Background The contribution of B cells to antitumor immunity remains controversial, with studies reporting varied effects across cancer types. Even less is known about the role of the endogenous humoral response, including when tumor-elicited antibodies are protective, when they are deleterious, and how they might be modulated to influence antitumor immunity. Critically, it remains unclear whether specific antigenic features govern the efficacy of humoral immune responses against cancer. We aim to define the conditions under which endogenous antibodies mediate antitumor immunity and to leverage these principles to improve neoantigen-directed immunotherapies, including cancer vaccines. Methods We performed a pan-cancer analysis of The Cancer Genome Atlas (TCGA) to assess associations between intratumoral IgG and clinical outcomes. Using syngeneic mouse tumor models expressing membrane or cytoplasmic neoantigens, we examined how antigen localization influences endogenous antibody responses. We evaluated strategies to enhance humoral immunity via cytokine and chemokine modulation and assessed antibody responses in the context of cancer vaccination. Results Our pan-cancer TCGA analysis revealed marked heterogeneity in the prognostic impact of intratumoral IgG. In vivo, expression of membrane-localized, but not cytoplasmic, neoantigens drove potent class-switched IgG responses, activated myeloid cells, and restricted tumor growth independently of CD8 T cells. Notably, membrane-restricted antigen expression was sufficient to convert tumor types such as colorectal cancer, where IgG correlates with poor prognosis, into settings in which antibodies mediate tumor suppression. These effects required CD4 T-cell help, antigen-specific IgG, and Fc receptor engagement. Enhancing B-cell recruitment (C-X-C motif chemokine ligand 13) or B-cell help (interleukin (IL)-21) further amplified antibody-mediated tumor control, with co-expression providing the strongest benefit. Finally, we establish that antigen localization dictates vaccine efficacy: the point-mutated trophoblast cell-surface antigen 2 (Trop2) T256R, which exhibits impaired membrane localization and reduced antibody binding relative to wild-type Trop2, failed to elicit comparable vaccine-induced tumor control. Conclusions These findings identify antigen subcellular localization as a key regulator of endogenous antibody-mediated antitumor immunity and cancer vaccine efficacy, providing a mechanistic framework for leveraging humoral immunity in immunotherapeutic strategies. CXCL13 and IL-21 emerge as candidate approaches to selectively enhance antibody-mediated tumor control in settings where membrane-bound antigens are present.

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.

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.

Artificial intelligence (AI) has the potential to transform nanoparticle development for drug delivery; however, existing strategies typically optimize either material selection or component ratios in isolation. To enable simultaneous optimization of both, we integrated an automated liquid handling platform with machine learning to systematically explore the nanoparticle formulation space. A data set comprising 1275 distinct formulations (spanning drug molecules, excipients, and synthesis molar ratios) was generated, resulting in a 42.9% increase in successful nanoparticle formation through composition optimization. We developed a bespoke hybrid kernel machine that couples molecular feature learning with relative compositional inference, enhancing the modeling of formulation outcomes across chemical spaces. This hybrid kernel significantly improved prediction performance across three kernel-based algorithms, with a support vector machine (SVM) achieving superior performance when using our kernel compared to standard kernels and outperforming all other machine learning architectures, including transformer-based deep neural networks. Using SVM-guided predictions, we successfully formulated the difficult-to-encapsulate venetoclax with optimized taurocholic acid ratios, yielding enhanced in vitro efficacy against Kasumi-1 leukemia cells. In a second case study, our AI-guided platform reduced excipient usage by 75% in a trametinib formulation while preserving the in vitro efficacy and in vivo pharmacokinetics relative to the standard formulation. Taken together, this study establishes a generalizable framework that combines robotic experimentation, kernel machine learning, and experimental validation to accelerate nanoparticle composition optimization for drug delivery.

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.

Delayed relapse is attributable to residual tumor cells that remain minimally proliferative/quiescent until favorable conditions for growth are met, but what determines the overall duration of remission is unresolved. Recent evidence suggests that epithelial-mesenchymal plasticity (EMP) facilitates phenotypic switching between dormant and proliferative cell states, in addition to enabling chemoresistance, metastasis, and immune evasion. However, which features of EMP are responsible for each or multiple of these phenotypes, and to what extent EMP is restricted to certain tumor populations remains unclear. To address these questions, we initially enriched for features of EMP (CD44hi, CD104low) in the mouse mammary tumor cell line TSAE1. Interestingly, no difference in tumor growth was observed between parental or EMP-enriched mammary fat pad (MFP) tumors in either syngeneic Balb/c or immune-deficient SCID-beige mice. Individual tumors were then cultured ex vivo, revealing a significant EMP-enrichment in parental tumors after one round of selection in vivo. Similar results were observed in the mouse mammary cell line D2.OR, suggesting that implantation alone gives rise to EMP which may mask important tumor cell-intrinsic differences in a heterogeneous tumor. Thus, EMP-enriched TSAE1 cells were further divided by isolating nine clonal lines. Although each expressed less epithelial markers than parental cells (Epcam, E-cadherin), individual TSAE1 clones displayed remarkably divergent growth rates and patterns in 3D culture. In vivo all clones were capable of forming tumors, but some were significantly delayed regardless of being implanted into Balb/c or SCID-beige animals. Furthermore, individual clones recruited distinct immune and stromal populations. To assess if intrinsic phenotypes change over time, 3D culture assays and bulk RNA sequencing was performed on three representative clones before implantation (“Pre”) and on three Balb/c MFP-derived tumors expanded ex-vivo from each clone (“Post”). Surprisingly, for each clone the Post tumors closely resembled the Pre clone and much of the difference in up/downregulated genes between individual clones and parental TSAE1 cells was already found in Pre samples. Finally, MFP tumors from EMP-enriched TSAE1 clones were differently sensitive to Doxorubicin therapy, suggesting that specific aspects of EMP may be important during different stages of BC survival and progression. Together, these data reveal that proliferation itself is intrinsically determined at the clonal level, independent of adaptive immunity, and highlight the potential for individual tumor clones in various EMP states to shape the tumor microenvironment for long-term survival. Timothy N. Trotter, Jason McBane, Xing Gao, Carina Dagotto, Tao Wang, H. Kim Lyerly, Zachary C. Hartman. Tumor clones display intrinsic and stable rates of progression and coordination of the tumor microenvironment in association with epithelial mesenchymal plasticity. [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 2675.

Lung cancer remains one of the most prevalent and deadly malignancies globally, with non-small cell lung cancer (NSCLC) comprising approximately 84% of all cases. Despite advances in treatment, the overall 5-year survival rate for NSCLC remains at 15%. Among NSCLC cases, 15-20% are driven by activating mutations (such as L858R) in the epidermal growth factor receptor (EGFR), which are treated with EGFR tyrosine kinase inhibitors (TKIs). Although three generations of TKIs have been developed, a number of genetic mutations (T790M and C797S) have been shown to evolve that confer resistance in most patients. This has spurred interest in combination therapies, particularly with PD-1 and PD-L1 immune checkpoint inhibitors (ICIs), however, these have limited efficacy in patients with EGFR-mutant NSCLC. Therefore, understanding the immune landscape of these tumors is critical. We developed a mouse model of EGFR-mutant lung cancer using a transgenic mouse model expressing an EGFR carrying L858R activating, as well as T790M and C797S resistance mutations. We demonstrate the expression of this gene after intranasal delivery of an adenovirus carrying GFP-Cre (Ad-GFP-Cre) from a CAG promoter, along with expression of Td-tomato and Luciferase to allow for tumor tracking and visualization. In a time-course experiment, we monitored tumor progression and immune infiltration. By 4-6 weeks post-Ad-GFP-Cre injection, we observed consistent and palpable tumor growth, establishing a critical window for therapeutic intervention. At 2 weeks post-injection, lung tissues showed robust infiltration of CD8+ T cells, highlighting a strong initial immune response. However, levels of regulatory T cells (Tregs) increased significantly over time. This accumulation of Tregs coincided with tumor growth and suppression of cytotoxic T cell activity, suggesting a shift toward an immunosuppressive microenvironment. CD4+ T cells remained relatively constant, underscoring the specific role of Tregs in facilitating immune evasion. Systemic adaptive immunity against EGFR was detected in all tumor bearing mice, demonstrating the potency of local immune suppression in EGFR+ tumor evolution. To evaluate the therapeutic relevance of these findings, we treated mice with a PD-1 inhibitor starting at 4 weeks post-injection. Tumor response was minimal, mirroring clinical observations of limited efficacy for ICIs in EGFR-mutant NSCLC. The early wave of cytotoxic immune activity followed by Treg-mediated suppression offers insights into potential therapeutic targets. This will be a useful model to study the impact of different EGFR TKIs, as well as the combination of these agents with different immunotherapeutic modalities to identify strategies to optimize combination therapies. This work has the potential to inform innovative approaches for treating EGFR-mutant NSCLC and improving patient outcomes. Anchit Bhagat, Cong-Xiao Liu, Xiao Yang, Melissa Gajda, Jason McBane, Herbert K. Lyerly, Zachary C. Hartman. Uncovering immune evasion in EGFR-mutant and treatment resistant NSCLC: insights from a novel mouse model [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 2197.

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

Causing nearly one in six deaths worldwide in 2020, cancer is one of the biggest health challenges of our time.Advancements in technology have led to improved treatments, one of which is using the body's immune system.Dr Zachary Hartman of Duke University's School of Medicine in the US is researching how to improve this approach and whether it has the potential to treat a variety of cancers.How can targeted antibodies and vaccines be used to treat cancer?Immunology

Abstract Trastuzumab deruxtecan (T-DXd), or fam-trastuzumab-deruxtecan-nxki, is a novel antibody-drug conjugate (ADC) targeting HER2 that has demonstrated profound clinical efficacy across HER2-positive breast cancers (BC) and other cancers. However, the precise mechanisms underlying its superiority over traditional ADCs, such as trastuzumab emtansine (T-DM1), remain poorly understood. In this study, we investigated the immune activation mechanisms mediated by T-DXd cytotoxicity in HER2-positive BC. We found that the T-DXd payload, Deruxtecan (DXd), induces a stronger tumor immunogenic cell death (ICD) phenotype compared to T-DM1. This is evidenced by the elevated release of multiple Damage-Associated Molecular Patterns (DAMPs) and the activation of nearby myeloid immune cells through TLR4 and STING pathways. Additionally, T-DXd’s antibody backbone engages with Fcγ-receptors to stimulate Antibody-Dependent Cellular Phagocytosis (ADCP), similar to trastuzumab. Coupled with DXd-induced ICD, T-DXd enhances tumor antigen uptake by macrophages and stimulates antigen-specific CD8+ T cells better than T-DM1. Importantly, DXd cytotoxicity also upregulates tumor CD47 expression, which engages with SIRPα to suppress ADCP. To assess the importance of this regulatory axis in T- DXd efficacy, we explored the use of CD47/SIRPα checkpoint blockade antibodies in combination with T-DXd. Our in vivo studies revealed that this combination synergistically enhanced anti-tumor efficacy in a HER2-transgenic BC mouse model, while inducing CD8+ T cell immune memory and preventing tumor recurrence after therapy cessation. In sum, these studies highlight the important role of T-DXd in stimulating innate and adaptive immune responses against HER2-positive BC and demonstrate the enhancement of this anti-tumor mechanism through combination with CD47/SIRPα checkpoint blockade. Citation Format: Li-Chung Tsao, John S. Wang, Xingru Ma, Sirajbir Sodhi, Herbert Kim Lyerly, Zachary C. Hartman. Enhancing Antitumor Immune Responses: Trastuzumab Deruxtecan (T- DXd) Induces Immunogenic Cell Death and Phagocytosis, Synergizing with CD47/SIRPα Checkpoint Blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B009.