarda-induced bacterial infection model. Results showed that zebrafish (Danio rerio) PD-L1 (DrPD-L1) and BTLA (DrBTLA) were differentially upregulated on MHC class II+ macrophages (Mϕs) and CD8+ T cells in response to E. tarda infection. DrPD-L1 has a strong ability to interact with DrBTLA, as shown by the high affinity (KD = 5.68 nM) between DrPD-L1/DrBTLA proteins. Functionally, the breakdown of DrPD-L1/DrBTLA interaction significantly increased the cytotoxicity of CD8+BTLA+ T cells to E. tarda-infected PD-L1+ Mϕ cells and reduced the immune escape of E. tarda from the target Mϕ cells, thereby enhancing the antibacterial immunity of zebrafish against E. tarda infection. Similarly, the engagement of DrPD-L1 by soluble DrBTLA protein diminished the tolerization of CD8+ T cells to E. tarda infection. By contrast, DrBTLA engagement by a soluble DrPD-L1 protein drives aberrant CD8+ T cell responses. These results were finally corroborated in a DrPD-L1-deficient (PD-L1-/-) zebrafish model. This study highlighted a primordial PD-L1/BTLA coinhibitory axis that regulates CD8+ T cell activation in teleost fish and may act as an alternative to the PD-L1/PD-1 axis in mammals. It also revealed a previously unrecognized strategy for E. tarda immune evasion by inducing CD8+ T cell tolerance to target Mϕ cells through eliciting the PD-L1/BTLA checkpoint pathway.

Dual inhibition of BTLA and PD-1 can enhance therapeutic efficacy of paclitaxel on intraperitoneally disseminated tumors

BackgroundExpression of immune checkpoints in the tumor microenvironment is one mechanism underlying paclitaxel (PTX) chemoresistance. This study aimed to investigate whether the addition of checkpoint blockade to PTX can improve...

Epitope topography of agonist antibodies to the checkpoint inhibitory receptor BTLA

B and T lymphocyte attenuator (BTLA) is an attractive target for a new class of therapeutics that attempt to rebalance the immune system by agonizing checkpoint inhibitory receptors (CIRs). Herpesvirus entry mediator (HVEM) binds BTLA in both trans- and cis-orientations. We report here the development and structural characterization of three humanized BTLA agonist antibodies, 22B3, 25F7, and 23C8. We determined the crystal structures of the antibody-BTLA complexes, showing that these antibodies bind distinct and non-overlapping epitopes of BTLA. While all three antibodies activate BTLA, 22B3 mimics HVEM binding to BTLA and shows the strongest agonistic activity in functional cell assays and in an imiquimod-induced mouse model of psoriasis. 22B3 is also capable of modulating HVEM signaling through the BTLA-HVEM cis-interaction. The data obtained from crystal structures, biochemical assays, and functional studies provide a mechanistic model of HVEM and BTLA organization on the cell surface and informed the discovery of a highly active BTLA agonist.

BTLA transcriptomic expression correlates with high levels of PD-1, PD-L2, and CTLA-4 checkpoints and with its ligand, HVEM, across diverse solid cancers.

e14641 Background: B and T lymphocyte attenuator (BTLA) is an immune checkpoint that is highly expressed on tumor-specific T cells and has been shown to be upregulated in several cancer types. The binding of BTLA to its ligand herpesvirus entry mediator (HVEM, also known as TNFRSF14) inhibits activation and proliferation of T lymphocytes, leading to decreased tumor killing. Early clinical trials of BTLA inhibitors are ongoing with preliminary results showing modest effects in advanced solid tumors. More recent clinical data suggests that combination checkpoint therapy with anti-BTLA and anti-PD-1 treatment provides greater benefit compared to monotherapy in patients with lymphoma. Methods: We analyzed RNA expression levels of 397 genes in various solid tumor types from 514 patients with various types of solid tumors seen at University of San Diego (UCSD), Moores Cancer Center. RNA expression was quantified at a CLIA-approved laboratory Omniseq (https://www.omniseq.com). Transcript abundance was normalized to internal housekeeping gene profiles and ranked (0-100 percentile) in a standardized manner to a reference population of 735 tumors spanning 35 histologies. We analyzed tumors with “High” (75-100 percentile rank) BTLA expression profiles. Odds ratio for high BTLA expression was calculated and multivariate analysis was utilized to determine significant correlations for multiple genes. Results: A total of 96 (18.7%) tumors had high BTLA (>75) expression across 32 different histologies. Cancers with the highest proportion of BTLA expression were small intestine (42%), breast (31%), pancreatic (29%), stomach (24%), neuroendocrine (20%), lung (20%), head and neck (17%) and colorectal (13%). There was significant association for BTLA and high PD-1 (p < 0.001), high CTLA-4 (p < 0.001), high PD-L2 (p = 0.05) and its ligand HVEM (p < 0.01). There was no significant association between BTLA expression and PD-L1, gender or stomach, neuroendocrine, head and neck, and lung histologies. Conclusions: High BTLA expression was found in a large number of tumor samples and significantly associated with other immune checkpoints with FDA-approved drugs. Furthermore, HVEM expression (ligand for BTLA) significantly correlates with high BTLA transcriptome levels. The BTLA/HVEM pathway has been associated with suppression of T cell-mediated anti-tumor activity and resistance to anti-PD-1 immunotherapy leading to poor clinical outcomes. The utilization of combination checkpoint therapy involving BTLA inhibitors and specific checkpoint blockade is currently being explored. Our results suggest that patient selection based on the RNA expression level of immune markers matched to corresponding immunotherapy agent, rather than a population-based approach, may be ideal for optimizing treatment response.