Member Of TNF Receptor Family Research Articles

Spatial characterization and quantification of CD40 expression across cancer types

BackgroundCD40, a TNF receptor family member, is expressed by a variety of immune cells and is involved in the activation of both adaptive and innate immune responses. Here, we used quantitative immunofluorescence (QIF) to evaluate CD40 expression on the tumor epithelium of solid tumors in large patient cohorts of lung, ovarian, and pancreatic cancers.MethodsTissue samples from nine different solid tumors (bladder, breast, colon, gastric, head and neck, non-small cell lung cancer (NSCLC), ovarian, pancreatic and renal cell carcinoma), constructed in tissue microarray format, were initially assessed for CD40 expression by QIF. CD40 expression was then evaluated on the large available patient cohorts for three of the tumor types demonstrating high CD40 positivity rate; NSCLC, ovarian and pancreatic cancer. The prognostic impact of CD40 expression on tumor cells was also investigated.ResultsCD40 expression on tumor cells was found to be common, with 80% of the NSCLC population, 40% of the ovarian cancer population, and 68% of the pancreatic adenocarcinoma population displaying some degree of CD40 expression on cancer cells. All of three of these cancer types displayed considerable intra-tumoral heterogeneity of CD40 expression, as well as partial correlation between expression of CD40 on tumor cells and on surrounding stromal cells. CD40 was not found to be prognostic for overall survival in NSCLC, ovarian cancer, or pancreatic adenocarcinoma.ConclusionsThe high percentage of tumor cells expressing CD40 in each of these solid tumors should be considered in the development of therapeutic agents designed to target CD40.

TRAF3/p38-JNK Signalling Crosstalk with Intracellular-TRAIL/Caspase-10-Induced Apoptosis Accelerates ROS-Driven Cancer Cell-Specific Death by CD40

The capacity to induce tumour-cell specific apoptosis represents the most unique feature of the TNF receptor (TNFR) family member CD40. Recent studies on the signalling events triggered by its membrane-presented ligand CD40L (mCD40L) in normal and malignant epithelial cells have started to unravel an exquisite context and cell type specificity for the functional effects of CD40. Here, we demonstrate that, in comparison to other carcinomas, mCD40L triggered strikingly more rapid apoptosis in colorectal carcinoma (CRC) cells, underpinned by its ability to entrain two concurrently operating signalling axes. CD40 ligation initially activates TNFR-associated factor 3 (TRAF3) and subsequently NADPH oxidase (NOX)/Apoptosis signal-regulating kinase 1 (ASK1)-signalling and induction of reactive oxygen species (ROS) to mediate p38/JNK- and ROS-dependent cell death. At that point, p38/JNK signalling directly activates the mitochondrial pathway, and triggers rapid induction of intracellular TNF-related apoptosis-inducing ligand (TRAIL) that signals from internal compartments to initiate extrinsic caspase-10-asscociated apoptosis, leading to truncated Bid (tBid)-activated mitochondrial signalling. p38 and JNK are essential both for direct mitochondrial apoptosis induction and the TRAIL/caspase-10/tBid pathway, but their involvement follows functional hierarchy and temporally controlled interplay, as p38 function is required for JNK phosphorylation. By engaging both intrinsic and extrinsic pathways to activate apoptosis via two signals simultaneously, CD40 can accelerate CRC cell death. Our findings further unravel the multi-faceted properties of the CD40/mCD40L dyad, highlighted by the novel TNFR crosstalk that accelerates tumour cell-specific death, and may have implications for the use of CD40 as a therapeutic target.

Abstract 2076: Opal is a conditional 4-1BB agonistic fusion protein comprising trimerized 4-1BB ligand and a high affinity variant of the extracellular domain of PD-1

Abstract 4-1BB is a co-stimulatory TNF receptor family member upregulated on activated T cells, where it supports proliferation and enhanced functionality. 4-1BB agonizing monoclonal antibodies have thus far failed to progress beyond early clinical development, either due to hepatic toxicities caused by FcγR-crosslinking (urelumab) or due to low clinical activity (utomilumab). An attractive next-generation approach to 4-1BB agonistic therapeutics are multi-functional biologics that conditionally activate 4-1BB only in the tumor microenvironment, thereby localizing activity and minimizing toxicity. Furthermore, there is a strong rationale to combine 4-1BB agonism with checkpoint inhibition. Opal is a novel multi-specific construct that, in a single molecule, combines (i) a single-chain, trimeric 4-1BB ligand (sc4-1BBL); (ii) an engineered variant of the PD1 extracellular domain (ECD) with affinity-enhancing mutations (mutPD1); and (iii) a serum half-life extension domain. By fusing sc4-1BBL and mutPD1, 4-1BB agonism became conditional on the presence of PDL1 or PDL2. By using the mutPD1 ECD rather than an anti-PDL1 antibody, the specificity of Opal is expanded to PDL2-expressing cancer cells with low to undetectable levels of PDL1. The use of trimeric 4-1BBL is expected to optimally agonize the 4-1BB receptor by engaging its natural binding site. Opal omits a functional Fc gamma domain to avoid the hepatic toxicities associated with urelumab. Opal properties were characterized using biophysical and cellular assays. We showed by surface plasmon resonance spectroscopy that compared to wildtype (wt) PD1, mutPD1 had affinities for PDL1 and PDL2 that were enhanced by >2000-fold and >200-fold, respectively. In a competition ELISA assay, Opal effectively blocked wt PD1 from binding to PDL1 and PDL2. PDL1/2-conditional activation of 4-1BB was shown in two independent assays. First, using 4-1BB reporter cells, 4-1BB activity was enhanced >5x when Opal was pre-complexed with recombinant PDL1-Fc. Second, when activated PBMCs were co-cultured in the presence of HEK293T-PDL1 or -PDL2 cells with serial dilutions of Opal, induction of IFNγ secretion was detectable only in the presence of PDL1 or PDL2. Importantly, variants of Opal with abrogated PD1 or 4-1BBL domains failed to trigger IFNγ production. To evaluate the efficacy and toxicity of Opal in vivo, MC38 tumor cells expressing human PDL1 were inoculated in the flanks of mice expressing human 4-1BB. When tumor-bearing mice were treated IP with Opal at 1 mg/kg, 7/10 mice exhibited durable cures without significant body weight loss. Taken together, the preclinical data suggests that Opal may exhibit single-agent activity in solid tumors by conditionally activating effector cells in the tumor microenvironment via 4-1BB agonism while concurrently blocking the PD1/PDL1/PDL2 axis. Citation Format: Naveen K. Mehta, Bochong Li, Patrick A. Baeuerle, Jennifer S. Michaelson. Opal is a conditional 4-1BB agonistic fusion protein comprising trimerized 4-1BB ligand and a high affinity variant of the extracellular domain of PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2076.

SRSF6 Regulates the Alternative Splicing of the Apoptotic Fas Gene by Targeting a Novel RNA Sequence.

Simple SummaryAlternative splicing (AS) produces multiple mRNA isoforms from a gene to make a large number of proteins. Fas (Apo-1/CD95) pre-mRNA, a member of TNF receptor family that mediates apoptosis, can generate pro-apoptotic and anti-apoptotic proteins through AS. Here, we identified SRSF6 as an essential regulator protein in Fas AS. We further located a new functional target sequence of SRSF6 in Fas splicing. In addition, our large-scale RNA-seq analysis using GTEX and TCGA indicated that while SRSF6 expression was correlated with Fas expression in normal tissues, the correlation was disrupted in tumors. Our results suggest a novel regulatory mechanisms of Fas AS.Alternative splicing (AS) is a procedure during gene expression that allows the production of multiple mRNAs from a single gene, leading to a larger number of proteins with various functions. The alternative splicing (AS) of Fas (Apo-1/CD95) pre-mRNA can generate membrane-bound or soluble isoforms with pro-apoptotic and anti-apoptotic functions. SRSF6, a member of the Serine/Arginine-rich protein family, plays essential roles in both constitutive and alternative splicing. Here, we identified SRSF6 as an important regulatory protein in Fas AS. The cassette exon inclusion of Fas was decreased by SRSF6-targeting shRNA treatment, but increased by SRSF6 overexpression. The deletion and substitution mutagenesis of the Fas minigene demonstrated that the UGCCAA sequence in the cassette exon of the Fas gene causes the functional disruption of SRSF6, indicating that these sequences are essential for SRSF6 function in Fas splicing. In addition, biotin-labeled RNA-pulldown and immunoblotting analysis showed that SRSF6 interacted with these RNA sequences. Mutagenesis in the splice-site strength alteration demonstrated that the 5′ splice-site, but not the 3′ splice-site, was required for the SRSF6 regulation of Fas pre-mRNA. In addition, a large-scale RNA-seq analysis using GTEX and TCGA indicated that while SRSF6 expression was correlated with Fas expression in normal tissues, the correlation was disrupted in tumors. Furthermore, high SRSF6 expression was linked to the high expression of pro-apoptotic and immune activation genes. Therefore, we identified a novel RNA target with 5′ splice-site dependence of SRSF6 in Fas pre-mRNA splicing, and a correlation between SRSF6 and Fas expression.

Protective Effect of TNFRSF11A rs7239667 G > C Gene Polymorphism on Coronary Outcome of Kawasaki Disease in Southern Chinese Population.

BackgroundThe main symptoms of Kawasaki disease (KD) are inflammatory vasculitis characterized by fever lasting 1–2 weeks, failure to respond to antibiotic treatment, conjunctivitis, redness of the lips and mouth, strawberry tongue, and painless enlargement of the neck lymph nodes. Studies have been shown that tumor necrosis factor (TNF) and TNF receptor family members are abnormally expressed in the acute phase of Kawasaki disease, also revealing that these two play a significant role in the pathogenesis of KD. The purpose of our study is to determine the relationship between TNFRSF11A rs7239667 and the pathogenesis of KD and Coronary artery lesions in KD.Methods and ResultsIn this study, TNFRSF11A (rs7239667) genotyping was performed in 1396 patients with KD and 1673 healthy controls. Our results showed that G > C polymorphism of TNFRSF11A (rs7239667) was not associated with KD susceptibility. In addition, the patients with KD were divided into CAA and NCAA groups according to whether they had coronary artery aneurysm (CAA) or not, and the TNFRSF11A rs7239667 genotyping was performed in the two groups. After gender and age calibration, We found that genotype CC of TNFRSF11A may be a protective factor in KD coronary artery damage (adjusted OR = 0.69 95% CI = 0.49–0.99 P = 0.0429) and is more significant in children with KD ≤ 60 months (adjusted OR = 0.49 95% CI = 0.49–0.93 P = 0.0173).ConclusionOur study suggests that TNFRSF11A rs7239667 G > C polymorphism maybe play a protective gene role for the severity of KD coronary artery injury and is related to age, which has not been previously revealed.

Abstract 1889: Dual-specific antibodies that bind to BCMA and TACI (HMBD-009) enable better targeting of multiple myeloma in pre-clinical models

Abstract Existing immunotherapies for multiple myelomas (MM), including bispecific antibodies, antibody-drug conjugates and chimeric antigen receptor-based strategies, have targeted the B-cell maturation antigen (BCMA) that is expressed at the surface of malignant plasma cells (PCs). Although promising, clinical outcomes are hampered by resistance mechanisms that include BCMA extracellular domain shedding and cell-surface downregulation. In addition, BCMA expression is known to be variable among MM patients. Transmembrane activator and CAML interactor (TACI), another member of the tumor necrosis factor receptor (TNFR) super family, represents another viable drug target. TACI expression largely overlaps with BCMA and expression is also increased in PCs of MM patients. Interestingly, TACI may have a role in maintaining an immunosuppressive tumor microenvironment as its expression was observed to be upregulated in Tregs in MM patient's tumor load. Activation of TACI in Tregs triggers their proliferation and survival further supporting a pro-tumoral role for this receptor. Altogether, the dual targeting of both BCMA and TACI should be beneficial to the efficient removal of malignant PCs. Despite low sequence homology, BCMA and TACI share a similar 3D structure, which is likely the reason that APRIL (A proliferation-Inducing ligand) binds to both receptors. We applied our proprietary Rational Antibody Discovery platform to successfully direct the generation of mouse IgG clones able to bind a conserved epitope on BCMA and TACI with sub-nanomolar affinities. In addition, these antibodies were able to inhibit the binding of BCMA to APRIL - an interaction known to favor MM tumor growth and survival. Two IgG clones were successfully humanized and retained their capacity to bind to both BCMA and TACI when heterologously expressed on HEK293, or endogenously expressed in MM cancer cell lines such as H929. No cross-reactivity to other TNFR family members was observed. In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, these clones were able to induce efficient cell killing by binding either BCMA or TACI. Moreover, our lead dual-specific anti-BCMA/anti-TACI antibody (HMBD-009) showed better tumor control compared to other anti-BCMA only antibodies in pre-clinical tumor models of MM. Dual-specific antibodies able to efficiently target both BCMA and TACI represent a promising new avenue to improve the elimination of malignant PCs from MM patients. Citation Format: Fabien M. Decaillot, Dipti Thakkar, Siyu Guan, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kikurp. Dual-specific antibodies that bind to BCMA and TACI (HMBD-009) enable better targeting of multiple myeloma in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1889.

Abstract 501: Double-humanized 4-1BB/4-1BBL mouse is an advanced model for liver toxicity and efficacy evaluation of 4-1BB therapeutic antibodies

Abstract 4-1BB is an activation-induced costimulatory TNF receptor family member which is expressed on a variety of cell types such as activated T cells, NK cells, DCs, B cells, monocytes and Neutrophils. 4-1BB/4-1BBL interaction leads to a series of activation effects including increased cytotoxic T lymphocytes (CTL) activity, cytokine induction, prevention of activation-induced cells death (AICD). Agonistic monoclonal antibodies targeting 4-1BB have shown robust anti-tumor activity but has also caused substantial liver toxicity which hampered their clinical development. To better assist the efficacy and toxicity evaluation of anti 4-1BB antibodies, Biocytogen developed a 4-1BB/4-1BBL double humanized knock in mice, which is the second generation of humanized 4-1BB mice. In the double humanized mouse, the exons of mouse 4-1BBL and 4-1BB genes that encode the transmembrane domain and extracellular domain were replaced by human counterpart exons. The expression of knocked in exons were confirmed by both RT-PCR and Flow cytometry. In vivo efficacy study showed that 4-1BB antibodies significantly inhibited tumor growth in mice bearing the colon cancer cell line MC38 and this effect was dose-dependent. The tumor growth inhibition (TGI) reached approximately 70% in the mice group dosed 1mg/kg. More importantly, Toxicity study showed that compared to the single humanized 4-1BB mice, double humanized 4-1BB/4-1BBL mice have higher sensitivity on liver toxicity testing, reflected by sera AST and ALT level as well as liver pathological analysis. These results indicate that the double humanized B-h4-1BB/h4-1BBL mouse model, by mimicking a more human like 4-1BB and 4-1BBL interaction, has been proved to be a more advanced option for preclinical toxicity and efficacy evaluation for 4-1BB therapeutic antibodies compared to single humanized 4-1BB mice. Citation Format: Lei Zhao, Linlin Wang, Dirui Li, Li Hui, Qingcong Lin. Double-humanized 4-1BB/4-1BBL mouse is an advanced model for liver toxicity and efficacy evaluation of 4-1BB therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 501.

TNFAIP8 (TIPE) Deficiency Exacerbates Acute Graft Versus-Host Disease in Murine Model

Background: Acute graft-versus-host disease (GVHD) occurs after allogeneic hematopoietic cell transplant (alloHCT) and presents a donor immune cell response against host tissues associated with significant morbidity and mortality. Up to 60% of alloHCT recipients will develop acute GVHD. Damage to the gastrointestinal tract from the conditioning is a key step in its pathogenesis by allowing for systemic translocation of lipopolysaccharide (LPS) and other danger signals, hereby promoting host APC activation. Tumor necrosis factor (TNF)–induced protein 8 (TNFAIP8) family is a newly identified group of proteins consisting of TNFAIP8 (TIPE), TIPE1 (TNFAIP8L1, TNF–induced protein 8-like 1), TIPE2 (TNFAIP8L2), and TIPE3 (TNFAIP8L3). TIPE acts as a negative mediator of apoptosis via inhibition of caspase-3 activation and also induces proliferation. TIPE signaling is involved in activation of NF-kappa-B and AP-1 pathways. TIPE-deficient mice have been shown to develop more severe colitis than wild type mice due to TIPE deficiency in non-hematopoietic cells along with higher levels of TNF and IL-6. Given the potential roles of TIPE in regulating these processes and considering the importance of these cytokines and of epithelial injury in the pathophysiology of GVHD, we hypothesized, that TIPE deficiency of allogeneic HCT recipients results in worsened GVHD.Methods: 9 - 12 week old naive C57BL/6 or TIPE knockout C57BL/6 mice were conditioned with 1000cGy single dose TBI, followed by transplantation of 10 million bone marrow cells and 20 million splenocytes from either syngeneic C57BL/6 or allogeneic BALB/c donors. Animals were monitored for clinical GVHD and survival and analyzed at week 6 after HCT for serum cytokines, T cell expansion, whole spleen cell chimerism and cell proliferation marker.Results: Allogeneic wild type recipients demonstrated significantly better survival when compared to allogeneic TIPE knockout recipients by week 6 (66.7% vs 40.0%, p =0.024). All syngeneic animals survived. Clinical GVHD scores were significantly higher in TIPE-deficient allogeneic recipients at Day +28 (p=0.0166), +35 (p=0.0349), +42 (p=0.0241) compared to allogeneic wild type recipients. Day +42 serum cytokine analysis revealed increased IL-17A (p=0.0027), TNF (p=0.0396), IL-6 (p=0.0064) levels in TIPE-deficient allogeneic recipients compared to allogeneic control. This was associated with increased splenic CD4+ (p=0.04) but not CD8+ (p= 0.0898) cell expansion when compared to allogeneic control. TIPE-deficient and allogeneic control mice showed no differences in splenic donor T cell chimerism (84.4 versus 89.4, p=0.178), indicating comparable donor T cell engraftment. Ki-67 expression was significantly decreased in epithelial cells of small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls, and a similar nonsignificant trend was seen in the colon as well. Active caspase-3 expression was significantly increased in the small intestine of TIPE knockout allogeneic recipients when compared to allogeneic controls and a similar nonsignificant trend was seen in the colon as well. This observation was associated with significantly increased GI tract pathology scores of TIPE-deficient allogeneic recipients compared to allogeneic control. No differences were observed in pathology of lung and liver between TIPE-deficient allogeneic recipients and allogeneic control. Histopathologic analysis of T cell infiltration in small intestine and colon by immunohistochemistry for CD3 antibody revealed no difference between allogeneic groups, suggesting a role of TIPE-deficient nonhematopoietic cells in GVHD pathophysiology.Conclusion TIPE deficiency in allogeneic C57BL/6 recipients resulted in increased mortality after transplant, which was associated with significant increase in pro-inflammatory cytokines expression and decreased proliferation and increased apoptosis of intestinal epithelial cells, suggesting not only exaggerated inflammatory damage but also decreased epithelial regeneration. Therefore, TIPE seems to be a TNF receptor family member with rather protective function in GVHD gut pathophysiology and overexpression may be a potential future target to prevent or treat this complication after allo HCT. DisclosuresNo relevant conflicts of interest to declare.

Coarse Grained Molecular Dynamic Simulations for the Study of TNF Receptor Family Members' Transmembrane Organization.

The Tumor Necrosis Factor (TNF) and the TNF receptor (TNFR) superfamilies are composed of 19 ligands and 30 receptors, respectively. The oligomeric properties of ligands, both membrane bound and soluble, has been studied most. However, less is known about the oligomeric properties of TNFRs. Earlier reports identified the extracellular, membrane-distal, cysteine-rich domain as a pre-ligand assembly domain which stabilizes receptor dimers and/or trimers in the absence of ligand. Nevertheless, recent reports based on structural nuclear magnetic resonance (NMR) highlight the intrinsic role of the transmembrane domains to form dimers (p75NTR), trimers (Fas), or dimers of trimers (DR5). Thus, understanding the structural basis of transmembrane oligomerization may shed light on the mechanism for signal transduction and the impact of disease-associated mutations in this region. To this end, here we used an in silico coarse grained molecular dynamics approach with Martini force field to study TNFR transmembrane homotypic interactions. We have first validated this approach studying the three TNFR described by NMR (p75NTR, Fas, and DR5). We have simulated membrane patches composed of 36 helices of the same receptor equidistantly distributed in order to get unbiassed information on spontaneous proteins assemblies. Good agreement was found in the specific residues involved in homotypic interactions and we were able to observe dimers, trimers, and higher-order oligomers corresponding to those reported in NMR experiments. We have, applied this approach to study the assembly of disease-related mutations being able to assess their impact on oligomerization stability. In conclusion, our results showed the usefulness of coarse grained simulations with Martini force field to study in an unbiased manner higher order transmembrane oligomerization.

Abstract IA04: Overcoming immune resistance with rationally designed combination immunotherapy

Abstract Over the past decade, blockade of the immune checkpoints CTLA-4, PD-1, and PD-L1 has been demonstrated to significantly extend survival of cancer patients across multiple tumor types, including metastatic melanoma, formally proving that immunotherapy is a viable option for the treatment of cancer. These successes have paved the way for the development of additional immune-modulatory antibodies, blocking alternative inhibitory receptors, or engaging costimulatory receptors such as the TNF receptor family member GITR. However, the clinical experience accumulated thus far with checkpoint blockade has clearly shown that only a limited fraction of patients achieve durable clinical benefit with these treatments. This highlights the need to deepen our understanding of the molecular mechanisms underlying response and resistance to immunotherapy and design more personalized and rational combinations based on these therapies. Immune-regulatory mechanisms are one of the major barriers limiting efficacy of immunotherapy. CTLA-4 blockade and GITR costimulation are two immunotherapeutic strategies known to interfere with conventional immunosuppressive regulatory T cells (Tregs). We thus investigated the effects of CTLA-4 blockade and GITR costimulation on suppressive T cells in in vivo mouse melanoma models resistant to these therapies with the aim to clarify the molecular mechanisms underlying refractoriness and provide the rationale to develop more effective therapeutic combinations. To understand the clinical relevance of these findings, we explored the same effects in cancer patients treated with CTLA-4 blockade or GITR costimulation. We found that CTLA-4 blockade, while counteracting conventional Tregs, promotes the expansion of a subset of CD4+Foxp3-T cells expressing high levels of PD-1 (4PD1hi), which constitute a new immunosuppressive T-cell population with T-follicular-helper-like features. Importantly, we observed that anti-CTLA-4 increases the frequency of circulating 4PD1hi in a dose-dependent manner. In contrast, PD-1 blockade decreases 4PD1hi in function of its clinical activity, underscoring the relevance of this cell subset as a pharmacodynamic and prognostic biomarker of checkpoint blockade. These findings indicate that optimizing checkpoint blockade doses and combination regimens so as to keep 4PD1hi in the right balance may favor a positive outcome. In mouse melanoma models of response (early tumors) and refractoriness (advanced tumors) to GITR agonism, we found that anti-GITR efficiently reduces Tregs and increases effector:Treg ratios in both curative and refractory treatment conditions. However, T-cell activation and cytotoxic functions are favored selectively in the presence of low tumor burden. Counteracting exhaustion with PD-1 blockade in combination with GITR agonism restored responsiveness of advanced tumors and CD8+ T-cell functionality. Aligned with the effects of anti-GITR in mice, we found that the agonist anti-human GITR antibody TRX518 decreases Tregs in peripheral blood and tumor to similar extents in patients treated in the first-in-human single-dose monotherapy trial (NCT01239134). However, coincident downregulation of Tregs in the peripheral blood and at the tumor site upon TRX518 was not sufficient to achieve substantial clinical responses in this population of advanced solid cancer patients. This suggests that Treg elimination from advanced tumors may not be sufficient to activate cytotoxic T-cell responses unless the T-cell exhaustion process is concurrently blocked. Based on these preclinical and clinical observations, we have started to explore anti-GITR in combination with PD-1 pathway blockade in patients with advanced solid tumors (NCT02628574). Taken as a whole, these findings illustrate the value of conventional and nonconventional immune-suppressive T cells as biomarkers of biologic and therapeutic activity of immunotherapy in melanoma and other tumor types. In addition, these results indicate that inhibition of immune-regulatory mechanisms, such as immunosuppressive T cells, may need to be associated with strategies able to positively activate T-cell responses to achieve significant clinical benefit. Citation Format: Roberta Zappasodi, Sadna Budhu, Cynthia Sirard, Jingjing Qi, Cailian Liu, Yanyun Li, Yasin Senbabaoglu, Sasikanth Manne, Billel Gasmi, Hong Zhong, Xia Yang, Moshen Abu-Akeel, David Schaer, Alexander Huang, Walter Newman, Philip Wong, Katherine S. Panageas, Michael A. Postow, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Matthew D. Hellmann, E. John Wherry, Taha Merghoub, Jedd D. Wolchok. Overcoming immune resistance with rationally designed combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA04.

Characterisation of a second gain of function EDAR variant, encoding EDAR380R, in East Asia

Ectodysplasin A1 receptor (EDAR) is a TNF receptor family member with roles in the development and growth of hair, teeth and glands. A derived allele of EDAR, single-nucleotide variant rs3827760, encodes EDAR:p.(Val370Ala), a receptor with more potent signalling effects than the ancestral EDAR370Val. This allele of rs3827760 is at very high frequency in modern East Asian and Native American populations as a result of ancient positive selection and has been associated with straighter, thicker hair fibres, alteration of tooth and ear shape, reduced chin protrusion and increased fingertip sweat gland density. Here we report the characterisation of another SNV in EDAR, rs146567337, encoding EDAR:p.(Ser380Arg). The derived allele of this SNV is at its highest global frequency, of up to 5%, in populations of southern China, Vietnam, the Philippines, Malaysia and Indonesia. Using haplotype analyses, we find that the rs3827760 and rs146567337 SNVs arose on distinct haplotypes and that rs146567337 does not show the same signs of positive selection as rs3827760. From functional studies in cultured cells, we find that EDAR:p.(Ser380Arg) displays increased EDAR signalling output, at a similar level to that of EDAR:p.(Val370Ala). The existence of a second SNV with partly overlapping geographic distribution, the same in vitro functional effect and similar evolutionary age as the derived allele of rs3827760, but of independent origin and not exhibiting the same signs of strong selection, suggests a northern focus of positive selection on EDAR function in East Asia.

TRAF3 can interact with GMEB1 and modulate its anti-apoptotic function

BackgroundMembers of Tumor Necrosis Factor (TNF) Receptor-Associated Factors (TRAFs) family interact with the cytoplasmic tails of TNF receptor family members to mediate signal transduction processes. TRAF3 has a major immunomodulatory function and TRAF3 deficiency has been linked to malignancies, such as multiple myeloma and lymphoid defects. In order to characterize the molecular mechanisms of TRAF3 signaling, the yeast two-hybrid system was used to identify proteins that interact with TRAF3.ResultsThe yeast two-hybrid screen of a human B cell cDNA library with TRAF3 as bait, identified Glucocorticoid Modulatory Element-Binding Protein 1 (GMEB1) as a TRAF3-interacting protein. Previous studies indicated that GMEB1 functions as a potent inhibitor of caspase activation and apoptosis. The interaction of TRAF3 and GMEB1 proteins was confirmed in mammalian cells lines, using immunoprecipitation assays. The RING and TRAF-C domains of TRAF3 were not essential for this interaction. The overexpression of TRAF3 protein enhanced the anti-apoptotic function of GMEB1 in HeLa cells. On the other hand, downregulation of TRAF3 by RNA interference decreased significantly the ability of GMEB1 to inhibit apoptosis. In addition, LMP1(1–231), a truncated form of the EBV oncoprotein LMP1, that can interact and oligomerize with TRAF3, was also able to cooperate with GMEB1, in order to inhibit apoptosis.ConclusionsOur protein-interaction experiments demonstrated that TRAF3 can interact with GMEB1, which is an inhibitor of apoptosis. In addition, cell viability assays showed that overexpression of TRAF3 enhanced the anti-apoptotic activity of GMEB1, supporting a regulatory role of TRAF3 in GMEB1-mediated inhibition of apoptosis. Better understanding of the molecular mechanism of TRAF3 function will improve diagnostics and targeted therapeutic approaches for TRAF3-associated disorders.

TNF Receptor-Associated Factor 6 Mediates TNFα-Induced Skeletal Muscle Atrophy in Mice During Aging.

During aging, muscle mass decreases, leading to sarcopenia, associated with low-level chronic inflammation (inflammaging), which induces sarcopenia by promoting proteolysis of muscle fibers and inhibiting their regeneration. Patients with a variety of pathologic conditions associated with sarcopenia, including rheumatoid arthritis (RA), have systemically elevated TNFα serum levels, and transgenic mice with TNFα overexpression (TNF-Tg mice, a model of RA) develop sarcopenia between adolescence and adulthood before they age. However, if and how TNFα contributes to the pathogenesis of sarcopenia during the normal aging process and in RA remains largely unknown. We report that TNFα levels are increased in skeletal muscles of aged WT mice, associated with muscle atrophy and decreased numbers of satellite cells and Type IIA myofibers, a phenotype that we also observed in adult TNF-Tg mice. Aged WT mice also have increased numbers of myeloid lineage cells in their skeletal muscles, including macrophages and granulocytes. These cells have increased TNFα expression, which impairs myogenic cell differentiation. Expression levels of TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, which mediates signaling by some TNF receptor (TNFR) family members, are elevated in skeletal muscles of both aged WT mice and adult TNF-Tg mice. TRAF6 binds to TNFR2 in C2C12 myoblasts and mediates TNFα-induced muscle atrophy through NF-κB-induced transcription of the muscle-specific E3 ligases, Atrogen1 and Murf1, which promote myosin heavy-chain degradation. Haplo-deficiency of TRAF6 prevents muscle atrophy and the decrease in numbers of satellite cells, Type IIA myofibers, and myogenic regeneration in TRAF6+/- ;TNF-Tg mice. Our findings suggest that pharmacologic inhibition of TRAF6 signaling in skeletal muscles during aging could treat/prevent age- and RA-related sarcopenia by preventing TNFα-induced proteolysis and inhibition of muscle fiber regeneration. © 2020 American Society for Bone and Mineral Research.

Serum BCMA Levels Predict Outcomes for Patients with MGUS and Smoldering Multiple Myeloma (SMM)

BCMA (B-cell maturation antigen) is a TNF receptor family member found on normal B-cells and malignant B-cells including multiple myeloma (MM). It plays a role in in proliferation pathways and antiapoptotic. Levels of serum (s)BMCA are increased in patients (pts) with plasma cell disorders (PCD) and higher with each stage of disease: healthy donor< MGUS.

Development of a Bispecific Antibody Targeting CD30 and CD137 on Hodgkin and Reed-Sternberg Cells

Hodgkin Lymphoma (HL) is a malignancy that frequently affects young adults. Although, there are effective treatments not every patient responds, necessitating the development of novel therapeutic approaches, especially for relapsed and refractory cases. The two TNF receptor family members CD30 and CD137 are expressed on Hodgkin and Reed Sternberg (HRS) cells, the malignant cells in HL. We found that this co-expression is specific for HRS cells. Based on this discovery we developed a bispecific antibody that binds preferentially to the CD30, CD137-double positive HRS cells. The CD30, CD137 bispecific antibody gets internalized into HRS cells opening up the possibility to use it as a carrier for a toxin. This antibody also induces antibody-dependent, cell-mediated cytotoxicity in CD30, CD137-double positive HRS cells. The enhances specificity of the CD30, CD137 bispecific antibody to HRS cells makes it a promising candidate for development as a novel HL treatment.

Serum BCMA levels to predict outcomes for patients with MGUS and smoldering multiple myeloma (SMM).

8020 Background: BCMA (B-cell maturation antigen) is a TNF receptor family member found on normal and malignant B-cells, including multiple myeloma (MM). It plays a role in proliferation and antiapoptotic pathways. Levels of serum (s)BMCA are elevated in patients (pts) with plasma cell disorders (PCD) and increase with each stage of disease: healthy donor&lt; MGUS&lt;SMM&lt; active untreated MM. The purpose of this study was to test whether sBCMA levels predict progression of MGUS or SMM to MM. Methods: There were 3 cohorts in this retrospective study: MGUS progressing to MM (n=42); MGUS not progressing to MM (n=49); SMM progressing to MM (n=32). sBCMA levels were measured using an ELISA-based assay with a polyclonal anti-BCMA antibody from R&amp;D Systems (Minneapolis, MN). The Kruskal-Wallis analysis was used to assess differences. The relationships between sBCMA and both time to progression and overall survival were also assessed using Cox proportional hazard models. Results: The highest values of sBCMA were seen among pts with more advanced PCD (Table). The lowest baseline levels were seen in pts with MGUS who did not progress; the change of sBCMA over time was lowest in the MGUS non-progressors. ROC analysis identified a cutoff of 74.4 ng/mL to be predictive of progression at 5 years. This cut-point was associated with a risk ratio of progression of 5.8 (95%CI 3.2, 11.3) for all comers, a risk ratio of death for all comers of 2.5 (95%CI 1.5, 4.2), and a risk ratio of death for MGUS pts of 3.3 (95%CI 1.9, 5.7). Conclusions: Serum BCMA levels were predictive of diagnosis, progression and death among pts with MGUS or SMM. Limitations of the current study are that only a minority of pts had baseline bone marrow exams or serum FLCs to place sBCMA risk in the context of other previously described risk factors. Serum FLC is now being determined on all patients. [Table: see text]

Immunodeficiency and EBV-induced lymphoproliferation caused by 4-1BB deficiency

The tumor TNF receptor family member 4-1BB (CD137) is encoded by TNFRSF9 and expressed on activated Tcells. 4-1BB provides a costimulatory signal that enhances CD8+ T-cell survival, cytotoxicity, and mitochondrial activity, thereby promoting immunity against viruses and tumors. The ligand for 4-1BB is expressed on antigen-presenting cells and EBV-transformed B cells. We investigated the genetic basis of recurrent sinopulmonary infections, persistent EBV viremia, and EBV-induced lymphoproliferation in 2 unrelated patients. Whole-exome sequencing, immunoblotting, immunophenotyping, and invitro assays of lymphocyte and mitochondrial function were performed. The 2 patients shared a homozygous G109S missense mutation in 4-1BB that abolished protein expression and ligand binding. The patients' CD8+ T cells had reduced proliferation, impaired expression of IFN-γ and perforin, and diminished cytotoxicity against allogeneic and HLA-matched EBV-B cells. Mitochondrial biogenesis, membrane potential, and function were significantly reduced in the patients' activated T cells. An inhibitory antibody against 4-1BB recapitulated the patients' defective CD8+ T-cell activation and cytotoxicity against EBV-infected B cells invitro. This novel immunodeficiency demonstrates the critical role of 4-1BB costimulation in host immunity against EBV infection.

Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase.

NF-κB-inducing kinase (NIK) is a protein kinase central to the noncanonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which has been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of lead chemical series of NIK inhibitors that were identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTβR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound 4f is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo.

CD70 Modulates Thymocyte Development and Type 1 Diabetes in NOD Mice

CD70 is a tumor necrosis factor (TNF) superfamily member expressed by activated T cells, B cells, dendritic cells, NK cells and mature macrophages. It interacts with CD27, a TNF receptor family member on T cells that regulates T cell activation, proliferation and survival. Type 1 diabetes (T1D) is caused by T cell mediated beta cell destruction, however, T1D-prone NOD mice developed accelerated disease in the absence of CD70. This was associated with altered proportions of double negative vs. double positive thymocytes in CD70 knockout mice, indicating that CD70 modulates global thymopoiesis. Also affected, but in opposing directions, was the development of regulatory T cells (Treg) and natural killer T (NKT) cells, two immunoregulatory lymphocyte subsets that are functionally and numerically deficient in NOD mice. Tregs frequencies were reduced by 23% and 32% in CD70 heterozygous (CD70-/+) and homozygous (CD70-/-) knockout mice. In contrast, NKT cell frequencies were respectively increased by 48% and 61% in CD70-/+ and CD70-/- mice compared to standard NOD mice. The higher level of NKT cells in CD70 knockout mice was unexpectedly associated with reduced levels of thymic CD1d, a molecule that is required for NKT cell development. Collectively, our results show that CD70 controls the development of several T cell populations that are known to modulate T1D development in NOD mice and that, in the absence of CD70, T1D is exacerbated. These finding may facilitate the development of clinical therapeutics aimed at enhancing the stimulatory function of CD70 as a way to suppress disease. Disclosure C. Ye: None. J. Driver: None. M.V. Wiles: None. D. Serreze: None.

Clinical significance of the TNF-α receptors, TNFRSF2 and TNFRSF9, on cell migration molecules Fascin-1 and Versican in acute leukemia

In human hematologic malignancies, some of the TNF receptor family members are up-regulated and have the ability to evoke reactions favoring tumor progression. Moreover, cell migration molecules, Fascin-1 and Versican are involved in proliferation, migration and invasion of cancer cells. They are linked to many human cancers. Therefore, we conducted this study to evaluate both the plasma and leukocytes concentrations of tumor necrosis factor receptor super family 2 (TNFRSF2), TNFRSF9, Fascin-1 and Versican in patients with acute leukemia, as well as to correlate these values with clinical features and treatment outcome. Therefore, forty-eight patients with acute myeloid leukemia (AML), thirty-two patients with acute lymphoblastic leukemia (ALL) and fifteen control subjects were included. TNFRSF2, TNFRSF9, Fascin-1 and Versican were measured in plasma and leukocytes of all subjects by enzyme-linked immunosorbent assay. We found that plasma TNFRSF9 was highly elevated in ALL and AML as compared with the control group. In addition, AML patients who failed to achieve complete remission showed a significant increase in leukocytes TNFRSF9 level. TNFRSF2 is significantly increased in plasma and leukocytes of ALL patients when compared with the control group and AML patients. Fascin-1 significantly increased in AML, but not in ALL cases. Plasma and leukocytes levels of Versican significantly increased in AML compared to both control and ALL subjects. Plasma Versican correlated with poor response to induction of chemotherapy in AML cases. In conclusion, TNFRSF2 and TNFRSF9 could act as a possible prognostic biomarkers for the outcomes of ALL patients and TNFRSF9 could be a potential target in AML. Versican may be used as a diagnostic biomarker and as a predictor of the response to chemotherapy in AML. In addition, plasma Fascin-1 is a potential biomarker for AML.