HER2-overexpressing Xenografts Research Articles

Discovery and development of pyrotinib: A novel irreversible EGFR/HER2 dual tyrosine kinase inhibitor with favorable safety profiles for the treatment of breast cancer

The discovery and development of a novel irreversible EGFR/HER2 dual tyrosine kinase inhibitor SHR1258 (pyrotinib) for the treatment of HER2-postive breast cancer is presented. The structure-activity relationship of lead series and their pharmacokinetic properties were evaluated to identify the potential candidates for further in vivo efficacy studies and preclinical safety assessments. Metabolic pathway and drug-drug interaction were also investigated in preclinical settings. In particular, major metabolites in human and animal species were assessed with regard to potential toxicity or off-target side effects. Overall, the potent and selective EGFR/HER2 dual inhibitor, pyrotinib, displayed robust anti-tumor effects on HER2-overexpressing xenograft models and sufficiently safety windows in animals as well as favorable pharmacokinetic properties in human, which substantially ensures current clinical development. Finally, recent advances of pyrotinib in clinical studies are highlighted with very encouraging outcomes in patients with HER2-postive advanced breast cancer.

Dual HER2 Targeting with Trastuzumab and Liposomal-Encapsulated Doxorubicin (MM-302) Demonstrates Synergistic Antitumor Activity in Breast and Gastric Cancer.

Trastuzumab is the standard of care for HER2-positive breast cancer patients, markedly improving disease-free and overall survival. Combined with chemotherapy, it enhances patient outcomes, but cardiotoxicity due to the trastuzumab treatment poses a serious adverse effect. MM-302 is a HER2-targeted PEGylated liposome that encapsulates doxorubicin to facilitate its delivery to HER2-overexpressing tumor cells while limiting exposure to nontarget tissues, including the heart. In this study, we evaluated the feasibility and preclinical activity of combining MM-302 with trastuzumab. MM-302 and trastuzumab target different domains of the HER2 receptor and thus could simultaneously bind HER2-overexpressing tumor cells in vitro and in vivo. Furthermore, trastuzumab did not disrupt the mechanism of action of MM-302 in delivering doxorubicin to the n0ucleus and inducing DNA damage. Reciprocally, MM-302 did not interfere with the ability of trastuzumab to block prosurvival p-Akt signaling. Interestingly, coadministration of the two agents acutely increased the deposition of MM-302 in human xenograft tumors and subsequently increased the expression of the DNA damage marker p-p53. Finally, the combination of MM-302 and trastuzumab induced synergistic antitumor activity in HER2-overexpressing xenograft models of breast and gastric cancer. Collectively, our findings highlight a novel combination therapy that efficiently targets HER2-overexpressing cells through multiple mechanisms and support the ongoing investigation of combined MM-302/trastuzumab therapy for HER2-positive metastatic breast cancer in a randomized phase II clinical trial.

Abstract B32: Microenvironmental distribution of trastuzumab in metastases and xenograft models is highly heterogeneous and decreases sharply when administered in combination with bevacizumab

Abstract Despite significant success, the response of Her2+ patients to trastuzumab (TzMAb, Herceptin ®) is varied, with many still experiencing tumor progression. We have used 3D tissue, tumor xenograft and metastatic models to examine the microregional distribution of TzMAb when administered alone or in combination with bevacizumab (BvMAb). Methods: Her2 positive (Her2+) SKOV-3 ovarian and MDA361, JIMT-1, BT474 mammary cancer cells were grown as 3D tissue discs, spheroids and as xenografts. Her2 expression was ranked as SKOV3 > BT474 > JIMT-1 > MDA361. Variable concentrations (25-100 µg/mL for in vitro; 2.5-10 mg/kg ip q3d for in vivo) of TzMAb, BvMAb or isotype IgG control antibodies were administered and tissues collected. Multiplexed immunohistochemistry generated maps of whole tissue sections for quantitative and qualitative analysis. In addition to direct visualization of fluorescent-tagged antibody therapeutics, features including Her2 expression (Her2), hypoxia (pimonidazole), blood vessels (CD31), vascular perfusion (carbocyanine fluorescent dye), pericytes (SMA, desmin), basal lamina (CIV) and tight junctions (ZO-1) were mapped relative to each other. Findings: In vitro: The rate of TzMAb distribution through 3D Her2+ tissue models was not significantly different to that of BvMAb or an isotype control (IgG). Inter-model variability was not correlated with the degree of Her2 expression. In vivo: All the xenograft models exhibited highly heterogeneous distribution of TzMAb, with variation at inter-vessel, inter-tumor and intra-tumor levels. There was no discernible pattern in the deposition of TzMAb; it was not limited to tumor margins or the central core, and was often bound up to 200 µm away from the nearest blood vessels. Other areas containing perfused vessels had little to no binding. Areas of limited TzMAb binding persisted after repeat dosing. SKOV-3 and BT474 metastatic lesions collected from liver and lung tissues had on average greater TzMAb staining intensity than was seen in subcutaneous tumors. However, there was a significant range in the ability of TzMAb to access and bind Her2+ve cells. Lesions ranged from less than 150 µm to several mm in diameter, with some metastases showing bound TzMAb on every cell and others showing no observable bound drug despite containing perfused vessels or being surrounded by highly perfused normal lung or liver tissue. For both subcutaneous tumors and metastatic lesions, no consistent, quantifiable difference or correlation was found between the amount of bound TzMAb and the presence of tight junctions, the density of nearest CD31 vessels, the location of hypoxic cells or the fraction of perfused, mature or immature vessels. Pretreatment with BvMAb for 24-72h resulted in significant reductions (up to 90% reduced) in tumor accumulation of TzMAb in all tumor models examined, including lung and liver metastases, despite the continued presence of perfused vessels. Summary: The extravascular distribution of TzMAb in Her2-overexpressing xenografts and metastases is heterogeneous and is not explained simply by the presence or absence of functioning vasculature. The relatively good distribution of TzMAb through 3D tissue models suggest that tight or irreversible binding to Her2 may not adequately explain the limited distribution of TzMAb seen in vivo. Pre-treatment with BvMAb dramatically decreases the access of TzMAb to the tumor microenvironment. Persistence of metastases and tissues with poor access to TzMAb suggests the possibility of inadequate drug exposure as a mechanism for resistance to TzMAb activity that could apply to additional targeted monoclonal antibody therapeutics. This resistance may be exacerbated when TzMAb or other antibodies are administered in combination with anti VEGF therapy. Citation Format: Jennifer H.E. Baker, Alastair H. Kyle, Jordan Cran, Haley Patrick, Maria-Jose Gandolfo, Andrew I. Minchinton. Microenvironmental distribution of trastuzumab in metastases and xenograft models is highly heterogeneous and decreases sharply when administered in combination with bevacizumab. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B32.

Abstract 2988: Microenvironmental distribution of trastuzumab is heterogeneous and decreases sharply when administered following a single dose of bevacizumab in Her2+ve xenografts and metastases models

Abstract We have previously shown that trastuzumab distributes heterogeneously within MDA-435-Her2 xenografts. Though peak accumulation occurs 24h after administration & trastuzumab was detected greater than 150 µm from many vessels, there remained substantial Her2+ve areas that were negative for bound drug. Methods: Her2+ve human carcinoma xenografts were grown in NOD/SCID mice as solid tumors or as metastatic models following ip or iv injection. Tumors were MR-imaged (T1-weighted) with a 500kDa Gd-labeled hyperbranched polyglycerol (HPG) contrast agent (CA). Treatments included bevacizumab (BvMAb, 10 mg/kg), trastuzumab (TsMab, 10 mg/kg) and human IgG (10 mg/kg). Multiplexed staining and imaging of whole tumor sections was used to evaluate the distribution of TsMAb in the context of the tumor microenvironment. Findings: BT474, MDA361 and SKOV3 primary tumor xenografts all show highly heterogeneous distribution of TsMAb at 24h, where some areas are highly saturated with TsMAb and others contain unbound Her2+ tissue. Small metastases in the peritoneal cavity and lung also exhibit substantial proportions of tissue that are negative for TsMAb. CD31-stained vessels dual-labeled for a perfusion marker are often seen to have little or no bound TsMAb on perivascular tumor cells. Pretreatment with BvMAb for 24-72h results in significant reductions in tumor accumulation of TsMAb, with tumors showing 60-90% of bound TsMAb relative to IgG-treated controls (p<0.05). BvMAb -treated xenografts had reduced fractions of perfused vessels and reduced measures of vascular function assessed using DCE-MRI (49% reduction in CA, p<0.05). Corresponding parameter maps of MR-imaged high vascular function and histological images of bound TsMAb do not consistently correlate. Detailed maps of the tumor microenvironment show that even with VEGF blockade, the distribution of TsMAb still does not correspond to vessels that are perfused. Significant numbers of perfused vessels persist with no perivascular bound TsMAb despite the presence of Her2+ cells; saturation with bound TsMAb is also not consistently correlated with the presence or absence of smooth muscle, collagen or ZO-1 stained tight junctions, examined using immunohistochemistry. Summary: The extravascular distribution of TsMAb in Her2-overexpressing xenografts, including micrometastases, is heterogeneous and is not explained simply by the presence or absence of vascular function. Pre-treatment with BvMAb for even short exposures of 24-72h worsens the distribution of TsMAb within the tumor microenvironment. The persistence of tissues that are unbound for trastuzumab suggests the possibility of inadequate drug exposure as a mechanism for drug resistance that may be exacerbated by pre-treatment with a VEGF blockade. Citation Format: Jennifer H. E. Baker, Alastair H. Kyle, Stefan A. Reinsberg, Firas Moosvi, Jordan Cran, Urs Hafeli, Katayoun Saatchi, Andrew I. Minchinton. Microenvironmental distribution of trastuzumab is heterogeneous and decreases sharply when administered following a single dose of bevacizumab in Her2+ve xenografts and metastases models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2014-2988

Utility of [18 F]FLT-PET to assess treatment response in trastuzumab-resistant and trastuzumab-sensitive HER2-overexpressing human breast cancer xenografts.

The objective of this study was to evaluate 3'-deoxy-3'-[(18) F]fluorothymidine ([(18) F]FLT) positron emission tomography (PET) as an early marker of trastuzumab response in HER2-overexpressing xenografts. Tumor-to-muscle ratios were compared between both trastuzumab-sensitive and trastuzumab-resistant cohorts prior to and after one and two treatments. A significant difference (P = 0.03) was observed between treated and control trastuzumab-sensitive xenografts after one treatment, which preceded between-group differences in tumor volume. Reduced Ki67 (P = 0.02) and thymidine kinase 1 (TK1) (P = 0.35) immunoreactivity was observed in the treated xenografts. No significant differences in volume, tumor-to-muscle ratio, or immunoreactivity were observed between treated and control trastuzumab-resistant cohorts. A significant difference (P = 0.02) in tumor-to-muscle ratio was observed between trastuzumab-sensitive and trastuzumab-resistant cohorts after two treatments; however, tumor volumes were also different (P = 0.04). Ki67 (P = 0.04) and TK1 (P = 0.24) immunoreactivity was ~50% less in trastuzumab-sensitive xenografts. [(18) F]FLT-PET provided early response assessment in trastuzumab-sensitive xenografts but only differentiated between trastuzumab-resistant and trastuzumab-sensitive xenografts concurrent with differences in tumor size.

Active Targeting of Block Copolymer Micelles with Trastuzumab Fab Fragments and Nuclear Localization Signal Leads to Increased Tumor Uptake and Nuclear Localization in HER2-Overexpressing Xenografts

Block copolymer micelles (BCMs) have been employed as effective drug delivery systems to solid tumors by virtue of their capacity to transport large therapeutic payloads and passively target tumor sites. Active targeting of nanoparticles (NPs) has been exploited as a means to increase the therapeutic efficacy of NP-based drugs by promoting their delivery to cellular sites of action. Effective whole tumor accumulation and cellular uptake constitute key objectives in the success of preclinical drug formulations, although they have seldom been investigated concurrently in vivo. The current study aims to elucidate the in vivo fate of 31-nm-sized block copolymer micelles (BCMs) targeted to the nucleus of HER2-overexpressing breast cancer cells. Pharmacokinetics, biodistribution, tumor uptake, and intratumoral distribution of BCMs were investigated in mice bearing subcutaneous BT-474 and MDA-MB-231 xenografts expressing high and low levels of HER2, respectively. Radiolabeling with (111)indium enabled quantitative assessment of BCM distribution at the whole body, tissue, and cellular levels. Surface-grafted trastuzumab Fab fragments (TmAb-Fab) facilitated binding and internalization of BCMs by HER2-positive breast cancer cells, while synthetic 13-mer nuclear localization signal (NLS) peptides conjugated to the TmAb-Fab conferred nuclear translocation capability. Active targeting of BCMs led to a 5-fold increase in tumor uptake in HER2-overexpressing BT-474 tumors, alongside a correspondingly greater level of cellular uptake and nuclear localization, relative to the nontargeted formulations. This study distinctively highlights the quantitative evaluation of active targeting on tumor, cellular and subcellular uptake of BCMs and presents a promising platform for the effective delivery of chemo- and/or radiotherapy in vivo.

Abstract P2-06-09: Mucin4 Is Associated with the Loss of Estrogen-Receptor-α in Breast Cancer

Abstract Data suggests that breast carcinomas that overexpress the HER2 oncoprotein are more resistant to endocrine treatments such as tamoxifen (Tam) or estrogen deprivation (ED) via aromatase inhibitors. To investigate possible mechanisms of endocrine resistance in vivo, we performed a gene microarray analysis on estrogen receptor-(ER)+, HER2-overexpressing xenograft tumors that are de novo resistant to tamoxifen treatment and acquire resistance to ED therapy. A large number of genes were upregulated, including several members of the mucin family of genes. This corroborated our finding from histological staining of the resistant tumors that indicated striking increases of mucin vacuoles when compared to sensitive tumors. These vacuoles were located mostly intracellularly and stained positive for mucins with mucicarmine. We were particularly interested in one particular mucin, mucin4 (MUC4) because previous studies have suggested its role in stabilizing and enhancing HER2 signaling. Quantitative realtime PCR confirmed the microarray results and showed that there was an approximately 156-fold increase in MUC4 transcript between the ED-sensitive and ED-resistant tumors, with a 65-fold increase between the early Tam and late Tam-resistant tumors. Immunohistochemistry confirmed that this upregulation in these two treatment groups was present also at the protein level. Furthermore, western blot analysis of these tumors demonstrated that resistant tumors have downregulation of ER protein and its downstream effectors with corresponding increase in total HER2 protein levels. These tumors have seemingly shifted away from the ER pathway to signaling via the growth factor signaling pathway. Preliminary experiments where MUC4 protein was stably overexpressed in ER+, HER2-overexpressing cells model these results in vitro with a downregulation of ER protein and downstream effectors, showing that ER expression and activity are affected. Furthermore, immunohistochemical analysis of 73 primary human invasive breast carcinomas suggests that MUC4 is associated with estrogen-receptor negative tumors (P=0.05). This clinical data validates the preclinical studies for investigating a possible mechanism for the loss of ER, and suggest MUC4 as an inviting biomarker for endocrine treatment and a potential therapeutic target for endocrine resistant breast cancer. Crosstabulation of MUC4 and ER Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-09.

Intracellular Src Family Kinases Mediate PI3K-Akt Pathway Activation and Resistance to Lapatinib in HER2-Overexpressing Human Breast Cancer Cells.

Abstract We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide-3 kinase (PI3K)-Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2-overexpressing breast cancer cells (BT-474, SKBR-3, HCC1954, MDA-MB-361, and SUM190) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib-resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P-HER2 immunoblots was undetectable in LR cells. However, they consistently showed recovery of PI3K-Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr-phosphorylated peptides and identification by tandem liquid chromatography-mass spectrometry (LC-MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P-tyr antibody followed by tandem LC-MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr-phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein subtrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P-SFK in BT-474 and HCC1954 LR cells compared to drug-sensitive cells. The MS phosphotyrosine profiling identified Yes in BT-474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT-PCR with SFK-specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT-PCR suggested that Lyn and/or Src are the active kinases. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P-Src, partial inhibition of S473 P-Akt, and inhibition of cell growth. Treatment of HCC1954 sensitive cells with AZD0530 and Lap for 14 days partially abrogated the emergence of lapatinib-resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K-Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2-overexpressing xenografts are ongoing at this time. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 707.

Abstract PR-6: Src family kinases mediate PI3K-Akt pathway activation and resistance to lapatinib in HER2-overexpressing human breast cancer cells

Abstract We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide-3 kinase (PI3K)-Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2-overexpressing breast cancer cells (BT-474, SKBR-3, HCC1954, MDA-MB-361, SUM190, and UACC893) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib-resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P-HER2 immunoblots remained inhibited in LR cells. However, they consistently showed recovery of PI3K-Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr-phosphorylated peptides and identification by tandem liquid chromatography-mass spectrometry (LC-MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P-tyr antibody followed by tandem LC-MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr-phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein substrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P-SFK in four of the LR lines: BT-474, MDA-MB-361, HCC1954, and UACC893. The MS phosphotyrosine profiling identified Yes in BT-474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT-PCR with SFK-specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT-PCR suggested that Lyn and/or Src are the active kinases, whereas Yes was the predominant SFK in MDA-MB-361 and UACC893 cells. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P-Src, partial inhibition of S473 P-Akt, and inhibition of cell growth. Treatment of HCC1954 or MDA-MB-361 sensitive cells with Src inhibitors and Lap for 21 days partially abrogated the emergence of lapatinib-resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K-Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2-overexpressing xenografts are ongoing at this time. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-6.

18F-FDG Small-Animal PET/CT Differentiates Trastuzumab-Responsive from Unresponsive Human Breast Cancer Xenografts in Athymic Mice

Breast cancers (BCs) with high human epidermal growth factor receptor type 2 (HER2) expression are most likely to respond to trastuzumab; however, the mechanisms of action of trastuzumab are complex and there are no established biomarkers to accurately monitor treatment outcome in individual patients. Therefore, our aim was to determine, in human BC xenografts in athymic mice treated with trastuzumab, whether there were any changes in (18)F-FDG uptake that were associated with response to the drug and that could have utility in monitoring response in patients. Baseline tumor uptake of (18)F-FDG was measured in mice with MDA-MB-361 HER2-overexpressing xenografts and MDA-MB-231 xenografts with low HER2 expression by small-animal PET imaging on day 0. Mice were treated with phosphate-buffered saline (PBS) or trastuzumab (4 mg/kg), and small-animal PET was repeated 2 d after treatment. Maintenance doses of trastuzumab (2 mg/kg) or PBS were administered on days 7 and 14, and mice were imaged again on days 9 and 16. Tumor uptake was measured as percentage injected dose per gram (%ID/g) by volume-of-interest analysis on days 0 (baseline), 2, 9, and 16, followed by biodistribution studies on day 16. Tumor growth was measured, and a tumor growth index was calculated. The treatment of mice with trastuzumab, compared with control mice treated with PBS, resulted in a significant decrease in tumor uptake of (18)F-FDG in HER2-overexpressing MDA-MB-361 xenografts after 16 d of treatment (2.6 +/- 0.8 %ID/g vs. 4.6 +/- 1.8 %ID/g, respectively; P < 0.03) but not after 2 or 9 d of treatment (P = 0.28-0.32). In contrast, there was no significant change in the tumor uptake of MDA-MB-231 xenografts with low HER2 expression during the entire course of therapy (4.4 +/- 1.7 %ID/g vs. 3.6 +/- 1.1 %ID/g, respectively; P = 0.31). Trastuzumab treatment, compared with PBS treatment of controls, resulted in significant growth inhibition of MDA-MB-361 xenografts as early as 10 d from the initiation of treatment (tumor growth index, 0.7 +/- 0.2 vs. 1.7 +/- 0.3, respectively; P < 0.0005), whereas no tumor growth inhibition was observed for MDA-MB-231 xenografts (5.3 +/- 2.7 and 5.2 +/- 3.0; P = 0.95). Changes in the tumor uptake of (18)F-FDG after therapy accurately identified responding and nonresponding human BC xenografts in athymic mice treated with trastuzumab; however, diminished glucose utilization did not precede changes in tumor volume.

Treatment of Human Epidermal Growth Factor Receptor 2-Overexpressing Breast Cancer Xenografts With Multiagent HER-Targeted Therapy

Human epidermal growth factor receptor 2 (HER2) is a member of the HER signaling pathway. HER inhibitors partially block HER signaling and tumor growth in preclinical breast cancer models. We investigated whether blockade of all HER homo- and heterodimer pairs by combined treatment with several inhibitors could more effectively inhibit tumor growth in such models. Mice carrying xenograft tumors of HER2-overexpressing MCF7/HER2-18 (HER2-transfected) or BT474 (HER2-amplified) cells were treated with estrogen supplementation or estrogen withdrawal, alone or combined with tamoxifen. One to three HER inhibitors (pertuzumab, trastuzumab, or gefitinib) could also be added (n > or = 8 mice per group). Tumor volumes, HER signaling, and tumor cell proliferation and apoptosis were assessed. Results were analyzed with the t test or Wilcoxon rank sum test and survival analysis methods. All statistical tests were two-sided. Median time to tumor progression was 21 days for mice receiving estrogen and 28 days for mice receiving estrogen and pertuzumab (difference = 7 days; P = .001; hazard ratio [HR] of progression in mice receiving estrogen and pertuzumab versus mice receiving estrogen = 0.27, 95% confidence interval [CI] = 0.09 to 0.77). Addition of gefitinib and trastuzumab to estrogen and pertuzumab increased this time to 49 days (difference = 21 days; P = .004; HR of progression = 0.28, 95% CI = 0.10 to 0.76). MCF7/HER2-18 tumors disappeared completely and did not progress (for > or = 189 days) after combination treatment with pertuzumab, trastuzumab, and gefitinib plus tamoxifen (19 of 20 mice) or plus estrogen withdrawal (14 of 15 mice). Both combination treatments induced apoptosis and blocked HER signaling and proliferation in tumor cells better than any single agent or dual combination. All BT474 tumors treated with pertuzumab, trastuzumab, and gefitinib disappeared rapidly, regardless of endocrine therapy, and no tumor progression was observed for 232 days. Combined treatment with gefitinib, trastuzumab, and pertuzumab to block signals from all HER homo- and heterodimers inhibited growth of HER2-overexpressing xenografts statistically significantly better than single agents and dual combinations.

177Lu]Pertuzumab: Experimental Therapy of HER-2–Expressing Xenografts

Pertuzumab (Omnitarg) is a novel antibody against HER-2, domain II. HER-2 is a tyrosine kinase receptor that is overexpressed in several carcinomas, especially breast cancer. Pertuzumab, labeled with the low-energy beta emitter (177)Lu, might be a candidate for targeted radiotherapy of disseminated HER-2-positive micrometastases. The radiolabeled antibody [(177)Lu]pertuzumab showed favorable targeting properties in BALB/c (nu/nu) mice with HER-2-overexpressing xenografts. The absorbed dose in tumors was more than five times higher than the absorbed dose in blood and more than seven times the absorbed dose in any other normal organ. Experimental therapy showed that [(177)Lu]pertuzumab delayed tumor progression compared with controls (no treatment, P < 0.0001; nonlabeled pertuzumab antibody, P < 0.0001; and (177)Lu-labeled irrelevant antibody, P < 0.01). No adverse side effects of the treatment could be detected. Thus, the experimental results support the planning of clinical studies applying [(177)Lu]pertuzumab for therapy.

Selective proapoptotic activity of a secreted recombinant antibody/AIF fusion protein in carcinomas overexpressing HER2

Apoptosis-inducing factor (AIF) represents a caspase-independent apoptotic pathway in the cell, and a mitochondrial localization sequence-truncated AIF (AIFDelta1-120) can be relocated from the cytoplasm to the nucleus and exhibit a constitutive proapoptotic activity. Here, we generated a chimeric immuno-AIF protein, which comprised an HER2 antibody, a Pseudomonas exotoxin translocation domain and AIFDelta1-120. Human Jurkat cells transfected with the immuno-AIF gene could express and secrete the chimeric protein, which selectively recognized HER2-overexpressing tumor cells and was endocytosed. Subsequent cleavage of truncated AIF from immuno-AIF and its release from the internalized vesicles resulted in apoptosis of tumor cells. Intramuscular injection of the immuno-AIF gene caused significant suppression of tumors and substantially prolonged mice survival in an HER2-overexpressing xenograft tumor model. Our study demonstrates the feasibility of the immuno-AIF gene as a novel approach to treating cancers that overexpress HER2.

Tumor targeting using anti-her2 immunoliposomes

We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes ( P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0–8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil ®). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.

Bispecific HER2 × CD3 Antibodies Enhance T-Cell Cytotoxicity in Vitro and Localize to HER2-Overexpressing Xenografts in Nude Mice

Recently, we reported the development of fully humanized bispecific F(ab′) 2 antibodies with dual binding specificities to human T-lymphocytes and to tumor cells overexpressing HER2. These antibodies were shown to effectively mediate targeted HER2-overexpressing tumor cell killing by freshly isolated human T-cells. In this report we extend our studies to describe the interaction of the bispecific antibody with activated T-lymphocytes (ATL) maintained in culture for an extended period of time. A microtiter plate radioreceptor assay was used to elucidate the affinity of bispecific antibody binding to ATL. The data show that ATL maintained in vitro for up to 5 weeks continued to express high-affinity CD3 surface markers that bound to bispecific antibody with a Kd of 2.49 nM and exerted cytolytic activities against targets overexpressing HER2. In addition, we demonstrated the specific localization of HER2 × CD3 bispecific antibody to HER2-overexpressing tumor xenografts in nude mice. Furthermore, HER2 × CD3 bispecific antibody has the ability to inhibit the proliferative activities of breast tumor (SKBR-3) cells in vitro. The clinical implications of these data are discussed.