e15155 Background: Activating mutations in the estrogen receptor alpha gene (ESR1) are a key mechanism of resistance to endocrine therapy in estrogen receptor–positive (ER+) breast cancer and represent an important unmet clinical need. While selective estrogen receptor degraders (SERDs) are under development for ESR1-mutant disease, the activity of (Z)-endoxifen, the active metabolite of tamoxifen, across clinically relevant ESR1 mutations has not been fully characterized. We evaluated ER signaling inhibition by (Z)-endoxifen in comparison with oral SERDs at clinically relevant concentrations. Methods: ER transcriptional activity was measured in human ER+ breast cancer cells using clinically relevant concentrations of (Z)-endoxifen (746–20 nM), elacestrant (224–56 nM), and imlunestrant (269–67.25 nM). ER signaling was quantified and expressed as percent activity relative to untreated control. Studies were conducted in ESR1 wild-type (WT) and mutant backgrounds, including Y537N, Y537S, and D538G as well as parental MCF7 cells. Results: In parental MCF-7 cells, endoxifen produced dose-dependent and statistically significant inhibition of ER signaling at all clinically relevant concentrations, reducing ER activity to 16–26% of control ( p < 0.001). Elacestrant and imlunestrant also significantly suppressed ER signaling in MCF-7 cells, achieving residual activity of approximately 13–14% and 10–11%, respectively ( p < 0.001). In ESR1-WT cells, all agents suppressed ER signaling to < 5% of control ( p < 0.001). In contrast, ESR1-mutant models demonstrated differential sensitivity. Endoxifen maintained statistically significant ER inhibition across Y537N, Y537S, and D538G mutations ( p < 0.01), whereas SERDs showed reduced efficacy in mutant settings, most notably in the D538G background, where residual ER signaling remained substantially higher despite treatment ( p < 0.05). Among ESR1 mutations, D538G exhibited the greatest resistance, while Y537N remained the most sensitive across therapies. Conclusions: At clinically relevant concentrations, (Z)-endoxifen demonstrates robust and consistent inhibition of ER signaling across key ESR1 mutations, including those associated with resistance to current endocrine therapies. These data support (Z)-endoxifen as a promising therapeutic approach in ESR1-mutant ER+ breast cancer and highlight its potential clinical relevance in a patient population with limited treatment options.

Self-replicating RNA vaccines delivered by viral replicon particles (VRPs) induce strong immunity, but repeated dosing on short intervals may be limited by vector responses. In patient samples and mice, VRP-srRNA vaccination generated VRP-neutralizing antibodies and T-cell responses to replicase. In mice, a third dose at two-week intervals minimally boosted transgene immunity, and prior VRP exposure reduced responses to a different-transgene VRP. Optimized schedules or heterologous prime-boost may sustain immunogenicity.

Trastuzumab deruxtecan (T-DXd), also known as fam-trastuzumab-deruxtecan-nxki, is a novel HER2-targeting antibody-drug conjugate (ADC) that has shown remarkable clinical efficacy in breast cancer (BC) and other cancers with varying levels of HER2 expression. However, the mechanisms underlying its activity in HER2-low and HER2-ultralow BC, and the role of immune activation, remain poorly understood. Here, we demonstrate that T-DXd’s efficacy in HER2-low/ultralow BC is predominantly driven by the extracellular tumor-specific protease Cathepsin L (CTSL), but not Ccathepsin B (CTSB). Our in vitro and in vivo studies reveal that extracellular CTSL cleaves T-DXd’s linker, releasing the DXd payload (validated by mass spectrometry) and inducing cytotoxicity within HER2-low/ultralow tumor microenvironments. This mechanism mediates potent anti-tumor effects across multiple HER2-negative cancers. Importantly, analysis of BC biopsies and tissue microarrays from T-DXd-treated patients revealed high CTSL expression in tumor and stromal compartments, independent of HER2 levels, suggesting broad therapeutic potential. In addition to extracellular cleavage, we also found that T-DXd elicits immune activation as an anti-tumor mechanism in HER2-positive BC. The DXd payload induces immunogenic cell death, activating nearby myeloid immune cells via TLR4 and STING pathways, while its antibody backbone engages Fcγ-receptors to stimulate Antibody-Dependent Cellular Phagocytosis (ADCP). This process enhances tumor antigen uptake and drives the expansion of antigen-specific CD8+ T cells. Furthermore, DXd upregulates CD47 on tumor cells, making them more susceptible to immune clearance. Combining CD47 blockade with T-DXd significantly enhanced anti-tumor efficacy in an immune suppressive HER2-transgenic BC mouse model, promoting CD8+ T cell memory and preventing tumor recurrence post-treatment. These studies suggest that strategies targeting the CD47-SIRPa axis may enhance the ability of ADCs to elicit tumor-specific immunity, thus extending the efficacy of these agents and generating long term memory responses against non-immunogenic cancers. Li-Chung Tsao, John S. Wang, Xingru Ma, Sirajbir Sodhi, Joey V. Ragusa, Bushangqing Liu, Jason McBane, Tao Wang, Junping Wei, Cong-Xiao Liu, Xiao-Yi Yang, Gang-jun Lei, Ivan Spasojevic, Ping Fan, Timothy N. Trotter, Michael A. Morse, Herbert K. Lyerly, Zachary C. Hartman. Uncovering the multiple therapeutic mechanisms of trastuzumab deruxtecan (T-DXd): effective extracellular payload release and immunomodulatory stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2128.

Tumor antigen vaccination represents an appealing approach for cancer but has failed to materialize as oncologic standard of care. To understand long-term vaccine efficacy, we conducted a retrospective analysis of patients with human epidermal growth receptor 2+ (HER2+) breast cancer who received HER2-targeting vaccines and survived for >18 years. PBMC analysis revealed HER2-specific CD27+ memory CD4 and CD8 T cells, suggesting that CD27 signaling supports long-term immune memory. In human CD27 transgenic mice, combining HER2 vaccination with anti-CD27 agonism enhanced HER2-specific responses, particularly long-lived CD4 memory T cells. Murine models demonstrated ~40% tumor regression with combined therapy compared with vaccine alone (~6%). Additional scRNA-seq analysis identified CD4 T cells with a distinct gene expression profile, and depletion/adoptive transfer studies validated that CD4 T cells were essential for this effect. These findings suggest that CD27 agonism enhances vaccine-induced antigen-specific CD4 T cell responses, enabling durable antitumor immunity not entirely dependent on CD8 T cells.

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC), with risk factors including greater frequency of recurrence and metastasis and lower overall survival rate compared to other BC subtypes. While the drivers of TNBC are unclear, Trop2 is a transmembrane protein that has been found to be highly expressed in TNBC, whose role in tumor progression is not well understood. High expression of Trop2 has led to the development of antibody-drug conjugates (ADCs) targeting wild-type Trop2, including Sacituzumab govitecan (SG) and Datopotamab deruxtecan (Dato-dxd). However, Trop2 is an intronless gene but can be posttranslationally cleaved by ADAM10 and other proteases in malignant tissues. Cleavage of Trop2 and its effect on tumor progression and drug resistance have not been previously explored, which is becoming increasingly crucial to understand as more Trop2-targeting therapies enter clinical practice. Experimental Methods and Results: To determine the oncogenic capacity of cleaved Trop2, our lab has generated a construct of N-terminally cleaved Trop2 ∆Q31-R87 called Trop2-∆N and have stably transduced mouse TNBC MM3MG cells, a cell line that is not well transformed and grows poorly in immune competent mice. SCID-beige mice who lack T and B cells and have non-functional NK cells and immune competent BALB/c were injected with wild-type Trop2, an uncleavable form of Trop2 called Trop2-87/88, Trop2-∆N, and control to find that Trop2-∆N conferred a proliferative phenotype, which was comparable to the known oncogene HER2∆16. Additionally, MM3MG cells were stained for SG, Dato-dxd, and their parental monoclonal antibodies and analyzed for binding via flow cytometry. We found that SG and Dato-dxd had lowered binding capacity towards Trop2-∆N expressing cells. To determine if the killing capacity of these ADCs were altered, we transduced the human breast epithelial cell line MCF10AT with our constructs of uncleavable Trop2-87/88 and Trop2-∆N to find that both SG and Dato-dxd to have lowered killing against Trop2-∆N expressing cells. Our studies found that N-terminally cleaved Trop2 induces a proliferative phenotype in vivo, reduce binding capacity of Trop2 ADCs, and are more resistance to ADC-induced killing in vitro. These data provide insight into the potential importance of N-terminally cleaved Trop2 in tumor progression and drug resistance that have not been explored. Future experiments will elucidate the mechanisms that underlie cleavage of Trop2 and refining Trop2-targeting therapies. Andrea E. Wilson, Junping Wei, Gangjun Lei, Tao Wang, Xiao-Yi Yang, Melissa Gajda, Cong-Xiao Liu, Zachary C. Hartman. The role of N-terminal cleavage of Trop2 in tumor progression and therapeutic resistance in triple negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3449.

Pancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate among major cancers, with over 50% of patients having liver metastases at diagnosis. Given that conventional T-cell based immunotherapies have been ineffective against PDAC liver metastases, it is vital to elucidate the mechanisms regulating tumor immunity within the hepatic microenvironment to enhance anti-tumor T cell. Notably, PDAC cells exhibit exceptional plasticity and intrinsic heterogeneity, contributing to the evolution of immunosuppressive primary and early liver PDAC metastases. Our study leverages an array of congenic PDAC clones with varying degrees of immunogenicity to delineate the signaling pathways driving primary and liver immunosuppression. We investigate the growth kinetics of PDAC clones both in vitro and in vivo, alongside their transcriptomic variations. Using model antigens (chicken ovalbumin), we assess how different clones modulate the immune microenvironment to elicit antigen-specific adaptive anti-tumor responses. Transcriptomic profiling informs CRISPR-based knockout and overexpression strategies to uncover intrinsic gene contributions to primary anti-tumor immunity in vivo. Additionally, we have developed stable PDAC liver metastasis models through portal vein injection to evaluate each clone’s metastatic efficiency and associated immune landscape within the hepatic microenvironment. In vivo engraftment of PDAC clones revealed differential infiltration of CD4+/CD8+ T cells and neutrophils. While all OVA+ PDAC clones triggered antigen-specific immune responses, only immune-sensitive tumors were eradicated, whereas immune-insensitive tumors persisted. Despite OVA expression, all clones successfully established liver metastases following intraportal injection. RNA-seq analysis identified upregulation of key pancreatic development regulators, including PDX1 and RBPJ, in immune-insensitive clones. CRISPR knockout of these transcription factors in vivo demonstrated altered immune cell composition and reduced tumor growth in immunocompetent mice expressing OVA. Using diverse PDAC models, we created tumors with immune heterogeneity and altered transcriptional profiles. We identified validated immune-altering cytokines (CXCL1 and CSF3) and immunomodulatory roles of PDX1 and RBPJ. Immune-insensitive clones evaded antigen-specific immunity in both subcutaneous and liver metastasis models, while immune-sensitive clones were eradicated subcutaneously but gained protection in the liver. Ongoing studies confirm the phenotypes of CRISPR-KO clones and delineate the immunomodulatory roles of these transcription factors in liver metastasis. Bushangqing Liu, Ethan Agritelley, Junping Wei, Gangjun Lei, Tao Wang, Jason McBane, Daniel Nussbaum, Zachary Hartman. Decoding the role of intrinsic pancreatic cellular signaling in shaping the immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 908.

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) targeting HER2, exhibiting significant clinical efficacy in breast cancer (BC) with varying HER2 expression, including HER2-low and HER2-ultralow. However, the precise mechanism underlying its efficacy and the contribution of immune activation in these settings remain unclear. Here, we demonstrate that T-DXd efficacy in HER2-low and HER2-negative BC is independent of HER2 engagement and ADC internalization. Instead, its activity relies on extracellular proteases, such as cathepsin L (CTSL), within the tumor microenvironment. Irrespective of their HER2 status, tumor and stromal compartments of invasive BC abundantly express CTSL, which efficiently cleaves the specialized linker of T-DXd, facilitating payload release and inducing cytotoxicity against HER2-low/negative tumors. In HER2-positive BC, the antibody backbone of T-DXd engages Fcγ-receptors and drives antibody-dependent cellular phagocytosis (ADCP). Concurrently, its cytotoxic payload (DXd) induces immunogenic cell death, further activating myeloid cells via TLR4 and STING pathways to enhance tumor antigen presentation to CD8+ T cells. Notably, T-DXd cytotoxicity also upregulates tumor CD47 expression, dampening immune activation. Combining T-DXd with CD47 checkpoint blockade significantly enhances anti-tumor immune responses in a HER2-transgenic BC mouse model, while also inducing durable CD8+ T cell memory to prevent tumor recurrence after therapy cessation.

T-Dxd (Enhertu) is a novel antibody-drug-conjugate (ADC) targeting HER2, which has demonstrated profound clinical efficacy across breast cancers (BC) and other cancers exhibiting varying degrees of HER2 expression. However, the precise mechanism underlying its efficacy in HER2-low cancers and the role of Fc-mediated immune activation in its efficacy remain unclear. Using bystander killing studies and pharmacokinetic assessment of its payload, Deruxtecan (Dxd), we found that T-Dxd efficacy in HER2-low and HER2-negative cancers was mediated by extracellular cathepsin L proteases within the tumor microenvironment (TME). This unique mechanism enables broader Dxd cytotoxicity, bypassing traditional ADC-resistance mechanisms like HER2 downregulation and resistance to ADC-internalization. Significantly, our studies revealed that the Dxd cytotoxic payload induces immunogenic cell death (ICD) with the secretion of specific Damage-Associated Molecular Patterns that activate myeloid cells through TLR4 and STING pathways. Additionally, the T-Dxd antibody backbone retains the capability to engage with Fcγ-receptors to stimulate Antibody-Dependent Cellular Phagocytosis (ADCP) that enhances tumor antigen uptake, which in concert with ICD responses, leads to augmented antigen presentation and expansion of tumor antigen-specific CD8+ T cells. Counteracting ADCP, we also found that T-Dxd stimulates tumor CD47 expression, which we subsequently blocked using an anti-CD47 antibody in combination with T-Dxd. This combination yielded a potent therapeutic synergy that enhanced anti-tumor adaptive immune responses, both systemically and within the TME. Critically, we found that combination therapy induces CD8+ T cell immune memory, preventing tumor recurrence post therapy discontinuation and suggests a critical therapeutic role for T-Dxd-mediated stimulation of adaptive immunity. Citation Format: Li-Chung Tsao, John S. Wang, Xingru Ma, Siraj Sodhi, Joey V. Ragusa, Bushangqing Liu, Jason McBane, Tao Wang, Junping Wei, Cong-Xiao Liu, Xiao Yang, Gangjun Liu, Ivan Spasojevic, Ping Fan, Timothy N. Trotter, Michael Morse, H. Kim Lyerly, Zachary C. Hartman. Unlocking the Therapeutic Potential of T DXd Enhertu Mechanisms of Action in HER2 Low Cancers and Immunomodulatory Effects [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-05-30.

BACKGROUND:Breast cancer (BC) continues to be a major health concern with 250,000 new cases diagnosed annually in the USA, 75% of which are hormone receptor positive (HR+), expressing estrogen receptor alpha (ER) and/or the progesterone receptor (PR). Although ER-targeted therapies are available, 30% of patients will develop resistance, underscoring the need for new non-ER/estrogen-based treatments. Notably, HR+BCs exhibit poor lymphocyte infiltration and contain an immunosuppressive microenvironment, which contributes to the limited efficacy of immunotherapies in HR+BC. In this study, we demonstrate that PR/progesterone signaling reduces major histocompatibility complex (MHC) Class I expression, facilitating immune evasion and escape from immune-based clearance of PR+tumors. METHODS:To determine the effect of PR/progesterone on MHC Class I expression, we treated human and mouse mammary tumor cell lines with progesterone and/or interferon (IFN) and measured expression of genes involved in antigen processing and presentation (APP), as well as surface MHC Class I expression. We used the OT-I/SIINFEKL model antigen system to measure the impact of progesterone on immune cell-mediated killing of modified tumor cells. We also analyzed two large BC clinical cohorts to determine how PR expression correlates with APP gene expression and MHC Class I expression in ER-positive tumors. RESULTS:In vitro, we show that PR/progesterone signaling reduces APP gene expression and MHC class I expression in human and breast mammary tumor cell lines. PR-mediated attenuation of APP/MHC Class I expression is more pronounced in the presence of IFN. In immune cell killing assays, PR-expressing mammary tumor cells treated with progesterone are protected from immune-mediated cytotoxicity. We demonstrate that PR expression in vivo prevents immune-mediated rejection of xenoantigen-modified mammary tumor cell lines through mechanisms involving MHC Class I expression and CD8 T cells. Data analysis of two large BC cohorts reveals lower APP gene expression and MHC Class I expression in ER/PR-positive tumors compared with ER-positive/PR-negative tumors. These findings show that HR+BCs, specifically PR+tumors, downregulate APP/MHC class I machinery through PR/progesterone signaling. Use of pharmacological PR/progesterone inhibitors may reverse these effects in patients with BC, thereby improving immunosurveillance and response to immunotherapies.

Tumor cells expressing GFP, rtTA, and Luciferase successfully engraft in NoGlow mice. A, Diagram of triple-transgenic (3 ×) E0771 cells. GFP and rtTA are constitutively expressed and Luc is induced with the addition of doxycycline. B, 3 × E0771 (106) were implanted into the mammary fat pad (MFP) of wild-type (WT) C57Bl/6 (n = 7), CAG-driven Full-body WT GFP/Luc expressing mice (n = 7), CMV cre littermates positive (n = 6) or negative (n = 7) for the NoGlow construct, or SCID beige (n = 4) mice. C, Overall (top) and individual (bottom) tumor growth in each group. No evidence of tumors was observed in Full-body GFP-Luc, NoGlow−, or C57Bl/6 animals by the end of experiment. D, Representative bioluminescence imaging of animals pre (left) or post (right) doxycycline diet introduction. Bottom, Luc was induced in SCID mice but to a lesser degree compared with NoGlow+ mice. E, Representative GFP imaging upon euthanasia of Noglow+, SCID, and Full-body GFP-Luc mice. Left, concentrated GFP signal in tumors of NoGlow+ and SCID compared with Full-body GFP-Luc mice. Right, visceral GFP signal only in Full-body GFP-Luc mice. F, Representative lungs of NoGlow+, NoGlow−, Full-body GFP-Luc, or SCID mice upon euthanasia. Lung metastasis can be visualized in NoGlow+ and SCID animals.

ER+ breast cancers (BC) are characterized by the elevated expression and signaling of estrogen receptor alpha (ESR1), which renders them sensitive to anti-endocrine therapy. While these therapies are clinically effective, prolonged treatment inevitably results in therapeutic resistance, which can occur through the emergence of gain-of-function mutations in ESR1. The central importance of ESR1 and development of mutated forms of ESR1 suggest that vaccines targeting these proteins could potentially be effective in preventing or treating endocrine resistance. To explore the potential of this approach, we developed several recombinant vaccines encoding different mutant forms of ESR1 (ESR1mut) and validated their ability to elicit ESR1-specific T cell responses. We then developed novel ESR1mut-expressing murine mammary cancer models to test the anti-tumor potential of ESR1mut vaccines. We found that these vaccines could suppress tumor growth, ESR1mut expression and estrogen signaling in vivo. To illustrate the applicability of these findings, we utilize HPLC to demonstrate the presentation of ESR1 and ESR1mut peptides on human ER+ BC cell MHC complexes. We then show the presence of human T cells reactive to ESR1mut epitopes in an ER+ BC patient. These findings support the development of ESR1mut vaccines, which we are testing in a Phase I clinical trial.

Abstract Background: Hormone receptor positive (HR⁺) breast cancer (BC) is a subtype of BC that is characterized by expression of estrogen receptor (ER) and progesterone receptor (PR). A cornerstone treatment for this subtype of BC is endocrine inhibition therapy (EIT), in which estrogen and ER are suppressed using antagonists to suppress tumor growth. However, patients eventually obtain resistance to EIT. Metastatic HR⁺BC is the deadliest form of BC, with no cure. Current therapies currently only utilize the ER-axis, with no PR-targeted therapies. Little is known about the role of PR in these BCs, but we have found evidence for an immunosuppressive role for PR in HR⁺BC. Yet, it is unclear how PR mediates immunosuppression and if this axis can be therapeutically targeted. We hypothesized that PR confers local immunosuppression through the stimulation of immunosuppressive transcriptional activity and that we can immunologically target this axis using a PR vaccine. Experimental methods: To assess the immune impact of PR, we expressed the isoforms of PR (A, B and A+B) in mouse mammary cancer cells and assessed their capacity to elicit PR signaling. We assessed the impact of PR expression on T cell activity in targeting PR⁺ mammary tumors that express GFP, along with using GFP-specific JEDI CD8⁺ T cells. We explored the ability of PR to protect against GFP xenoantigen immune responses by engrafting PR⁺ mammary cells into immune competent and deficient mice. Lastly, we engineered 1st generation adenoviral vectors encoding mouse and human PR and utilized two antiprogestins (Mifepristone and Onapristone) in vivo. Using our vectors, we vaccinated BALB/c in the footpad and assessed PR-specific immunity using IFNγ ELISPOT. Results: Using PR signaling reporters, we found canonical PR signaling activity in the absence of progesterone (P4) by PR-A and in the presence of P4 by PR-B, but the most profound activation of PR signaling was seen through combined expression of PR-A and B. While expression of these genes did not confer striking cytotoxic T cell resistance in vitro, combination of PR-A+PR-B in 67NR cells permitted successful tumor engraftment in immune competent mice, despite xenoantigen expression. GFP expression did not alter tumor growth in immunodeficient mice, suggesting a key role in modulating local anti-tumor immunity. The use of either Mifepristone or Onapristone significantly suppressed tumor engraftment in immune competent mice in vivo. We found that an Ad-PR vaccine could elicit T cell responses against human PR-specific epitopes, a potential step towards immune targeting these BCs. Conclusions: We found that murine PR-A and PR-B can confer robust P4 signaling that is linked to an immunosuppressive phenotype in vivo. We developed a PR vaccine that elicited PR-specific T cell responses in vivo. We are currently examining the mechanisms that underlie PR-mediated immunosuppression and exploring the potential of PR vaccines in HR⁺BC. Citation Format: Andrea E. Wilson, Junping Wei, Gangjun Lei, Xiao-Yi Yang, Cong-Xiao Liu, Melissa Gajda, Tao Wang, Christy Hagan, Zachary C. Hartman. Immune targeting progesterone receptor in hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3981.

Abstract Background: Androgen receptor (AR) signaling inhibition is part of the mechanism behind many effective prostate cancer therapies, including androgen deprivation and next generation anti-androgens. However in castration resistant prostate cancer up to 50% of treatment resistance has been linked to AR amplification or expression of the constitutively active RNA splice variant, AR-V7. Although the use of immune checkpoint blockade has failed to demonstrate clinical success, immunotherapeutic interventions remain attractive as prostate cancer’s relatively slow progression provides time for anti-tumor immune responses to develop. Cancer specific vaccines have the potential to enable and enhance tumor specific immune responses by stimulating and directing T cell immunity to important oncologic targets. We hypothesized that a cancer vaccine designed to induce a T cell response against cells expressing high levels of AR would result in an anti-tumor effect and potentially prevent or delay the development of therapeutic resistance. Methods: Adenoviral vaccines against AR (Ad-AR) or AR splice variant 7 (Ad-AR-V7) were used to evaluate the potential of AR targeting vaccination in mouse models. We evaluated AR and AR variant specific adaptive responses produced by our vaccines with naïve and tumor-bearing mice by ex vivo peptide restimulation. We challenged vaccinated mice with multiple syngeneic subcutaneous tumor models that we engineered to express AR-V7, including a PTEN-/- p53-/- prostate tumor cell line. Finally we tested our vaccines in a therapeutic regimen, vaccinating mice after implanting our subcutaneous AR-V7 expressing models. Results: Across multiple strains of mice, AR or AR-V7 targeting adenoviral vaccination induced robust adaptive immune responses against the N-terminal domain of AR. Vaccination with either Ad-AR or Ad-AR-V7 resulted in significant anti-tumor responses, providing a significant overall survival benefit and complete tumor rejection by the majority of mice vaccinated against AR or AR-V7. Similarly tumor bearing mice treated with Ad-AR or Ad-AR-V7 vaccination displayed tumor growth inhibition or complete tumor regression resulting in an overall survival benefit. Additionally, we found that vaccine responses were unaffected by the use of the approved antiandrogen drug enzalutamide, suggesting that vaccinations could be deployed in tandem with standard-of-care therapies. Conclusions: Vaccination against AR and AR splice variants can induce AR specific adaptive immune responses capable of inhibiting the growth of AR-V7 expressing tumor models, illustrating the potential of AR and its splice variants as immunologic targets in prostate cancer. Citation Format: Robert D. Marek, Junping Wei, Tao Wang, Xiao-Yi Yang, Gangjun Lei, Zachary C. Hartman. Treatment with androgen receptor targeting vaccines provides an overall survival benefit in AR-V7 expressing tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4102.

NoGlow mice reveal the impact of sex and tissue distribution on immune tolerance. A, Male WT (n = 3), CAG Luc-GFP (n = 3), and NoGlow+ (n = 6) or NoGlow− (n = 10) littermates were vaccinated with an Ad encoding WT GFP-Luc and serum was collected after two weeks for anti-Luc or anti-GFP responses. Unvaccinated naïve WT (n = 2) animals were used as baseline controls. B, Luc staining showing protein expression in mammary epithelium of female MMTV Cre NoGlow+ or NoGlow− littermates. C, Luc staining showing protein expression in prostate epithelium of male Pbsn Cre NoGlow+ or NoGlow− littermates. D, Luc staining demonstrating expression in acini and islets in pancreases of male Pdx1 Cre NoGlow+ or NoGlow− littermates. E–G, MMTV Cre (E; n = 4 NoGlow- F, n = 5 NoGlow+ F, n = 2 NoGlow- M, n = 7 NoGlow+ M, n = 3 naïve), Pbsn Cre (F; n = 6 NoGlow- F, n = 6 NoGlow+ F, n = 4 NoGlow- M, n = 8 NoGlow+ M, n = 1 naïve), or Pdx1 Cre (G; n = 2 NoGlow- F, n = 4 NoGlow+ F, n = 4 NoGlow- M, n = 5 NoGlow+ M, n = 2 naïve) littermates were vaccinated with an Ad encoding WT eGFP and Luc. Serum antibody responses to Luc and GFP were determined by ELISA two weeks postvaccination. P values for A, E, F, G are displayed at 1:50 and determined by two-way ANOVA with Tukey correction. H, Normalized ratio of serum anti-Luc or -GFP antibody responses in independent NoGlow+ versus mean of NoGlow− littermates across two independent experiments demonstrating highest tolerance in CMV Cre and Pdx1 Cre animals. P values were determined by one-way ANOVA with Tukey correction. All P values represent mean ± SEM.

Validating the NoGlow construct in vitro

Abstract The ability to temporally regulate gene expression and track labeled cells make animal models powerful biomedical tools. However, sudden exposure to xenobiotic genes (e.g. GFP, luciferase (luc), or rtTA3) triggers inadvertent immune responses that can significantly suppress foreign gene expression or result in complete rejection of transplanted cells. Although germline expression of foreign genes somewhat addresses these challenges, native fluorescence and bioluminescence abrogates their utility as cellular reporters and spatiotemporally restricted expression can lead to suboptimal xenoantigen tolerance. Even in endogenous cancer models, we find that the means of tissue-directed expression of the same foreign gene yield significant differences in overall tolerance. Thus, the degree of tolerance to foreign genes strongly impacts the utility of different genetic modification and can strongly influence experimental outcomes and interpretations. To overcome these unwanted immune responses and enable reliable cell tracking/gene regulation, we developed a novel mouse model that selectively expresses antigen-intact but non-functional forms of GFP, luc, and rtTA3. These "NoGlow" mice possess no detectable background fluorescence or luminescence and mount limited adaptive immune responses against transgenic xenoantigens even after vaccination. Furthermore, we demonstrate that NoGlow mice allow tracking and tetracycline inducible gene regulation of triple-transgenic cells expressing GFP/luc/rtTA3, in contrast to transgene-negative immune competent mice which completely eliminate these cells. Notably, this model enables de novo metastasis from orthotopically implanted, triple-transgenic tumor cells, despite high xenoantigen expression. Finally, we observed model and sex-based differences in immune tolerance to the encoded xenoantigens after activation by different tissue-specific CREs, illustrating the obstacles of tolerizing animals to foreign genes and validating the utility of NoGlow mice to dissect the mechanisms of central and peripheral tolerance. Altogether, the NoGlow model provides a critical resource for in vivo studies across multiple disciplines, including oncology, developmental biology, infectious disease, autoimmunity, and transplantation. Citation Format: Timothy N. Trotter, Andrea Wilson, Jason McBane, Carina Dagotto, Xiao Yang, Junping Wei, Gangjun Lei, Hannah Thrash, H. Kim Lyerly, Zachary Hartman. Immune-competent “NoGlow” transgenic mice overcome restrictive tolerance to multiple xenoantigens and simultaneously permit identification, tracking, and temporal gene expression in tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3985.

Additional E0771 tumor growth in WT, GH, and CAG Luc-GFP mice; MMTV CAG HER2 Ad-HER2 vaccination T cell responses.

Abstract Background: T-DXd is an antibody-drug conjugate (ADC) targeting HER2 that has demonstrated superior clinical efficacy over previous HER2-targeting agent Trastuzumab emtansine (T-DM1). Notably, T-Dxd is clinically effective in breast cancers (BC) with a range of HER2 expression. While its purported mechanism of action (MOA) involves its cleavable linker leading to bystander killing, it remains unclear how this potential MOA accounts for efficacy in HER2-low cancers or the role FCGR-mediated signaling and immune induction plays in its clinical efficacy. Method: In vitro studies utilized co-culture, internalization, FCGR activation, ADCP, flow cytometry, and gene expression by QRT-PCR and ELISA assays. Assessments of tumor antigen-specific T cell stimulation involved JEDI T cells. In vivo studies were conducted using in vivo implantation of HER2+ and HER2- cells, PK assessments of T-Dxd/DM1 and therapeutic effects determined using an endogenous HER2 transgenic mouse model that measured HER2-specific adaptive immune responses, in combination with immune checkpoint blockade anti-CD47. Results: T-Dxd elicited a systemic bystander effect that was only partially dependent on HER2 expression, mediated by specific cathepsins in the extracellular TME. Notably, T-Dxd cytotoxicity induced immunogenic cell death (ICD), while also retaining the ability to engage FCGRs to promote ADCP and innate immune activation. ICD was mediated by secretion of specific Dxd driven DAMPs (HMGB1 and eATP), resulting in TLR4/STING activation. ICD and FCGR activation along with antigen ADCP together elicited myeloid cell activation (CD80, CD40) and antigen presentation, resulting in effective expansion of tumor antigen-specific T cells responses, demonstrated by co-culture assays using EGFP/HER2+ BC cells and CD8+ JEDI T cells. T cell activation required TLR4 and STING pathways while in vivo, T-Dxd anti-tumor responses associated with augmented HER2-specific T and B cell responses. However, T-Dxd also induced CD47 to limit phagocytosis. But, the use of CD47/SIRPa blocking antibodies dramatically enhanced CD8+ T cell expansion in vitro and anti-tumor efficacy in vivo upon T-Dxd treatment. Conclusion: Our study demonstrates that T-Dxd is cleavable in HER2 negative BC in vivo, which permits more widespread Dxd activity against HER2 low BC. Critically, Dxd elicits ICD, resulting in innate immune stimulation of myeloid cells, which when combined with FCGR activation and ADCP, leads to a striking induction of tumor-specific adaptive immunity. This combination was unique to T-Dxd, which may underlie its clinical superior efficacy. However, T-Dxd activity also elicits suppressive CD47 expression which could be negated using CD47/SIRPA blockade. We found that this combination dramatically enhanced T-Dxd resulted therapeutic efficacy, thus supporting clinical exploration of these combinations. Citation Format: Li-Chung Tsao, Xingru Ma, John S. Wang, Timothy N. Trotter, Tao Wang, Jun-Ping Wei, Gang-jun Lei, Jason McBane, Ping Fan, Ivan Spasojevic, Zachary C. Hartman. HER2-ADC efficacy is rooted in its induction of immunogenic cell death with FcGR activation and a systemic bystander effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2377.

Background/Objectives: Androgen receptor (AR) expression and signaling are critical for the progression of prostate cancer and have been the therapeutic focus of prostate cancer for over 50 years. While a variety of agents have been developed to target this axis, many of these fail due to the emergent expression of AR RNA splice variants, such as AR-V7, that can signal independently of ligand binding. Other therapies, such as vaccination against prostate-specific antigens, have achieved FDA approvals but have fallen short of being incorporated as standard-of-care therapies for advanced prostate cancer. This may be due to the elevated level of immunosuppression observed in prostate cancer, which remains largely refractory to immune checkpoint blockade. Methods: We developed a vaccine targeting AR-V7, a common isoform associated with treatment resistance, and demonstrated its ability to elicit AR-V7-specific immunity and enable anti-tumor responses against AR-V7+ cancers in subcutaneous tumor models. Results: Our studies also revealed that AR-V7 expression conferred an immune suppressive phenotype that was significant in a non-AR-dependent prostate cancer model. Notably, in this model, we found that vaccination in combination with enzalutamide, an AR antagonist, suppressed these aggressive immune suppressive cancers and resulted in enhanced survival in comparison to control vaccinated and enzalutamide-treated mice. While anti-PD-1 immune checkpoint inhibition (ICI) alone slowed tumor growth, the majority of vaccinated mice that received anti-PD-1 therapy showed complete tumor elimination. Conclusions: Collectively, these results validate the importance of AR signaling in prostate cancer immune suppression and suggest the potential of AR-V7-specific vaccines as therapeutic strategies against prostate cancer, offering significant protective and therapeutic anti-tumor responses, even in the presence of androgen signaling inhibitors.