Search

Summary The authors of a recent study identified noncanonical peptides (NCP) presented by cancer cells’ HLA and observed lack of reactivity to these antigens by endogenous tumor-reactive T cells. In vitro sensitization generated NCP-reactive T cells that recognized epitopes shared by a majority of cancers tested, providing opportunities for novel therapies to shared antigens. See related article by Lozano-Rabella et al., p. xxx

A patient with progressive metastatic pancreatic cancer was treated with a single infusion of 16.2×10(9) autologous T cells that had been genetically engineered to clonally express two allogeneic HLA-C*08:02–restricted T-cell receptors (TCRs) targeting mutant KRAS G12D expressed by the tumors. The patient had regression of visceral metastases (overall partial response of 72% according to the Response Evaluation Criteria in Solid Tumors, version 1.1); the response was ongoing at 6 months. The engineered T cells constituted more than 2% of all the circulating peripheral-blood T cells 6 months after the cell transfer. In this patient, TCR gene therapy targeting the KRAS G12D driver mutation mediated the objective regression of metastatic pancreatic cancer. (Funded by the Providence Portland Medical Foundation.)

The 2022 Immunotherapy Bridge congress (November 30–December 1, Naples, Italy) featured a Great Debate session which addressed three contemporary topics in the field of immunotherapy. The debates included counterpoint views from leading experts and considered whether adoptive cell therapy (ACT) has a role in the treatment of solid tumors, the use of peripheral/blood biomarkers versus tumor microenvironment biomarkers for cancer immunotherapy and the role of chimeric antigen receptor T cell versus natural killer cell therapy. As is the tradition in the Immunotherapy Bridge Great Debates, speakers are invited by the meeting Chairs to express one side of the assigned debate and the opinions given may not fully reflect their own personal views. Audiences voted in favour of either side of the topic both before and after each debate.

Supplementary Table 1

Supplementary Table 2

Microsatellite-stable (MSS) colorectal cancers are characterized by low mutation burden and limited immune-cell infiltration and thereby respond poorly to immunotherapy. Here, we report a case of metastatic MSS colorectal cancer with a robust anticancer immune response. The primary tumor was resected in 2012, and the patient received several cycles of chemotherapy until 2017. In 2018, the patient underwent a left hepatectomy to remove a new metastasis. Analysis of the metastatic tumor revealed a strong CD8+ T-cell response. A high frequency of CD8+ T cells coexpressed CD39 and CD103, a phenotype characteristic of tumor-reactive cells. Using whole-exome sequencing, we identified somatic mutations that generated peptides recognized by CD39+CD103+CD8+ T cells. The observed reactivity against the tumor was dominated by the response to a single mutation that emerged in the metastasis. Somatic mutations that were not immunogenic in the primary tumor led to robust CD8+ T-cell expansion later during disease progression. Our data suggest that the cytotoxic treatment regimen received by the patient might be responsible for this effect. Hence, the capacity of cytotoxic regimens to prime the immune system in colorectal cancer patients should be investigated further and might provide a rationale for combination with immunotherapy.

Phenotypic analysis of the CD4 compartment in patients with CRLM

Expression levels of genes involved in the MHC class I pathway in the primary tumor and CRLM of patient CRI 3280

PDF file - 103K, Supplemental Figure 1. Frequency of HIBCH alteration across different studies available on cBioPortal for Cancer Genomics (http://www.cbioportal.org/public-portal/cross_cancer.do?).

We present the case of a man with metastatic, castration-resistant prostate cancer, who had a complete prostate-specific antigen (PSA) response after 2½ doses of ipilimumab. His treatment course was complicated by diarrhea and autoimmune hepatitis, both of which resolved within 4 months. Sera and biopsy specimens were accessed, and sera from pretreatment and day 113 were analyzed. Augmented antibody responses were detected against 11 potential tumor antigens, with responses ranging from 5- to 20-fold in day 113 sera compared with baseline. Genes that were targets of a strong antibody response (arbitrarily set at 10-fold or greater increase) were analyzed by real-time PCR for expression in the tumor biopsy cDNA. Of the top 5 genes, only 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) could be identified in the amplified tumor biopsy cDNA. Using an antibody to HIBCH, immunohistochemical analysis documented strong expression of the protein. Together, these data suggest that an augmented antibody response to HIBCH, an antigen that was expressed by the patient's prostate cancer, could have contributed to the clinical response. After 16 months of PSA stability, he discontinued his androgen-suppression therapy. With the return of his testosterone, his PSA increased slightly, likely originating from his intact prostate. He has been disease free for the past 6 years without any additional therapy. Cancer Immunol Res; 2(5); 399–403. ©2014 AACR.

PDF file - 613K

Purpose: We previously reported that autophagy in tumor cells plays a critical role in cross-presentation of tumor antigens and that autophagosomes are efficient antigen carriers for cross-priming of tumor-reactive CD8+ T cells. Here, we sought to characterize further the autophagosome-enriched vaccine named DRibble (DRiPs-containing blebs), which is derived from tumor cells after inhibition of protein degradation, and to provide insights into the mechanisms responsible for their efficacy as a novel cancer immunotherapy.Experimental Design: DRibbles were characterized by Western blot and light or transmission electron microscopy. The efficiency of cross-presentation mediated by DRibbles was first compared with that of whole-tumor cells and pure proteins. The mechanisms of antigen cross-presentation by DRibbles were analyzed, and the antitumor efficacy of the DRibble vaccine was tested in 3LL Lewis lung tumors and B16F10 melanoma.Results: The DRibbles sequester both long-lived and short-lived proteins, including defective ribosomal products (DRiP), and damage-associated molecular pattern molecules exemplified by HSP90, HSP94, calreticulin, and HMGB1. DRibbles express ligands for CLEC9A, a newly described C-type lectin receptor expressed by a subset of conventional and plasmacytoid dendritic cells (DC), and cross-presentation was partially CLEC9A dependent. Furthermore, this autophagy-assisted antigen cross-presentation pathway involved both caveolae- and clathrin-mediated endocytosis and endoplasmic reticulum–associated degradation machinery. It depends on proteasome and TAP1, but not lysosome functions of antigen-presenting cells. Importantly, DCs loaded with autophagosome-enriched DRibbles can eradicate 3LL Lewis lung tumors and significantly delay the growth of B16F10 melanoma.Conclusions: These data documented the unique characteristics and potent antitumor efficacy of the autophagosome-based DRibble vaccine. The efficacy of DRibble cancer vaccine will be further tested in clinical trials. Clin Cancer Res; 17(22); 7047–57. ©2011 AACR.

Figure S5

Treatment related changes in cell type scores

Supplementary Figures 1-8

Figure S1

PDF file - 476K

Purpose: Effective cancer vaccines must both drive a strong CTL response and sustain long-term memory T cells capable of rapid recall responses to tumor antigens. We sought to characterize the phenotype and function of gp100 peptide-specific memory CD8+ T cells in melanoma patients after primary gp100209-2M immunization and assess the anamnestic response to boosting immunization.Experimental Design: Eight-color flow cytometry analysis of gp100-specific CD8+ T cells was done on peripheral blood mononuclear cells collected shortly after the primary vaccine regimen, 12 to 24 months after primary vaccination, and after boosting immunization. The anamnestic response was assessed by comparing the frequency of circulating gp100-specific T cells before and after boosting. Gp100 peptide-induced in vitro functional avidity and proliferation responses and melanoma-stimulated T-cell CD107 mobilization were compared for cells from all three time points for multiple patients.Results: The frequency of circulating gp100-specific memory CD8+ T cells was comparable with cytomegalovirus-specific and FLU-specific T cells in the same patients, and the cells exhibited anamnestic proliferation after boosting. Their phenotypes were not unique, and individual patients exhibited one of two distinct phenotype signatures that were homologous to either cytomegalovirus-specific or FLU-specific memory T cells. Gp100-specific memory T cells showed some properties of competent memory T cells, such as heightened in vitro peptide-stimulated proliferation and increase in central memory (TCM) differentiation when compared with T-cell responses measured after the primary vaccine regimen. However, they did not acquire enhanced functional avidity usually associated with competent memory T-cell maturation.Conclusions: Although vaccination with class I–restricted melanoma peptides alone can break tolerance to self-tumor antigens, it did not induce fully competent memory CD8+ T cells—even in disease-free patients. Data presented suggest other vaccine strategies will be required to induce functionally robust long-term memory T cells.

Purpose:Tumors with low frequencies of checkpoint positive tumor-infiltrating lymphocytes (cpTIL) have a low likelihood of response to PD-1 blockade. We conducted a prospective multicenter phase II trial of intratumoral plasmid IL-12 (tavokinogene telseplasmid; “tavo”) electroporation combined with pembrolizumab in patients with advanced melanoma with low frequencies of checkpoint positive cytotoxic lymphocytes (cpCTL).Patients and Methods:Tavo was administered intratumorally days 1, 5, and 8 every 6 weeks while pembrolizumab (200 mg, i.v.) was administered every 3 weeks. The primary endpoint was objective response rate (ORR) by RECIST, secondary endpoints included duration of response, overall survival and progression-free survival. Toxicity was evaluated by the CTCAE v4. Extensive correlative analysis was done.Results:The combination of tavo and pembrolizumab was well tolerated with adverse events similar to those previously reported with pembrolizumab alone. Patients had a 41% ORR (n = 22, RECIST 1.1) with 36% complete responses. Correlative analysis showed that the combination enhanced immune infiltration and sustained the IL-12/IFNγ feed-forward cycle, driving intratumoral cross-presenting dendritic cell subsets with increased TILs, emerging T cell receptor clones and, ultimately, systemic cellular immune responses.Conclusions:The combination of tavo and pembrolizumab was associated with a higher than expected response rate in this poorly immunogenic population. No new or unexpected toxicities were observed. Correlative analysis showed T cell infiltration with enhanced immunity paralleling the clinical activity in low cpCTL tumors.

Lesion response data

Figure S6

Purpose: Tumor-specific antigens of 3-methylcholanthrene (MCA)-induced sarcomas were defined by the narrow immune responses they elicited, which uniquely rejected the homologous tumor, with no cross-reactions between independently derived syngeneic MCA-induced tumors. This study examines whether an autophagosome-enriched vaccine derived from bortezomib-treated sarcomas can elicit an immune response that cross-reacts with other unique sarcomas.Experimental Design: Mice were vaccinated with either MCA-induced sarcomas or autophagosomes derived from those tumors and later challenged with either homologous or nonhomologous sarcomas. In addition, 293 cells expressing a model antigen were used to understand the necessity of short-lived proteins (SLiP) in this novel vaccine. These findings were then tested in the sarcoma model. Autophagosomes were characterized by Western blotting and fluorescent microscopy, and their ability to generate immune responses was assessed in vitro by carboxyfluorescein succinimidyl ester dilution of antigen-specific T cells and in vivo by monitoring tumor growth.Results: In contrast to a whole-cell tumor vaccine, autophagosomes isolated from MCA-induced sarcomas treated with a proteasome inhibitor prime T cells that cross-react with different sarcomas and protect a significant proportion of vaccinated hosts from a nonhomologous tumor challenge. Ubiquitinated SLiPs, which are stabilized by proteasome blockade and delivered to autophagosomes in a p62/sequestosome-dependent fashion, are a critical component of the autophagosome vaccine, as their depletion limits vaccine efficacy.Conclusion: This work suggests that common short-lived tumor-specific antigens, not physiologically available for cross-presentation, can be sequestered in autophagosomes by p62 and used as a vaccine to elicit cross-protection against independently derived sarcomas. Clin Cancer Res; 17(20); 6467–81. ©2011 AACR.

Purpose: Adoptive cell transfer (ACT) is a promising immunotherapeutic approach for cancer. Host lymphodepletion is associated with favorable ACT therapy outcomes, but it may cause detrimental effects in humans. We tested the hypothesis that IL15 administration enhances ACT in the absence of lymphodepletion. We previously showed that bioactive IL15 in vivo comprises a stable complex of the IL15 chain with the IL15 receptor alpha chain (IL15Rα), termed heterodimeric IL15 (hetIL15).Experimental Design: We evaluated the effects of the combination regimen ACT + hetIL15 in the absence of lymphodepletion by transferring melanoma-specific Pmel-1 T cells into B16 melanoma-bearing mice.Results: hetIL15 treatment delayed tumor growth by promoting infiltration and persistence of both adoptively transferred Pmel-1 cells and endogenous CD8+ T cells into the tumor. In contrast, persistence of Pmel-1 cells was severely reduced following irradiation in comparison with mice treated with hetIL15. Importantly, we found that hetIL15 treatment led to the preferential enrichment of Pmel-1 cells in B16 tumor sites in an antigen-dependent manner. Upon hetIL15 administration, tumor-infiltrating Pmel-1 cells showed a “nonexhausted” effector phenotype, characterized by increased IFNγ secretion, proliferation, and cytotoxic potential and low level of PD-1. hetIL15 treatment also resulted in an improved ratio of Pmel-1 to Treg in the tumor.Conclusions: hetIL15 administration improves the outcome of ACT in lymphoreplete hosts, a finding with significant implications for improving cell-based cancer immunotherapy strategies. Clin Cancer Res; 23(11); 2817–30. ©2016 AACR.

Table S1

Figure S3

GH-secreting PAs starburst plot gene details. The top 50 most significantly differentially expressed genes contained within Figure 5 for GH-secreting PAs as compared to endocrine-inactive PAs. The table has five columns; 1) gene symbol, 2) Entrez ID number, 3) the log fold change value from the RNA-seq differential expression analysis comparing GH-secreting PAs to endocrine-inactive PAs, 4) the change in methylation beta values between GH-secreting PAs and endocrine-inactive PAs, and 5) the genomic feature for the methylation data.

PDF file - 44K, Irradiated whole cell sarcoma-pulsed APCs do not protect mice from a challenge with non-homologous sarcomas. Kaplan-Meier survival curves of mice vaccinated with irradiated whole cell sarcoma-pulsed APCs or autophagosome-pulsed APCs. Autophagosome vaccination was performed as in Figure 2. Subcutaneous whole cell vaccinations consisted of 3x106 antigen-presenting cells pulsed with 5x106 irradiated tumor cells injected into the lower right flank of the mouse. Mice were challenged with 30,000 viable MCA-304 sarcomas. Tumor growth was monitored at least twice weekly. Each survival plot represents 2 independent experiments with 5 mice per group (n=10). Survival curves denoted with � represent statistically significant (p>0.05) protection from a tumor challenge compared to no vaccine.

Table S3

Figure S4

Figure S1 shows combination treatment induces splenomegaly and T cells Apoptosis Figure S2 shows PD-L1 is increased on splenic T cells with combination treatment Figure S3 shows combination treatment increases costimulatory receptors on T cells Figure S4 shows delayed anti-PD-1 and anti-PD-L1 are ineffective at tumor control Figure S5 shows sequential combination provides anti-tumor immunity in the 4T1 model. Figure S6 shows sequential combination does not induce inflammatory cytokines Figure S7 shows sequential combination does not increase costimulatory receptors Figure S8 shows combination treatment induces functional PyMT-specific T cells

Table of significant SCNAs among PAs. This table highlights the top 10 most significant GISTIC2 calls for all PAs in this study, each PA subtype, and invasive versus non-invasive PAs.

ACTH-secreting PAs starburst plot gene details. The top 50 most significantly differentially expressed genes contained within Figure 5 for ACTH-secreting PAs as compared to endocrine-inactive PAs. The table has five columns; 1) gene symbol, 2) Entrez ID number, 3) the log fold change value from the RNA-seq differential expression analysis comparing ACTH-secreting PAs to endocrine-inactive PAs, 4) the change in methylation beta values between ACTH-secreting PAs and endocrine-inactive PAs, and 5) the genomic feature for the methylation data.

Supplementary Figure Legends

PDF file - 650K

Supplementary Data from Partial CD4 Depletion Reduces Regulatory T Cells Induced by Multiple Vaccinations and Restores Therapeutic Efficacy

Supplementary Figure Legends

Fluorescence-conjugated antibody used for flow cytometry

Supplementary methods

Purpose: Antibodies specific for inhibitory checkpoints PD-1 and CTLA-4 have shown impressive results against solid tumors. This has fueled interest in novel immunotherapy combinations to affect patients who remain refractory to checkpoint blockade monotherapy. However, how to optimally combine checkpoint blockade with agents targeting T-cell costimulatory receptors, such as OX40, remains a critical question.Experimental Design: We utilized an anti-PD-1–refractory, orthotopically transplanted MMTV-PyMT mammary cancer model to investigate the antitumor effect of an agonist anti-OX40 antibody combined with anti-PD-1. As PD-1 naturally aids in immune contraction after T-cell activation, we treated mice with concurrent combination treatment versus sequentially administering anti-OX40 followed by anti-PD-1.Results: The concurrent addition of anti-PD-1 significantly attenuated the therapeutic effect of anti-OX40 alone. Combination-treated mice had considerable increases in type I and type II serum cytokines and significantly augmented expression of inhibitory receptors or exhaustion markers CTLA-4 and TIM-3 on T cells. Combination treatment increased intratumoral CD4+ T-cell proliferation at day 13, but at day 19, both CD4+ and CD8+ T-cell proliferation was significantly reduced compared with untreated mice. In two tumor models, sequential combination of anti-OX40 followed by anti-PD-1 (but not the reverse order) resulted in significant increases in therapeutic efficacy. Against MMTV-PyMT tumors, sequential combination was dependent on both CD4+ and CD8+ T cells and completely regressed tumors in approximately 30% of treated animals.Conclusions: These results highlight the importance of timing for optimized therapeutic effect with combination immunotherapies and suggest the testing of sequencing in combination immunotherapy clinical trials. Clin Cancer Res; 23(20); 6165–77. ©2017 AACR.See related commentary by Colombo, p. 5999

Purpose: Pituitary adenomas are one of the most common benign neoplasms of the central nervous system. Although emerging evidence suggests roles for both genetic and epigenetic factors in tumorigenesis, the degree to which these factors contribute to disease remains poorly understood.Experimental Design: A multiplatform analysis was performed to identify the genomic and epigenomic underpinnings of disease among the three major subtypes of surgically resected pituitary adenomas in 48 patients: growth hormone (GH)–secreting (n = 17), adrenocorticotropic hormone (ACTH)–secreting (n = 13, including 3 silent-ACTH adenomas), and endocrine-inactive (n = 18). Whole-exome sequencing was used to profile the somatic mutational landscape, whole-transcriptome sequencing was used to identify disease-specific patterns of gene expression, and array-based DNA methylation profiling was used to examine genome-wide patterns of DNA methylation.Results: Recurrent single-nucleotide and small indel somatic mutations were infrequent among the three adenoma subtypes. However, somatic copy-number alterations (SCNA) were identified in all three pituitary adenoma subtypes. Methylation analysis revealed adenoma subtype-specific DNA methylation profiles, with GH-secreting adenomas being dominated by hypomethylated sites. Likewise, gene-expression patterns revealed adenoma subtype-specific profiles. Integrating DNA methylation and gene-expression data revealed that hypomethylation of promoter regions are related with increased expression of GH1 and SSTR5 genes in GH-secreting adenomas and POMC gene in ACTH-secreting adenomas. Finally, multispectral IHC staining of immune-related proteins showed abundant expression of PD-L1 among all three adenoma subtypes.Conclusions: Taken together, these data stress the contribution of epigenomic alterations to disease-specific etiology among adenoma subtypes and highlight potential targets for future immunotherapy-based treatments. This article reveals novel insights into the epigenomics underlying pituitary adenomas and highlights how differences in epigenomic states are related to important transcriptome alterations that define adenoma subtypes. Clin Cancer Res; 24(17); 4126–36. ©2018 AACR.

PCA analysis of genome-wide methylation profiles. PCA analysis using all probes in the methylation data set. Points are colored by the percent of the genome altered by SCNAs. The three PA subtypes are indicated by the shape of the points, with GH-secreting PAs represented by dots, ACTH-secreting PAs represented by triangles, and endocrine-inactive PAs represented by squares. Ellipses indicate 95% confidence intervals. Overall, PAs cluster by PA subtype and not by the percent of the genome disrupted by SCNAs.

Purpose: A single vaccination of intact or reconstituted-lymphopenic mice (RLM) with a granulocyte macrophage colony-stimulating factorsecreting B16BL6-D5 melanoma cell line induces protective antitumor immunity and T cells that mediate the regression of established melanoma in adoptive immunotherapy studies. We wanted to study if multiple vaccinations during immune reconstitution of the lymphopenic host would maintain a potent antitumor immune response.Experimental Design: RLM were vaccinated multiple times over a 40-day period. Spleens were isolated from these mice, activated in vitro, and adoptively transferred into mice bearing 3-day experimental pulmonary metastases.Results: Multiple vaccinations, rather than boosting the immune response, significantly reduced therapeutic efficacy of adoptive immunotherapy and were associated with an increased frequency and absolute number of CD3+CD4+Foxp3+ T regulatory (Treg) cells. Anti-CD4 administration reduced the absolute number of Treg cells 9-fold. Effector T-cells generated from anti-CD4treated mice were significantly (P < 0.0001) more therapeutic in adoptive transfer studies than T cells from multiply vaccinated animals with a full complement of CD4+ cells.Conclusion: These results suggest that CD4+ Treg cells limit the efficacy of multiple vaccinations and that timed partial depletion of CD4+ T cells may reduce suppression and tip-the-balance in favor of therapeutic antitumor immunity. The recent failure of large phase III cancer vaccine clinical trials, wherein patients received multiple vaccines, underscores the potential clinical relevance of these findings. (Clin Cancer Res 2009;15(22):688190)

Table S2

Purpose:Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed.Patients and Methods:Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets.Results:Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8+ T-cell infiltration following treatment. One patient, previously unresponsive to anti–PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti–PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response.Conclusions:These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC.

PDF file - 539K

PCA analysis of genome-wide gene expression profiles. PCA analysis using all genes in the gene expression data set. Points are colored by the percent of the genome altered by SCNAs. The three PA subtypes are indicated by the shape of the points, with GH-secreting PAs represented by dots, ACTH-secreting PAs represented by triangles, and endocrine-inactive PAs represented by squares. Ellipses indicate 95% confidence intervals. Overall, PAs cluster by PA subtype and not by the percent of the genome disrupted by SCNAs.

Table of clinical parameters for each patient sample.

Treatment Related Adverse Events by Grade

Figure S2

Supplementary Figure 1. IL-15 availability promotes proliferation and maintenance of transferred CD8+ T in the spleen of recipient mice; Supplementary Figure 2. Gating strategy for the identification of adoptively transferred Pmel-1 cells and endogenous CD8+ T cells infiltrating the tumor; Supplementary Figure 3. Absolute counts of splenic Pmel-1 and CD8+ T cells are profoundly affected by hetIL-15 treatment; Supplementary Figure 4. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating Pmel-1 cells; Supplementary Figure 5. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating CD8+ T cells; Supplementary Table 1. Hematological parameters during ACT treatments.