Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent "NoGlow" Mice.

The ability to temporally regulate gene expression and track labeled cells makes animal models powerful biomedical tools. However, sudden expression of xenobiotic genes [e.g., GFP, luciferase (Luc), or rtTA3] can trigger inadvertent immunity that suppresses foreign protein expression or results in complete rejection of transplanted cells. Germline exposure to foreign antigens somewhat addresses these challenges; however, native fluorescence and bioluminescence abrogates the utility of reporter proteins and highly spatiotemporally restricted expression can lead to suboptimal xenoantigen tolerance. 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 nonfunctional forms of GFP and Luc, as well as rtTA3, after CRE-mediated recombination. Using tissue-specific CREs, we observed model and sex-based differences in immune tolerance to the encoded xenoantigens, illustrating the obstacles of tolerizing animals to foreign genes and validating the utility of these "NoGlow" mice to dissect mechanisms of central and peripheral tolerance. Critically, tissue unrestricted NoGlow mice possess no detectable background fluorescence or luminescence and exhibit limited adaptive immunity against encoded transgenic xenoantigens after vaccination. Moreover, 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 that eliminate these cells or prohibit metastatic seeding. Notably, this model enables de novo metastasis from orthotopically implanted, triple-transgenic tumor cells, despite high xenoantigen expression. Altogether, the NoGlow model provides a critical resource for in vivo studies across disciplines, including oncology, developmental biology, infectious disease, autoimmunity, and transplantation. SIGNIFICANCE: Multitolerant NoGlow mice enable tracking and gene manipulation of transplanted tumor cells without immune-mediated rejection, thus providing a platform to investigate novel mechanisms of adaptive immunity related to metastasis, immunotherapy, and tolerance.

Vaccines targeting ESR1 activating mutations elicit anti-tumor immune responses and suppress estrogen signaling in therapy resistant ER+ breast cancer.

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.

CEA vaccines.

Carcinoembryonic antigen (CEA) is a glycosylated cell surface oncofetal protein involved in adhesion, proliferation, and migration that is highly upregulated in multiple carcinomas and has long been a promising target for cancer vaccination. This review summarizes the progress to date in the development of CEA vaccines, examining both pre-clinical and clinical studies across a variety of vaccine platforms that in aggregate, begin to reveal some critical insights. These studies demonstrate the ability of CEA vaccines to break immunologic tolerance and elicit CEA-specific immunity, which associates with improved clinical outcomes in select individuals. Approaches that have combined replicating viral vectors, with heterologous boosting and different adjuvant strategies have been particularly promising but, these early clinical trial results will require confirmatory studies. Collectively, these studies suggest that clinical efficacy likely depends upon harnessing a potent vaccine combination in an appropriate clinical setting to fully realize the potential of CEA vaccination.

Dormant tumors circumvent tumor-specific adaptive immunity by establishing a Treg-dominated niche via DKK3.

Approximately 30% of breast cancer survivors deemed free of disease will experience locoregional or metastatic recurrence even up to 30 years after initial diagnosis, yet how residual/dormant tumor cells escape immunity elicited by the primary tumor remains unclear. We demonstrate that intrinsically dormant tumor cells are indeed recognized and lysed by antigen-specific T cells in vitro and elicit robust immune responses in vivo. However, despite close proximity to CD8+ killer T cells, dormant tumor cells themselves support early accumulation of protective FoxP3+ T regulatory cells (Tregs), which can be targeted to reduce tumor burden. These intrinsically dormant tumor cells maintain a hybrid epithelial/mesenchymal state that is associated with immune dysfunction, and we find that the tumor-derived, stem cell/basal cell protein Dickkopf WNT signaling pathway inhibitor 3 (DKK3) is critical for Treg inhibition of CD8+ T cells. We also demonstrate that DKK3 promotes immune-mediated progression of proliferative tumors and is significantly associated with poor survival and immunosuppression in human breast cancers. Together, these findings reveal that latent tumors can use fundamental mechanisms of tolerance to alter the T cell microenvironment and subvert immune detection. Thus, targeting these pathways, such as DKK3, may help render dormant tumors susceptible to immunotherapies.

Beyond Neoantigens: Antigens Derived from Tumor Drivers as Cancer Vaccine Targets.

A vaccine targeting HER2, a nonmutated but overexpressed tumor antigen, readily primed T cells for ex vivo expansion and adoptive transfer with minimal toxicity. This regimen led to intramolecular epitope spreading in a majority of patients and offers a treatment modality that may improve outcomes for patients with metastatic breast cancer expressing HER2. See related article by Disis et al., p. 3362.

Clinical trials of self-replicating RNA-based cancer vaccines.

Therapeutic cancer vaccines, designed to activate immune effectors against tumor antigens, utilize a number of different platforms for antigen delivery. Among these are messenger RNAs (mRNA), successfully deployed in some prophylactic SARS-CoV2 vaccines. To enhance the immunogenicity of mRNA-delivered epitopes, self-replicating RNAs (srRNA) that markedly increase epitope expression have been developed. These vectors are derived from positive-strand RNA viruses in which the structural protein genes have been replaced with heterologous genes of interest, and the structural proteins are provided in trans to create single cycle viral replicon particles (VRPs). Clinical stage srRNA vectors have been derived from alphaviruses, including Venezuelan Equine Encephalitis (VEE), Sindbis, and Semliki Forest virus (SFV) and have encoded the tumor antigens carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), prostate specific membrane antigen (PSMA), and human papilloma virus (HPV) antigens E6 and E7. Adverse events have mainly been grade 1 toxicities and minimal injection site reactions. We review here the clinical experience with these vaccines and our recent safety data from a study combining a VRP encoding HER2 plus an anti-PD1 monoclonal antibody (pembrolizumab). This experience with VRP-based srRNA supports recent development of fully synthetic srRNA technologies, where the viral structural proteins are replaced with protective lipid nanoparticles (LNP), cationic nanoemulsions or polymers.

Oncofetal protein glypican-3 is a biomarker and critical regulator of function for neuroendocrine cells in prostate cancer.

Neuroendocrine (NE) cells comprise ~1% of epithelial cells in benign prostate and prostatic adenocarcinoma (PCa). However, they become enriched in hormonally treated and castration-resistant PCa (CRPC). In addition, close to 20% of hormonally treated tumors recur as small cell NE carcinoma (SCNC), composed entirely of NE cells, which may be the result of clonal expansion or lineage plasticity. Since NE cells do not express androgen receptors (ARs), they are resistant to hormonal therapy and contribute to therapy failure. Here, we describe the identification of glypican-3 (GPC3) as an oncofetal cell surface protein specific to NE cells in prostate cancer. Functional studies revealed that GPC3 is critical to the viability of NE tumor cells and tumors displaying NE differentiation and that it regulates calcium homeostasis and signaling. Since our results demonstrate that GPC3 is specifically expressed by NE cells, patients with confirmed SCNC may qualify for GPC3-targeted therapy which has been developed in the context of liver cancer and displays minimal toxicity due to its tumor-specific expression. © 2023 The Pathological Society of Great Britain and Ireland.

Cancer vaccine strategies using self-replicating RNA viral platforms.

The development and success of RNA-based vaccines targeting SARS-CoV-2 has awakened new interest in utilizing RNA vaccines against cancer, particularly in the emerging use of self-replicating RNA (srRNA) viral vaccine platforms. These vaccines are based on different single-stranded RNA viruses, which encode RNA for target antigens in addition to replication genes that are capable of massively amplifying RNA messages after infection. The encoded replicase genes also stimulate innate immunity, making srRNA vectors ideal candidates for anti-tumor vaccination. In this review, we summarize different types of srRNA platforms that have emerged and review evidence for their efficacy in provoking anti-tumor immunity to different antigens. These srRNA platforms encompass the use of naked RNA, DNA-launched replicons, viral replicon particles (VRP), and most recently, synthetic srRNA replicon particles. Across these platforms, studies have demonstrated srRNA vaccine platforms to be potent inducers of anti-tumor immunity, which can be enhanced by homologous vaccine boosting and combining with chemotherapies, radiation, and immune checkpoint inhibition. As such, while this remains an active area of research, the past and present trajectory of srRNA vaccine development suggests immense potential for this platform in producing effective cancer vaccines.

A Non-Invasive Deep Photoablation Technique to Inhibit DCIS Progression and Induce Antitumor Immunity.

Ductal carcinoma in situ (DCIS) of the breast is often managed by lumpectomy and radiation or mastectomy, despite its indolent features. Effective non-invasive treatment strategies could reduce the morbidity of DCIS treatment. We have exploited the high heat shock protein 90 (HSP90) activity in premalignant and malignant breast disease to non-invasively detect and selectively ablate tumors using photodynamic therapy (PDT). PDT with the HSP90-targeting photosensitizer, HS201, can not only ablate invasive breast cancers (BCs) while sparing non-tumor tissue, but also induce antitumor immunity. We hypothesized that HS201-PDT would both non-invasively ablate DCIS and prevent progression to invasive BC. We tested in vitro selective uptake and photosensitivity of HS201 in DCIS cell lines compared to the non-selective parental verteporfin, and assessed in vivo antitumor efficacy in mammary fat pad and intraductal implantation models. Selective uptake of HS201 enabled treatment of intraductal lesions while minimizing toxicity to non-tumor tissue. The in vivo activity of HS201-PDT was also tested in female MMTV-neu mice prior to the development of spontaneous invasive BC. Mice aged 5 months were administered HS201, and their mammary glands were exposed to laser light. HS201-PDT delayed the emergence of invasive BC, significantly prolonged disease-free survival (DFS) (p = 0.0328) and tended to improve overall survival compared to the no-treatment control (p = 0.0872). Systemic administration of anti-PD-L1 was combined with HS201-PDT and was tested in a more aggressive spontaneous tumor model, HER2delta16 transgenic mice. A single PDT dose combined with anti-PD-L1 improved DFS compared to the no-treatment control, which was significantly improved with repetitive HS201-PDT given with anti-PD-L1 (p = 0.0319). In conclusion, a non-invasive, skin- and tissue-sparing PDT strategy in combination with anti-PD-L1 antibodies effectively prevented malignant progression of DCIS to invasive BC. This non-invasive treatment strategy of DCIS may be safe and effective, while providing an option to reduce the morbidity of current conventional treatment for patients with DCIS. Clinical testing of HS201 is currently underway.

Combination of a novel heat shock protein 90-targeted photodynamic therapy with PD-1/PD-L1 blockade induces potent systemic antitumor efficacy and abscopal effect against breast cancers.

BACKGROUND: We previously demonstrated potent antitumor activity against human breast cancer xenografts using photodynamic therapy (PDT) targeting a novel tumor-specific photosensitizer (HS201), which binds heat shock protein 90 (HS201-PDT). However, induction of systemic antitumor immunity by HS201-PDT alone or by the combination strategy with immune checkpoint blockade has yet to be determined. METHODS: Using unilateral and bilateral implantation models of syngeneic breast tumors (E0771, MM3MG-HER2, and JC-HER3) in mice, we assessed whether HS201-PDT could induce local and systemic antitumor immunity. In an attempt to achieve a stronger abscopal effect for distant tumors, the combination strategy with anti-PD-L1 antibody was tested. Tumor-infiltrating leukocytes were analyzed by single cell RNA-sequencing and receptor-ligand interactome analysis to characterize in more detailed the mechanisms of action of the treatment and key signaling pathways involved. RESULTS: HS201-PDT demonstrated greater tumor control and survival in immune competent mice than in immunocompromised mice, suggesting the role of induced antitumor immunity; however, survival was modest and an abscopal effect on distant implanted tumor was weak. A combination of HS201-PDT with anti-PD-L1 antibody demonstrated the greatest antigen-specific immune response, tumor growth suppression, prolonged mouse survival time and abscopal effect. The most significant increase of intratumoral, activated CD8+T cells and decrease of exhausted CD8+T cells occurred following combination treatment compared with HS201-PDT monotherapy. Receptor-ligand interactome analysis showed marked enhancement of several pathways, such as CXCL, GALECTIN, GITRL, PECAM1 and NOTCH, associated with CD8+T cell activation in the combination group. Notably, the expression of the CXCR3 gene signature was the highest in the combination group, possibly explaining the enhanced tumor infiltration by T cells. CONCLUSIONS: The increased antitumor activity and upregulated CXCR3 gene signature induced by the combination of anti-PD-L1 antibody with HS201-PDT warrants the clinical testing of HS201-PDT combined with PD-1/PD-L1 blockade in patients with breast cancer, and the use of the CXCR3 gene signature as a biomarker.

Sensitizing immune unresponsive colorectal cancers to immune checkpoint inhibitors through MAVS overexpression.

BACKGROUND: The majority of colorectal carcinomas (CRCs) are insensitive to programmed death protein-1/programmed death-ligand 1 (anti-PD-1/PD-L1) immune checkpoint inhibitor (ICI) antibodies. While there are many causes for ICI insensitivity, recent studies suggest that suppression of innate immune gene expression in tumor cells could be a root cause of this insensitivity and an important factor in the evolution of tumor immunosuppression. METHODS: We first assessed the reduction of mitochondrial antiviral signaling gene (MAVS) and related RIG-I pathway gene expression in several patient RNA expression datasets. We then engineered MAVS expressing tumor cells and tested their ability to elicit innate and adaptive anti-tumor immunity using both in vitro and in vivo approaches, which we then confirmed using MAVS expressing viral vectors. Finally, we observed that MAVS stimulated PD-L1 expression in multiple cell types and then assessed the combination of PD-L1 ICI antibodies with MAVS tumor expression in vivo. RESULTS: MAVS was significantly downregulated in CRCs, but its re-expression could stimulate broad cellular interferon-related responses, in both murine and patient-derived CRCs. In vivo, local MAVS expression elicited significant anti-tumor responses in both immune-sensitive and insensitive CRC models, through the stimulation of an interferon responsive axis that provoked tumor antigen-specific adaptive immunity. Critically, we found that tumor-intrinsic MAVS expression triggered systemic adaptive immune responses that enabled abscopal CD8 +T cell cytotoxicity against distant CRCs. As MAVS also induced PD-L1 expression, we further found synergistic anti-tumor responses in combination with anti-PD-L1 ICIs. CONCLUSION: These data demonstrate that intratumoral MAVS expression results in local and systemic tumor antigen-specific T cell responses, which could be combined with PD-L1 ICI to permit effective anti-tumor immunotherapy in ICI resistant cancers.

Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis.

Two HER2-specific mAbs, trastuzumab and pertuzumab (T+P), combined with chemotherapy comprise standard-of-care treatment for advanced HER2+ breast cancers (BC). While this antibody combination is highly effective, its synergistic mechanism-of-action (MOA) remains incompletely understood. Past studies have suggested that the synergy underlying this combination occurs through the different mechanisms elicited by these antibodies, with pertuzumab suppressing HER2 heterodimerization and trastuzumab inducing antitumor immunity. However, in vivo evidence for this synergy is lacking. In this study, we found that the therapeutic efficacy elicited by their combination occurs through their joint ability to activate the classical complement pathway, resulting in both complement-dependent cytotoxicity and complement-dependent cellular phagocytosis of HER2+ tumors. We also demonstrate that tumor C1q expression is positively associated with survival outcome in HER2+ BC patients and that complement regulators CD55 and CD59 were inversely correlated with outcome, suggesting the clinical importance of complement activity. Accordingly, inhibition of C1q in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant antitumor MOA for T+P therapy that may be functionally enhanced to potentially augment clinical therapeutic efficacy.

HSP90-Specific nIR Probe Identifies Aggressive Prostate Cancers: Translation from Preclinical Models to a Human Phase I Study.

A noninvasive test to discriminate indolent prostate cancers from lethal ones would focus treatment where necessary while reducing overtreatment. We exploited the known activity of heat shock protein 90 (Hsp90) as a chaperone critical for the function of numerous oncogenic drivers, including the androgen receptor and its variants, to detect aggressive prostate cancer. We linked a near-infrared fluorescing molecule to an HSP90 binding drug and demonstrated that this probe (designated HS196) was highly sensitive and specific for detecting implanted prostate cancer cell lines with greater uptake by more aggressive subtypes. In a phase I human study, systemically administered HS196 could be detected in malignant nodules within prostatectomy specimens. Single-cell RNA sequencing identified uptake of HS196 by malignant prostate epithelium from the peripheral zone (AMACR+ERG+EPCAM+ cells), including SYP+ neuroendocrine cells that are associated with therapeutic resistance and metastatic progression. A theranostic version of this molecule is under clinical testing.

Mechanisms of Therapeutic Antitumor Monoclonal Antibodies.

Monoclonal antibodies (mAb) are a major component of cancer therapy. In this review, we summarize the different therapeutic mAbs that have been successfully developed against various tumor-expressed antigens and examine our current understanding of their different mechanisms of antitumor action. These mechanisms of action (MOA) largely center on the stimulation of different innate immune effector processes, which appear to be principally responsible for the efficacy of most unconjugated mAb therapies against cancer. This is evident in studies of mAbs targeting antigens for hematologic cancers, with emerging data also demonstrating the critical nature of innate immune-mediated mechanisms in the efficacy of anti-HER2 mAbs against solid HER2+ cancers. Although HER2-targeted mAbs were originally described as inhibitors of HER2-mediated signaling, multiple studies have since demonstrated these mAbs function largely through their engagement with Fc receptors to activate innate immune effector functions as well as complement activity. Next-generation mAbs are capitalizing on these MOAs through improvements to enhance Fc-activity, although regulation of these mechanisms may vary in different tumor microenvironments. In addition, novel antibody-drug conjugates have emerged as an important means to activate different MOAs. Although many unknowns remain, an improved understanding of these immunologic MOAs will be essential for the future of mAb therapy and cancer immunotherapy.

HER2 Isoforms Uniquely Program Intratumor Heterogeneity and Predetermine Breast Cancer Trajectories During the Occult Tumorigenic Phase.

HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype (WTHER2), exon-16 null (d16HER2), and N-terminally truncated (p95HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. Although WTHER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast, d16HER2 and p95HER2 induced rapid tumor development. d16HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereas p95HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed for d16HER2 and p95HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer. IMPLICATIONS: Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment.

Progesterone promotes immunomodulation and tumor development in the murine mammary gland.

BACKGROUND: Clinical studies have linked usage of progestins (synthetic progesterone [P4]) to breast cancer risk. However, little is understood regarding the role of native P4, signaling through the progesterone receptor (PR), in breast tumor formation. Recently, we reported a link between PR and immune signaling pathways, showing that P4/PR can repress type I interferon signaling pathways. Given these findings, we sought to investigate whether P4/PR drive immunomodulation in the mammary gland and promote tumor formation. METHODS: To determine the effect of P4 on immune cell populations in the murine mammary gland, mice were treated with P4 or placebo pellets for 21 days. Immune cell populations in the mammary gland, spleen, and inguinal lymph nodes were subsequently analyzed by flow cytometry. To assess the effect of PR overexpression on mammary gland tumor development as well as immune cell populations in the mammary gland, a transgenic mouse model was used in which PR was overexpressed throughout the entire mouse. Immune cell populations were assessed in the mammary glands, spleens, and inguinal lymph nodes of 6-month-old transgenic and control mice by flow cytometry. Transgenic mice were also monitored for mammary gland tumor development over a 2-year time span. Following development of mammary gland tumors, immune cell populations in the tumors and spleens of transgenic and control mice were analyzed by flow cytometry. RESULTS: We found that mice treated with P4 exhibited changes in the mammary gland indicative of an inhibited immune response compared with placebo-treated mice. Furthermore, transgenic mice with PR overexpression demonstrated decreased numbers of immune cell populations in their mammary glands, lymph nodes, and spleens. On long-term monitoring, we determined that multiparous PR-overexpressing mice developed significantly more mammary gland tumors than control mice. Additionally, tumors from PR-overexpressing mice contained fewer infiltrating immune cells. Finally, RNA sequencing analysis of tumor samples revealed that immune-related gene signatures were lower in tumors from PR-overexpressing mice as compared with control mice. CONCLUSION: Together, these findings offer a novel mechanism of P4-driven mammary gland tumor development and provide rationale in investigating the usage of antiprogestin therapies to promote immune-mediated elimination of mammary gland tumors.

Long-term survival of patients with stage III colon cancer treated with VRP-CEA(6D), an alphavirus vector that increases the CD8+ effector memory T cell to Treg ratio.

BACKGROUND: There remains a significant need to eliminate the risk of recurrence of resected cancers. Cancer vaccines are well tolerated and activate tumor-specific immune effectors and lead to long-term survival in some patients. We hypothesized that vaccination with alphaviral replicon particles encoding tumor associated antigens would generate clinically significant antitumor immunity to enable prolonged overall survival (OS) in patients with both metastatic and resected cancer. METHODS: OS was monitored for patients with stage IV cancer treated in a phase I study of virus-like replicon particle (VRP)-carcinoembryonic antigen (CEA), an alphaviral replicon particle encoding a modified CEA. An expansion cohort of patients (n=12) with resected stage III colorectal cancer who had completed their standard postoperative adjuvant chemotherapy was administered VRP-CEA every 3 weeks for a total of 4 immunizations. OS and relapse-free survival (RFS) were determined, as well as preimmunization and postimmunization cellular and humoral immunity. RESULTS: Among the patients with stage IV cancer, median follow-up was 10.9 years and 5-year survival was 17%, (95% CI 6% to 33%). Among the patients with stage III cancer, the 5-year RFS was 75%, (95%CI 40% to 91%); no deaths were observed. At a median follow-up of 5.8 years (range: 3.9-7.0 years) all patients were still alive. All patients demonstrated CEA-specific humoral immunity. Patients with stage III cancer had an increase in CD8 +TEM (in 10/12) and decrease in FOXP3 +Tregs (in 10/12) following vaccination. Further, CEA-specific, IFNγ-producing CD8+granzyme B+TCM cells were increased. CONCLUSIONS: VRP-CEA induces antigen-specific effector T cells while decreasing Tregs, suggesting favorable immune modulation. Long-term survivors were identified in both cohorts, suggesting the OS may be prolonged.

Novel Small-Molecule Inhibitor for the Oncogenic Tyrosine Phosphatase SHP2 with Anti-Breast Cancer Cell Effects.

The oncogenic property of the Src homology phosphotyrosine phosphatase 2 (SHP2) is well-known, but developing specific inhibitors has been very difficult. Based on our previous reports that showed the importance of acidic residues surrounding SHP2 substrate phosphotyrosines for specific recognition, we have rationally designed and chemically synthesized a small-molecule SHP2 inhibitor named 4,4'-(4'-carboxy)-4-nonyloxy-[1,1'-biphenyl]-3,5-diyl)dibutanoic acid (CNBDA). Molecular modeling predicted that CNBDA packs well into the SHP2 active site and makes extended interactions primarily with positively charged and polar amino acids surrounding the active site. In vitro PTPase assays showed that CNBDA inhibits SHP2 with an IC50 of 5 µM. However, the IC50 of CNBDA toward SHP1, the close structural homologue of SHP2, was 125 µM, suggesting an approximately 25-fold effectiveness against SHP2 than SHP1. Because SHP2 is known for its positive role in breast cancer (BC) cell biology, we tested the effect of SHP2 inhibition with CNBDA in HER2-positive BC cells. Treatment with CNBDA suppressed cell proliferation in 2D culture, anchorage-independent growth in soft agar, and mammosphere (tumorisphere) formation in suspension cultures in a concentration-dependent manner. Furthermore, CNBDA inhibited EGF-induced signaling and expression of HER2 by inhibiting the PTPase activity of SHP2 in BC cells. These findings suggest that CNBDA is a promising anti-SHP2 lead compound with anti-BC cell effects.