Estrogen Receptor Mutations as Novel Targets for Immunotherapy in Metastatic Estrogen Receptor-positive Breast Cancer.

Estrogen receptor-positive (ER+) breast cancer is not considered immunogenic and, to date, has been proven resistant to immunotherapy. Endocrine therapy remains the cornerstone of treatment for ER+ breast cancers. However, constitutively activating mutations in the estrogen receptor alpha (ESR1) gene can emerge during treatment, rendering tumors resistant to endocrine therapy. Although these mutations represent a pathway of resistance, they also represent a potential source of neoepitopes that can be targeted by immunotherapy. In this study, we investigated ESR1 mutations as novel targets for breast cancer immunotherapy. Using machine learning algorithms, we identified ESR1-derived peptides predicted to form stable complexes with HLA-A*0201. We then validated the binding affinity and stability of the top predicted peptides through in vitro binding and dissociation assays and showed that these peptides bind HLA-A*0201 with high affinity and stability. Using tetramer assays, we confirmed the presence and expansion potential of antigen-specific CTLs from healthy female donors. Finally, using in vitro cytotoxicity assays, we showed the lysis of peptide-pulsed targets and breast cancer cells expressing common ESR1 mutations by expanded antigen-specific CTLs. Ultimately, we identified five peptides derived from the three most common ESR1 mutations (D538G, Y537S, and E380Q) and their associated wild-type peptides, which were the most immunogenic. Overall, these data confirm the immunogenicity of epitopes derived from ESR1 and highlight the potential of these peptides to be targeted by novel immunotherapy strategies. SIGNIFICANCE: Estrogen receptor (ESR1) mutations have emerged as a key factor in endocrine therapy resistance. We identified and validated five novel, immunogenic ESR1-derived peptides that could be targeted through vaccine-based immunotherapy.

Biotinylated Streptavidin Surface Coating Improves the Efficacy of a PLGA Microparticle-Based Cancer Vaccine.

Triple-negative breast cancer (TNBC) is an immune-enriched subset of breast cancer that has recently demonstrated clinical responsiveness to combinatorial immunotherapy. However, the lack of targeted interventions against hormone receptors or HER2 continues to limit treatment options for these patients. To begin expanding available interventions for patients with metastatic TNBC, we previously reported a therapeutic vaccine regimen that significantly reduced spontaneous lung metastases in a preclinical TNBC model. This heterologous vaccine approach "primed" mice with tumor lysate antigens encapsulated within poly(lactic-co-glycolic) acid microparticles (PLGA MPs), and then "boosted" mice with tumor lysates plus adjuvant. The use of the PLGA MP prime as monotherapy demonstrated no efficacy, suggesting that improving this component of our therapy would achieve greater vaccine efficacy. Here, we functionally improved the PLGA MP prime by coating microparticles with biotinylated streptavidin-conjugated using 1-ethyl-3-(3-dimethylaminoproplyl) carbodiimide/N-hydroxysuccinimide (EDC/Sulfo-NHS) linkers. This modification enhanced the immunostimulatory potential of our PLGA MPs, as evidenced by increased phagocytosis, maturation, and stimulatory ligand expression by antigen-presenting cells (APCs). Therapeutic prime/boost vaccination of TNBC-bearing mice with surfaced-coated PLGA MPs significantly reduced spontaneous lung metastases by an average of 56% relative to mice primed with unmodified PLGA MPs, and a significant 88% average reduction in spontaneous lung metastases relative to untreated control mice. These findings illustrate that relatively common biotin-streptavidin conjugation formulations can positively affect microparticle-based vaccine immunogenicity resulting in enhanced therapeutic efficacy against established preclinical mammary tumors.

Obesity diminishes response to PD-1-based immunotherapies in renal cancer.

BACKGROUND: Obesity is a major risk factor for renal cancer, yet our understanding of its effects on antitumor immunity and immunotherapy outcomes remains incomplete. Deciphering these associations is critical, given the growing clinical use of immune checkpoint inhibitors for metastatic disease and mounting evidence for an obesity paradox in the context of cancer immunotherapies, wherein obese patients with cancer have improved outcomes. METHODS: We investigated associations between host obesity and anti-programmed cell death (PD-1)-based outcomes in both renal cell carcinoma (RCC) subjects and orthotopic murine renal tumors. Overall survival (OS) and progression-free survival (PFS) were determined for advanced RCC subjects receiving standard of care anti-PD-1 who had ≥6 months of follow-up from treatment initiation (n=73). Renal tumor tissues were collected from treatment-naive subjects categorized as obese (body mass index, 'BMI' ≥30 kg/m2) or non-obese (BMI <30 kg/m2) undergoing partial or full nephrectomy (n=19) then used to evaluate the frequency and phenotype of intratumoral CD8+ T cells, including PD-1 status, by flow cytometry. In mice, antitumor immunity and excised renal tumor weights were evaluated ±administration of a combinatorial anti-PD-1 therapy. For a subset of murine renal tumors, immunophenotyping was performed by flow cytometry and immunogenetic profiles were evaluated via nanoString. RESULTS: With obesity, RCC patients receiving anti-PD-1 administration exhibited shorter PFS (p=0.0448) and OS (p=0.0288). Treatment-naive renal cancer subjects had decreased frequencies of tumor-infiltrating PD-1highCD8+ T cells, a finding recapitulated in our murine model. Following anti-PD-1-based immunotherapy, both lean and obese mice possessed distinct populations of treatment responders versus non-responders; however, obesity reduced the frequency of treatment responders (73% lean vs 44% obese). Tumors from lean and obese treatment responders displayed similar immunogenetic profiles, robust infiltration by PD-1int interferon (IFN)γ+CD8+ T cells and reduced myeloid-derived suppressor cells (MDSC), yielding favorable CD44+CD8+ T cell to MDSC ratios. Neutralizing interleukin (IL)-1ß in obese mice improved treatment response rates to 58% and reduced MDSC accumulation in tumors. CONCLUSIONS: We find that obesity is associated with diminished efficacy of anti-PD-1-based therapies in renal cancer, due in part to increased inflammatory IL-1ß levels, highlighting the need for continued study of this critical issue.

Enhancing Dendritic Cell-based Immunotherapy with IL-2/Monoclonal Antibody Complexes for Control of Established Tumors.

U.S. Food and Drug Administration-approved high-dose IL-2 therapy and dendritic cell (DC) immunization offer time-tested treatments for malignancy, but with defined issues of short in vivo t1/2, toxicity, and modest clinical benefit. Complexes of IL-2 with specific mAbs (IL-2c) exhibit improved stability in vivo with reduced toxicity and are capable of stimulating NK cell and memory phenotype CD8 T cell proliferation. In this study, we demonstrate that IL-2c treatment in tumor-bearing mice can enhance NK cell and tumor-specific CD8 T cell numbers. Importantly, DC immunization coupled with stabilized IL-2c infusion drastically improves the tumor-specific effector CD8 T cell response. DC + IL-2c treatment enhances number, 41BB and GITR expression, granzyme B production, CTL/regulatory T cell ratio, and per-cell killing capacity of CD8 T cells without increasing inhibitory molecule expression. Notably, IL-2c treatment of anti-CD3-stimulated human CD8 T cells resulted in higher number and granzyme B production, supporting the translational potential of this immunotherapy strategy for human malignancy. DC + IL-2c treatment enhances both endogenous NK cell and tumor Ag-specific CD8 T cell immunity to provide a marked reduction in tumor burden in multiple models of pre-existing malignancy in B6 and BALB/c mice. Depletion studies reveal contributions from both tumor-specific CD8 T cells and NK cells in control of tumor burden after DC + IL-2c treatment. Together, these data suggest that combination therapy with DC and IL-2c may be a potent treatment for malignancy.

Biodegradable microparticles loaded with doxorubicin and CpG ODN for in situ immunization against cancer.

In situ immunization is based on the concept that it is possible to break immune tolerance by inducing tumor cell death in situ in a manner that provides antigen-presenting cells such as dendritic cells (DCs) with a wide selection of tumor antigens that can then be presented to the immune system and result in a therapeutic anticancer immune response. We designed a comprehensive approach to in situ immunization using poly(lactic-co-glycolic acid) (PLGA)-biodegradable microparticles (MPs) loaded with doxorubicin (Dox) and CpG oligodeoxynucleotides (CpG) that deliver Dox (chemotherapy) and CpG (immunotherapy) in a sustained-release fashion when injected intratumorally. Dox induces immunogenic tumor cell death while CpG enhances tumor antigen presentation by DCs. PLGA MPs allow their safe co-delivery while evading the vesicant action of Dox. In vitro, we show that Dox/CpG MPs can kill B and T lymphoma cells and are less toxic to DCs. In vivo, Dox/CpG MPs combined with antibody therapy to enhance and maintain the T cell response generated systemic immune responses that suppressed injected and distant tumors in a murine B lymphoma model, leading to tumor-free mice. The combination regimen was also effective at reducing T cell lymphoma and melanoma tumor burdens. In conclusion, Dox/CpG MPs represent an efficient and safe tool for in situ immunization that could provide a promising component of immunotherapy for patients with a variety of types of cancer.

A therapeutic microparticle-based tumor lysate vaccine reduces spontaneous metastases in murine breast cancer.

Metastatic breast cancer is currently incurable, and available therapies are associated with severe toxicities. Induction of protective anti-tumor immunity is a promising therapeutic approach for disseminated breast cancer, as immune responses are (i) systemic; (ii) antigen-specific; and (iii) capable of generating long-lived "memory" populations that protect against future tumor recurrences. Pursuant with this approach, we have developed a novel heterologous prime/boost vaccination regimen that reduces spontaneous lung metastases in mice with established murine 4T1 adenocarcinoma breast tumors. In our studies, mice were orthotopically challenged with luciferase-expressing 4T1 tumor cells; luciferase expression was retained in vivo, enabling us to quantitatively track metastatic tumor growth via bioluminescent imaging. On day 6 post-challenge, mice received a therapeutic "prime" consisting of bulk tumor lysates encapsulated in poly(lactic-co-glycolic) acid (PLGA) microparticles (MPs). On day 11, mice received a "boost" composed of free tumor lysates plus a cocktail of Toll-like receptor (TLR)-stimulating adjuvants. Tumor progression was monitored in vaccinated and untreated mice for 25 days, a time at which 100% of untreated mice had detectable lung tumors. PLGA MPs injected subcutaneously trafficked to draining lymph nodes and were efficiently phagocytosed by dendritic cells (DCs) within 48 h. Our combination therapy reduced metastatic lung tumor burdens by 42% and did not induce autoimmunity. These findings illustrate that vaccines based upon MP delivery of tumor lysates can form the basis of an effective treatment for metastatic breast cancer and suggest that similar approaches may be both efficacious and well-tolerated in the clinic.

Tumor lysate-loaded biodegradable microparticles as cancer vaccines.

Cancer vaccines that use tumor lysate (TL) as a source of tumor-associated antigens (TAAs) have significant potential for generating therapeutic anti-tumor immune responses. Vaccines encompassing TL bypass the limitations of single antigen vaccines by simultaneously stimulating immunity against multiple TAAs, thereby broadening the repertoire of TAA-specific T-cell clones available for activation. Administration of TL in particulate form, such as when encapsulated in biodegradable microparticles, increases its immunostimulatory capacity and produces more robust immune responses than when TL is given in soluble form. These effects can be further enhanced by co-administering TL with adjuvants. A number of recent studies using polymeric microparticle delivery of TL, with or without adjuvants, have produced promising results in preclinical studies. In this review, we will discuss current experimental approaches involving TL being pursued in the oncoimmunology field, and comment on strategies such as combining specific chemotherapeutic agents with TL microparticle delivery that may eventually lead to improved survival outcomes for cancer patients.