Tumor-associated Self-antigens Research Articles

T-Switch: A specificity-based engineering platform for developing safe and effective Tcell therapeutics.

Many promising targets for adoptive Tcell therapy (ACT) are self-antigens, but self-reactive Tcells are generally eliminated during thymic selection or diverted to regulatory phenotypes. To bypass Tcell tolerance and obtain potent and safe Tcell therapeutics, we developed T-Switch, an invitro Tcell receptor (TCR) engineering platform for the creation, modification, and comprehensive profiling of TCRs that can target self-antigens. T-Switch first expands Tcells that recognize a "foreign" peptide closely related to a self-antigen. The fine specificity of the TCR is then modified by directed evolution of the peptide binding region to switch its specificity to the self-antigen of interest. We applied T-Switch to engineer synthetic TCRs reactive to a tumor-associated self-antigen, validated the safety and efficacy of this approach, and detected no off-target recognition as measured against the human proteome. Thus, T-Switch represents a resource for the creation of collections of highly sensitive synthetic TCRs for Tcell-based immunotherapies.

A replicating LCMV-based vaccine for the treatment of solid tumors

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T-cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of LCMV-based vectors to break tolerance and induce effective antigen-specific CD8+ T-cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T-cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T-cell priming, which renders previously “cold” tumors open to immune infiltration and reprograms the tumor microenvironment to “hot”. Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

Liquid nitrogen frozen cells for chemotherapy drug delivery and vaccination of melanoma.

Cancer vaccine (CV) has thrived as a promising tool for cancer prevention and treatment. However, how to maintain the integrity and diversity of individualized vaccine antigens and activate the adaptive immune system is still challenging. Herein, a preventive and therapeutic vaccine platform for in situ effective multi-model synergistic therapy is developed. In our study, we process B16F10 cells by liquid nitrogen frozen (LNF) to obtain LNF cells, the characterizationof LNF cells were conducted. Moreover,the anti-tumor effect and immune activation ability were studied, and the role as a CV were investigated. The LNF cells preserve intact cellular structure and tumor-associated self-antigen gp100. Moreover, LNF cells have the ability of loading and releasing doxorubicin (DOX). Except for the anti-tumor effect of chemotherapy brought by DOX, the LNF cells can promote the maturation of dendritic cells (DCs) and induce immune response by activating CD4+ and CD8+ T cells, particularly with the existence of adjuvant, R848. Specifically, the CD8+ T cells of mice in LNF-DOX/R848 group are 6 times of that in PBS group in tumor microenvironment, and twice in spleen. Therefore, LNF cells can also be utilized as a CV. Vaccination with LNF/R848 cells effectively suppress the tumor growth in mice by fivefold as compared to the control group. Inthis work, we obtain the LNF cells with a simple procedure. The LNF cells not only provides a tumor cells-based multi-modal system for cancer therapy but inspires new insights into future development of individualized CVs strategies. This study processes live B16F10 cells by liquid nitrogen frozen to obtain LNF cells, which preserve cell integrity and homologous targeting ability. The LNF cells can load and deliver drug and can serve as tumor vaccine. Results demonstrated the LNF cells have effective prophylactic ability, and ideal anti-tumor ability with the loaded drug and adjuvant.

Autoreactive napsin A-specific T cells are enriched in lung tumors and inflammatory lung lesions during immune checkpoint blockade.

Cancer treatment with immune checkpoint blockade (ICB) often induces immune-related adverse events (irAEs). We hypothesized that proteins coexpressed in tumors and normal cells could be antigenic targets in irAEs and herein described DITAS (discovery of tumor-associated self-antigens) for their identification. DITAS computed transcriptional similarity between lung tumors and healthy lung tissue based on single-sample gene set enrichment analysis. This identified 10 lung tissue-specific genes highly expressed in the lung tumors. Computational analysis was combined with functional T cell assays and single-cell RNA sequencing of the antigen-specific T cells to validate the lung tumor self-antigens. In patients with non-small cell lung cancer (NSCLC) treated with ICB, napsin A was a self-antigen that elicited strong CD8+ T cell responses, with ICB responders harboring higher frequencies of these CD8+ T cells compared with nonresponders. Human leukocyte antigen (HLA) class I ligands derived from napsin A were present in human lung tumors and in nontumor lung tissues, and napsin A tetramers confirmed the presence of napsin A-specific CD8+ T cells in blood and tumors of patients with NSCLC. Napsin A-specific T cell clonotypes were enriched in lung tumors and ICB-induced inflammatory lung lesions and could kill immortalized HLA-matched NSCLC cells ex vivo. Single-cell RNA sequencing revealed that these T cell clonotypes expressed proinflammatory cytokines and cytotoxic markers. Thus, DITAS successfully identified self-antigens, including napsin A, that likely mediate effective antitumor T cell responses in NSCLC and may simultaneously underpin lung irAEs.

Preclinical development of a vaccine-based immunotherapy regimen (VBIR) that induces potent and durable T cell responses to tumor-associated self-antigens.

The development of therapeutic cancer vaccines remains an active area, although previous approaches have yielded disappointing results. We have built on lessons from previous cancer vaccine approaches and immune checkpoint inhibitor research to develop VBIR, a vaccine-based immunotherapy regimen. Assessment of various technologies led to selection of a heterologous vaccine using chimpanzee adenovirus (AdC68) for priming followed by boosts with electroporation of DNA plasmid to deliver T cell antigens to the immune system. We found that priming with AdC68 rapidly activates and expands antigen-specific T cells and does not encounter pre-existing immunity as occurs with the use of a human adenovirus vaccine. The AdC68 vector does, however, induce new anti-virus immune responses, limiting its use for boosting. To circumvent this, boosting with DNA encoding the same antigens can be done repetitively to augment and maintain vaccine responses. Using mouse and monkey models, we found that the activation of both CD4 and CD8 T cells was amplified by combination with anti-CTLA-4 and anti-PD-1 antibodies. These antibodies were administered subcutaneously to target their distribution to vaccination sites and to reduce systemic exposure which may improve their safety. VBIR can break tolerance and activate T cells recognizing tumor-associated self-antigens. This activation lasts more than a year after completing treatment in monkeys, and inhibits tumor growth to a greater degree than is observed using the individual components in mouse cancer models. These results have encouraged the testing of this combination regimen in cancer patients with the aim of increasing responses beyond current therapies.

A Critical Role of CD40 and CD70 Signaling in Conventional Type 1 Dendritic Cells in Expansion and Antitumor Efficacy of Adoptively Transferred Tumor-Specific T Cells

In vivo expansion of adoptively transferred CD8+ T cells is a critical determinant of successful adoptive T cell therapy. Emerging evidence indicates Batf3-dependent conventional type 1 dendritic cells (cDC1s) rarely found within the tumor myeloid compartment are crucial for effector T cell recruitment to the tumor microenvironment. However, the role of cDC1s in expansion of tumor-specific CD8+ T cells remains unclear. In this article, we addressed the role of cDC1s and their costimulatory molecules, CD40, CD70, and CD80/CD86, in expansion and antitumor efficacy of adoptively transferred in vitro-primed CD8+ T cells recognizing nonmutated tumor-associated self-antigens. We found that TLR/CD40-mediated expansion and antitumor efficacy of adoptively transferred tumor-specific CD8+ T cells were abrogated in Batf3-/- mice. Further mechanistic studies using mixed bone marrow chimeric mice identified that CD40 and CD70 but not CD80/CD86 signaling in cDC1s played a critical role in expansion and antitumor efficacy of adoptively transferred CD8+ T cells. Moreover, induction and activation of cDC1s by administration of FMS-like tyrosine kinase 3 ligand (Flt3L) and TLR/CD40 agonists augmented expansion of adoptively transferred CD8+ T cells, delayed tumor growth, and improved survival. These findings reveal a key role for CD40 and CD70 signaling in cDC1s and have major implications for the design of new vaccination strategies with adoptive T cell therapy.

Tumor-Specific CD8+ T-Cell Responses Induced by DNA Vaccination.

DNA vaccines assisted by electroporation efficiently trigger antitumor cytotoxic CD8+ T cell responses in preclinical cancer models and hold potential for human use. They can be easily engineered to express either tumor-associated self-antigens, which are broadly expressed among tumor patients but also in healthy tissue, or tumor-specific neoantigens, which are uniquely expressed in tumors and differ among patients. Recently, it has been demonstrated that DNA vaccination generates both circulating and tissue-resident compartments of CD8+ T cells, which act concertedly against tumors. Here we describe the steps to obtain and test DNA vaccines against models of self-antigens and neoantigens in mice. It includes the evaluation of effector and memory CD8+ Tcell responses, as well as assessing the antitumor potential in vivo using transplantable syngeneic tumor models.

Thymic Function Associated With Cancer Development, Relapse, and Antitumor Immunity - A Mini-Review.

The thymus is the central lymphoid organ for T cell development, a cradle of T cells, and for central tolerance establishment, an educator of T cells, maintaining homeostatic cellular immunity. T cell immunity is critical to control cancer occurrence, relapse, and antitumor immunity. Evidence on how aberrant thymic function influences cancer remains largely insufficient, however, there has been recent progress. For example, the involuted thymus results in reduced output of naïve T cells and a restricted T cell receptor (TCR) repertoire, inducing immunosenescence and potentially dampening immune surveillance of neoplasia. In addition, the involuted thymus relatively enhances regulatory T (Treg) cell generation. This coupled with age-related accumulation of Treg cells in the periphery, potentially provides a supportive microenvironment for tumors to escape T cell-mediated antitumor responses. Furthermore, acute thymic involution from chemotherapy can create a tumor reservoir, resulting from an inflammatory microenvironment in the thymus, which is suitable for disseminated tumor cells to hide, survive chemotherapy, and become dormant. This may eventually result in cancer metastatic relapse. On the other hand, if thymic involution is wisely taken advantage of, it may be potentially beneficial to antitumor immunity, since the involuted thymus increases output of self-reactive T cells, which may recognize certain tumor-associated self-antigens and enhance antitumor immunity, as demonstrated through depletion of autoimmune regulator (AIRE) gene in the thymus. Herein, we briefly review recent research progression regarding how altered thymic function modifies T cell immunity against tumors.

Virus-Like Particles as an Immunogenic Platform for Cancer Vaccines.

Virus-like particles (VLP) spontaneously assemble from viral structural proteins. They are naturally biocompatible and non-infectious. VLP can serve as a platform for many potential vaccine epitopes, display them in a dense repeating array, and elicit antibodies against non-immunogenic substances, including tumor-associated self-antigens. Genetic or chemical conjugation facilitates the multivalent display of a homologous or heterologous epitope. Most VLP range in diameter from 25 to 100 nm and, in most cases, drain freely into the lymphatic vessels and induce antibodies with high titers and affinity without the need for additional adjuvants. VLP administration can be performed using different strategies, regimens, and doses to improve the immunogenicity of the antigen they expose on their surface. This article summarizes the features of VLP and presents them as a relevant platform technology to address not only infectious diseases but also chronic diseases and cancer.

Generation of affinity ranged antigen-expressing tumor cell lines.

The interaction strength between CD8+ T cells' TCR and cognate peptide-MHC (pMHC) impacts on the CD8+ T cell response against pathogens and tumors (Martinez-Usatorre, Donda, Zehn, & Romero, 2018; Zehn, Lee, & Bevan, 2009). CD8+ T cell responses against tumors are characterized by the presence of low affinity CD8+ T cells specific for nonmutated tumor associated self-antigens (TAA) and potentially high affinity tumor specific CD8+ T cells recognizing mutated self-antigens (Gros et al., 2016; Kvistborg et al., 2012; McMahan & Slansky, 2007). High affinity T cells display enhanced survival, expansion capacity and tumor control (Martinez-Usatorre et al., 2018; Schmid et al., 2010). In fact, recent clinical trials using neoantigen tumor vaccines showed prolonged progression free survival in melanoma patients (Ott et al., 2017; Sahin et al., 2017), while only modest clinical efficacy was obtained with TAA vaccines (Romero et al., 2016). However, the highly individual nature of neoantigens constitutes a major technical and economical hurdle for routine clinical application. Thus, the characterization of TAA-specific CD8+ T cell responses may reveal new strategies to enhance their anti-tumor properties. In parallel, the identification of high affinity antigens and CD8+ T cells may be essential to design effective tumor vaccines and adoptive cell transfer therapies. Therefore, in this chapter, we describe how to generate tumor cell lines with stable expression of affinity-ranged antigens and methods to assess T-cell affinity.

An HER2 DNA vaccine with evolution-selected amino acid substitutions reveals a fundamental principle for cancer vaccine formulation in HER2 transgenic mice.

Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.

Arenavirus-based vector platform for massive tumor self-antigen-specific CD8 T cell immunity.

e14297 Background: The induction of powerful CD8+ T cell immunity to tumor associated self-antigens (TAAs) represents a critical yet challenging goal. Here we report on the development of an arenavirus-based delivery platform meeting this challenge. Previously we have shown that genetically engineered replication-attenuated lymphocytic choriomeningitis virus (LCMV) vectors, TheraT(LCMV), induce strong TAA-specific CD8 T cell immunity, but these responses can not be substantially augmented upon TheraT(LCMV) readministration. Counter to expectations, vector-neutralizing antibodies were not accountable for limited homologous prime-boosting capacity. Instead, dominant viral backbone-reactive CD8+ T cells competed against subdominant TAA-specific responses, limiting their magnitude. Methods: Herein we engineered and characterized delivery systems based on the arenaviruses Mopeia, Candid#1 and Pichinde (TheraT(MOP), TheraT(CAND), TheraT(PIC)). Results: We demonstrate that heterologous TheraT(CAND) – TheraT(LCMV) and TheraT(PIC)-TheraT(LCMV) prime-boost substantially augment TAA-specific CD8 T cell responses by rendering them immunodominant. Accordingly, intravenous administration of mice triggered up to 50% TAA epitope-specific CD8+ T cells and cured established tumors. Conversely, TheraT(MOP) – TheraT(LCMV) prime-boost was poorly immunogenic owing to cross-reactive T cell epitopes in the respective viral backbones. Conclusions: These findings establish heterologous arenavirus prime-boost combinations as a powerful new modality in tumor immunotherapy and highlight CD8 T cell epitope dominance as a significant hurdle to overcome in the vectored delivery of TAAs.

The death receptor Fas mediates local bystander killing of antigen-negative variants by antigen-specific CD8 T cells in a heterogeneous tumor

Abstract Antigen-specificity is one of the hallmarks of the adaptive immune system, and CD8 T cells are particularly crucial for eliminating malignant cells with aberrant expression of tumor-associated self-antigens or neoantigens. We established a high-throughput pooled CRISPR/Cas9 screen for functionally assessing cancer cell genes involved in modulating the cytotoxic activity of antigen-specific CD8 T cells. As validation of our approach, we identified many genes known to be altered in cancers refractory to immunotherapy, including MHC component B2m and the checkpoint molecule Cd274. Interestingly, the cell surface death receptor Fas was identified as a strong hit in mediating the killing of antigen-positive lymphoma and was further verified with breast cancer, despite the availability of other cytotoxic effectors such as perforin/granzymes. Using a 2-antigen system, we surprisingly observed that Fas was also mediating the killing of neighboring GFP-negative cancer cells by T cells highly specific for a GFP epitope. This phenomenon was not dependent on MHC Class I expression but was potentiated by interferon gamma, which is known to upregulate Fas expression. Even for T cells that were previously primed, this bystander cytotoxicity required the presence of neighboring antigen-positive cells, suggesting a very hyperlocal and temporally regulated off-target effect that was not dependent on natural killer cells in vivo. We believe this phenomenon to be crucial to the durable remissions seen with highly antigen-specific approaches such as CAR-T cell therapy. Modulating this pathway may be a viable approach for preventing immune escape of rare antigen-loss variants or treating heterogeneous tumors with no clear single target antigen.

Abstract A024: Identifying a T-cell receptor for immunotherapy against a leukemia-associated self-antigen in an allogeneic setting

Abstract Our aim was to identify a T-cell receptor (TCR) that recognizes a leukemia-associated self-antigen that is highly expressed in acute myeloid leukemia and that is restricted by HLA-A2 (expressed in approximately 50% of Caucasians) for future adoptive T-cell therapy. Tumor-associated self-antigens (self-TAA) make attractive targets for adoptive T-cell therapy in cancer. However, autologous T-cells are tolerant to self-TAA expressed on self-HLA if antigen expression is sufficiently high. In contrast, T-cells are not tolerant to self-antigens presented on non-self HLA. By identifying alloreactive T-cells reactive to self-peptide in complex with foreign HLA and subsequently cloning their TCR, it is possible to redirect patient T-cells against self-TAA. The advantage of targeting self-antigens is the potential application of TCR-engineered T-cells in all cancer patients with a certain cancer type and expressing the restricting HLA molecule. We identified a protein that is selectively expressed in normal myeloid cells and highly expressed by leukemic cells in patients with acute myeloid leukemia (AML). HLA-A2 negative CD8 T-cells from healthy donors were stimulated with HLA-A2 positive dendritic cells that were expressing the full-length target antigen. CD8 T-cells recognizing peptides from the target antigen in complex with HLA-A2 were subsequently identified and sorted using fluorescently labeled HLA-A2-peptide-multimers. T-cell clones obtained from sorted multimer-positive T-cells responded to HLA-A2 positive patient leukemia cells and cell lines expressing the antigen, whereas no response was seen to HLA-A2 positive antigen negative targeT-cells, unless loaded with the relevant peptide. Responses were measured as secretion of interferon gamma, expression of the T-cell activation marker CD137 or expression of the degranulation marker CD107. The TCR sequences were identified, and we next evaluated specificity and functionality of TCR-engineered cells. Indeed, TCR-transduced healthy donor T-cells were able to selectively kill HLA-A2 positive AML patient-derived leukemic cells as well as antigen positive cell lines in vitro. We also performed a comprehensive peptide scan to determine potential cross reactivity of the obtained TCR. For this scan, the amino acids (AA) in each position of the target peptide-epitope were individually replaced by every other natural amino acid (19 AA for each position x 9 postions = 171 peptides). In addition, potential responses to other variants of the 9mer target peptide based on the natural antigen protein sequence were evaluated (8mer, 10mers, 11mers and 12mers). Based on the results obtained with TCR-transduced T-cells (interferon gamma ELISA), candidate peptides naturally occurring in the proteome to which our TCR could cross-react were not identified. These data warrant further evaluation of the TCR for in vivo efficacy and specificity. Citation Format: Maxi-Lu Böschen, Weiwen Yang, Erlend Strønen, Johanna Olweus. Identifying a T-cell receptor for immunotherapy against a leukemia-associated self-antigen in an allogeneic setting [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A024.

Paraneoplastic NMOSD associated with EG junction adenocarcinoma expressing unprotected AQP4.

Paraneoplastic neurologic disorder (PND) is a remote manifestation triggered by altered immune response against neoplasms. Emerging discovery of antineuronal antibodies and diagnostic tool development, such as PET-CT, allow us the early recognition and treatment of these disorders.1 Neuromyelitis optica spectrum disorder (NMOSDs) is an inflammatory disease affecting the optic nerve and spinal cord, where autoantibodies against aquaporin 4 (AQP4-IgG) supposed to play a pathogenic role.2 Several tumors, such as breast cancer or carcinoid, were reported to be associated with paraneoplastic NMOSD based on immunoreactivity to de novo AQP4 in the tumor.3 One may have thought because AQP4 is expressed not only on CNS but also on muscle, kidney, and stomach,4 alteration of tumor associated self-antigens expression can cause paraneoplastic NMOSD in the tumors of these organs. However, AQP4 expression is known to decline during carcinogenesis in the gastric cancer.5 Contrary, programmed cell death ligand 1 (PD-L1) on tumor that couples with PD-1, an inhibitory receptor on both CD4+ and CD8+ T cells, to play an important role in the ability of tumor cells to escape from the host immune system.6 Indeed, 40% of patients with advanced gastric cancer express PD-L1 and proved clinical benefit by an immune check-point therapy targeting PD-1/PD-L1 axis.7 Here, we report a paraneoplastic NMOSD case with adenocarcinoma of the esophagogastric (EG) junction that exhibited immunoreactivity to AQP4 and negative for PD-L1 expression. The authors thank people in the Department of Diagnostic Pathology at Kobe University Graduate School of Medicine for their help in preparing histology sections.

T cell therapy targeting a public neoantigen in microsatellite instable colon cancer reduces in vivo tumor growth

ABSTRACTT-cell receptor (TCR) transfer is an attractive strategy to increase the number of cancer-specific T cells in adoptive cell therapy. However, recent clinical and pre-clinical findings indicate that careful consideration of the target antigen is required to limit the risk of off-target toxicity. Directing T cells against mutated proteins such as frequently occurring frameshift mutations may thus be a safer alternative to tumor-associated self-antigens. Furthermore, such frameshift mutations result in novel polypeptides allowing selection of TCRs from the non-tolerant T-cell repertoire circumventing the problem of low affinity TCRs due to central tolerance. The transforming growth factor β Receptor II frameshift mutation (TGFβRIImut) is found in Lynch syndrome cancer patients and in approximately 15% of sporadic colorectal and gastric cancers displaying microsatellite instability (MSI). The -1A mutation within a stretch of 10 adenine bases (nucleotides 709–718) of the TGFβRII gene gives rise to immunogenic peptides previously used for vaccination of MSI+ colorectal cancer patients in a Phase I clinical trial. From a clinically responding patient, we isolated a cytotoxic T lymphocyte (CTL) clone showing a restriction for HLA-A2 in complex with TGFβRIImut peptide. Its TCR was identified and shown to redirect T cells against colon carcinoma cell lines harboring the frameshift mutation. Finally, T cells transduced with the HLA-A2-restricted TGFβRIImut-specific TCR were demonstrated to significantly reduce the growth of colorectal cancer and enhance survival in a NOD/SCID xenograft mouse model.

Abstract A42: Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen

Abstract While cancer immunotherapies like checkpoint inhibitors have resulted in unprecedented clinical success, they only benefit a subset of patients. To improve therapeutic outcomes for greater numbers of patients, one strategy is to rationally combine different immunotherapy modalities. We recently demonstrated that attaching albumin-binding lipophilic tails to peptide antigens or molecular adjuvants (creating amphiphile vaccines) results in enhanced T-cell responses. Additionally, we observed significant tumor regression upon combining tumor-antigen targeting antibodies with extended half-life IL-2 (exPK-IL-2) in mouse models of melanoma and prostate cancer. In the present work, we combined both approaches to treat a spontaneous model of lung adenocarcinoma expressing carcinoembryonic antigen (CEA), an oncofetal protein expressed in some human lung cancers. KrasLSL-G12D/+;;p53fl/fl mice were crossed with transgenic mice expressing human-CEA to generate a CEA-tolerant background. Lung tumors were induced by infection with a lentivirus expressing Cre recombinase and human CEA. Due to the extended latency of tumor initiation in this model, we also generated a CEA-expressing cell line from these mice to test the efficacy of different combination immunotherapy regimens. We discovered that weekly treatments combining a CEA-targeting amphiphile-vaccine, exPK-IL-2, and an anti-CEA antibody with checkpoint inhibitors (anti-PD-1 and -CTLA4) resulted in sustained tumor regression in 50% of mice bearing established tumors. We are currently testing this combination immunotherapy on autochthonous lung tumors. Our results suggest that breaking tolerance to a tumor-associated self-antigen (CEA) and combining immunotherapies to recruit both innate and adaptive immune effectors can have a potent therapeutic effect in intractable tumors like lung cancer. Citation Format: Kavya Rakhra, Eric F. Zhu, Wuhbet Abraham, Kelly D. Moynihan, Naveen Mehta, Karl D. Wittrup, Darrell J. Irvine. Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A42.

Abstract B120: Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen

Abstract Previous work from our lab has demonstrated that a combination of anti-tumor antibody and an IL-2 fusion protein that exhibits extended serum half-life elicits an immune response that can effectively control a wide variety of tumor models. We have since then combined this therapeutic regimen with a vaccine exhibiting efficient lymph node trafficking that can generate an impressive population of tumor-antigen specific CD8+ T-cells but by itself does not provide good anti-tumor efficacy. The efficacy of this combination immunotherapy is further boosted by immune checkpoint blockade, leading to a robust four-component therapy: 1) anti-tumor antigen antibody; 2) IL-2 fusion protein; 3) anti-tumor antigen vaccine; 4) anti-PD-1 antibody. Although this four-pronged approach is demonstrably effective in the syngeneic subcutaneous melanoma model B16F10, we wish to test its efficacy in a more physiological model. To this end, we have turned to a model developed by the Jacks Lab, known as the KP model: an inducible lung tumor model where lentivirus-driven integration and expression of Cre is able to activate oncogenic Kras and completely remove p53 function. Because our therapeutic regimen requires a targetable tumor-associated antigen with respect to both the antibody and vaccine, we chose to induce expression of human carcinoembryonic antigen (CEA) in these tumors, as CEA has a well-studied structure and biology, and frequently expresses aberrantly in many forms of human adenocarcinomas. Additionally, our lab has previously engineered an antibody targeting CEA possessing picomolar affinity. Finally, to remove any endogenous immunological response against human CEA as a foreign antigen in our mouse system, we have crossed the KP model with a mouse model transgenic for human CEA, which in the literature has been described to have identical spatiotemporal expression of CEA as found in humans and should allow for central tolerance of this antigen. In the course of this work, we have successfully introduced human CEA into our lentivirus constructs and shown tumorigenesis by these constructs in the KP model coincides with expression of tumor-associated CEA, as detected by qPCR. On the therapeutic side, we have tailored the vaccine to successfully drive an anti-CEA CD8+ T-cell response. Performing preliminary therapeutic experiments in a transplant model of the KP tumor with our four-component therapy, we saw tumor control compared to untreated tumors. Upon interrogating the CD8+ T-cell response against CEA, we found 1-15% of CD8+ T-cells in the blood respond to CEA stimulation by intracellular cytokine staining. With regards to the lung tumor model, in the course of establishing the system we have also observed that the growth kinetics of tumors expressing CEA lags behind those tumors without CEA, even in the transgenic background. Preliminary immunophenotyping work by flow cytometry suggests that tumors with CEA seem to have a reduced myeloid-derived suppressor cell (MDSC) population and a higher CD8a+ dendritic cell (DC) population compared to tumors without CEA, suggesting that the former may have a less immunosuppressive tumor microenvironment that is better able to prime an anti-tumor CD8+ T-cell response. We will be planning to conduct therapeutic trials in the more physiological lung tumor KP model in the near future, as well as investigate the differences in the immune response with tumors expressing or lacking CEA. Citation Format: Eric F. Zhu, Kavya Rakhra, Naveen Mehta, Kelly D. Moynihan, Cary F. Opel, Darrell J. Irvine, K. Dane Wittrup. Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B120.

The combined molecular adjuvant CASAC enhances the CD8+ T cell response to a tumor-associated self-antigen in aged, immunosenescent mice.

BackgroundIneffective induction of T cell mediated immunity in older individuals remains a persistent challenge for vaccine development. Thus, there is a need for more efficient and sophisticated adjuvants that will complement novel vaccine strategies for the elderly. To this end, we have investigated a previously optimized, combined molecular adjuvant, CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity), incorporating two complementary Toll-like receptor agonists, CpG and polyI:C, a class-II epitope, and interferon (IFN)-γ in aged mice.FindingsIn aged mice with typical features of immunosenescence, antigen specific CD8+ T cell responses were stimulated after serial vaccinations with CASAC or Complete/Incomplete Freund’s Adjuvant (CFA/IFA) and a class I epitope, deriving either from ovalbumin (SIINFEKL, SIL) or the melanoma-associated self-antigen, tyrosinase-related protein-2 (SVYDFFVWL, SVL). Pentamer analysis revealed that aged, CASAC/SIL-vaccinated animals had substantially higher frequencies of H-2Kb/SIL-specific CD8+ T cells compared to the CFA/IFA-vaccinated groups. Similarly, higher frequencies of H-2Kb/SVL-pentamer+ and IFN-γ+ CD8+ T cells were detected in the aged, CASAC + SVL-vaccinated mice than in their CFA/IFA-vaccinated counterparts. In both antigen settings, CASAC promoted significantly better functional CD8+ T cell activity.ConclusionThese studies demonstrate that functional CD8+ T cells, specific for both foreign and tumour-associated self-antigens, can be effectively induced in aged immunosenescent mice using the novel multi-factorial adjuvant CASAC.

CD8 T cell tolerance to a tumor-associated self-antigen is reversed by CD4 T cells engineered to express the same T cell receptor.

Ag receptors used for cancer immunotherapy are often directed against tumor-associated Ags also expressed in normal tissues. Targeting of such Ags can result in unwanted autoimmune attack of normal tissues or induction of tolerance in therapeutic T cells. We used a murine model to study the phenotype and function of T cells redirected against the murine double minute protein 2 (MDM2), a tumor-associated Ag that shows low expression in many normal tissues. Transfer of MDM2-TCR–engineered T cells into bone marrow chimeric mice revealed that Ag recognition in hematopoietic tissues maintained T cell function, whereas presentation of MDM2 in nonhematopoietic tissues caused reduced effector function. TCR-engineered CD8+ T cells underwent rapid turnover, downmodulated CD8 expression, and lost cytotoxic function. We found that MDM2-TCR–engineered CD4+ T cells provided help and restored cytotoxic function of CD8+ T cells bearing the same TCR. Although the introduction of the CD8 coreceptor enhanced the ability of CD4+ T cells to recognize MDM2 in vitro, the improved self-antigen recognition abolished their ability to provide helper function in vivo. The data indicate that the same class I–restricted TCR responsible for Ag recognition and tolerance induction in CD8+ T cells can, in the absence of the CD8 coreceptor, elicit CD4 T cell help and partially reverse tolerance. Thus MHC class I–restricted CD4+ T cells may enhance the efficacy of therapeutic TCR-engineered CD8+ T cells and can be readily generated with the same TCR.