Cell-mediated Tumor Rejection Research Articles

Tumor Burden Dictates the Neoantigen Features Required to Generate an Effective Cancer Vaccine.

Tumor neoantigens (nAg) represent a promising target for cancer immunotherapy. The identification of nAgs that can generate T-cell responses and have therapeutic activity has been challenging. Here, we sought to unravel the features of nAgs required to induce tumor rejection. We selected clinically validated Great Ape-derived adenoviral vectors (GAd) as a nAg delivery system for differing numbers and combinations of nAgs. We assessed their immunogenicity and efficacy in murine models of low to high disease burden, comparing multi-epitope versus mono-epitope vaccines. We demonstrated that the breadth of immune response is critical for vaccine efficacy and having multiple immunogenic nAgs encoded in a single vaccine improves efficacy. The contribution of each single neoantigen was examined, leading to the identification of 2 nAgs able to induce CD8+ T cell-mediated tumor rejection. They were both active as individual nAgs in a setting of prophylactic vaccination, although to different extents. However, the efficacy of these single nAgs was lost in a setting of therapeutic vaccination in tumor-bearing mice. The presence of CD4+ T-cell help restored the efficacy for only the most expressed of the two nAgs, demonstrating a key role for CD4+ T cells in sustaining CD8+ T-cell responses and the necessity of an efficient recognition of the targeted epitopes on cancer cells by CD8+ T cells for an effective antitumor response. This study provides insight into understanding the determinants of nAgs relevant for effective treatment and highlights features that could contribute to more effective antitumor vaccines. See related Spotlight by Slingluff Jr, p. 382.

CD19-Specific CAR NK Cells Coexpressing IL-21 Exhibit Superior Expansion and Antitumor Activity Against CD19-Postive Lymphoma

Background Chimeric antigen receptor (CAR) expressing NK cells induce robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. IL-15 and IL-21 promote NK cell expansion, survival, and possess both overlapping and distinctive functions. CAR NK cells engineered to express IL-15 have shown long-term persistence and potent antitumor activity, however, the pleiotropic effects of IL-21 on CAR NK cell-mediated tumor rejection are lacking. Therefore, we compared the therapeutic potency of CD19-specific human NK cells that constitutively express IL-15 or IL-21. Methods The codon-optimized minigene encoding cytokine IL-21 or IL-15, linked with CD19-CAR containing 4-1BB costimulatory domain by a P2A sequence were synthesized and sub-cloned into the pSFG retroviral vector yielding the BBz.IL21 or BBz.IL15 retroviral construct. To produce CD19-CAR retrovirus, 293T/17 cells were transfected with a combination of plasmids containing CAR constructs, RDF plasmid and PegPam3 plasmid. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. The expression of CAR, phenotype, degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 3 post transduction. To measure IL-15 and IL-21 production, supernatants were collected from CAR NK cells cultured in the absence of IL-2 for 24h and evaluated by ELISA. Results After transduction, all constructs were stably expressed by NK cells, with construct containing IL-21 (71.23 ± 4.05 %, n = 3) demonstrating higher transduction efficiency compared with the BBz.IL15 (61.13 ± 3.43 %), but slightly lower than the BBz construct (79.63 ± 3.83 %). High levels of IL-21 were found in the supernatant of BBz.IL21 NK cells after resting 24h (3464 ± 199 pg/ml), while no detectable IL-21 was observed on BBz and BBz.IL15 NK cells. NK cells expressing BBz.IL15 produced significantly more IL-15 than BBz.IL21 and BBz NK cells as baseline (BBz.IL15: 213 ± 14 pg/ml, BBz.IL21: 61.86± 4 pg/ml, BBz: 60.56 ± 11 pg/ml). Given that IL-15 or IL-21 could potentially induce antigen-independent NK cell proliferation, we compared the ability of each NK-cell group to maintain growth in the presence of IL-2. The expansion of CAR NK cells expressing IL-21 was significantly higher than that of CAR NK cells expressing IL-15, while the number of BBz.IL15 NK cells and BBz NK cells were comparable (fold expansion in 5 days: BBz.IL21: 26.6 fold, BBz.IL15: 15.9 fold, BBz: 17.2 fold), suggesting a synergistic effect of IL-21 and IL-2 on CAR NK cell expansion. BBz.IL21 NK cells shared a similar immunophenotypic profile to BBz.IL15 NK cells, as the expression of inhibitory receptors PD1, LAG3, TIM3 and NKG2A, as well as activation receptors NKG2D and CD69 were comparable. CAR engagement by CD19-postive Raji cells specifically induced cytokines production by CD19-CAR NK cells but not by control NK cells. IL-21 coexpression significantly enhanced IFN-γ and TNF-α production compared with CAR NK cells as well as CAR NK cells expressing IL-15 (BBz.IL21 vs. BBz.IL15, P < 0.05; BBz.IL21 vs. BBz, P < 0.05; BBz.IL15 vs. BBz, P > 0.05). Moreover, a higher percentage of degranulated NK cells was observed on BBz.IL21 NK cells compared to BBz.IL15 NK cells and BBz NK cells. To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. CAR NK cells expressing IL-21 showed superior cytotoxicity, compared to CAR NK cells expressing IL-15. Conclusion CD19-CAR NK cells engineered to express IL-21 gene produced high levels of IL-21 during cultivation and enabled greater ex vivo expansion of CAR NK cells compared with CAR NK cells expressing IL-15. IL-21 coexpression induced a significant increase in IFN-γ, TNF-α production and degranulation compared with IL-15 coexpression, resulting in enhanced antitumor activity of CD19-CAR NK cells against CD19-postive lymphoma.

Abstract PR008: Overcoming PDAC T cell therapy barriers with CD47-targeted costimulatory fusion proteins

Abstract T cell therapy is a promising immunotherapy treatment option that uses genetically modified immune cells (T cells) to eliminate tumors. This approach uses the patients’ own immune cells to generate a “living drug” and can avoid the toxic side effects of other common therapies, such as radiation or chemotherapies. However, the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) can establish several obstacles to protect the tumor from T cells, including delivery of inhibitory and death signals that shut down T cells, usurping metabolic nutrients, and recruiting and/or converting immune cells to inhibitory phenotypes that block the T cell response. CD47 is upregulated in PDAC tumors, and patients with CD47hi tumors exhibit worse survival. CD47 serves as a “don’t eat me” signal by binding to the SIRPα receptor on macrophages to block phagocytosis. CD47 expression also inhibits T cell-mediated tumor rejection by blocking cross-presentation of tumor antigens by SIRPα+ dendritic cells. Reagents that interfere with SIRPα-CD47 signaling, such as monoclonal antibodies, are currently in clinical trials and have been shown to synergize with chemotherapy in PDA models, but native CD47 expression can produce erythrocyte and platelet depletion. Costimulatory signals delivered by surface-bound receptors can initiate gene expression programs that address multiple issues in the PDAC TME by mechanisms such as lowering the threshold of activation, altering metabolic programming, and reducing exhaustion. We develop engineering strategies to deliver costimulatory signals to engineered T cells. Immunomodulatory fusion proteins (IFPs) combine the ectodomain of an inhibitory T cell receptor with an intracellular costimulatory signaling domain and we have shown in proof-of-concept studies that this effectively “replaces a brake with an accelerator” in CD8 T cells. We have found that generating IFPs is not as simple as indiscriminately fusing any two proteins together and we have developed critical design concepts that inform the selection of the fusion site, ectodomain and costimulatory signaling domain pairing, and immunological synapse localization to enhance signaling. To develop a CD47-targeted IFP, we combined the SIRPa ectodomain with the CD28 signaling endodomain and tested this technology with our mesothelin-targeted TCR-T cell platform. In in vitrostudies, SIRPa-CD28 T cells exhibited enhanced proliferation, accumulation, and tumor cytotoxicity. SIRPa IFP-T cells exhibited increased intratumoral accumulation and therapeutic efficacy in the KrasLSL-G12D/+; Trp53LSL-R172H/+;p48Cre/+ (KPC) model, without toxicity or erythrocyte depletion. Here we describe how a thoughtfully engineered fusion protein can “armor” T cells against multiple obstacles and significantly improve therapeutic efficacy against PDAC. Citation Format: Shannon Oda, Leah Schmidt, Ashley Thelen, Cody Jenkins, Edison Chiu, Aitong Ruan, Philip Greenberg. Overcoming PDAC T cell therapy barriers with CD47-targeted costimulatory fusion proteins [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR008.

Mechanisms of CD40-dependent cDC1 licensing beyond costimulation.

CD40 signaling in classical type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1s, including Cd70, Tnfsf9, Ptgs2 and Bcl2l1, and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T cell expansion. Loss of 4-1BB, alone or in Cd27-/- mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased caspase activation in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation by Cd40-/- cDC1s to CD8+ T cells, which were reversed by re-expression of Bcl2l1. Thus, CD40 signaling in cDC1s not only induces costimulatory ligands for CD8+ T cells but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses.

Paving the Way to Solid Tumors: Challenges and Strategies for Adoptively Transferred Transgenic T Cells in the Tumor Microenvironment.

Simple SummaryAdoptive transfer of T cells targeting tumors still remains mainly ineffective in solid entities. In this review we discuss challenges related to the tumor microenvironment (TME) and promising strategies to improve tumor control by engineering the TME or the transferred T cells themselves.T cells are important players in the antitumor immune response. Over the past few years, the adoptive transfer of genetically modified, autologous T cells—specifically redirected toward the tumor by expressing either a T cell receptor (TCR) or a chimeric antigen receptor (CAR)—has been adopted for use in the clinic. At the moment, the therapeutic application of CD19- and, increasingly, BCMA-targeting-engineered CAR-T cells have been approved and have yielded partly impressive results in hematologic malignancies. However, employing transgenic T cells for the treatment of solid tumors remains more troublesome, and numerous hurdles within the highly immunosuppressive tumor microenvironment (TME) need to be overcome to achieve tumor control. In this review, we focused on the challenges that these therapies must face on three different levels: infiltrating the tumor, exerting efficient antitumor activity, and overcoming T cell exhaustion and dysfunction. We aimed to discuss different options to pave the way for potent transgenic T cell-mediated tumor rejection by engineering either the TME or the transgenic T cell itself, which responds to the environment.

Abstract 3563: CD8+ T cell-mediated tumor rejection by an Adeno-associated virus-like particle (AAVLP) vaccine

Abstract The utilization of adeno-associated viruses (AAVs) as vaccines has primarily been focused on the induction of antibody responses. Displaying antigens derived from HER2 or HPV on the AAV capsid surface lead to strong humoral immune responses. However, the potential of AAVs as T cell inducing vaccines has been poorly investigated. Model antigens have been inserted into the capsid of AAVs to analyze T cell responses, but these studies rather focused on preventing vector immunity for an improved tolerance of gene therapies. By using this as a starting point, we set out to test AAVs as T cell inducing vaccines with the prospect of targeting cancer neoantigens. In first experiments, the general properties of the vaccination strategy were estimated by displaying the ovalbumin-derived model antigen SIINFEKL on the surface of adeno-associated virus-like particles (AAVLPs). Upon injection of the AAVLP-SIINFEKL vaccine, mice developed strong CD8+ T cell responses against the displayed antigen. Highest immune responses were achieved by subcutaneous hock injection of AAVLPs, adjuvanted by Montanide ISA 51 VG. SIINFEKL-specific T cell responses peaked around three weeks after vaccination, whereas a memory subset remained present for long term. The anti-tumor efficacy of the vaccine was shown by injecting SIINFEKL-expressing B16F10 melanoma cells subcutaneously into mice, in which the tumor was completely rejected after vaccination. Interestingly, the induction of CD8+ T cell responses and the tumor protection depended on the presence of CD4+ T cells. Accordingly, T helper epitopes were identified in the AAVLP capsid sequence. In addition to the initial tests, a set of murine neoantigens, derived from B16F10 cells, were displayed on AAVLPs. In direct comparison to a peptide vaccine, which did not have an effect on tumor growth, the AAVLP vaccine had a significant impact on the tumor growth rate. In conclusion, AAVLPs show promising effects as T cell vaccines. The vaccination strategy can be used to induce cytotoxic T cell responses in general and anti-tumor effects in particular. AAV capsid-specific helper epitopes is one inherent advantage, since the AAVLP vaccine delivers immune stimulation within the viral particle itself. Thus, antigen-displaying AAVLPs could be an alternative to current gold standards in the field of neoantigen vaccines and have a prospect for future clinical applications. Citation Format: Lasse Neukirch, Patrick Schmidt, Inka Zörnig, Dirk Jäger, Silke Uhrig-Schmidt. CD8+ T cell-mediated tumor rejection by an Adeno-associated virus-like particle (AAVLP) vaccine [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 3563.

Abstract 1309: Patient-derived tumor organoids as a platform to study radiation-exposed neoantigens

Abstract Focal radiation therapy (RT) can increase tumor immunogenicity and T cell-mediated tumor rejection when combined with immune checkpoint blockade (ICB). Our prior findings in patients and in preclinical studies suggest that one mechanism whereby RT increases tumor immunogenicity is by enhancing the expression of neoantigens.1,2 In response to RT, cells rapidly transcribe and translate hundreds of genes associated with the DNA damage response (DDR), protein turnover and cellular stress. Genes in these pathways are frequently mutated in cancer. Thus, we hypothesized that the presence of immunogenic mutations in genes upregulated by radiation could help predict the benefits of RT used in combination with ICB. In order to develop such a predictor, we built an in vitro system to study the RT-induced transcriptional response of primary human tumors cultured as patient-derived tumor organoids (PDO). PDOs were established from 2 breast cancer (BC), 4 non-small cell lung cancer (NSCLC) and 3 colorectal cancer (CRC) patients. PDOs were irradiated with doses of 5 to 8 Gy daily for 3 days (n=4) or left untreated (UT, n=4) and cultured for 24h prior to RNA extraction. RNA sequencing was carried out using a NovaSeq6000 sequencer (Illumina) to a depth of 30 million reads. Differential expression analysis between RT and UT samples was performed using DESeq2 and significantly perturbed genes were defined as genes with fold change greater than 1.5 and adjusted p-value cutoff of 0.05. In total we detected an average of 15,000+/-800 protein coding genes per sample and the number of genes modulated by RT was found to vary with cancer type. This ranged from an average of 3.9+/-1.6% genes upregulated by RT in NSCLC to 12.1+/-2.5% in CRC organoids. There were 104 and 490 genes commonly upregulated by RT in NSCLC and CRC, respectively. One of the BC PDOs is a triple-negative BC and the other HR+ and despite this difference, we identified 24 commonly upregulated genes. Gene Set Enrichment Analysis (GSEA), revealed enrichments in DDR and pro-inflammatory signaling pathways among the upregulated genes. Whole exome sequencing and paired normal tissue was used to determine the variants and predicted neoantigens for these PDOs. In one of the EGFR mutated NSCLC PDOs we found that among the genes transcriptionally upregulated by RT, 18 carried somatic mutations predicted to be antigenic. The presentation of these neoantigens is being investigated by mass spectrometry analysis of the immunopeptidome eluted from surface MHC-I molecules. Similar investigations are being carried out in the other models. Overall, results support the feasibility of identifying a common signature of RT upregulated genes in PDOs. This will provide a system to predict RT- exposed neoantigens. [1] Formenti et al. Nat. Med. 2018;24(12):1845-1851, [2] Lhuillier et al. J. Clin. Invest. 2021;131(5):e138740 Citation Format: Samantha J. Van Nest, Jared Capuano, Bhavneet Bhinder, Martin G. Klatt, Tuo Zhang, M. Laura Martin, Silvia C. Formenti, Olivier Elemento, Nils-Petter Rudqvist, Sandra Demaria. Patient-derived tumor organoids as a platform to study radiation-exposed neoantigens [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 1309.

Mechanisms of CD40-dependent cDC1 licensing beyond co-stimulation

Abstract CD40 signaling in conventional type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1 and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T-cell expansion. Loss of 4-1BB, alone or in Cd27−/− mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased apoptosis in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation, which were reversed by re-expression of Bcl-xL. Thus, CD40 signaling in cDC1 not only induces co-stimulatory ligands for CD8 T cells, but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses. This work was supported by grants from the NIH (R01AI150297, R01CA248919, and R21AI164142 to K.M.M., and F30CA247262 to R.W.).

Targeting transforming growth factor-β2 by antisense oligodeoxynucleotide accelerates T cell-mediated tumor rejection in a humanized mouse model of triple-negative breast cancer.

Transforming growth factor-beta (TGF-β) pathway mediates suppression of antitumor immunity and is associated with poor prognosis in triple-negative breast cancer (TNBC). In this study, we generated a humanized animal model by transplanting human peripheral blood mononuclear cells into immunodeficient mice followed by inoculation of MDA-MB-231 cells and subsequently analyzed the role of TGF-β2 in the interaction between human T cells and human tumor cells. Following reconstitution of the human immune system, inhibition of TGF-β signaling by TGF-β2 antisense oligodeoxynucleotide (TASO) resulted in accelerated tumor growth inhibition. TGF-β2 inhibition also resulted in downregulation of peripheral Foxp3 + regulatory T cells (Treg), whereas no effect was seen in the expression of CD8 + cytotoxic T cells. Analysis of the TASO-treated mice serum revealed elevated levels of human IFN-γ and reduced levels of human IL-10 and TGF-β2. Moreover, TGF-β2 inhibition resulted in increased CD8 + T cell infiltration, whereas the reduced infiltration of Tregs into the tumor partly resulted from decreased expression of CCL22. Decreased intratumoral Tregs facilitated the activation of cytotoxic T cells, associated with increased granzyme B expression. These results indicate that TASO potentiated T cell-mediated antitumor immunity, and it is proposed that TGF-β2 may be a promising target in the immunotherapeutic strategy of TNBC.

Neoantigen vaccination induces clinical and immunologic responses in non-small cell lung cancer patients harboring EGFR mutations

BackgroundNeoantigen (NeoAg) peptides displayed at the tumor cell surface by human leukocyte antigen molecules show exquisite tumor specificity and can elicit T cell mediated tumor rejection. However, few NeoAgs are...

Non-redundant activity of GSK-3α and GSK-3β in T cell-mediated tumor rejection

SummaryGlycogen synthase kinase-3 (GSK-3) is a positive regulator of PD-1 expression in CD8+ T cells and GSK-3 inhibition enhances T cell function and is effective in the control of tumor growth. GSK-3 has two co-expressed isoforms, GSK-3α and GSK-3β. Using conditional gene targeting, we demonstrate that both isoforms contribute to T cell function to different degrees. Gsk3b−/− mice suppressed tumor growth to the same degree as Gsk3a/b−/− mice, whereas Gsk3a−/− mice behaved similarly to wild-type, revealing an important role for GSK-3β in regulating T cell-mediated anti-tumor immunity. The individual GSK-3α and β isoforms have differential effects on PD-1, IFNγ, and granzyme B expression and operate in synergy to control PD-1 expression and the infiltration of tumors with CD4 and CD8 T cells. Our data reveal a complex interplay of the GSK-3 isoforms in the control of tumor immunity and highlight non-redundant activity of GSK-3 isoforms in T cells, with implications for immunotherapy.

Abstract IA05: Innate immune defenses against cancer: Potential for mobilizing NK cells for cancer immunotherapy

Abstract Immunotherapies that mobilize CD8 T cells have revolutionized cancer therapy in recent years. A likely limitation of CD8 T cells in immunotherapy is that many tumors display few neoantigens and/or lack MHC I expression. Natural killer cells have emerged as exciting targets for new generations of immunotherapeutics. We investigated innate cues necessary for mobilizing spontaneous anti-tumor responses against tumors in mice and identified the cGAS-STING pathway as a critical pathway that is activated by cancer cells and mobilizes both NK cell and T cell mediated tumor rejection. Cyclic dinucleotides (CDNs) are STING agonists that, when injected into tumors, superactivate cellular immunity. We investigated the efficacy of CDNs in mobilizing anti-tumor activity in 6 independent gene edited models of MHC I-deficient tumors. Due to the absence of MHC I, CD8 T cells played no role in these responses. When transplanted syngeneic tumors of all 6 models were established and then treated intratumorally with CDNs (a single treatment, or in some cases 3 early treatments), tumor rejection rapidly ensued, which was NK-dependent and independent of CD8 T cells. In 5 of 6 MHC I-deficient tumor models tested, CDN therapy resulted in the indefinite survival of between 30% and 100% of the animals, with no evidence of residual tumors, suggesting the animals were cured. In one model (MHC I-deficient B16 melanoma cells) early rejection of the tumors was followed by relapse in all of the animals. Tumor rejection induced by CDNs was IFN-I-dependent, and conditional knockout experiments indicated that IFN-I acted directly on NK cells but also indirectly on DCs. After CDN therapy, DCs in the draining lymph nodes upregulated IL-15/IL-15R complexes on the membrane, and tumor rejection was blocked by neutralizing IL-15/IL15R. These data suggested that CDNs induce trans-presentation of IL15 by DCs to NK cells to support the tumor rejection response. CDN-induced tumor rejection was markedly enhanced by combining CDNs with IL-2 superkine (“super2”, in collaboration with C. Garcia), resulting in long-term tumor free survival in nearly all of the mice in the MHC I-deficient B16 and MC-38 models, indicating striking synergy between CDNs and super2. Our results suggest great potential for combining agonists of innate immune responses with superkines for treating cancers that are invisible to CD8 T cells, including MHC I-deficient cancers and potentially cancers with low neoantigen expression. Citation Format: David H. Raulet. Innate immune defenses against cancer: Potential for mobilizing NK cells for cancer immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr IA05.

Metastasis-associated macrophages constrain antitumor capability of natural killer cells in the metastatic site at least partially by membrane bound transforming growth factor β

BackgroundMetastatic breast cancer is a leading cause of cancer-related death in women worldwide. Infusion of natural killer (NK) cells is an emerging immunotherapy for such malignant tumors, although elimination of...

All-trans Retinoic Acid (atRA) promotes tumor resistence to NK cells by downregulating tumor B7-H6 expression

Abstract The interaction between the activating receptor NKp30 and its major tumor ligand B7-H6 is important for NK cell mediated tumor rejection while the regulation of B7-H6 by tumor therapeutics remains largely unknown. In this study, we investigated the regulation of B7-H6 by all-trans Retinoic acid (atRA), which is a terminal differentiation inducer of tumor cells and in use for tumor therapy. We found that B7-H6 expression on tumor cell suface was downregulated upon atRA treatment. We further found that atRA downregulated B7-H6 at the mRNA level. Epgenetic modifications such as DNA methylation and HDAC was not responsible as inhibitors of these pathways did not restore the B7-H6 mRNA expression. Instead, atRA downregulated c-myc and inhibited the transcription of B7-H6. NK92 and NKG cells mediated U937 cell killing was mainly dependent on the NKp30-B7-H6 interaction. Thus atRA treatment dampened U937 lysis by NK92 and NKG cells. Together, our data showed that atRA treatment downregulated B7-H6 expression and promoted tumor cell resistence to NK cells.

Abstract B78: Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade

Abstract Malignant melanoma is the most aggressive form of skin cancer, accounting for 10,000 deaths annually. Largely resistant to conventional cytotoxic chemotherapy and radiation, immune checkpoint blockade (ICB) therapies have revolutionized patient care, accounting for partial or complete responses in up to 70% of patients. An important aspect of cancer elimination is activation of the cytotoxic T lymphocyte (CTL) response. Recent data suggest that ICB mediated broadening of the peripheral blood CTL repertoire correlates with good clinical outcomes. Thus, recent studies in cancer immunotherapy have focused on targeting tumor neoantigens (tNeoAg) with cancer vaccines for use in combination with ICB. Computational algorithm-driven predictions of immunogenic tNeoAgs yield vast numbers of potential peptide targets, only a fraction of which may be immunogenic in patients. This likely contributed to the modest success of cancer vaccines targeting predicted, high-avidity tNeoAgs, emphasizing the importance of identifying true tumor rejection antigens, including sub-dominant antigens, outside the scope of predictive models. In addition to promoting T cell-mediated tumor rejection, ICB often comes at the expense of treatment-induced immune-related adverse effects (irAE) that frequently require discontinuation of treatment. Modulation of ICB towards antitumor immunity and away from autoimmunity may dramatically improve the therapeutic index of modern cancer therapy. This appears feasible considering the clinical observation that antitumor efficacy does not correlate with type/severity of irAE, suggesting that the mechanisms of both processes, though likely related (CTL mediated), may not be identical. We aim to identify and separate non-cross-reacting antigenic targets mediating tumor rejection from those mediating irAEs to enable therapeutic interventions that maximize the efficacy of ICB, expanding tumor-specific CTLs with vaccines while minimizing irAEs through desensitization. We have access to samples from responding/nonresponding melanoma patients subjected to ICB with varying degrees of irAEs. We have designed an experimental approach that combines established mass spectrometry and sequencing techniques to identify peptides and matching TCR clones with a novel strategy that targets the TCR-associated CD3 complex to allow inclusion of subdominant antigens in our studies. T-cell “co-potentiation” is achieved when anti-CD3 monovalent Fabs induce a conformational change in the CD3 complex that sustains the T-cell response to weak antigenic stimulation. We have successfully used anti-human CD3 Fabs to co-potentiate in vitro the activation and subsequent response to weak peptide-HLA/TCR interactions of human T cells found in PBMCs isolated from healthy donors. Our preliminary data suggest T-cell co-potentiation may allow identification of ICB-induced CTL clones specific for subdominant tNeoAgs and irAE targets in patients with melanoma undergoing active immunotherapy. Citation Format: Laura Elsbernd, Alfreda Nelson, Hien Huynh, Michele Hoffmann, Christopher Parks, Wendy Nevala, Shari Sutor, Larry Pease, Adam Schrum, Diana Gil, Svetomir N. Markovic. Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B78.

A Combination of Positive Tumor HLA-I and Negative PD-L1 Expression Provides an Immune Rejection Mechanism in Bladder Cancer.

Tumor human leukocyte antigen class I (HLA-I) expression plays an important role in T cell-mediated tumor rejection. Loss of HLA-I is associated with cancer progression and resistance to immunotherapy, including antibodies blocking programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) signaling. Our objective was to analyze a correlation between HLA-I, tumor immune infiltration, and PD-L1/PD-1 axis in bladder cancer in association with the clinicopathologic features of patients. We analyzed 85 cryopreserved bladder tumors by immunohistochemistry to investigate the expression of HLA-I, PD-L1, PD-1, CD3,CD8, and CXC chemokine receptor 4 (CXCR4). The results were correlated with tumor stage and other clinicopathologic variables of patients. We found a strong positive correlation between tumor HLA-I expression and infiltration with CD3+ and CD8 + T cells. PD-L1 expression was positive in 15.5% of tumors and heterogeneous in 40.5%, and was linked to a more advanced tumor stage. The majority of HLA-I-positive/heterogeneous tumors also expressed PD-L1 and PD-1, which were significantly correlated with each other and with lymphocyte infiltration. Interestingly, the analysis of the simultaneous expression of both markers revealed that 85.2% of tumors with a positive/heterogeneous HLA-I phenotype and negative for PD-L1 were mostly non-invasive, representing a 'tumor rejection' immune phenotype. High tumor HLA-I expression with absence of PD-L1 provides bladder cancer with an immune rejection mechanism. Evaluation of PD-L1 and HLA-I together should be considered in bladder cancer and may provide a new predictive biomarker of tumor invasiveness and of the response to 'immune checkpoint' therapy.

Harnessing Tumor Mutations for Truly Individualized Cancer Vaccines.

T cells are key effectors of anticancer immunity. They are capable of distinguishing tumor cells from normal ones by recognizing major histocompatibility complex-bound cancer-specific peptides. Accumulating evidence suggests that peptides associated with T cell-mediated tumor rejection arise predominantly from somatically mutated proteins and are unique to every patient's tumor. Knowledge of an individual's cancer mutanome (the entirety of cancer mutations) allows harnessing this enormous tumor cell-specific repertoire of highly immunogenic antigens for individualized cancer vaccines. This review outlines the preclinical and clinical state of individualized cancer vaccine development and the challenges ahead.

Combination of Sunitinib and PD-L1 Blockade Enhances Anticancer Efficacy of TLR7/8 Agonist-Based Nanovaccine.

Cancer vaccines composed of tumor-associated antigens (TAAs) and toll-like receptor (TLR) agonists have shown promising antitumor efficacy in preclinical studies by generating antigen-specific CD8 T cells, but translation of cancer vaccines to the clinic has been limited due to variables responses and development of resistance. The tumor microenvironment deploys various immune escape mechanisms that neutralize CD8 T cell-mediated tumor rejection. Therefore, we hypothesized that modulation of the tumor microenvironment can augment CD8 T cell activation and enhance therapeutic efficacy of cancer vaccines. To accomplish this, we aimed to eliminate immune suppressive cells and block their inhibitory signaling. Combination of the tyrosine kinase inhibitor (TKI) sunitinib with a nanoparticle-based cancer vaccine (nanovaccine) resulted in the reduction of immune-suppressive myeloid-derived suppressive cells (MDSCs) and regulatory T cells (Tregs). Blockade of programmed death-ligand 1 (PD-L1) using anti-PD-L1 antibody was used to reduce CD8 T cell exhaustion. Combination of nanovaccine+sunitinib+PD-L1 antibody treatment reduced PD-L1high M2 macrophages and MDSCs and upregulated activation of CD8 T cells in the tumor. Nanovaccine+sunitinib+PD-L1 antibody treatment also stimulated antigen-specific CD8 T cell response, which led to improved therapeutic efficacy in MB49 and B16F10 murine tumor models. These results suggest that modulation of tumor microenvironment using sunitinib and PD-L1 blockade can significantly enhance the antitumor efficacy of cancer nanovaccine.

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Image : https://commons.wikimedia.org/wiki/File%3AMitochondria.svg. Nevit (own work)In tumor microenvironments and during chronic inflammation, cytotoxic T cells become “exhausted.” Bengsch et al. and Scharping et al. found that the exhaustion involved repression of mitochondrial replication and energy production, orchestrated by reductions in PGC1α expression.Bengsch B,…, Wherry, EJ. Immunity 2016 Aug 16;45:358–73.Scharping NE,….Delgoffe GM. Immunity 2016 Aug 16; 45:374–88.Image : https://commons.wikimedia.org/wiki/File:Medulloblastoma_HE_x200.jpg. Jensflorian (own work)Cdk5 is expressed in medulloblastomas. Deleting Cdk5 allowed a potent CD4+ T cell–mediated tumor rejection to develop. The tumor immunoevasive mechanism evoked by interferon-γ that increases PD-L1 expression was suppressed in medulloblastomas that did not express Cdk5.Dorand RD,…, Petrosiute A. Science 2016 July 22; 353:399–403.Image : Jacoby et al., Nat. Comms. Fig. 2fWhen CD19 CAR T-cell therapy is used against ALL, the CD19 protein is often downregulated. This was found not to be the mere disappearance of an epitope, but represented cellular reprogramming and a switch to the myeloid lineage.Jacoby E,…, Fry TJ. Nat Comms 2016 Jul 27. doi: 10.1038/ncomms12320.Image : https://commons.wikimedia.org/w/index.php?curid=4463752. Gleiberg (own work)Less than 2% of CD8+ T cells express CXCR5. These cells were found to be generated in chronically infected animals and to have stem cell-like renewal properties. They could also differentiate into CXCR5− cells and proliferate extensively upon PD-1 blockade with checkpoint inhibitors.Leong YA,…, Yu D. Nat Immunol 2016 Aug 3. doi:10.1038/ni.3543.Im SJ,…, Ahmed R. Nature 2016 Aug 2. doi: 10.1038/nature19330.He R,…, Ye L. Nature 2016 Aug 2. doi: 10.1038/nature19317.Image : https://en.wikipedia.org/wiki/Oxygen_sensor#/media/File:Diving_oxygen_analyser.JPGTumor metastasis requires the maintenance of favorable protumor niches. T cells use the oxygen-sensitive PHD proteins to regulate differentiation and effector function. In lung tumors, PHD proteins promoted T-cell localization and Treg proliferation and repressed antitumor activity.Clever D,…, Restifo NP. Cell 2016 Aug 25;166:1117–31.e14.Image : https://commons.wikimedia.org/w/index.php?curid=9051744. Nephron (own work)T cells specific for tumors are often functionally disabled, and this has been attributed to the tumor microenvironment. Schietinger et al. found that this dysfunctional state is induced early in tumorigenesis, is intrinsic, and becomes irreversible over time.Schietinger A,…, Greenberg PD. Immunity 2016;45:389–401.

Interleukin-7 Modulates Anti-Tumor CD8+ T Cell Responses via Its Action on Host Cells.

The adoptive transfer of antigen-specific CD8+ T cells is a promising approach for the treatment of chronic viral and malignant diseases. In order to improve adoptive T cell therapy (ATT) of cancer, recent strategies aim at the antibody-based blockade of immunosuppressive signaling pathways in CD8+ T cells. Alternatively, adjuvant effects of immunostimulatory cytokines might be exploited to improve therapeutic CD8+ T cell responses. For example, Interleukin-7 (IL-7) is a potent growth, activation and survival factor for CD8+ T cells that can be used to improve virus- and tumor-specific CD8+ T cell responses. Although direct IL-7 effects on CD8+ T cells were studied extensively in numerous models, the contribution of IL-7 receptor-competent (IL-7R+) host cells remained unclear. In the current study we provide evidence that CD8+ T cell-mediated tumor rejection in response to recombinant IL-7 (rIL-7) therapy is strictly dependent on IL-7R+ host cells. On the contrary, CD8+ T cell expansion is independent of host IL-7R expression. If, however, rIL-7 therapy and peptide vaccination are combined, host IL-7R signaling is crucial for CD8+ T cell expansion. Unexpectedly, maximum CD8+ T cell expansion relies mainly on IL-7R signaling in non-hematopoietic host cells, similar to the massive accumulation of dendritic cells and granulocytes. In summary, we provide evidence that IL-7R+ host cells are major targets of rIL-7 that modulate therapeutic CD8+ T cell responses and the outcome of rIL-7-assisted ATT. This knowledge may have important implications for the design and optimization of clinical ATT protocols.