HER2 Transgenic Mice Research Articles

Abstract 2377: HER2-ADC efficacy is rooted in its induction of immunogenic cell death with FcGR activation and a systemic bystander effect

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.

Abstract 4000: A novel pegylated bispecific antibody-drug conjugate (P-BsADC) targeting Her2+ cancers with improved efficacy and therapeutic window

Abstract Despite the fact that ADCs improve the efficacy and target selectivity comparing to the non-specific small molecule cytotoxicity drugs in cancer treatment, traditional ADCs still suffer from many issues which include low tumor penetration and accumulation, inefficient internalization, undesired efflux of ADC from tumor cells, significant on-target off-tumor toxicity, Fc mediated uptake that results in off-target toxicity, limited extravasation across capillary walls due to big molecular size, poor diffusion into the tumor masses due to increased tumor interstitial fluid pressure, and the binding-site-barrier. To address these issues, we previously reported that the compound JY201, a Polyethylene Glycol (PEG)-based bispecific ADC (P-BsADC) targeting two epitopes of Her2, demonstrated advantages in tumor penetration, internalization efficiency, lysosome trafficking effectiveness, no Fc related toxicity, and better efficacy in tumor inhibition than transtuzumab deruxtecan (Ds-8201). Continuing from our previous study, here we further reveal that JY201 can penetrate the tumor deeply and distribute more homogeneously in entire tumor masses while Ds-8201 limits its diffusion to the regions very close to the blood vessels in the tumor. Furthermore, JY201 shows better efficacy than Ds-8201 in inhibiting tumors with low expression of Her2 in pdx (patient derived xenograft) and cdx models. In addition, JY201 can effectively inhibit tumors resistant to Ds-8201. In an in-vitro plasma stability test, JY201 demonstrated high stability in cynomolgus monkey and human serums. JY201 also has a biodistribution profile advocating better safety than Ds-8201 in tumor bearing mice. In the repeated-dosing toxicological study in Her2 transgenic mice, JY201 with the dose of 50mg/kg was well tolerated and did not induce any tissue/organ damage to the animals. Due to much shorter half-life (5 times shorter) in mice for PEGylated proteins than in primates, we expect JY201 will have much higher tolerated dose than the 50mg/kg in primates. In summary, the findings from this study provide solid preclinical evidence for JY201 to be developed further as an efficacious and safe clinical treatment for patients with Her2 positive cancers. Citation Format: Yu (Yvonne) Wen, Shuqiang Yin, Weidong Lyu, Yang Lei, Qiudong Zhuo, Zibin Wu, Bin Sun, Shuangyu Tan, Lidong Jiang, Teng Zhang, Bo Gao, Rui Xu, Yong Li, Liling Zheng, Shumin Liu, David (Dechun) Wu. A novel pegylated bispecific antibody-drug conjugate (P-BsADC) targeting Her2+ cancers with improved efficacy and therapeutic window. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4000.

Prevention and Therapy of Metastatic HER-2+ Mammary Carcinoma with a Human Candidate HER-2 Virus-like Particle Vaccine.

Vaccines are a promising therapeutic alternative to monoclonal antibodies against HER-2+ breast cancer. We present the preclinical activity of an ES2B-C001, a VLP-based vaccine being developed for human breast cancer therapy. FVB mice challenged with HER-2+ mammary carcinoma cells QD developed progressive tumors, whereas all mice vaccinated with ES2B-C001+Montanide ISA 51, and 70% of mice vaccinated without adjuvant, remained tumor-free. ES2B-C001 completely inhibited lung metastases in mice challenged intravenously. HER-2 transgenic Delta16 mice developed mammary carcinomas by 4–8 months of age; two administrations of ES2B-C001+Montanide prevented tumor onset for >1 year. Young Delta16 mice challenged intravenously with QD cells developed a mean of 68 lung nodules in 13 weeks, whereas all mice vaccinated with ES2B-C001+Montanide, and 73% of mice vaccinated without adjuvant, remained metastasis-free. ES2B-C001 in adjuvant elicited strong anti-HER-2 antibody responses comprising all Ig isotypes; titers ranging from 1–10 mg/mL persisted for many months. Antibodies inhibited the 3D growth of human HER-2+ trastuzumab-sensitive and -resistant breast cancer cells. Vaccination did not induce cytokine storms; however, it increased the ELISpot frequency of IFN-γ secreting HER-2-specific splenocytes. ES2B-C001 is a promising candidate vaccine for the therapy of tumors expressing HER-2. Preclinical results warrant further development towards human clinical studies.

HER2 and APC Mutations Promote Altered Crypt-Villus Morphology and Marked Hyperplasia in the Intestinal Epithelium.

Background and AimsThe Cancer Genome Atlas (TCGA) project has identified HER2 mutations or amplification in 7% of colon cancers. In addition to HER2 mutations, colon cancer patients also possess co-occurring mutations in genes such as APC. Here, we investigated the role of HER2 and APC mutations on the crypt-villus architecture of the intestinal epithelium, localization of secretory cells, and expression of intestinal stem cell markers.MethodsWe generated a HER2 transgenic mouse (HER2V777L Tg) possessing an activating mutation commonly found in colorectal cancer patients, HER2V777L, using transcription activator-like effector nucleases–based gene editing technology. We expressed the HER2V777L transgene in mouse small intestine and colon using Lgr5-Cre and Villin-Cre recombinases. In addition, we analyzed Lgr5-Cre; APCmin; HER2V777L Tg mice by morphologic and gene expression assays on intestinal sections and organoids derived from the epithelium.ResultsHER2V777L expression resulted in hypertrophic crypt formation with expanded zones of proliferation. Proximal intestinal villi showed increased abundance of multiple differentiated lineages including extensive intermediate cell differentiation, as evidenced by MUC2/MMP7 co-immunofluorescence and transmission electron microscopy. HER2V777L expression in the context of APC loss resulted in further enhancement and expansion of the proliferative crypt compartment.ConclusionsWe established an epithelial intrinsic role for HER2V777L on enhanced cellular proliferation. Additionally, we determined that HER2 and APC mutations, when combined, promote enhanced proliferation of intestinal crypts.

Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity.

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose-dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in chromosome 17 that was responsible for regulating the response. A/J and NOD segments of genes in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC-IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies.

Abstract LB-023: Dynamic real time in vivo CAR T cell tracking: Clinical and preclinical studies using a novel dual PET-MR imaging agent

Abstract Objective: The aim is to demonstrate dynamic in-vivo tracking of CAR T cell therapy for treatment of solid tumors using Cu-64 labeled superparamagnetic iron oxide nanoparticles (SPION) as novel dual PET-MR imaging agent. Methodology: Cu-64 SPION: Cu-64 radioisotope is bound to silica coated SPION using enhanced electrolysis plating techniques with tin and palladium seeding. Preclinical Model: Mouse splenic T cells were activated with anti-CD3, anti-CD28 & cultured with IL-2 and IL-7, prior to being transduced with second generation anti-Her-2 CAR (scFv-CD28-CD3ζ). 5 x 105 E0771-hHER2 breast tumor cells were implanted subcutaneously into male C57Bl/6-human HER2 transgenic mice. 107 labeled CAR T or control T cells (Cu-64 5-8 MBq) were injected into tail vein. Clinical Model: Activated T cells using antibody CD3 (OKT3) & IL-2 are transduced with retroviral vector constructs encoding for chimeric T-cell receptor specific for Lewis Y antigen. Modified T-cells are further expanded ex-vivo and reinfused. 3 x 108 CAR T cells were labeled with Cu-64 (200 - 300 MBq). Labeling of CAR T cells with Cu-64 SPION: Transfecting agent protamine sulphate facilitated cellular uptake of Cu-64 SPION within cells. Functional assays: 51Chromium release, cytometric bead array and cell viability showed that labeling process did not affect CAR T cell cytotoxicity, cytokine secretion (TNFα and IFN-γ) and viability. CAR T Cell Tracking: Scanning was performed using clinical grade dual PET-MR scanner. Preliminary Data: In this clinical trial (HREC/16/PMCC/30) patients are being enrolled for first in human in vivo study to determine how many cells distribute to solid tumor sites within first few days of CAR T cell therapy. This is first data that demonstrates that CAR-T cells can be consistently and efficiently labeled (≤60%) with cell viability (≥85%) and at sensitivity comparable to detecting at least z cells at tumor site using clinical grade dual PET-MR scanner. SUVmean values provides insight into individual response to therapy. The observed increase in SUVmax over time specifies localization of CAR T cells at tumor sites. Clinical data at early time point showed moderate uptake in lungs posterior basal segments without increased activity over next few days, thus suggesting transient process. Mild, diffuse bone marrow and relatively intense uptake in the liver and spleen suggests margination of cells to the reticulo-endothelial system. Distinct PET signal suggests localization of labeled cells in the secondary tumor sites. Little background uptake in important organs such as brain and heart indicate the safety profile of imaging agent. Absence of signal in bladder indicates hepatobiliary excretion, which may allow re-absorption from GI tract and re-circulation. Distinct PET signal within tumor in preclinical studies confirms trafficking of CAR T cells to tumor site as compared to controls. A negative contrast in the liver on T2 weighted MRI in both the preclinical and clinical studies. Preliminary Conclusion:This is first in human in vivo study to show CAR T cell distribution in real time and provides insight into individual responses of tumors to therapy. CAR T cell functionality largely remain unchanged due to labeling process. The preliminary findings indicate that labeled cells traffic to tumor sites in first few hours of infusion and remain persistent for extended period. Citation Format: Ritu Singla, Dominic Wall, Samuel Anderson, Nicholas Zia, James C. Korte, Lucy Kravets, Gerard McKiernan, Jeanne Butler, Amanda Gammilonghi, Jyoti Arora, Ben Solomon, Rodney Hicks, Timothy Cain, Phillip Darcy, Carleen Cullinane, Paul Neeson, Rajesh Ramanathan, Ravi Shukla, Vipul Bansal, Simon Harrison. Dynamic real time in vivo CAR T cell tracking: Clinical and preclinical studies using a novel dual PET-MR imaging agent [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-023.

STUDY OF THE EFFECT OF LOMUSTIN ON HER2-POSITIVE BREAST CANCER IN FVB/N HER-2 TRANSGENIC MICE

Because of the high risk of brain metastases from HER2-positive breast cancer, the study of the anticancer activity of drugs used to treat brain tumors, in particular lomustine, is of great importance. In the FVB/N Her-2 transgenic mice bearing HER2-positive breast cancer (BC HER2+), a single oral administration of lomustine at a dose of 50 mg/kg resulted in a significant tumor growth inhibition (up to 96 %, p<0.0001). The tumor growth index (TGI) expressed as a ratio between the areas under the kinetic curves of tumor growth in the study and control groups and amounted to 33 % (p<0.001) indicated the high activity of lomustine. However, the effect of lomustine on intramuscularly transplanted Ehrlich tumor was insignificant (tumor growth inhibition and tumor growth index were <39 % and 68 %, respectively). Lomustine administered orally at a single dose of 50 mg/kg 24 hours after intracranial transplantation of BC HER2+ increased the median survival time up to 30 days in FVB/N mice compared to 21 days in the control group mice (p<0.001). The high therapeutic effect of lomustine in HER2-positive breast cancer mice is likely can be explained by the biological characteristics of this tumor; therefore clinical trials of lomustine for HER2-positive tumors are needed.

Abstract 3731: A novel heat shock protein 90-targeted photosensitizer (HS-201) enables enhanced tumor-specific photodynamic therapy of inflammatory breast cancers

Abstract Background: Photodynamic therapy (PDT) is an effective local anti-cancer modality applied for the treatment of early stage disease and palliation of advanced disease, and could be used in managing inflammatory breast cancer (IBC). Although PDT can be focused by limiting the area of near infrared (nIR) light exposure, normal tissue injury due to the photosensitizer (PS) uptake by non-tumor tissues remains a barrier to broader applicability. We reported that selective delivery of a compound to malignant cells by exploiting their heat shock protein 90 (Hsp90) activity could be used for imaging breast cancers. This work was extended by the development of an Hsp90 targeted PS (HS201), in which a well characterized PS, verteporfin (VP), has been chemically tethered to a small molecule Hsp90 inhibitor and assessed the tumor-specificity and antitumor efficacy of HS201-based PDT (HS201-PDT). Methods & Results: HS201 was taken up by human breast cancer cell lines, including IBC in vitro more readily than VP, resulting in increased tumor killing in vitro after exposure to a laser with a 690 nm wavelength. The in vivo selectively of HS201 uptake in tumor tissue was confirmed in both human breast cancer xenograft models and spontaneous tumors from HER2-transgenic mice. HS201 accumulation in the tumor was dramatically enhanced after the first laser exposure due to the feed forward increase in Hsp90 expression in the treated tumor cells, and thus a repeated laser exposure was conducted to enhance antitumor effects. This strategy enhanced antitumor effects, as MDA-MB-231 tumors implanted into SCID-beige mice responded to HS201-PDT but not to VP-PDT. Human xenograft breast cancer cell lines, including the IBC cell lines SUM149 and KPL-4, as well as non-IBC cell line BT474M1 and the patient-derived breast cancer xenograft HCI-013, were also tested in SCID-beige mice and demonstrated significantly suppressed tumor growth by HS201-PDT. Conclusions: HS201 showed selective uptake and longer retention by tumor cells, including IBC, especially when the tumors were exposed to local laser treatment. Due to the advantage of this feed-forward control of HS201 distribution, HS201-PDT demonstrated superior anti-tumor effects compared to VP-PDT against various human breast cancer xenografts in mice. These results suggest that HS201-PDT may be used in managing IBC, and may have an important role in anti-cancer therapy. Citation Format: Kensuke Kaneko, Takuya Osada, Philip F. Hughes, Timothy A. Haystead, Michael A. Morse, Herbert K. Lyerly. A novel heat shock protein 90-targeted photosensitizer (HS-201) enables enhanced tumor-specific photodynamic therapy of inflammatory breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3731.

An HER2 DNA vaccine with evolution-selected amino acid substitutions reveals a fundamental principle for cancer vaccine formulation in HER2 transgenic mice.

Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.

19 Autophagy induction as a new therapy for HER2+ breast tumorigenesis

IntroductionHER2 is a receptor tyrosine kinase whose gene is amplified in ~20% human breast cancer patients. Allelic loss of the autophagy gene, beclin 1/BECN1, is associated with HER2 amplification in breast cancer; low beclin 1 mRNA expression is associated with increased risk of HER2-positive breast cancer; and overexpressed HER2 and Beclin 1 interact in cultured cells. However, the functional significance of HER2/Beclin 1 interaction and of altered autophagy in HER2-driven tumorigenesis and whether autophagy induction may be beneficial in preventing HER2-positive breast tumour growth is unknown.Material and methodsWe explored the regulation of autophagy in breast cancer cells by HER2 in vitro and the effects as well as genetic and pharmacological approaches to increase autophagy on HER2-driven breast cancer growth in vivo.Results and discussionsHere we show that endogenous HER2 interacts with Beclin 1 in multiple HER2 +breast cancer cells and inhibits autophagy. Mice with a knock-in mutation in Becn1 (Becn1F121A) that leads to increased basal autophagy are protected from mammary tumorigenesis when crossed with mammary-specific HER2 transgenic mice, and HER2 fails to inhibit autophagy in primary cells derived from these mice.Moreover, treatment of mice with HER2-positive human breast cancer xenografts with the Tat-Beclin 1 autophagy-inducing peptide inhibits tumour growth as effectively as a clinically used HER2 tyrosine kinase inhibitor (TKI). This inhibition of tumour growth is associated with a robust induction of autophagy, a disruption of HER2/Beclin 1 binding, and a transcriptional signature in the tumours that is distinct from that observed with HER2 TKI treatment.ConclusionThese findings indicate that the HER2-mediated inhibition of Beclin 1 and autophagy likely contributes to HER2-mediated tumorigenesis. They also suggest that strategies to block HER2/Beclin 1 binding and/or increase autophagy may represent a new therapeutic approach for HER2-positive breast cancers.

Linkage between TIM3 genotype and human HER2 vaccine response in Diversity Outbred (DO) HER2 transgenic (Tg) mice

Abstract Regulatory T cell (Treg) depletion boosts HER2 vaccine response in human HER2 transgenic (Tg) mice. To test whether polymorphisms of Treg-associated genes impact vaccine response, individually distinct F1(DOxB6 HER2 Tg) mice were vaccinated 2× with Adv/HER2 to generate highly variable responses. Single nucleotide polymorphisms (SNP) of 16 candidate genes were analyzed by MonsterPlex sequencing or TaqMan assay, with TIM3 showing an association with vaccine response. There are14 SNPs in the 2kb proximal promoter region and 10 SNPs in a 1kb region containing TIM3 exon 7. Four distinct TIM3 genotypes were identified for either region with one genotype consistently matching between the promoter and exon 7 loci, i.e. the CAST/EiJ strain genotype. Analysis of 84 vaccinated DOxB6 HER2 Tg mice using a tag SNP rs28230613 showed that mice with homozygous CC produce HER2 Ab at 26.9±28.9 μg/mL, and those with heterozygous CA (CAST/Eij genotype) 12.8±19.4 μg/mL (p=0.0075). T cell response was absent in most CA heterozygotes. Relative transcript levels of splenocyte TIM3 over CD4 showed heterozygotes at 2.06±2.27 and homozygotes 0.54±0.51 (p=0.0073, n=24), indicating higher TIM3 transcript in CA heterozygotes. Thus, CAST/EiJ TIM3 genotype is associated with higher TIM3 transcript level and lower vaccine response to self HER2. This correlation is absent in mice depleted of CD25+ Treg, implicating Treg and their TIM3 in regulating HER2 vaccine response. The segregation of TIM3 genotype with vaccine response is not absolute, suggesting additional molecular regulation in HER2 vaccine response. CA76340

Mitochondrial Haplotype Alters Mammary Cancer Tumorigenicity and Metastasis in an Oncogenic Driver-Dependent Manner.

Using a novel mouse model, a mitochondrial-nuclear exchange model termed MNX, we tested the hypothesis that inherited mitochondrial haplotypes alter primary tumor latency and metastatic efficiency. Male FVB/N-Tg(MMTVneu)202Mul/J (Her2) transgenic mice were bred to female MNX mice having FVB/NJ nuclear DNA with either FVB/NJ, C57BL/6J, or BALB/cJ mtDNA. Pups receiving the C57BL/6J or BALB/cJ mitochondrial genome (i.e., females crossed with Her2 males) showed significantly (P < 0.001) longer tumor latency (262 vs. 293 vs. 225 days), fewer pulmonary metastases (5 vs. 7 vs. 15), and differences in size of lung metastases (1.2 vs. 1.4 vs. 1.0 mm diameter) compared with FVB/NJ mtDNA. Although polyoma virus middle T-driven tumors showed altered primary and metastatic profiles in previous studies, depending upon nuclear and mtDNA haplotype, the magnitude and direction of changes were not the same in the HER2-driven mammary carcinomas. Collectively, these results establish mitochondrial polymorphisms as quantitative trait loci in mammary carcinogenesis, and they implicate distinct interactions between tumor drivers and mitochondria as critical modifiers of tumorigenicity and metastasis. Cancer Res; 77(24); 6941-9. ©2017 AACR.

Multiplex three-dimensional optical mapping of tumor immune microenvironment

Recent developments in optical tissue clearing and microscopic imaging have advanced three-dimensional (3D) visualization of intact tissues and organs at high resolution. However, to expand applications to oncology, critical limitations of current methods must be addressed. Here we describe transparent tissue tomography (T3) as a tool for rapid, three-dimensional, multiplexed immunofluorescent tumor imaging. Cutting tumors into sub-millimeter macrosections enables simple and rapid immunofluorescence staining, optical clearing, and confocal microscope imaging. Registering and fusing macrosection images yields high resolution 3D maps of multiple tumor microenvironment components and biomarkers throughout a tumor. The 3D maps can be quantitatively evaluated by automated image analysis. As an application of T3, 3D mapping and analysis revealed a heterogeneous distribution of programmed death-ligand 1 (PD-L1) in Her2 transgenic mouse mammary tumors, with high expression limited to tumor cells at the periphery and to CD31+ vascular endothelium in the core. Also, strong spatial correlation between CD45+ immune cell distribution and PD-L1 expression was revealed by T3 analysis of the whole tumors. Our results demonstrate that a tomographic approach offers simple and rapid access to high-resolution three-dimensional maps of the tumor immune microenvironment, offering a new tool to examine tumor heterogeneity.

Genetic linkage analysis of cancer vaccine response in Diversity Outbred mice

Abstract Recent advances in cancer immunotherapy demonstrate the power of anti-tumor immunity, however only a fraction of patients maintain durable responses. To test the hypothesis that genetic variation determines cancer immunotherapy response, we conducted genetic linkage analysis of HER2 vaccine response in Diversity Outbred (DO) mice. Our HER2 DNA vaccine has shown varying anti-tumor activity in mice, cats and human patients. Differential vaccine response was found in HER2 transgenic (Tg) mice of BALB/c versus C57BL/6 backgrounds. DO mice, produced by non-sibling crossing of 8 mouse stains (A/J, C57BL/6, 129S1/Svlm, NOD, NZO, CAST/EiJ, PWK/PhJ and WSB/EiJ) incorporate &amp;gt;90% of all genetic variation in mice. The fully sequenced founder strain genomes enable genetic linkage analysis using Mouse Universal Genotyping Array (GigaMUGA), which consists of &amp;gt;150,000 genetic markers, mostly single nucleotide variants. DO mice were bred with C57BL/6 HER2 Tg mice, generating HER2 DO mice that express human HER2 as a self-antigen. A wide range of IgG (1.8 – 50 μg/mL) and IFNg producing T cells (0 – 348/106 splenocytes) were detected after 2× vaccination with an adenoviral vaccine encoding human HER2. Genetic loci associated with positive response (p&amp;lt;10−5) were sought by linear regression analysis. Immune-related genes within identified loci were examined. Preliminary analysis links IgG response to NFkB, IL6, and IFNg-related gene clusters, and IFNg producing T cell response to NFkB, IL6, IL10, Akt and p38 MAPK-related gene clusters. Further analysis will define and rank genetic variants of candidate immune regulators to help identify vaccine responsive individuals.

HER2 isoforms co-expression differently tunes mammary tumor phenotypes affecting onset, vasculature and therapeutic response.

Full-length HER2 oncoprotein and splice variant Delta16 are co-expressed in human breast cancer. We studied their interaction in hybrid transgenic mice bearing human full-length HER2 and Delta16 (F1 HER2/Delta16) in comparison to parental HER2 and Delta16 transgenic mice. Mammary carcinomas onset was faster in F1 HER2/Delta16 and Delta16 than in HER2 mice, however tumor growth was slower, and metastatic spread was comparable in all transgenic mice. Full-length HER2 tumors contained few large vessels or vascular lacunae, whereas Delta16 tumors presented a more regular vascularization with numerous endothelium-lined small vessels. Delta16-expressing tumors showed a higher accumulation of i.v. injected doxorubicin than tumors expressing full-length HER2. F1 HER2/Delta16 tumors with high full-length HER2 expression made few large vessels, whereas tumors with low full-length HER2 and high Delta16 contained numerous small vessels and expressed higher levels of VEGF and VEGFR2. Trastuzumab strongly inhibited tumor onset in F1 HER2/Delta16 and Delta16 mice, but not in full-length HER2 mice. Addiction of F1 tumors to Delta16 was also shown by long-term stability of Delta16 levels during serial transplants, in contrast full-length HER2 levels underwent wide fluctuations. In conclusion, full-length HER2 leads to a faster tumor growth and to an irregular vascularization, whereas Delta16 leads to a faster tumor onset, with more regular vessels, which in turn could better transport cytotoxic drugs within the tumor, and to a higher sensitivity to targeted therapeutic agents. F1 HER2/Delta16 mice are a new immunocompetent mouse model, complementary to patient-derived xenografts, for studies of mammary carcinoma onset, prevention and therapy.

Abstract 2361: Novel animal models for prediction of cancer vaccine response

Abstract Predicting cancer vaccine response is a challenge that must be addressed in immune competent animals that recapitulate human disease and human response to therapy. Furthermore, genetically heterogeneous humans respond differently to vaccination due, in part, to their varying MHC haplotypes and polymorphisms including those found in costimulatory or checkpoint genes. We previously showed that human HER2 transgenic (Tg) mice of different genetic backgrounds have distinct response to HER2 vaccines in the order of BALB &amp;gt; (BALBxB6)F1 &amp;gt; (B6xDR3)F1 &amp;gt; B6 (Radkevich-Brown 2010). We anticipate even greater variance among human populations, and seek genetically diverse animal models to project human cancer vaccine response. Domestic felines develop mammary tumors with similar pathology and etiology as human cancer, and expression of EGFR, HER2 and HER3 genes that are highly homologous to their human counterparts. Intramuscular electrovaccination of healthy outbred domestic shorthair cats with heterologous (xenogeneic) or single point-mutated self HER2 DNA induces HER2-specific T cells in ∼30% of vaccinated cats (Gibson 2015). Humoral immune responses were induced by both vaccine constructs, with point-mutated feline HER2 vaccine producing antibodies to unique epitopes on feline mammary carcinoma (FMC). The latter inhibits FMC growth in vitro and delays tumor growth in SCID mice. Vaccination with wild type feline HER2 DNA fails to induce specific immunity and even hinders feline HER2 immune activation by other vaccines. Studies in domestic cats provide novel insight on the range and nature of vaccine response in healthy cats which can also be tested in feline cancer patients. Strategic modification of minimal residues may be most effective in inducing reactivity to cancer associated self antigens. To address genetic regulation of vaccine response, diversity outbred (DO) mice offer a pragmatic option. These mice were generated by cross-breeding 8 inbred mouse strains to ensure broad genetic diversity among the mice (Svenson 2012). The human HER2 gene is introduced by breeding DO mice with our human HER2 transgenic (Tg) mice. F1 mice represent a genetically heterogeneous population that expresses human HER2 as a self antigen. Specific genes impacting vaccine response will be identified by genome-wide quantitative trait locus (QTL) scan to associate haplotype or single nucleotide variants with vaccine response. Findings in these mice may lead to the identification of critical genes that regulate cancer vaccine response. Candidate vaccines identified in DO mice can be further validated in cats and expanded to human trials. With these novel animal models, cancer vaccines can be designed and tested with greater precision, and patients with reactive genotypes may be identified before vaccination. (Supported by CA76340) Citation Format: Heather Gibson, Richard F. Jones, Joyce D. Reyes, Wei-Zen Wei. Novel animal models for prediction of cancer vaccine response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2361.

Abstract 1200: HER-2 isoform interaction in mammary carcinoma onset and progression

Abstract Human breast cancer cells express full-length HER-2 along with proteins resulting from mutation, alternative splicing, alternative initiation of translation and post-translational modification. The Delta16 splice variant, lacking exon 16, has the properties of an activated oncogene, but it could also play beneficial roles in the response to targeted therapeutic agents. To study mammary carcinogenesis in a mouse model that mimics human coexpression of full-length HER-2 and Delta16 isoforms, we produced hybrid mice bearing heterozygous copies of both human transgenes (F1 mice), and we compared them to parental mice (Delta16 and HER-2 transgenic mice, respectively). Tumor onset in F1 and Delta16 mice was much faster than in HER-2 mice (30 vs &amp;gt;80 weeks), but the growth of established tumors and metastatic spread were not enhanced. Each mammary carcinoma of F1 mice expressed both isoforms at variable ratios. Most (∼80%) expressed high levels of Delta16 and low levels of full length HER-2, a few (∼5%) expressed full-length HER-2 and little, if any, Delta 16, and the remainder (∼15%) coexpressed at high level both isoforms. The study of tumor vascularization showed that full-length HER-2 tumors mainly contained few large vessels or vascular lacunae, whereas Delta16 tumors were perfused by numerous endothelium-lined small vessels. F1 tumors with high full-length HER-2 expression made few large vessels, whereas tumors with low full-length HER-2 and high Delta16 contained numerous small vessels and expressed high levels of both VEGF and VEGFR2. Administration of trastuzumab to young F1 mice effectively prevented mammary carcinoma onset in ∼85% of mice at 1 year of age. The preventive effect of trastuzumab was stronger in F1 mice than in both parental strain. To analyze the intrinsic sensitivity of F1 mammary carcinoma cells to targeted drugs in 3D, we established cell lines expressing different HER-2 isoform ratios. High Delta16 expression caused high sensitivity to HER-2 inhibitors (trastuzumab, neratinib, lapatinib), whereas high full-length HER-2 was associated with a lower sensitivity. Interestingly, high levels of both isoforms was associated with resistance to trastuzumab, but sensitivity to small molecule inhibitors. In conclusion, the coexpression of full-length HER-2 and Delta16 controls several determinants of mammary carcinoma development, progression and sensitivity to targeted agents, as revealed by the study of F1 mice. Citation Format: Arianna Palladini, Massimiliano Dall’Ora, Tania Balboni, Giordano Nicoletti, Marianna Ianzano, Roberta Laranga, Lorena Landuzzi, Veronica Giusti, Alessia Lamolinara, Carla De Giovanni, Augusto Amici, Serenella M. Pupa, Manuela Iezzi, Patrizia Nanni, Pier-Luigi Lollini. HER-2 isoform interaction in mammary carcinoma onset and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1200.

Isolation of Primary Breast Cancer Cells from HER2 Transgenic Mice

Isolation of Primary Breast Cancer Cells from HER2 Transgenic Mice

Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene.

The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2+/- mice appear to develop in a similar manner to wild type mice (Her2-/-), it has proven difficult to generate homozygous Her2+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2+/+ mice, similar to Pds5b-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.

Bovine herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2+ mammary cancer

ABSTRACTThe epidermal growth factor receptor 2 (HER-2) oncogene is a major target for the immunotherapy of breast cancer. Following up to the therapeutic success achieved with Her-2-targeting monoclonal antibodies, immune-prophylactic approaches directed against Her-2 have also been investigated taking into account, and trying to overcome, Her-2 self-tolerance. Perhaps due to safety (and efficacy) concerns, the least explored anti-Her-2 active immunization strategy so far has been the one relying on viral-vectored vaccine formulations. Taking advantage of the favorable properties of bovine herpesvirus 4 (BoHV-4) in terms of safety and ease of manipulation as well as its previously documented ability to transduce and confer immunogenicity to heterologous antigens, we tested the ability of different recombinant HER-2-BoHV-4 immunogens to 8break tolerance and elicit a protective, anti-mammary tumor antibody response in HER-2 transgenic BALB-neuT mice. All the tested constructs expressed the HER-2 transgenes at high levels and elicited significant cellular immune responses in BALB/c mice upon administration via either DNA vaccination or viral infection. In BALB-neuT mice, instead, only the viral construct expressing the membrane-bound chimeric form of Her-2 protein (BoHV-4-RHuT-gD) elicited a humoral immune response that was more intense and earlier-appearing than that induced by DNA vaccination. In keeping with this observation, two administrations of BoHV-4-RHuT-gD effectively protected BALB-neuT mice from tumor formation, with 50% of vaccinated animals tumor-free after 30 weeks from immunization compared to 100% of animals exhibiting at least one palpable tumor in the case of animals vaccinated with the other BoHV-4-HER-2 constructs.