Checkpoint Modulators Research Articles

P04.19 Analysis of tumor immune microenvironment and immune checkpoint modulators across infantile and pediatric pilocytic astrocytomas to elucidate the role of immunotherapy in these tumors

Abstract BACKGROUND Pilocytic astrocytomas are the most common central nervous system tumors in pediatric age-group. Although grade I, some of the cases show recurrence and progression, and few might not be amenable to surgery due to location or size, and hence have a less favorable prognosis. Drugs blocking immune check-point interactions such as those including programmed cell death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are now in clinical use for certain tumors. We performed this study to understand the potential candidature of pilocytic astrocytomas in infants and children for immunotherapy by analyzing the expression of immune checkpoint proteins and immune infiltrate, and correlating with clinical details, wherever possible. MATERIALS AND METHODS Cases with adequate tissue (2010–2017) diagnosed in pediatric age-group (<18 years) were retreived from the archives of Department of Pathology, AIIMS, New Delhi. Immunohistochemistry for PD-L1 (SP263, Ventana), CTLA-4, CD3, CD8, CD4 and CD68 was performed. Quantification of cytotoxic lymphocytes was done using digital imaging in the core of the tumor. RESULTS A total of 50 pilocytic astrocytomas were included, 14 of them were <3 years (infants), while 36 were of pediatric age-group (3–18 years). Overall, male preponderance was noted. Cerebellum was the most common location, followed by 4th venrticle, optic pathway, hypothalamus, cerebrum and thalamus. Almost all CD3 lymphocytes were cytotoxic T-lymphocyes (CD8 positive, CTLs). Helper T-lymphocyte infiltration was not seen. Median CTL density/mm3 was 13/mm3(Range:1–85/mm3). CTLA-4 was positive in 4 cases, positivity ranged from 1–4 cells/lpf. PD-L1 was found to be positive in 7 cases, and the positivity ranged from 1+ to 2+ in 1 to 5% of tumor cells. A median TAM (tumor associated macrophages) density of 44/hpf (range: 1–98/hpf) was noted. There was no correlation of CTL density with PD-L1 or CTLA-4 expression, and neither with TAM density. On correlation with clinical parameters, a higher density of CTLs and TAMs was noted in infants, and a higher proportion of cases revealed PD-L1 positivity, though not statistically significant. There was no correlation of TILs or TAMs with the tumor location. CONCLUSION Immune check point blockade using PD-(L)1 or CTLA4 inhibitors may not be a potential therapeutic option for unresectable or recurrent pilocytic astrocytomas, as low positivity rate as well as extremely low percentage of tumor/ immune cells found to be positive. However, alternate forms of immunotherapy might be helpful as most of the cases showed immune infiltrates and a high density of tumor-associated macrophages (TAMs). Large scale studies with larger numbers and longer follow-up periods including in-vitro and clinical studies are warranted for decoding the tumor immunogram.

Immunization with mannosylated nanovaccines and inhibition of the immune-suppressing microenvironment sensitizes melanoma to immune checkpoint modulators.

A low response rate, acquired resistance and severe side effects have limited the clinical outcomes of immune checkpoint therapy. Here, we show that combining cancer nanovaccines with an anti-PD-1 antibody (αPD-1) for immunosuppression blockade and an anti-OX40 antibody (αOX40) for effector T-cell stimulation, expansion and survival can potentiate the efficacy of melanoma therapy. Prophylactic and therapeutic combination regimens of dendritic cell-targeted mannosylated nanovaccines with αPD-1/αOX40 demonstrate a synergism that stimulates T-cell infiltration into tumours at early treatment stages. However, this treatment at the therapeutic regimen does not result in an enhanced inhibition of tumour growth compared to αPD-1/αOX40 alone and is accompanied by an increased infiltration of myeloid-derived suppressor cells in tumours. Combining the double therapy with ibrutinib, a myeloid-derived suppressor cell inhibitor, leads to a remarkable tumour remission and prolonged survival in melanoma-bearing mice. The synergy between the mannosylated nanovaccines, ibrutinib and αPD-1/αOX40 provides essential insights to devise alternative regimens to improve the efficacy of immune checkpoint modulators in solid tumours by regulating the endogenous immune response.

Abstract 3794: Understanding H. pylori-related gastric MALToma using multi-omics data

Abstract Mucosa-associated lymphoid tissue lymphoma (MALToma) is an extranodal B cell lymphoma, predominantly associated with bacterial infection of the stomach by Helicobacter pylori (H. pylori). Infection of H. pylori can be divided into four steps: 1) entrance into host and survival; 2) motility and chemotaxis; 3) Interaction between adhesion-receptors and colonization, and 4) release of toxins and damage to host. To date, pathogenesis of bacteria related lymphoma and individual functions of H. pylori genes are not yet unearthed. Hence, we aim to characterize the carcinogenic mechanism of H. pylori-related MALToma throughout this study.We obtained fresh gastric tissues of both tumor and adjacent normal region from MALToma patients. Using the tissue and saliva samples, we generated 12 sets of whole exome and transcriptome sequencing data. To identify the gene expression pattern, we aligned the raw data using STAR algorithm and quantified expression of genes in both H. pylori and human genome. Next, we estimated the relative abundance of immune cells in each tissue, employing a linear support vector regression (SVR) based analytical algorithm, CIBERSORT. Besides, we investigated somatic mutations in the patients using our internal pipeline.Gene expression profiling of H. pylori enabled us to identify frequently expressed genes in MALToma, and among them, aliphatic amidase (aimE) was expressed in 9 of 21 lesion tissues. The role of aimE is to contribute to ammonia production, which is analogous to that urease. Then, we explored gene expression pattern of immune checkpoint modulators. We determined that major groups of high CD112, CD155, and VISTA expression, and minor groups of high CD86 and PD-L1 expression, signifying cell adhesion and regulation of T cell activation.From CIBERSORT data, we found that increment of resting CD4 memory T cells and decrease of plasma cells in pair-wise MALToma donors. Since CD4+ T cells are abundantly distributed in the gastric cellular infiltrates, this immune cell composition implies that CD4 memory T cells might have been re-activated by exposure to bacterial antigens. In addition, majority of B cells including plasma cells die after they fight against infected cells, and this supports the decreased plasma cells in the tumor tissues.Using exome sequencing data, we identified two recurrent missense mutations (K169R and N175S) of a cell cycle gene, CDC27, in ~20% of MALToma patients. across the donors. Previous studies mentioned that impaired functions of CDC27 facilitate cell growth as it fails to regulate the cell growth inhibition.To understand carcinogenesis of H. pylori-related MALToma, we focused on determining gene expression patterns, immune cell composition, and recurrent somatic mutations. In conclusion, we demonstrated expression profiling of both H. pylori genes and human checkpoint modulators, estimation of immune cell abundance, and verification of recurrent DNA mutations, in MALToma. Citation Format: Hyojin Song, Eun Ae Kang, Hosim Soh, Sungyoung Lee, Hongseok Yun, Hyunsoo Chung, Jaeyoung Chun, Sung-Soo Yoon, Youngil Koh. Understanding H. pylori-related gastric MALToma using multi-omics data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3794.

Abstract 2390: FcgR co-engagement by anti-TIGIT monoclonal antibodies enhances T cell functionality and antitumor immune responses

Abstract T-cell immunoreceptor with Ig and ITIM domains (TIGIT) has emerged as an important regulator of the cancer-immunity cycle. Preclinical studies have demonstrated that TIGIT antibodies can enhance T and NK cell anti-tumor immunity, and therapeutic antibodies targeting TIGIT are advancing in the clinic. Nonetheless, questions on the optimal format for TIGIT therapeutics remain unresolved. Preclinical studies have demonstrated that anti-TIGIT antibodies enhance anti-tumor immunity by (1) blocking inhibitory signaling downstream of TIGIT/PVR and TIGIT/PVRL2 receptor-ligand interactions and (2) redirecting PVR/PVRL2 ligand binding to the co-stimulatory receptor CD226. Here we describe a novel and unanticipated mechanism of action of anti-TIGIT antibodies in which interactions between the antibody Fc domain and select FcγRs, particularly FcγRIIIA, dramatically improve T cell activation and effector function. In mouse tumor models, TIGIT antibodies that promote FcγR interactions enhance anti-tumor responses, whereas Fc-inert TIGIT antibodies show no such benefit. Moreover, TIGIT antibodies that enhance binding to human FcγRIIIA or mouse FcγRIV promote superior single agent activity and combination activity with other checkpoint modulators in antigen-stimulation assays and mouse tumor models respectively. Notably, this mechanism of action is independent of regulatory T cell depletion. Our findings support a dependence on Fc-FcγR interaction for promoting T cell responsiveness and effector function upon TIGIT antagonism. We further demonstrate that this novel mechanism also extends to anti-CTLA-4, but not anti-PD-1 or anti-LAG-3 antibodies. Altogether, our data describe a novel FcγR-dependent mechanism of action that may enhance the therapeutic activity of anti-TIGIT antibodies, deepen our understanding of this class of therapies, and inform on the optimal design for a new class of Fc-engineered antibodies that could be leveraged to potentially enhance antitumor immune responses. Citation Format: Dhan Chand, Jeremy D. Waight, Elena Paltrinieri, Sylvia Dietrich, Mark Bushell, Mathew Costa, Randi Gombos, Nicholas S. Wilson, Jennifer S. Buell, Robert B. Stein, Alexander Duncan, David A. Savitsky. FcgR co-engagement by anti-TIGIT monoclonal antibodies enhances T cell functionality and antitumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2390.

Abstract LB-020: Neoantigen targeted vaccine converts pancreatic tumors into immune sensitive tumors

Abstract Immune checkpoint inhibitors have been most successful for the treatment of patients with cancers, such as melanoma and smoking associated non-small cell lung cancer - cancers that are inherently rich in infiltrating T cells and harbor a high number of somatic mutations. A subset of these somatic mutations express neoantigens, which are tumor-specific proteins produced during cancer progression and are recognized as foreign to the immune system. However, the majority of aggressive cancers (>70%) which include pancreatic adenocarcinoma, do not respond to immune checkpoint therapy. These tumors typically have a low burden of mutations, express much fewer neoantigens, and therefore fail to attract infiltrating T cells into their immunosuppressive or “cold” tumor microenvironment. An ongoing challenge has thus been to harness an immune response in such insensitive tumors. We show that a neoantigen-targeted vaccine in combination with checkpoint modulators can effectively induce de novo neoantigen-specific T cells into the tumor and result in tumor clearance in a murine pancreatic cancer model, Panc02 (Kinkead et al., JCI Insight 2018). Using Panc02 cell line, 12 immunogenic neoantigens have been identified by combining whole exome sequencing and RNAseq with neoantigen prediction algorithms; the latter provide estimates of MHC binding affinity (NetMHC). 20mer long synthetic long peptides (SLPs) were generated with the mutated residue in the center of the peptide. When the pool of peptides were admixed with a STING-targeted adjuvant, transient tumor regression was evoked. However, when combined with an agonist OX40 antibody and an anti-PD-1 antibody, most mice receiving both vaccine and OX40 antibody cleared established tumors. Flow cytometry of these triple-treatment tumors also revealed a less exhausted population of CD8+ T cells within the tumor (evident as a decreased population of PD1+Lag-3+ CD8+ T-cells). We thus provide proof-of-concept for using a neoantigen-targeted vaccine to activate and expand the limited repertoire of T cells specific for the expressed neoantigens with one or more checkpoint modifiers to induce clinically relevant anti-tumor responses in low mutation load tumors. Further work is focused on identifying ways to better identify which neoantigens to include in vaccine. Notably, current algorithms oftentimes “miss” immunogenic epitopes that have a lower binding affinity. We have thus utilized in silico structural modeling to identify structural correlates of immunogenicity with our pool of Panc02 neoepitopes. Importantly, we implicate the importance of solvent-exposed area of the mutated residue with immunogenicity. We note that non-immunogenic epitopes are often buried within the MHC-I molecule and not “seen” the T cell receptor. Finally, we are currently enriching our neoepitope-targeted vaccine with SLPs that would evoke a preferential CD4+ T cell response, which we suspect will further enhance CD8+ T cell cytotoxicity. We hypothesize that these strategies will further enhance the immunogenicity of neoantigen-targeting vaccines and provide new insights into neoantigen prediction for vaccine immunotherapy. * 1. Kinkead, HL, H... Armstrong TD, Jaffee EM, Zaidi N (2018). Combining STING-based neoantigen-targeted vaccine with checkpoint modulators enhances antitumor immunity in murine pancreatic cancer. JCI Insight 18:20. Citation Format: Neeha Zaidi, Mariya Soban, Shozeb Haider, Fangluo Chen, Todd D. Armstrong, Elizabeth M. Jaffee. Neoantigen targeted vaccine converts pancreatic tumors into immune sensitive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-020.

Abstract 2128: Simple Western Size (SWS) in combination with precision cut cancer tissue slices: An excellent patient-derived platform to support drug development

Abstract The goal of personalized medicine is to provide individual patients with the most appropriate treatment. This approach strongly depends on extensive characterization of individual tumors and their sensitivity to therapeutics. We previously have shown that our drug testing platform based on Precision Cut Cancer Tissue Slices (PCCTS) is applicable to analyze individual responses of patient's tumor tissue to defined compounds such as checkpoint modulators or bispecific antibodies. PCCTS from tumors of CRC and NSCLC cases were generated using a Krumdieck Slicer and incubated in triplicates with immunomodulatory compounds such as OKT3® (Muromonab) or Nivolumab with and without autologous PBMCs. After incubation in 24-well plates, tissue slices were snap frozen and subsequently lysed for analysis of protein expression by Simple Western™ Size (SWS) analysis. In addition to the tissue slices, immune cells were collected from each well and characterized by flow cytometry. Cytokines as an important read-out for immune cell stimulation were analyzed using the Meso Scale Discovery® technology. Combining the different technologies enables a comprehensive characterization of drug effects in individual patient samples. The SWS technology by ProteinSimple™, a highly standardized capillary western blot, using minute amounts of protein (ng), has become extremely useful for the low amount of protein generated from small, 500 µm tissue slices. We developed protocols for several signaling molecules such as PD-1, PD-L1, pAkt, Akt, pErk, Erk, and pS6RP running on a Peggy Sue™ instrument. In addition, a SWS assay for the detection of cleaved caspase-3 has been applied for the detection of apoptosis. All targets have been analyzed in duplicates within one run, a very efficient procedure for the limited amount of material available. PCCTS treated with OKT3® or Nivolumab showed significant changes in the expression level of pERK, pAKT, and pS6RP in the cases analyzed. An increase of cleaved caspase-3 was detectable after treatment compared to untreated PCCTS. These data are in accordance to the down-regulation of signaling molecules. The analysis of cytokines in the supernatants using a ten-plex panel from Meso Scale Discovery® confirmed the activation of T-cells after OKT3® treatment. In all patients a significant increase of IFN gamma, IL-2, and TNF alpha was measured. Treatment with Nivolumab resulted in an increase of IFN gamma. This model of PCCTS represents a unique opportunity to test immunemodulatory compounds in a fully human, patient-derived model that is close to the in vivo situation. In combination with sensitive methods such as SWS a detailed characterization of compound induced molecular intracellular signaling pathways is possible and enables a better understanding of the mechanism of action in the process of compound development. Citation Format: Nicole Grabinski, Kristina Bernoth, Mirja Piller, Moiken Petersen, Jana Krüger, Olivia Timm, Hartmut Juhl, Kerstin A. David. Simple Western Size (SWS) in combination with precision cut cancer tissue slices: An excellent patient-derived platform to support drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2128.

Abstract 2816: SB1703 has therapeutic effect on syngeneic mouse model as an immune modulator in TME

Abstract Although stage I melanoma is highly curable, the prognosis of late stage melanoma is poor and treatment options are limited to immune checkpoint modulators (ICMs). In this study, we synthesized a novel small molecule, SB1703, and identified high-mobility group box (HMGB) proteins as the direct binding target proteins. SB1703 inhibited melanoma growth in B16F10 syngeneic mouse model. To elucidate the tumor growth inhibitory mechanism, we determined the changes in myeloid-derived suppressor cell (MDSC) number during drug treatment. MDSCs expand in disease states such as cancer and have immune suppressive functions. Serum HMGB1 and interleukin (IL)-6 levels were increased in vehicle administered mice and decreased in SB1703 administered mice. We examined the possibility of combinatorial treatment with SB1703 and ICMs. SB1703 showed synergistic effect with ICMs. In conclusion, SB1703 exhibited therapeutic effects against melanoma in both single and combination treatments with ICMs, which might be due to modulation of myeloid cells such as MDSCs Citation Format: Hyo Young Kim, Hee-Jung Kim, Jae Hoon Jung, So Young Jo, Ju Hee Kim, Wansang Cho, Mingi Kim, Junhyeong Yim, Seung Bum Park. SB1703 has therapeutic effect on syngeneic mouse model as an immune modulator in TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2816.

Abstract 2393: Bridging MOA-based reporter bioassays with PBMC based ADCC for immunotherapy drug development

Abstract Having a functional bioassay that is MOA-based, accurate, precise, robust and reproducible is critical for the development of antibody-based biologics. We have developed reporter bioassays that meet these criteria for a broad range of antibody modalities including Fc effector function, immune checkpoint modulation, bispecific antibody engagement, cytokine modulation, and others. Here we will present the latest technology advancements and bridging of the PBMC based ADCC cell killing assay with reporter based ADCC Bioassay. Citation Format: Mei Cong, Pete Stecha, Aileen paguio, brock binkowski, Zhi-Jie Cheng, Frank Fan. Bridging MOA-based reporter bioassays with PBMC based ADCC for immunotherapy drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2393.

Abstract 3241: Novel anti-CD40 antibodies demonstrate anti-tumor activity in humanized mouse models

Abstract CD40 (Cluster of Differentiation 40) is a member of the TNF-receptor superfamily. CD40 is a co-stimulatory protein found primarily on antigen presenting cells, including B cells, macrophages and dendritic cells. Its ligand, CD40L (CD154) is predominantly expressed on T helper cells, whose engagement activates antigen presenting cells and induces a variety of positive downstream effects. Whereas most immune checkpoint receptors are expressed on the surface of T cells, CD40 stands out as it is expressed by antigen presenting cells and controls the proper activation of these cells. In turn, CD40 activation exerts a significant impact on T cell-mediated activity against cancer cells. Several pharmaceutical companies are pursuing anti-CD40 therapies for treating hematological and solid cancers. Therefore, it is imperative to develop novel CD40 antibodies to boost cancer patients’ own immunity in light of other successful immune checkpoint modulators. We asked whether we could find suitable anti-CD40 reagents that successfully combat developed tumors in preclinical settings. We designed a powerful screening strategy based on Biocytogen’s in vivo drug screening platform. Candidate antibodies were generated by immunizing wild-type mice with recombinant hCD40 protein. Hybridomas were then screened by high-throughput flow cytometry. Instead of examining the immune modulating activity in cell culture, we tested these antibodies in B-hCD40 mice bearing MC38 tumors and monitored the tumor growth. Through this straightforward screening strategy, several clones stood out by their unparalleled benefits. These clones were further selected for humanization. Furthermore, we identified that one clone works in concert with Keytruda in the dual humanized model of h-CD40 and h-PD1. In conclusion, we uncovered CD40 antibody candidates with the most potent anti-tumor activity which are promising as tools for cancer treatment in the preclinical setting. Citation Format: Chaoshe Guo, Benny(Yi) Yang, Jian Ni, Yanan Guo, Tian Gan. Novel anti-CD40 antibodies demonstrate anti-tumor activity in humanized mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3241.

BRAF Inhibition: Bridge or Boost to T-cell Therapy?

The concept of treatment-refractory disease has evolved as checkpoint modulation has changed the therapeutic landscape for patients with metastatic melanoma. Developing meaningful salvage strategies will involve the exploration of combination therapies and new immunotherapeutics, including adoptive transfer of tumor-infiltrating lymphocytes or other T-cell-based therapy.See related article by Atay et al., p. 2783.

Immune infiltration in renal cell carcinoma.

Immune infiltration of tumors is closely associated with clinical outcome in renal cell carcinoma (RCC). Tumor‐infiltrating immune cells (TIICs) regulate cancer progression and are appealing therapeutic targets. The purpose of this study was to determine the composition of TIICs in RCC and further reveal the independent prognostic values of TIICs. CIBERSORT, an established algorithm, was applied to estimate the proportions of 22 immune cell types based on gene expression profiles of 891 tumors. Cox regression was used to evaluate the association of TIICs and immune checkpoint modulators with overall survival (OS). We found that CD8+ T cells were associated with prolonged OS (hazard ratio [HR] = 0.09, 95% confidence interval [CI].01‐.53; P = 0.03) in chromophobe carcinoma (KICH). A higher proportion of regulatory T cells was associated with a worse outcome (HR = 1.59, 95% CI 1.23‐.06; P < 0.01) in renal clear cell carcinoma (KIRC). In renal papillary cell carcinoma (KIRP), M1 macrophages were associated with a favorable outcome (HR = .43, 95% CI .25‐.72; P < 0.01), while M2 macrophages indicated a worse outcome (HR = 2.55, 95% CI 1.45‐4.47; P < 0.01). Moreover, the immunomodulator molecules CTLA4 and LAG3 were associated with a poor prognosis in KIRC, and IDO1 and PD‐L2 were associated with a poor prognosis in KIRP. This study indicates TIICs are important determinants of prognosis in RCC meanwhile reveals potential targets and biomarkers for immunotherapy development.

Immune checkpoint modulators in cancer immunotherapy: Recent advances and combination rationales.

As a new hallmark of cancer, immune surveillance evading plays a critical role in carcinogenesis. Through modulating the immune checkpoints, immune cells in tumor microenvironment can be harnessed to battle cancer cells. In recent years, the administration of anti-CTLA or/and anti-PD-1/L1 antibody has exhibited unexpected antitumor effect in multiple types of cancer, motivating the researchers to find more potential immune checkpoints as clinical targets. A wealth of clinical trials have been done to evaluate the safety and efficacy of monotherapy or combination therapy with immune checkpoint modulators. However, there still exist problems such as low response rate and adverse events in the clinical, which in turn leads us to the basic study. The better understanding of the crosstalk between the immune cells and the cancer cells within the microenvironment may inspire us new ideas for cancer treatment. In this review, we mainly summarize the recent advances in application of immune checkpoint modulators and the combination rationales, and discuss the problems existing in the precision therapy with immune checkpoint modulators.

Critical role of kinase activity of hematopoietic progenitor kinase 1 in anti-tumor immune surveillance.

Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of—function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens.

Role of cytokine agonists and immune checkpoint inhibitors toward HIV remission.

The current article describes the current status of the use of cytokines and immune-checkpoint inhibitors as therapeutic strategies toward HIV remission. Clinical trials using IL-2 and IL-7 showed increased levels of circulating T cells, although no reduction to the viral reservoir was observed. Studies in nonhuman primates (NHP) demonstrated that experimental IL-15 administration increased proliferation and cytotoxicity of simian immunodeficiency virus (SIV)-specific CD8 T cells, and promoted their localization to the lymph node (LN) B cell follicles. Immune checkpoint modulators targeting programed cell death-1 and cytotoxic T-lymphocyte associated protein 4, successfully used in oncologic diseases, have shown potential to restore HIV-specific function in early stage clinical trials, while also transiently increasing plasma and cell-associated viral RNA. Due to the complexity of the mechanisms regulating HIV persistence, it is very likely that combinatorial approaches, including cytokines with immune checkpoint blockades (ICBs), will be needed to achieve HIV remission. The present review covers approaches based on cytokine agonists and immune checkpoint inhibitors that have shown promise toward therapeutic pathways for HIV remission. These strategies have been tested preclinically in animal models of HIV infection to determine their safety, activity, and mechanisms of action, with the goal to inform the design of the most synergistic combinatorial strategies. Several of these interventions are included in ongoing or planned clinical trials in HIV infection; these trials will elucidate the clinical efficacy of these innovative immunotherapy approaches toward HIV remission.

The expression of the immune checkpoint regulator VISTA correlates with improved overall survival in pT1/2 tumor stages in esophageal adenocarcinoma

ABSTRACTImmune checkpoint modulation in cancer has been demonstrated as a high-value therapeutic strategy in many tumor entities. VISTA is an immune checkpoint receptor regulating T-cell function. To the best of our knowledge, nothing is known about the expression and prognostic impact of VISTA on tumor infiltrating lymphocytes (TILs) in the tumor microenvironment of esophageal adenocarcinoma (EAC). We analyzed in total 393 EACs within a test-cohort (n = 165) and a validation-cohort (n = 228) using a monoclonal antibody (clone D1L2G). These data were statistically correlated with clinical as well as molecular data. 22.2% of the tumor cohort presented with a VISTA expression on TILs. These patients demonstrated an improved median overall survival compared to patients without VISTA expression (202.2 months vs. 21.6 months; p < 0.0001). The favorable outcome of VISTA positive tumors is significant in the entire cohort but mainly driven by the general better prognosis of T1/T2 tumors. However, in the pT1/2 group, VISTA positive tumors show a tremendous survival benefit compared to VISTA negative tumors revealing real long-term survivors in this particular subgroup. The survival difference is independent of the T-stage. This unique characteristic could influence neoadjuvant therapy concepts for EAC, since a profit of therapy could be reduced in the already favorable subgroup of VISTA positive tumors. VISTA emerges as a prognostic biomarker for long-term survival especially in the group of early TNM-stages. Future studies have to show the relevance of VISTA positive TILs within a tumor concerning response to specific immune checkpoint inhibition.

The multiple faces of CD5.

Since its discovery, over 30 years ago, CD5 has been used as a marker to identify T cells, B1-a cells, and B cell chronic lymphocytic leukemia cells. Throughout the years, many studies have described the functional relevance of CD5 as a modulator of T and B cell receptor signaling. However, it has not been until recent years that CD5 has emerged as a functional receptor in other areas of the immune system. Here, we review some of the most important aspects of CD5 as a modulator of TCR and BCR signaling, cell survival receptor both in T and B cells during health and disease, as well as the newly discovered roles of this receptor in thymocyte selection, T cell effector differentiation, and immune tolerance. CD5 was found to promote T cell survival by protecting autoreactive T cell from activation-induced cell death, to promote de novo induction of regulatory T cells in the periphery, to modulate Th17 and Th2 differentiation, and to modulate immune responses by modulating dendritic cell functions. CD5 is overexpressed in Tregs and Bregs, which are fundamental to maintain immune homeostasis. The newly established roles of CD5 in modulating different aspects of immune responses identify this receptor as an immune checkpoint modulator, and therefore it could be used as a target for immune intervention in different pathologies such as cancer, autoimmune diseases or infections.

Immunotherapy in breast cancer.

The idea of using the immune system to fight cancer is over 100 years old. A new molecular approach led to a better understanding of the immune system. Checkpoint regulation, understanding the roles of Tregs, Th1, and Th2, development of Chimeric antigen receptor (CAR)-T cells, as well as regulation of dendritic cells and macrophages, are just a few examples of our understating that has also led to the discovery of immune checkpoint inhibitors (ICIs) and modulators. This led the Nobel Prize committee in 2018, to award Dr. James P. Allison the Nobel Prize in medicine for the discovery of Cytotoxic T-lymphocyte-associated antigen-4, and Dr. Tasuku Honjo for the discovery of programmed cell death-1 (PD-1)/PD-1-ligand (PDL-1). Several ICIs are already approved by the regulatory authorities, and many more are currently used in studies of several solid tumors and hematologic malignancies. Positive studies have led to the US Food and Drug Administration (FDA) and European Medicines Agency approval of a number of these compounds, but none to date are approved in breast cancer (BC). Moreover, PD-1/PDL-1, MSI high (and dMMR), and tumor mutational burden are the currently “best” predictive markers for benefit from immunotherapy. BCs have some of these markers positive only in subsets but less frequently expressed than most other solid tumors, for example, malignant melanoma or non-small cell lung cancer. To improve the potential efficacy of ICI in BC, the addition of chemotherapy was one of the strategies. Many early and large clinical trials in all phases are underway in BC. We will discuss the role of immune system in BC editing, and the potential impact of immunotherapy in BC outcomes.

Druggable Molecular Targets for the Treatment of Triple Negative Breast Cancer.

Breast cancer (BC) is still the most common cancer among women worldwide. Amongst the subtypes of BC, triple negative breast cancer (TNBC) is characterized by deficient expression of estrogen, progesterone, and human epidermal growth factor receptor 2 receptors. These patients are therefore not given the option of targeted therapy and have worse prognosis as a result. Consequently, much research has been devoted to identifying specific molecular targets that can be utilized for targeted cancer therapy, thereby limiting the progression and metastasis of this invasive tumor, and improving patient outcomes. In this review, we have focused on the molecular targets in TNBC, categorizing these into targets within the immune system such as immune checkpoint modulators, intra-nuclear targets, intracellular targets, and cell surface targets. The aim of this review is to introduce and summarize the known targets and drugs under investigation in phase II or III clinical trials, while introducing additional possible targets for future drug development. This review brings a tangible benefit to cancer researchers who seek a comprehensive comparison of TNBC treatment options.

Immune checkpoint modulation: Tenets and implications in glioblastoma

Glioblastoma (GBM) is the most common primary central nervous system tumor, and despite advances made in traditional chemotherapy and radiation, it continues to carry a poor prognosis. The discovery of the profound immunosuppressive microenvironment created by GBM has given insight on the aggressiveness of this recalcitrant disease. This has led many to believe that immune therapy may yield the improvement in survival that the neuro-oncology community is seeking. In other cancers, the targeting of immune checkpoints has been the most promising immunotherapeutic strategy to date. Immune checkpoints modulate the function of the immune system by increasing or decreasing immune activity. Checkpoint inhibitors and more recently agonists target molecules that regulate immune response to increase immune function either directly or by removal of inhibitory signals. These molecules modulate immunity in the physiologic state to maintain homeostasis, but they are co-opted by cancer to avoid immune detection and attack. The use of checkpoint inhibition to improve cancer therapy has revolutionized the field of oncology, leading to unprecedented improvements in survival from many systemic malignancies. Utilizing PubMed and ClinicalTrials.gov to compile published findings and ongoing trials, we review immune checkpoints and their modulators from bench to bedside over several decades. In this review, the discovery of different checkpoint molecules and the development of drugs used to target them are addressed. In addition, the current state of checkpoint inhibition in GBM, presenting completed and ongoing preclinical and clinical studies utilizing these therapies, is discussed. Finally, we conclude by reviewing the current limitations and potential future directions for the use of checkpoint blockade in the treatment of GBM.

The Prognostic Landscape of Tumor-Infiltrating Immune Cells and Immune Checkpoints in Glioblastoma.

Tumor-infiltrating immune cells are part of a complex microenvironment and associated with improved clinical outcomes in a broad range of tumor types. However, a detailed map for the prognostic landscape of tumor-infiltrating immune cells and immune checkpoint modulators in glioblastoma is still lacking. Here, with the web-accessible resource, The Cancer Immunome Archive, 28 types of both adaptive and innate tumor-infiltrating immune cells were characterized in glioblastoma. Tumors lacking central memory CD4 T cells or natural killer cells were associated with better prognosis in glioblastoma, as verified by immunohistochemical analysis. Moreover, Kaplan-Meier analysis for a total of 71 key immune checkpoint molecules revealed that the expression level of inducible T cell costimulators, tumor necrosis factor superfamily member 14, and UL16 binding protein 1 were negatively correlated with the clinical outcome of patients with glioblastoma. In addition, there was a significant difference between nontumor and glioblastoma samples of several immune checkpoint modulators based on the expression level of their corresponding gene. Collectively, the annotation of tumor-infiltrating immune cells and immune checkpoint modulators in glioblastoma provides a valuable resource for identifying their involvement in tumor escape mechanisms and response to therapy.