Tumor-associated Antigen-specific CD8 Research Articles

The predominant Quillaja Saponaria fraction, QS-18, is safe and effective when formulated in a liposomal murine cancer peptide vaccine

Extracts of the Chilean soapbark tree, Quillaja Saponaria (QS) are the source of potent immune-stimulatory saponin compounds. This study compared the adjuvanticity and toxicity of QS-18 and QS-21, assessing the potential to substitute QS-18 in place of QS-21 for vaccine development. QS-18, the most abundant QS saponin fraction, has been largely overlooked due to safety concerns. We found that QS-18 spontaneously inserted into liposomes, thereby neutralizing hemolytic activity, and following administration did not induce local reactogenicity in a footpad swelling test in mice. With high-dose intramuscular administration, transient weight loss was minor, and QS-18 did not induce significantly more weight loss compared to a liposome vaccine adjuvant system lacking it. Two days after administration, no elevation of inflammatory cytokines was detected in murine serum. In a formulation including cobalt-porphyrin-phospholipid (CoPoP) for short peptide sequestration, QS-18 did not impact the formation of peptide nanoparticles. With immunization, QS-18 peptide particles induced higher levels of cancer neoepitope-specific and tumor-associated antigen-specific CD8+ T cells compared to QS-21 particles, without indication of greater toxicity based on mouse body weight. T cell receptor sequencing of antigen-specific CD8+ T cells showed that QS-18 induced significantly more T cell transcripts. In two murine cancer models, vaccination with QS-18 peptide particles induced a similar therapeutic effect as QS-21 particles, without indication of increased toxicity. Antigen-specific CD8+ T cells in the tumor microenvironment were found to express the exhaustion marker PD-1, pointing to the rationale for exploring combination therapy. Taken together, these data demonstrate that QS-18, when formulated in liposomes, can be a safe and effective adjuvant to induce tumor-inhibiting cellular responses in murine models with potential to facilitate or diminish costs of production for vaccine adjuvant systems. Further studies are warranted to assess liposomal QS-18 immunogic, reactogenic and toxicological profiles in mice and other animal species.

Identification and characterization of a dominated T cell population in tumor associated antigen specific CD8 +T cells of tumor microenvironment

Abstract The characteristics and roles of tumor associated antigens (TAAs) specific tumor infiltrative T cells (TILs) are not well understood. Alpha-enolase is one TAA of oral squamous cell carcinoma (OSCC) and pancreatic cancer, and are highly expressed in many cancer types by analyzing the TCGA database. We detected α-enolase responsive CD8 +T cells in peripheral blood mononuclear cells (PBMCs) and TILs of OSCC patients. The α-enolase responsive CD8 +T cells significantly increased in TILs than those in PBMCs. We developed two tetramers to identify two different α-enolase peptides specific CD8 +T cells. Two types of tetramer positive CD8 +TILs, total CD8 +TILs, and total CD8 +T cells from PBMC of three OSCC patients were sorted. Single cell RNA sequencing was performed. Unbiased clustering revealed 11 distinct CD8 +T cell clusters. To our surprise, the cell population patterns of both tetramer positive CD8 +TILs were similar, but were much different from total blood CD8 +T cells or total CD8 +TILs. The percentages of relatively exhausted T cells were similar in three TIL groups, but was nearly absent in blood. The blood CD8 +T cells had the highest percentage of effector memory T cells and naïve T cells. A specific cell cluster dominated (>50% population) in the two tetramer positive CD8 +TILs. The expression of important immune related genes in the specific cell cluster were all relatively low. That hinted this T cell subset was more quiescent than other cell clusters, and may have potentials to be activated. Further clarify the roles of the special T cell subset in tumor immunology is warranted. Supported by grants from National Science and Technology Council, Taiwan (110-2314-B-002-299)

Abstract 3278: Identification of epigenetic therapy induced tumor antigens to promote proliferation and infiltration of antigen specific T cells in non-small cell lung cancer

Abstract Among the hallmarks of non-small cell lung cancer (NSCLC) is cancer immunoediting. Immunoediting results when cancer cells evade immune recognition via positive selection for cells that do not present immunogenic antigens recognized by CD8+ T cells. An epigenetic therapy-based approach has been developed in our lab to enhance expression of tumor antigens using combinatorial DNA methyltransferase inhibition (DNMTi) paired with histone deacetylase inhibition (HDACi). However, durable responses from the application of epigenetic therapy alone or in combination with other therapies have remained elusive in NSCLC. This may be because enhanced proliferation and infiltration of tumor-associated antigen specific CD8+ T cells are necessary to recognize epigenetically induced antigens to halt disease progression. In the current study, we identified epigenetic therapy inducible self-antigens that can potentially be used to activate and guide the differentiation of CD8+ T cells specific to NSCLC tumor antigens. The identification of these candidates was derived from analyses of paired RNAseq data from NSCLC patients treated with combination epigenetic therapy. From these analyses, differentially expressed transcripts selected from total transcriptome data were filtered to remove non-protein coding genes and genes with detectable expression in normal tissues as indicated by GTEx. HLA binding affinity (HLA-A*0201) of selected targets was defined by NetMHC 4.0 artificial neural network-based prediction to identify strong binding peptide candidates. The combined results of these data filtering facilitated the identification of 34 epigenetic therapy induced genes and associated peptides (9mers) with predicted high binding affinity for HLA-A*0201. The predicted peptides of target genes were selected for single peptide pulsing in allogenic dendritic cells to generate antigen specific CD8+ T cells for TCR sequencing (TCRseq). TCRseq libraries derived from peptide stimulation experiments revealed distinct V and J gene frequencies and emergence of antigen specific clonotypes, which suggest clonal expansion. To validate whether epigenetic therapy could induce target genes expression in an independent experimental model, in vitro human NSCLC cell lines were treated with DNMTi and HDACi to mimic the clinical drug paradigm and transcriptional augmentation was assessed by qRT-PCR. In this orthogonal system, we validated eight target genes that were induced by epigenetic therapy in vitro. Altogether, these data suggest that a conserved subset of genes, whose derived proteins are predicted to be presented in class I MHC and act as T cell epitopes, is induced by combination epigenetic treatment. Our studies provide a basis for the development of novel and personalized strategies to improve outcomes and quality of life for more patients with NSCLC. Citation Format: Julia An, Valsamo Anagnostou, Kristen A. Marrone, Victor E. Velculescu, Julie R. Brahmer, Stephen B. Baylin, Michael J. Topper. Identification of epigenetic therapy induced tumor antigens to promote proliferation and infiltration of antigen specific T cells in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3278.

Targeting Neuropilin-1 with Nanobodies Reduces Colorectal Carcinoma Development.

Simple SummaryNeuropilin-1 is a co-receptor for semaphorins and vascular endothelial growth factor family members. Neuropilin-1 can be expressed on tumor cells, tumor-infiltrating myeloid and lymphoid cells and has been linked to a tumor-promoting environment. We investigated nanobodies (Nbs) targeting neuropilin-1 for their potential to hamper colorectal carcinoma development in mice. Our data suggest that targeting neuropilin-1 in cancer using neuropilin-1 blocking Nbs delays tumor growth and extends the survival through a shift in the anti-tumor macrophage/pro-tumor macrophage ratio and activation of colorectal cancer-specific CD8+ T cells. These findings provide a rationale for the further development of Nbs targeting human neuropilin-1 and bringing them from the bench to the bedside.Neuropilin-1 (NRP-1) is a co-receptor for semaphorins and vascular endothelial growth factor (VEGF) family members that can be expressed on cancer cells and tumor-infiltrating myeloid, endothelial and lymphoid cells. It has been linked to a tumor-promoting environment upon interaction with semaphorin 3A (Sema3A). Nanobodies (Nbs) targeting NRP-1 were generated for their potential to hamper the NRP-1/Sema3A interaction and their impact on colorectal carcinoma (CRC) development was evaluated in vivo through the generation of anti-NRP-1-producing CRC cells. We observed that tumor growth was significantly delayed and survival prolonged when the anti-NRP-1 Nbs were produced in vivo. We further analyzed the tumor microenvironment and observed that the pro-inflammatory MHC-IIhigh/trophic MHC-IIlow macrophage ratio was increased in tumors that produce anti-NRP-1 Nbs. This finding was corroborated by an increase in the expression of genes associated with MHC-IIhigh macrophages and a decrease in the expression of MHC-IIlow macrophage-associated genes in the macrophage pool sorted from anti-NRP-1 Nb-producing tumors. Moreover, we observed a significantly higher percentage of tumor-associated antigen-specific CD8+ T cells in tumors producing anti-NRP-1 Nbs. These data demonstrate that an intratumoral expression of NRP-1/Sema3A blocking biologicals increases anti-tumor immunity.

Association Between Expression Level of PD1 by Tumor-Infiltrating CD8+ T Cells and Features of Hepatocellular Carcinoma

T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). We investigated features of exhaustion in CD8+ T cells isolated from HCC specimens. We obtained HCC specimens, along with adjacent nontumor tissues and blood samples, from 90 patients who underwent surgical resection at Asan Medical Center (Seoul, Korea) from April 2016 through April 2018. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8+ T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA sequencing. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8+ T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8+ T cells were identified using HLA-A*0201 dextramers. PDL1 expression on tumor tissue was assessed by immunohistochemistry. PD1-high, PD1-intermediate, and PD1-negative CD8+ T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and low proportions of TCF1+, TBEThigh/eomesoderminlow, and CD127+. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in the PD1 expression patterns of CD8+ T cells led to the identification of 2 subgroups of HCCs: HCCs with a discrete population of PD1-high cells were more aggressive than HCCs without a discrete population of PD1-high cells. HCCs with a discrete population of PD1-high cells had higher levels of predictive biomarkers of response to anti-PD1 therapy. Incubation of CD8+ T cells from HCCs with a discrete population of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3. We found HCC specimens to contain CD8+ T cells that express different levels of PD1. HCCs with a discrete population of PD1-high CD8+ T cells express TIM3 and/or LAG3 and produce low levels of IFNG andTNF in response to anti-CD3. Incubation of these cells withantibodies against PD1 and TIM3 or LAG3 further restoreproliferation and production of cytokines; HCCs with a discrete population of PD1-high CD8+ T cells might be more susceptible to combined immune checkpoint blockade-based therapies.

An adenoviral cancer vaccine co-encoding a tumor associated antigen together with secreted 4-1BBL leads to delayed tumor progression

Previous studies have shown promising results when using an agonistic anti-4-1BB antibody treatment against established tumors. While this is promising, this type of treatment can induce severe side effects. Therefore, we decided to incorporate the membrane form of 4-1BB ligand (4-1BBL) in a replicative deficient adenovirus vaccine expressing the invariant chain (Ii) adjuvant fused to a tumor associated antigen (TAA). The Ii adjuvant increases and prolongs TAA specific CD8+ T cells as previously shown and local expression of 4-1BBL was chosen to avoid the toxicity associated with systemic antibody administration. Furthermore, adenovirus encoded 4-1BBL expression has previously been successfully used to enhance responses toward Plasmodium falciparum and Influenza A antigens. We showed that the incorporation of 4-1BBL in the adenovirus vector led to surface expression of 4-1BBL on antigen presenting cells, but it did not enhance T cell responses in mice towards the Ii linked antigen. In tumor-bearing mice, our vaccine was found to decrease the frequency of TAA specific CD8+ T cells, but this difference did not alter the therapeutic efficacy. In order to reconcile our findings with the previous reports of increased anti-cancer efficacy using systemically delivered 4-1BB agonists, we incorporated a secreted version of 4-1BBL (Fc-4-1BBL) in our vaccine and co-expressed it with the Ii linked to TAA. In tumor bearing mice, this vaccine initially delayed tumor growth and slightly increased survival compared to the vaccine expressing the membrane form of 4-1BBL. Accordingly, secreted 4-1BBL co-encoded with the Ii linked antigen may offer a simplification compared to administration of drug and vaccine separately.

CD40 ligand-expressing recombinant vaccinia virus promotes the generation of CD8(+) central memory Tcells.

Central memory CD8(+) Tcells (TCM ) play key roles in the protective immunity against infectious agents, cancer immunotherapy, and adoptive treatments of malignant and viral diseases. CD8(+) TCM cells are characterized by specific phenotypes, homing, and proliferative capacities. However, CD8(+) TCM -cell generation is challenging, and usually requires CD4(+) CD40L(+) T-cell "help" during the priming of naïve CD8(+) Tcells. We have generated a replication incompetent CD40 ligand-expressing recombinant vaccinia virus (rVV40L) to promote the differentiation of human naïve CD8(+) Tcells into TCM specific for viral and tumor-associated antigens. Soluble CD40 ligand recombinant protein (sCD40L), and vaccinia virus wild-type (VV WT), alone or in combination, were used as controls. Here, we show that, in the absence of CD4(+) Tcells, a single "in vitro" stimulation of naïve CD8(+) Tcells by rVV40L-infected nonprofessional CD14(+) antigen presenting cells promotes the rapid generation of viral or tumor associated antigen-specific CD8(+) Tcells displaying TCM phenotypic and functional properties. These observations demonstrate the high ability of rVV40L to fine tune CD8(+) mediated immune responses, and strongly support the use of similar reagents for clinical immunization and adoptive immunotherapy purposes.

Targeting the tumor-stromal-immune cell axis.

Targeting the tumor-stromal-immune cell axis.

Cancer-associated fibroblast-targeted strategy enhances antitumor immune responses in dendritic cell-based vaccine.

Given the close interaction between tumor cells and stromal cells in the tumor microenvironment (TME), TME-targeted strategies would be promising for developing integrated cancer immunotherapy. Cancer-associated fibroblasts (CAFs) are the dominant stromal component, playing critical roles in generation of the pro-tumorigenic TME. We focused on the immunosuppressive trait of CAFs, and systematically explored the alteration of tumor-associated immune responses by CAF-targeted therapy. C57BL/6 mice s.c. bearing syngeneic E.G7 lymphoma, LLC1 Lewis lung cancer, or B16F1 melanoma were treated with an anti-fibrotic agent, tranilast, to inhibit CAF function. The infiltration of immune suppressor cell types, including regulatory T cells and myeloid-derived suppressor cells, in the TME was effectively decreased through reduction of stromal cell-derived factor-1, prostaglandin E2, and transforming growth factor-β. In tumor-draining lymph nodes, these immune suppressor cell types were significantly decreased, leading to activation of tumor-associated antigen-specific CD8+ T cells. In addition, CAF-targeted therapy synergistically enhanced multiple types of systemic antitumor immune responses such as the cytotoxic CD8+ T cell response, natural killer activity, and antitumor humoral immunity in combination with dendritic cell-based vaccines; however, the suppressive effect on tumor growth was not observed in tumor-bearing SCID mice. These data indicate that systemic antitumor immune responses by various immunologic cell types are required to bring out the efficacy of CAF-targeted therapy, and these effects are enhanced when combined with effector-stimulatory immunotherapy such as dendritic cell-based vaccines. Our mouse model provides a novel rationale with TME-targeted strategy for the development of cell-based cancer immunotherapy.

Tumor-associated antigen specific CD8+ T cells in hepatocellular carcinoma – a promising target for immunotherapy

Immunotherapy is a promising treatment option for patients with hepatocellular carcinoma (HCC). Indeed, CD8+ T-cell responses against various tumor antigens occur in these patients. However, these antitumoral T cells show a severely impaired effector function. Several immunosuppressive mechanisms contribute to this T-cell failure, including regulatory T cells and inhibitory receptors.

Screening and enumeration of virus- and tumor-associated antigen-specific CD8+ T lymphocytes using streptamer technology and single-platform flowcytometry assay

Screening and enumeration of virus- and tumor-associated antigen-specific CD8+ T lymphocytes using streptamer technology and single-platform flowcytometry assay

Adaptive Antiviral Immunity Is a Determinant of the Therapeutic Success of Oncolytic Virotherapy

Oncolytic virotherapy, the selective killing of tumor cells by oncolytic viruses (OVs), has emerged as a promising avenue of anticancer research. We have previously shown that KM100, a Herpes simplex virus type-1 (HSV) deficient for infected cell protein 0 (ICP0), possesses substantial oncolytic properties in vitro and has antitumor efficacy in vivo, in part by inducing antitumor immunity. Here, we illustrate through T-cell immunodepletion studies in nontolerized tumor-associated antigen models of breast cancer that KM100 treatment promotes antiviral and antitumor CD8(+) cytotoxic T-cell responses necessary for complete tumor regression. In tolerized tumor-associated antigen models of breast cancer, antiviral CD8(+) cytotoxic T-cell responses against infected tumor cells correlated with the induction of significant tumoristasis in the absence of tumor-associated antigen-specific CD8(+) cytotoxic T-cells. To enhance oncolysis, we tested a more cytopathic ICP0-null HSV and a vesicular stomatitis virus M protein mutant and found that despite improved in vitro replication, oncolysis in vivo did not improve. These studies illustrate that the in vitro cytolytic properties of OVs are poor prognostic indicators of in vivo antitumor activity, and underscore the importance of adaptive antiviral CD8(+) cytotoxic T-cells in effective cancer virotherapy.

TRP-2 TCR Transgenic Mice as a Model to Study CD8+ T cell Responses to Melanoma Antigens (50.15)

Abstract To study the effects of T cell avidity on tumor immunity and autoimmunity, we have generated mice that bear T cell receptor (TcR) transgenes specific for a peptide from tyrosinase-related protein 2 (TRP-2), a melanosomal enzyme expressed by both normal melanocytes and melanoma cells. We have begun characterizing a founder mouse line derived from an intermediate avidity T cell line, designated clone 37. Interestingly, the clone 37 TcR transgenic (Tg) mice did not develop autoimmune depigmentation despite the presence of TRP-2-reactive T cells in the lymph nodes and systemic expression of TRP-2. These Tg T cells displayed a naïve phenotype (CD44low, CD62hi, CD69neg, and CD25neg) and proliferated upon stimulation in vitro with cognate antigen. No difference in T cell responsiveness or frequency was observed in the development of Clone 37 T cells following bone marrow reconstitution of wild type C57Bl/6 or TRP-2−/− mice. Adoptive transfer of clone 37 TCR Tg cells into C57BL/6 showed that these T cells do not encounter the endogenous TRP-2 antigen as well as a subcutaneous B16/BL6 melanoma tumor (i.e., they are “ignorant”). However, vaccination with TRP-2 peptide-pulsed dendritic cells induced proliferation of these adoptively transferred CD8+ T cells and subsequent migration into subcutaneous B16/BL6 tumors, but did not inhibit progressive tumor growth. Similarly, B16/BL6 tumors grew progressively in clone 37 TCR Tg mice despite the large number of tumor-associated antigen-specific CD8+ T cells. These findings demonstrate the ability of tumors to evade an anti-tumor immune response despite the presence of tumor-specific T cells. Our on-going studies will characterize two additional mouse lines bearing TcR transgenes from high and low avidity T cell clones and study the impact of different T cell avidities on tumor immunity and autoimmunity.

Plasmacytoid Dendritic Cells Induce CD8+ Regulatory T Cells In Human Ovarian Carcinoma

To directly dissect the role of each immune component in human tumor immunopathogenesis, we have studied the interaction between dendritic cells and T cells in the tumor environment of patients with ovarian carcinoma. We previously reported that functional plasmacytoid dendritic cells, but not functionally mature myeloid dendritic cells, accumulated in tumor microenvironments. We now show that tumor ascites macrophage-derived dendritic cells induced tumor-associated antigen-specific CD8+ T cells with effector functions. Strikingly, tumor ascites plasmacytoid dendritic cells induced interleukin-10+ CCR7+ CD45RO+ CD8+ regulatory T cells. Four characteristics have been identified in tumor plasmacytoid dendritic cell-induced CD8+ regulatory T cells: (a) induction of CD8+ regulatory T cells is independent of CD4+ CD25+ T cells; (b) CD8+ regulatory T cells significantly suppress myeloid dendritic cell-mediated tumor-associated antigen-specific T cell effector functions through interleukin-10; (c) repetitive myeloid dendritic cell stimulation can recover CD8+ regulatory T cell-mediated poor T cell proliferation, but not T cell effector function; (d) CD8+ regulatory T cells express functional CCR7, and efficiently migrate with lymphoid homing chemokine MIP-3beta. Primary suppressive CCR7+ CD45RO+ CD8+ T cells are found in the tumor environment of patients with ovarian cancers. Thus, tumor-associated plasmacytoid dendritic cells contribute to the tumor environmental immunosuppressive network. Collectively, tumors manipulate tumor microenvironmental dendritic cell subset distribution and function to subvert tumor immunity. The data are relevant to understanding tumor immunopathology as well as reevaluating tumor immunotherapeutic strategies.