Regulation of HHLA2 expression in kidney cancer and myeloid cells.

BACKGROUND: The immune checkpoint HERV-H LTR-associating 2 (HHLA2) is expressed in kidney cancer and various other tumor types. Therapeutics targeting HHLA2 or its inhibitory receptor KIR3DL3 are being developed for solid tumors, including renal cell carcinoma (RCC). However, the regulation of HHLA2 expression remains poorly understood. A better understanding of HHLA2 regulation in tumor cells and the tumor microenvironment is crucial for the successful translation of these therapeutic agents into clinical applications. METHODS: Flow cytometry and quantitative real-time PCR were used to analyze HHLA2 expression in primary kidney tumors ex vivo and during in vitro culture. HHLA2 expression in A498 and 786-O ccRCC cell lines was examined in vitro and in subcutaneous tumor xenografts in NSG mice. Monocytes and dendritic cells were analyzed for HHLA2 expression. We tested a range of cytokines and culture conditions, including hypoxia, to induce HHLA2 expression. RESULTS: Analysis of HHLA2 expression revealed that HHLA2 is expressed on tumor cells in primary kidney tumors ex vivo; however, its expression gradually diminishes during a 4-week in vitro culture period. A498 and 786-O ccRCC tumor cell lines do not express HHLA2 in vitro, but HHLA2 expression was observed when grown as subcutaneous xenografts in NSG immunodeficient mice. Induction experiments using various cytokines and culture conditions failed to induce HHLA2 expression in A498 and 786-O tumor cell lines in vitro. Analysis of HHLA2 expression in monocytes and dendritic cells demonstrated that only IL-10 and BMP4, along with IL-1ß and IL-6 to a lesser extent, modestly enhanced HHLA2 protein and mRNA expression. CONCLUSIONS: HHLA2 expression is induced on kidney cancer cells in vivo by a tumor microenvironmental signal that is not present in vitro. HHLA2 expression is differentially regulated in kidney cancer epithelial cells and monocytes. Cytokines, particularly IL10, that induce HHLA2 expression in monocytes fail to upregulate HHLA2 expression in tumor cell lines in vitro. These findings underscore the importance of the interplay between tumor cell and tumor microenvironmental signals in the regulation of HHLA2. Further investigation is warranted to elucidate the mechanisms involved in HHLA2 regulation and its implications for therapeutic development.

Clinical and research updates on the VISTA immune checkpoint: immuno-oncology themes and highlights.

Immune checkpoints limit the activation of the immune system and serve an important homeostatic function but can also restrict immune responses against tumors. Inhibition of specific immune checkpoint proteins such as the B7:CD28 family members programmed cell death protein-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) has transformed the treatment of various cancers by promoting the anti-tumor activation of immune cells. In contrast to these effects, the V-domain immunoglobulin suppressor of T-cell activation (VISTA) regulates the steady state of the resting immune system and promotes homeostasis by mechanisms distinct from PD-1 and CTLA-4. The effects of VISTA blockade have been shown to include a decrease in myeloid suppression coupled with proinflammatory changes by mechanisms that are separate and distinct from other immune checkpoint proteins; in some preclinical studies these immune effects appear synergistic. Given the potential benefits of VISTA blockade in the context of cancer therapy, the second Annual VISTA Symposium was convened virtually on September 23, 2022, to review new research from investigators and immuno-oncology experts. Discussions in the meeting extended the knowledge of VISTA biology and the effects of VISTA inhibition, particularly on cells of the myeloid lineage and resting T cells, as three candidate anti-VISTA antibodies are in, or nearing, clinical development.

Phase 1 first-in-human study of dalutrafusp alfa, an anti-CD73-TGF-ß-trap bifunctional antibody, in patients with advanced solid tumors.

BACKGROUND: Cluster of differentiation (CD)73-adenosine and transforming growth factor (TGF)-ß pathways are involved in abrogated antitumor immune responses and can lead to protumor conditions. This Phase 1 study (NCT03954704) evaluated the safety, pharmacokinetics, pharmacodynamics, and efficacy of dalutrafusp alfa (also known as GS-1423 and AGEN1423), a bifunctional, humanized, aglycosylated immunoglobulin G1 kappa antibody that selectively inhibits CD73-adenosine production and neutralizes active TGF-ß signaling in patients with advanced solid tumors. METHODS: Dose escalation started with an accelerated titration followed by a 3+3 design. Patients received dalutrafusp alfa (0.3, 1, 3, 10, 20, 30, or 45 mg/kg) intravenously every 2 weeks (Q2W) up to 1 year or until progressive disease (PD) or unacceptable toxicity. RESULTS: In total, 21/22 patients received at least one dose of dalutrafusp alfa. The median number of dalutrafusp alfa doses administered was 3 (range 1-14). All patients had at least one adverse event (AE), most commonly fatigue (47.6%), nausea (33.3%), diarrhea (28.6%), and vomiting (28.6%). Nine (42.9%) patients had a Grade 3 or 4 AE; two had Grade 5 AEs of pulmonary embolism and PD, both unrelated to dalutrafusp alfa. Target-mediated drug disposition appears to be saturated at dalutrafusp alfa doses above 20 mg/kg. Complete CD73 target occupancy on B cells and CD8+ T cells was observed, and TGF-ß 1/2/3 levels were undetectable at dalutrafusp alfa doses of 20 mg/kg and higher. Free soluble (s)CD73 levels and sCD73 activity increased with dalutrafusp alfa treatment. Seventeen patients reached the first response assessment, with complete response, partial response, stable disease, and PD in 0, 1 (4.8%), 7 (33.3%), and 9 (42.9%) patients, respectively. CONCLUSIONS: Dalutrafusp alfa doses up to 45 mg/kg Q2W were well tolerated in patients with advanced solid tumors. Additional evaluation of dalutrafusp alfa could further elucidate the clinical utility of targeting CD73-adenosine and TGF-ß pathways in oncology.

Soluble PD-L1 as an early marker of progressive disease on nivolumab.

BACKGROUND: Soluble PD-L1 (sPD-L1) has been associated with worse prognosis in numerous solid tumors. We determined sPD-L1 levels before and during nivolumab treatment in two prospective clinical trials of metastatic clear cell renal cell carcinoma (RCC) and melanoma patients, and investigated its relationship to clinical factors, biomarkers, and outcome. METHODS: Using a new Single Molecule Array assay, serum sPD-L1 level were determined in RCC (CheckMate 009, n=91) and melanoma (CheckMate 038-Part 1, n=78) prior to, and at two time points on treatment. Gene expression data was obtained from biopsies taken prior to, and at day 28 on treatment. Results were integrated with clinical variables, tumor PD-L1 status from immuno-histochemistry, and genomic mutation status. RESULTS: In RCC patients, sPD-L1 levels were higher in patients with progressive disease as their best response. For both RCC and melanoma patients, progressive or stable disease was associated with an increase in sPD-L1 on nivolumab therapy, whereas mean sPD-L1 levels did not change or declined in patients with objective responses. By categorizing RCC patients into transcriptomic molecular subtypes, we identified a subgroup where the associations between sPD-L1 and progressive disease were particularly evident. In baseline biopsies, we identified six biological processes that were associated with sPD-L1 level in both RCC and melanoma: higher sPD-L1 is associated with lower tumor expression of the Hallmark gene sets 'hypoxia', 'fatty acid metabolism', 'glycolysis', 'MTORC1 signaling' and 'androgen response', and with higher expression of 'KRAS signaling_Down'. CONCLUSION: Baseline and on-therapy sPD-L1 levels in RCC have the potential to predict progressive disease on PD-1 inhibitor nivolumab. In a hypothesis-generating analysis of tumor gene expression, high baseline sPD-L1 is associated with a tumor metabolic state reflecting potentially targetable processes in both melanoma and RCC. In both trials, we observed associations between change in sPD-L1 on treatment and outcome metrics. sPD-L1 levels may further refine a nivolumab-refractory subtype of RCC within transcriptionally based subtypes of RCC.

Monitoring PD-1 Phosphorylation to Evaluate PD-1 Signaling during Antitumor Immune Responses.

PD-1 expression marks activated T cells susceptible to PD-1-mediated inhibition but not whether a PD-1-mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho-PD-1). We showed PD-1+ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1+TIM-3+ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8+ TILs. Phospho-PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.

Progressive immune dysfunction with advancing disease stage in renal cell carcinoma.

The tumor immune microenvironment plays a critical role in cancer progression and response to immunotherapy in clear cell renal cell carcinoma (ccRCC), yet the composition and phenotypic states of immune cells in this tumor are incompletely characterized. We performed single-cell RNA and T cell receptor sequencing on 164,722 individual cells from tumor and adjacent non-tumor tissue in patients with ccRCC across disease stages: early, locally advanced, and advanced/metastatic. Terminally exhausted CD8+ T cells were enriched in metastatic disease and were restricted in T cell receptor diversity. Within the myeloid compartment, pro-inflammatory macrophages were decreased, and suppressive M2-like macrophages were increased in advanced disease. Terminally exhausted CD8+ T cells and M2-like macrophages co-occurred in advanced disease and expressed ligands and receptors that support T cell dysfunction and M2-like polarization. This immune dysfunction circuit is associated with a worse prognosis in external cohorts and identifies potentially targetable immune inhibitory pathways in ccRCC.

Leukemia vaccine overcomes limitations of checkpoint blockade by evoking clonal T cell responses in a murine acute myeloid leukemia model.

We have developed a personalized vaccine whereby patient derived leukemia cells are fused to autologous dendritic cells, evoking a polyclonal T cell response against shared and neo-antigens. We postulated that the dendritic cell (DC)/AML fusion vaccine would demonstrate synergy with checkpoint blockade by expanding tumor antigen specific lymphocytes that would provide a critical substrate for checkpoint blockade mediated activation. Using an immunocompetent murine leukemia model, we examined the immunologic response and therapeutic efficacy of vaccination in conjunction with checkpoint blockade with respect to leukemia engraftment, disease burden, survival and the induction of tumor specific immunity. Mice treated with checkpoint blockade alone had rapid leukemia progression and demonstrated only a modest extension of survival. Vaccination with DC/AML fusions resulted in the expansion of tumor specific lymphocytes and disease eradication in a subset of animals, while the combination of vaccination and checkpoint blockade induced a fully protective tumor specific immune response in all treated animals. Vaccination followed by checkpoint blockade resulted in upregulation of genes regulating activation and proliferation in memory and effector T cells. Long term survivors exhibited increased T cell clonal diversity and were resistant to subsequent tumor challenge. The combined DC/AML fusion vaccine and checkpoint blockade treatment offers unique synergy inducing the durable activation of leukemia specific immunity, protection from lethal tumor challenge and the selective expansion of tumor reactive clones.

Enzalutamide With Radiation Therapy for Intermediate-Risk Prostate Cancer: A Phase 2 Study.

PURPOSE: Androgen deprivation therapy (ADT) is often used as adjuvant treatment with radiation therapy (RT) for intermediate-risk prostate cancer. ADT is associated with multiple side effects, including weight gain, loss of libido, and hot flashes. In contrast, antiandrogen monotherapy has been generally better tolerated. This study aimed to assess the effectiveness of enzalutamide (an antiandrogen) monotherapy with RT for the treatment of intermediate-risk prostate cancer. METHODS AND MATERIALS: This trial was an open-label, phase 2 study of 6 months of enzalutamide monotherapy with external beam RT for intermediate-risk prostate cancer. Enzalutamide was initiated 2 months before external beam RT. The primary endpoint was prostate-specific antigen (PSA) response measured at the end of enzalutamide administration at the 6-month timepoint. Secondary endpoints included assessment of toxicity and changes in anthropomorphic body measurement, sexual function, and metabolism. The sample size was 64 patients. The hypothesis was that if ≥60% of the patients did not achieve a PSA nadir of ≤0.2 ng/mL, the study results would be deemed negative. RESULTS: The results met the prespecified endpoint for efficacy in that PSA values ≤0.2 ng/mL were observed in 49 of 64 patients (77%), and 60 of 64 patients (94%) had PSA values ≤0.5ng/mL. The most frequent adverse events were hypertension and gynecomastia. There were no changes in anthropomorphic body measurements and only modest erectile dysfunction. CONCLUSIONS: Using PSA response as an endpoint, enzalutamide monotherapy may be as effective as ADT in combination with external beam RT for patients with intermediate-risk prostate cancer, and it is associated with fewer side effects. Randomized trials comparing enzalutamide with ADT are justified.

VISTA: A Mediator of Quiescence and a Promising Target in Cancer Immunotherapy.

V-domain Ig suppressor of T cell activation (VISTA) is a B7 family member that maintains T cell and myeloid quiescence and is a promising target for combination cancer immunotherapy. During inflammatory challenges, VISTA activity reprograms macrophages towards reduced production of proinflammatory cytokines and increased production of interleukin (IL)-10 and other anti-inflammatory mediators. The interaction of VISTA with its ligands is regulated by pH, and the acidic pH ~6.0 in the tumor microenvironment (TME) facilitates VISTA binding to P-selectin glycoprotein ligand 1 (PSGL-1). Targeting intratumoral pH might be a way to reduce the immunoinhibitory activity of the VISTA pathway and enhance antitumor immune responses. We review differences among VISTA therapeutics under development as candidate immunotherapies, focusing on VISTA binding partners and the unique structural features of this interaction.

KIR3DL3 Is an Inhibitory Receptor for HHLA2 that Mediates an Alternative Immunoinhibitory Pathway to PD1.

Blockade of the PD1 pathway is a broadly effective cancer therapy, but additional immune-inhibitory pathways contribute to tumor immune evasion. HERV-H LTR-associating 2 (HHLA2; also known as B7H5 and B7H7) is a member of the B7 family of immunoregulatory ligands that mediates costimulatory effects through its interaction with the CD28 family member transmembrane and immunoglobulin domain containing 2 (TMIGD2). However, HHLA2 has also been known to have inhibitory effects on T cells. Here, we report that we have identified killer cell immunoglobulin-like receptor, three immunoglobulin domains and long cytoplasmic tail 3 (KIR3DL3) as an inhibitory receptor for HHLA2 in T cells and natural killer (NK) cells and have generated HHLA2 and KIR3DL3 antibodies that block the immune-inhibitory activity of HHLA2, preserving the costimulatory signal. It is known that HHLA2 is frequently expressed in several tumor types, including clear cell renal cell carcinoma (ccRCC). We found that HHLA2 expression was nonoverlapping with PDL1 expression in ccRCC, suggesting that HHLA2 mediates a mechanism of tumor immune evasion that is independent from PDL1. Blockade of both the PD1 and KIR3DL3 pathways may be a more effective way to reverse tumor immune evasion.See related Spotlight on p. 128.

The importance of exosomal PDL1 in tumour immune evasion.

The interaction of programmed cell death 1 ligand 1 (PDL1) with its receptor programmed cell death 1 (PD1) inhibits T cell responses, and blockade of this interaction has proven to be an effective immunotherapy for several different cancers. PDL1 can be expressed on the surface of tumour cells, immune cells and other cells in the tumour microenvironment but is also found in extracellular forms. Recent studies have explored the importance of different forms of extracellular PDL1, such as on exosomes or as a freely soluble protein, and have shown that PDL1-expressing exosomes can inhibit antitumour immune responses. In patients with melanoma, exosomal PDL1 is also a marker of immune activation early after initiation of therapy with PD1-blocking antibodies and predicts a clinical response to PD1 blockade. In this Progress article, we highlight recent insights into the role of exosomal PDL1 in immune oncology and how it may be useful as a biomarker for the management of cancer or to define a subset of patients who would benefit from therapeutics that block exosome production.

irRECIST for the Evaluation of Candidate Biomarkers of Response to Nivolumab in Metastatic Clear Cell Renal Cell Carcinoma: Analysis of a Phase II Prospective Clinical Trial.

PURPOSE: Immune-related RECIST (irRECIST) were designed to capture atypical responses seen with immunotherapy. We hypothesized that, in patients with metastatic clear cell renal cell carcinoma (mccRCC), candidate biomarkers for nivolumab response would show improved association with clinical endpoints capturing atypical responders (irRECIST) compared with standard clinical endpoints (RECISTv1.1). EXPERIMENTAL DESIGN: Endpoints based on RECISTv1.1 [objective response rate (ORR)/progression-free survival (PFS)] or irRECIST [immune-related ORR (irORR)/immune-related PFS (irPFS)] were compared in patients enrolled in the CheckMate-010 trial. Pretreatment tumors were analyzed by PD-L1 and PD-L2 IHC, and by multiplex immunofluorescence for CD8, PD-1, TIM-3, and LAG-3. T-cell activation signatures were assessed by RNA sequencing. RESULTS: Median irPFS was significantly longer than median PFS. irORR was not significantly different from ORR, but immune-related progressive disease (irPD) rate was significantly lower than progressive disease (PD) rate. Tumor cell (TC) PD-L1 expression was not associated with PFS or ORR, but patients with TC PD-L1 ≥1% had longer median irPFS and higher irORR. High percentage of CD8+ tumor-infiltrating cells (TIC) that are PD-1+TIM-3-LAG-3- (% CD8+PD-1+TIM-3-LAG-3- TIC) correlated with high levels of T-cell activation and was associated with longer median irPFS and higher irORR. Notably, combination of TC PD-L1 expression with % CD8+PD-1+TIM-3-LAG-3- TIC identified three groups of patients for which irPFS and irORR were significantly different. CONCLUSIONS: Atypical responders to nivolumab were identified in the CheckMate-010 trial. We observed improved association of candidate biomarkers for nivolumab response with endpoints defined by irRECIST compared with RECISTv1.1. TC PD-L1 expression in combination with PD-1 expression on CD8+ TIC may predict outcome on nivolumab in mccRCC.

A secreted PD-L1 splice variant that covalently dimerizes and mediates immunosuppression.

Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.

Expression of PD-L1 in Hormone-naïve and Treated Prostate Cancer Patients Receiving Neoadjuvant Abiraterone Acetate plus Prednisone and Leuprolide.

Purpose: Programmed cell death ligand-1 (PD-L1)/programmed cell death-1 (PD-1) blockade has been unsuccessful in prostate cancer, with poor immunogenicity and subsequent low PD-L1 expression in prostate cancer being proposed as an explanation. However, recent studies indicate that a subset of prostate cancer may express significant levels of PD-L1. Furthermore, the androgen antagonist enzalutamide has been shown to upregulate PD-L1 expression in prostate cancer preclinical models. In this study, we evaluated the effect of neoadjuvant androgen deprivation therapy with abiraterone acetate plus prednisone and leuprolide (Neo-AAPL) on PD-L1 expression in prostate cancer.Experimental Design: Radical prostatectomy (RP) tissues were collected from 44 patients with intermediate- to high-risk prostate cancer who underwent RP after Neo-AAPL treatment. Untreated prostate cancer tissues were collected from 130 patients, including 44 matched controls for the Neo-AAPL cases. Tumor PD-L1 expression was detected by IHC using validated anti-PD-L1 antibodies. Tumor-infiltrating CD8+ cells were analyzed in trial cases and matched controls. Expression of DNA mismatch repair genes was examined in PD-L1-positive tumors.Results: Neo-AAPL-treated tumors showed a trend toward decreased PD-L1 positivity compared with matched controls (7% vs. 21% having ≥1% positive tumor cells; P = 0.062). Treated tumors also harbored significantly fewer tumor-infiltrating CD8+ cells (P = 0.029). In 130 untreated prostate cancers, African American ethnicity, elevated serum PSA, and small prostate independently predicted tumor PD-L1 positivity. Loss of MSH2 expression was observed in 1 of 21 PD-L1-positive tumors.Conclusions: A subset of prostate cancer expresses PD-L1, which is not increased by Neo-AAPL treatment, indicating that combining Neo-AAPL treatment with PD-L1/PD-1 blockade may not be synergistic. Clin Cancer Res; 23(22); 6812-22. ©2017 AACR.

Soluble PD-L1 as a Biomarker in Malignant Melanoma Treated with Checkpoint Blockade.

Blockade of the pathway including programmed death-ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) has produced clinical benefits in patients with a variety of cancers. Elevated levels of soluble PD-L1 (sPD-L1) have been associated with worse prognosis in renal cell carcinoma and multiple myeloma. However, the regulatory roles and function of sPD-L1 particularly in connection with immune checkpoint blockade treatment are not fully understood. We identified four splice variants of PD-L1 in melanoma cells, and all of them are secreted. Secretion of sPD-L1 resulted from alternate splicing activities, cytokine induction, cell stress, cell injury, and cell death in melanoma cells. Pretreatment levels of sPD-L1 were elevated in stage IV melanoma patient sera compared with healthy donors. High pretreatment levels of sPD-L1 were associated with increased likelihood of progressive disease in patients treated by CTLA-4 or PD-1 blockade. Although changes in circulating sPD-L1 early after treatment could not distinguish responders from those with progressive disease, after five months of treatment by CTLA-4 or PD-1 blockade patients who had increased circulating sPD-L1 had greater likelihood of developing a partial response. Induction of sPD-L1 was associated with increased circulating cytokines after CTLA-4 blockade but not following PD-1 blockade. Circulating sPD-L1 is a prognostic biomarker that may predict outcomes for subgroups of patients receiving checkpoint inhibitors. Cancer Immunol Res; 5(6); 480-92. ©2017 AACR.

Learning from PD-1 Resistance: New Combination Strategies.

Only a minority of cancer patients respond to anti PD-1 immunotherapy. A recent study demonstrates that PD-1 therapy-resistant melanoma patients present distinct signatures of upregulated genes involved in immunosuppression, angiogenesis, monocyte and macrophage chemotaxis, extracellular matrix remodeling, and epithelial-mesenchymal transition (EMT). Combination targeting of these pathways with PD-1 may help overcome PD-1 resistance, thus producing effective antitumor immunity.

Phase 2 Study of Bevacizumab and Temsirolimus After VEGFR TKI in Metastatic Renal Cell Carcinoma.

BACKGROUND: Inhibiting VEGF and mammalian target of rapamycin (mTOR) pathways are standard treatment approaches for patients with metastatic renal cell carcinoma (mRCC). Here we report the activity and safety of the VEGF ligand inhibitor bevacizumab and the mTOR inhibitor temsirolimus combination in patients with clear cell (CC) and non-clear cell (NCC) mRCC whose disease had failed to respond to prior VEGF blockade. PATIENTS AND METHODS: In this phase 2 investigator-initiated multicenter study, patients received bevacizumab and temsirolimus. The primary end point was 4-month progression-free survival (PFS) rate. Secondary end points included overall response rate, median overall survival (OS), toxicity, and correlative studies of biomarkers downstream of mTOR. RESULTS: Forty patients received at least 1 dose of therapy. Thirty-three (82.5%) had favorable/intermediate risk disease according to International Metastatic Renal Cell Carcinoma Database Consortium criteria, 13 (32.5%) with nccRCC histology. Nineteen (48.7%) had primary vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI)-refractory disease. The 4-month PFS rate was 65%. Overall median PFS and OS were 5.6 and 12.2 months. Median PFS and OS were 6.5 and 9.6 months in patients with primary VEGFR TKI-refractory disease, and 5.6 months and 13.1 months in patients with nccRCC. Dose reductions were needed in 80% of patients. Most frequent toxicities included fatigue, hypertension, dyslipidemia, and proteinuria. Dose discontinuation due to adverse events occurred in 27.5% of patients. Baseline tumor immunohistochemistry for phospho-S6 protein was not associated with clinical benefit. CONCLUSION: Combining bevacizumab and temsirolimus in patients previously treated with VEGFR TKI was possible but with dose reductions and treatment discontinuations. This combination resulted in modest activity, including in patients with primary VEGF-refractory disease and NCC histology.

PD-L1 Antibodies to Its Cytoplasmic Domain Most Clearly Delineate Cell Membranes in Immunohistochemical Staining of Tumor Cells.

Blocking the programmed death-1 (PD-1) pathway has clinical benefit in metastatic cancer and has led to the approval of the mAbs pembrolizumab and nivolumab to treat melanoma and nivolumab for non-small cell lung cancer. Expression of PD-L1 on the cell surface of either tumor cells or infiltrating immune cells is associated with a higher likelihood of response to PD-1 blockade in multiple studies. Most mAbs to PD-L1 in use are directed to its extracellular domain and immunohistochemically stain tumor tissue with a mixture of cytoplasmic and membrane staining. Cytoplasmic staining obscures the interpretation of a positive reaction on the tumor cell membrane, and thus affects the accuracy of PD-L1 scoring systems. We developed a mAb to the cytoplasmic domain of PD-L1, 405.9A11 (9A11), which is both more selective for membranous PD-L1 and more sensitive in IHC and Western blotting, compared with previous mAbs specific for the PD-L1 extracellular domain. Here, we compare immunohistochemical staining patterns of PD-L1 expression in five types of tumors, using five PD-L1 mAbs: 9A11, 7G11, and three commercially available mAbs. We demonstrate that 9A11, as well as two other cytoplasmic domain-specific mAbs, E1L3N and SP142, can clearly delineate the membrane of PD-L1-positive cells in formalin-fixed paraffin-embedded tissue and facilitate interpretation of staining results.

Combination cancer immunotherapy and new immunomodulatory targets.

Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory signals and enabling patients to produce an effective antitumour response. Inhibitors of CTLA4, PD1 or PDL1 administered as single agents have resulted in durable tumour regression in some patients, and combinations of PD1 and CTLA4 inhibitors may enhance antitumour benefit. Numerous additional immunomodulatory pathways as well as inhibitory factors expressed or secreted by myeloid and stromal cells in the tumour microenvironment are potential targets for synergizing with immune checkpoint blockade. Given the breadth of potential targets in the immune system, critical questions to address include which combinations should move forward in development and which patients will benefit from these treatments. This Review discusses the leading drug targets that are expressed on tumour cells and in the tumour microenvironment that allow enhancement of the antitumour immune response.