Cora D. Arthur

Checkpoint Blockade Cancer Immunotherapy Targets Tumour-Specific Mutant Antigens

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

Disruption of the Jak1 Gene Demonstrates Obligatory and Nonredundant Roles of the Jaks in Cytokine-Induced Biologic Responses

Herein we report the generation of mice lacking the ubiquitously expressed Janus kinase, Jak1. Jak1-/- mice are runted at birth, fail to nurse, and die perinatally. Although Jak1-/- cells are responsive to many cytokines, they fail to manifest biologic responses to cytokines that bind to three distinct families of cytokine receptors. These include all class II cytokine receptors, cytokine receptors that utilize the gamma(c) subunit for signaling, and the family of cytokine receptors that depend on the gp130 subunit for signaling. Our results thus demonstrate that Jak1 plays an essential and nonredundant role in promoting biologic responses induced by a select subset of cytokine receptors, including those in which Jak utilization was thought to be nonspecific.

Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting

Cancer immunoediting, the process by which the immune system controls tumour outgrowth and shapes tumour immunogenicity, is comprised of three phases: elimination, equilibrium and escape. Although many immune components that participate in this process are known, its underlying mechanisms remain poorly defined. A central tenet of cancer immunoediting is that T-cell recognition of tumour antigens drives the immunological destruction or sculpting of a developing cancer. However, our current understanding of tumour antigens comes largely from analyses of cancers that develop in immunocompetent hosts and thus may have already been edited. Little is known about the antigens expressed in nascent tumour cells, whether they are sufficient to induce protective antitumour immune responses or whether their expression is modulated by the immune system. Here, using massively parallel sequencing, we characterize expressed mutations in highly immunogenic methylcholanthrene-induced sarcomas derived from immunodeficient Rag2−/− mice that phenotypically resemble nascent primary tumour cells. Using class I prediction algorithms, we identify mutant spectrin-β2 as a potential rejection antigen of the d42m1 sarcoma and validate this prediction by conventional antigen expression cloning and detection. We also demonstrate that cancer immunoediting of d42m1 occurs via a T-cell-dependent immunoselection process that promotes outgrowth of pre-existing tumour cell clones lacking highly antigenic mutant spectrin-β2 and other potential strong antigens. These results demonstrate that the strong immunogenicity of an unedited tumour can be ascribed to expression of highly antigenic mutant proteins and show that outgrowth of tumour cells that lack these strong antigens via a T-cell-dependent immunoselection process represents one mechanism of cancer immunoediting.

Type I interferon is selectively required by dendritic cells for immune rejection of tumors

Dendritic cell responsiveness to type I interferon is required for the generation of antitumor T cell responses and tumor rejection.

Cancer immunoediting by the innate immune system in the absence of adaptive immunity

In the absence of adaptive immunity, NK cells polarize M1 macrophages to facilitate cancer immunoediting.

STAT1-deficient mice spontaneously develop estrogen receptor α-positive luminal mammary carcinomas

IntroductionAlthough breast cancers expressing estrogen receptor-α (ERα) and progesterone receptors (PR) are the most common form of mammary malignancy in humans, it has been difficult to develop a suitable mouse model showing similar steroid hormone responsiveness. STAT transcription factors play critical roles in mammary gland tumorigenesis, but the precise role of STAT1 remains unclear. Herein, we show that a subset of human breast cancers display reduced STAT1 expression and that mice lacking STAT1 surprisingly develop ERα+/PR+ mammary tumors.MethodsWe used a combination of approaches, including histological examination, gene targeted mice, gene expression analysis, tumor transplantaion, and immunophenotyping, to pursue this study.ResultsForty-five percent (37/83) of human ERα+ and 22% (17/78) of ERα- breast cancers display undetectable or low levels of STAT1 expression in neoplastic cells. In contrast, STAT1 expression is elevated in epithelial cells of normal breast tissues adjacent to the malignant lesions, suggesting that STAT1 is selectively downregulated in the tumor cells during tumor progression. Interestingly, the expression levels of STAT1 in the tumor-infiltrating stromal cells remain elevated, indicating that single-cell resolution analysis of STAT1 level in primary breast cancer biopsies is necessary for accurate assessment. Female mice lacking functional STAT1 spontaneously develop mammary adenocarcinomas that comprise > 90% ERα+/PR+ tumor cells, and depend on estrogen for tumor engraftment and progression. Phenotypic marker analyses demonstrate that STAT1-/- mammary tumors arise from luminal epithelial cells, but not myoepithelial cells. In addition, the molecular signature of the STAT1-/- mammary tumors overlaps closely to that of human luminal breast cancers. Finally, introduction of wildtype STAT1, but not a STAT1 mutant lacking the critical Tyr701 residue, into STAT1-/- mammary tumor cells results in apoptosis, demonstrating that the tumor suppressor function of STAT1 is cell-autonomous and requires its transcriptional activity.ConclusionsOur findings demonstrate that STAT1 suppresses mammary tumor formation and its expression is frequently lost during breast cancer progression. Spontaneous mammary tumors that develop in STAT1-/- mice closely recapitulate the progression, ovarian hormone responsiveness, and molecular characteristics of human luminal breast cancer, the most common subtype of human breast neoplasms, and thus represent a valuable platform for testing novel treatments and detection modalities.

Temporally Distinct PD-L1 Expression by Tumor and Host Cells Contributes to Immune Escape

PD-L1 induction on tumor cells is IFNγ-dependent and transient, but becomes IFNγ-independent and long-lived on tumorassociated macrophages. Thus, assessing PD-L1 expression on both tumor and host cells may better stratify patients undergoing PD-1/PD-L1 blockade therapy. Antibody blockade of programmed death-1 (PD-1) or its ligand, PD-L1, has led to unprecedented therapeutic responses in certain tumor-bearing individuals, but PD-L1 expressions prognostic value in stratifying cancer patients for such treatment remains unclear. Reports conflict on the significance of correlations between PD-L1 on tumor cells and positive clinical outcomes to PD-1/PD-L1 blockade. We investigated this issue using genomically related, clonal subsets from the same methylcholanthrene-induced sarcoma: a highly immunogenic subset that is spontaneously eliminated in vivo by adaptive immunity and a less immunogenic subset that forms tumors in immunocompetent mice, but is sensitive to PD-1/PD-L1 blockade therapy. Using CRISPR/Cas9-induced loss-of-function approaches and overexpression gain-of-function techniques, we confirmed that PD-L1 on tumor cells is key to promoting tumor escape. In addition, the capacity of PD-L1 to suppress antitumor responses was inversely proportional to tumor cell antigenicity. PD-L1 expression on host cells, particularly tumor-associated macrophages (TAM), was also important for tumor immune escape. We demonstrated that induction of PD-L1 on tumor cells was IFNγ-dependent and transient, but PD-L1 induction on TAMs was of greater magnitude, only partially IFNγ dependent, and was stable over time. Thus, PD-L1 expression on either tumor cells or host immune cells could lead to tumor escape from immune control, indicating that total PD-L1 expression in the immediate tumor microenvironment may represent a more accurate biomarker for predicting response to PD-1/PD-L1 blockade therapy, compared with monitoring PD-L1 expression on tumor cells alone. Cancer Immunol Res; 5(2); 106–17. ©2017 AACR.

High-Dimensional Analysis Delineates Myeloid and Lymphoid Compartment Remodeling during Successful Immune-Checkpoint Cancer Therapy

Although current immune-checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of high-dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor-infiltrating cells by single-cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d, and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.

Abstract A001: Tumor-specific mutant antigens in cancer immunotherapy

Monoclonal antibody (mAb) blockade of immune checkpoints such as PD-1 and CTLA-4 can stimulate therapeutic, T cell-dependent anti-tumor activity in mice and humans. We asked whether exome sequencing and epitope prediction algorithms could identify antigens in progressively growing tumors that render them susceptible to anti-PD-1- or anti-CTLA-4-induced immune elimination. Expressed mutations in progressively growing, T3 methylcholanthrene (MCA)-sarcomas were identified by exome sequencing and prioritized based primarily on potential MHC class I binding affinity and predicted peptide processing. The two best candidate tumor-specific mutant antigens (TSMA) were identified from mutations in Laminin α subunit 4 (Lama4) and a glucosyltransferase called Alg8. When tested in vitro, mutant Lama4 (mLama4) and mutant Alg8 (mAlg8) peptides were the only top predicted TSMA that stimulated T3-specific T cells. The relevance of these findings was validated by four in vivo studies: (i) mLama4 and mAlg8 peptides bound to H-2Kb were biochemically detected on the surface of T3 tumors, (ii) as shown by tetramer staining, CD8+ tumor infiltrating lymphocytes (TIL) expressing TCRs for mLama4 and mAlg8 accumulated over time in T3 tumors in checkpoint blockade treated, tumor-bearing mice, reaching maximum levels just prior to tumor rejection, (iii) vaccination of naive wild type mice with mutant but not WT forms of Lama4 or Alg8 induced strong CD8+ T cell responses and (iv) prophylactic or therapeutic vaccination with mLama4 plus mAlg8 synthetic long peptides (SLP) resulted in rejection of T3 cells. The therapeutic protection provided by the SLP vaccine was equal to that afforded by checkpoint blockade therapy. Since we did not see evidence of T cell responses to any other TSMA, we then explored whether the immunodominant antigens masked responses to other TSMA. Using CRISPR/Cas9, we introduced point mutations to revert mLama4 and mAlg8 in T3 back to their nonimmunogenic wild type counterparts. T3 tumor cells with either mLama4 or mAlg8 reverted to wild type were still susceptible to anti-PD-1 or anti-CTLA-4 immune-mediated control with skewing of the T cell response in the TIL towards the remaining TSMA. Additionally, we have obtained T3 tumor cells with both TSMA reverted to wild type and are currently testing whether these “fixed” T3 tumor cells are still subject to anti-PD-1 or anti-CTLA-4 and whether SLP vaccines comprised of subdominant TSMA still present in fixed T3 tumor cells can protect mice against tumor growth. This system will allow us to address the many questions surrounding poorly understood phenomenon of immunodominance and how this may influence patient-specific personalized cancer immunotherapy directed against TSMA. Citation Format: Matthew M. Gubin, Jeffrey P. Ward, Takuro Noguchi, Xiuli Zhang, Cora Arthur, Willem-Jan Krebber, Gwenn E. Mulder, Cornelis J.M. Melief, William E. Gillanders, Maxim Artyomov, Elaine R. Mardis, Robert D. Schreiber. Tumor-specific mutant antigens in cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A001.