HER2-expressing Tumor Cells Research Articles

Antitumor activity of phenethyl isothiocyanate in HER2-positive breast cancer models

BackgroundHER2 is an oncogene, expression of which leads to poor prognosis in 30% of breast cancer patients. Although trastuzumab is apparently an effective therapy against HER2-positive tumors, its systemic toxicity and resistance in the majority of patients restricts its applicability. In this study we evaluated the effects of phenethyl isothiocyanate (PEITC) in HER2-positive breast cancer cells.MethodsMDA-MB-231 and MCF-7 breast cancer cells stably transfected with HER2 (high HER2 (HH)) were used in this study. The effect of PEITC was evaluated using cytotoxicity and apoptosis assay in these syngeneic cells. Western blotting was used to delineate HER2 signaling. SCID/NOD mice were implanted with MDA-MB-231 (HH) xenografts.ResultsOur results show that treatment of MDA-MB-231 and MCF-7 cells with varying concentrations of PEITC for 24 h extensively reduced the survival of the cells with a 50% inhibitory concentration (IC50) of 8 μM in MDA-MB-231 and 14 μM in MCF-7 cells. PEITC treatment substantially decreased the expression of HER2, epidermal growth factor receptor (EGFR) and phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr-705. The expression of BCL-2-associated × (BAX) and BIM proteins were increased, whereas the levels of B cell lymphoma-extra large (BCL-XL) and X-linked inhibitor of apoptosis protein (XIAP) were significantly decreased in both the cell lines in response to PEITC treatment. Substantial cleavage of caspase 3 and poly-ADP ribose polymerase (PARP) were associated with PEITC-mediated apoptosis in MDA-MB-231 and MCF-7 cells. Notably, transient silencing of HER2 decreased and overexpressing HER2 increased the effects of PEITC. Furthermore, reactive oxygen species (ROS) generation, mitochondrial depolarization and apoptosis by PEITC treatment were much higher in breast cancer cells expressing higher levels of HER2 (HH) as compared to parent cell lines. The IC50 of PEITC following 24 h of treatment was reduced remarkably to 5 μM in MDA-MB-231 (HH) and 4 μM in MCF-7 (HH) cells, stably overexpressing HER2. Oral administration of 12 μM PEITC significantly suppressed the growth of breast tumor xenografts in SCID/NOD mice. In agreement with our in vitro results, tumors from PEITC-treated mice demonstrated reduced HER2, EGFR and STAT3 expression and increased apoptosis as revealed by cleavage of caspase 3 and PARP. In addition our results show that PEITC can enhance the efficacy of doxorubicin.ConclusionsOur results show a unique specificity of PEITC in inducing apoptosis in HER2-expressing tumor cells in vitro and in vivo and enhancing the effects of doxorubicin. This unique specificity of PEITC offers promise to a subset of breast cancer patients overexpressing HER2.

Anti-Her2 single-chain antibody mediated DNMTs-siRNA delivery for targeted breast cancer therapy

The targeted delivery of small interfering RNA (siRNA) to specific tumor tissues and tumor cells remains as one of the key challenges in the development of RNA interference as a therapeutic application. To target breast cancer, we developed a therapeutic delivery system using a fusion protein of an anti-Her2 single-chain antibody fragment with a positively charged protamine, namely F5-P, as the carrier to specifically deliver siRNA-targeting DNA methyltransferases 1 and/or 3b genes (siDNMTs) into Her2-expressing breast tumor cells. The carrier F5-P, expressed by the Escherichia coli system, was able to bind siRNA molecules and specifically deliver the siRNA to Her2-expressing BT474 breast cancer cells but not Her2-nonexpressing MDA-MB-231 breast cancer cells, while delivery of siDNMTs to BT474 cells successfully silenced the expression of targeted DNA methyltransferases (DNMTs) and facilitated the de-methylation of the RASSF1A tumor suppressor gene promoter, leading to the suppression of tumor cell proliferation. Moreover, as demonstrated in the BT474 xenograft murine model, F5-P successfully delivered siRNA into a Her2-expressing breast tumor, and tumor growth inhibition was mediated by an intravenous injection of F5-P/siDNMTs complex by down-regulating the expression of DNMTs and restoring tumor suppressor gene expression. These data suggest that the delivery of siDNMTs by F5-P could be used to treat Her2-expressing breast cancer.

Development of 177Lu‐nanobodies for radioimmunotherapy of HER2‐positive breast cancer: evaluation of different bifunctional chelators

Nanobodies show favourable pharmacokinetic characteristics for tumor targeting, including high tumor-to-background-ratios. Labelled with a therapeutic radionuclide, nanobodies could be used as an adjuvant treatment option for HER2-overexpressing minimal residual disease. The therapeutic radionuclide Lutetium-177 is linked to the nanobody using a bifunctional chelator. The choice of the bifunctional chelator could affect the in vivo behaviour of the radiolabeled nanobody. Consequently, we compared four different bifunctional chelators - p-SCN-Bn-DOTA, DOTA-NHS-ester, CHX-A"-DTPA or 1B4M-DTPA - in order to select the optimal chemical link between Lutetium-177 and a HER2 targeting nanobody. MS results revealed different degrees of chelator-conjugation. High stability in time was observed, together with nanomolar affinities on HER2-expressing tumor cells. Ex vivo biodistributions as well as SPECT/micro-CT analyses showed high activities in tumors expressing medium HER2 levels with low background activity except for the kidneys. The 1B4M-DTPA-coupled conjugate was further evaluated in a high HER2-expressing tumor model. Here, tumor uptake values of 5.99 ± 0.63, 5.12 ± 0.17, 2.83 ± 0.36 and 2.47 ± 0.38 %IA/g were obtained at 1, 3, 24 and 48h p.i., which coincided with exceptionally low background values, except for the kidneys, and unprecedented tumor-to-background ratios. No specific binding was observed in a HER2-negative model. In conclusion, the in-house developed anti-HER2 nanobody 2Rs15dHIS can be successfully labeled with (177) Lu using different bifunctional chelators. Both macrocyclic and acyclic chelators show high stability in time. High specific tumor uptake combined with the lowest background uptake was measured using the 1B4M-DTPA-based conjugate.

Combining Oncolytic Vaccinia Virotherapy with Adoptive T Cell Therapy,

Abstract 4042 Background.Immunotherapy with cytotoxic T lymphocytes (CTLs) has proved safe and effective for the treatment of post-transplant viral re-activation and lymphoproliferative diseases. T cells specific for non-viral tumor antigens (TA) have been effective for certain tumors but their efficacy is limited. To improve T cell activity and circumvent tumor evasion T cells have been genetically modified with chimeric antigen receptors (CARs), that consist of extracellular variable domains of antibodies specific for tumor cell surface markers linked via a transmembrane domain to the z chain of the T cell receptor. Although the persistence of CAR-T cell can be improved by the inclusion of signaling domains from various costimulatory molecules, these rarely produce sufficient proliferation and persistence in vivo. An alternative way to promote T cell expansion in vivo is by vaccination. If the T cells carrying the transgenic CAR are naturally specific for a vaccine antigen, then the vaccine could be used in vivo to enhance proliferation and anti-tumor activity.The oncolytic vaccinia virus (OVV) JX-594 produces reduction in tumor bulk, but complete remissions have not been published to date. JX-594 is tumor selective due to its dependency on increased EGFP-Ras pathway and sensitivity to IFN and it potently activates innate immunity by transgenic expression of human GM-CSF and TLR stimulation. We hypothesize that JX-594 could enhance the antitumor efficacy of vaccinia virus (VV)-specific T cells expressing tumor-specific CARs while at the same time reducing tumor mass allowing increased T cell penetration and survival.To identify VV vaccine antigens that induce T cell proliferation in vivo, we selected 6 different VV antigens and asked first if they could activate memory T cells from vaccinated individuals and second if the frequency of these T cells in vivo increased in response to vaccination both in healthy donors and in patients with hepatocellular carcinoma (HCC) who had received OVV treatment. We then determined if VV-specific T cells could be induced to express a CAR specific for human epidermal growth factor receptor 2, HER2 that is expressed on a range of solid tumors. Methods.PBMCs were obtained from healthy donors and patients who had received intratumoral JX-594 injections at the University of California in San Diego, USA, and McMaster University Medical Center, Canada. PBMCs were stimulated with overlapping peptide libraries (20aa overlapping by 15aa) spanning the entire protein sequences of the A10L, D8L, H3L, G5R, B22R, and D8L antigens of VV. Three days after stimulation, cells were transduced with retroviral vector encoding HER2.CAR and then expanded as for non-transduced T cells. After 9 days T cells were tested for their dual specificity and function in ELIspot and cytotoxicity assays. Results.We activated VV-specific T cells from 20/21 healthy donors vaccinated from 1 month to over 40 years previously. VV-specific T cell lines recognized a median of 4 of the 6 VV antigens (range 0 to 6). The frequency of VV-specific T cells increased in response to vaccination in healthy seronegative donors and in patients receiving multiple OVV injections. Transduced VV-specific T cells expressed the HER2.CAR in 40 to 50% of cells and CAR(+) T cells secreted g-IFN in response to stimulation with VV peptides in intracellular cytokine assays, validating their dual specificity. HER2.CAR-transduced VV-specific T cells killed both VV peptide pulsed activated T cells and HER2-expressing tumor cells in an HLA-independent manner.In most donors we were not able to detect VV-specific T cells without prior stimulation. However VV-specific T cells from two healthy donors could be detected in blood and expanded 120 and 90 fold, respectively, after one stimulation. In all the other donors, VV-specific T cells expanded to comparable frequencies at the end of the first stimulation, range 0.1% to 5% (with a median of 1.5%) of the final T cell population as measured by ELISpot assay. Conclusions and future plans.We have identified 6 VV antigens that consistently reactivate and expand T cells after vaccination. T cells specific for these antigens will therefore be suitable hosts for tumor-specific CARs that can be reactivated and expanded in vivo after adoptive transfer until the tumor is eliminated. We will test our combined OVV & adoptive T cell strategy in an immunocompetent murine model prior to evaluation in clinical trials. Disclosures:No relevant conflicts of interest to declare.

Abstract 3637: MM-302, a HER2-targeted liposomal doxorubicin, shows binding/uptake and efficacy in HER2 2+ cells and xenograft models

Abstract Introduction: MM-302 is an ErbB2/HER2-targeted liposomal doxorubicin formulation designed to target doxorubicin to HER2-overexpressing cancer cells while limiting uptake into non-target cells. The characterization of the binding and uptake of MM-302 as a function of HER2 surface levels on various cell lines and in tumor models will help determine the minimum HER2 receptor levels required for MM-302 patient selection. Methods: Cellular models included 16+ established human tumor cell lines as well as 4T1 murine breast cancer and HeLa human cervical cancer cells engineered to overexpress HER2 from 1E05-1E06 receptors/cell. Cells were incubated with MM-302, untargeted liposomes or free doxorubicin and binding/uptake was measured by HPLC or flow cytometry. In vivo efficacy was determined in select mouse xenograft models. Tumors were mechanically and enzymatically dissociated, followed by flow cytometry to quantify the uptake of targeted and untargeted liposomes into HER2-expressing tumor cells, macrophages and other cell types. Results: Binding and uptake of MM-302 increased with rising HER2 surface levels in cells of common parental origin (4T1, HeLa), with significant uptake into cells with >2E05 HER2/cell. Further, total cell binding/uptake of MM-302 also increased with rising HER2 expression across a panel of 16+ cell lines. There was negligible uptake of untargeted liposomes under all conditions that showed no dependence on HER2 expression. Improved anti-tumor efficacy of MM-302 was observed in BT474-M3 and NCI-N87 mouse xenograft models relative to untargeted liposomal doxorubicin and free doxorubicin. Further, using disaggregated tumors, the relationship between HER2 expression and MM-302 uptake was confirmed in vivo in both models. Conclusions: Our studies identified a HER2 threshold for uptake of MM-302 of approximately 2E05 receptors/cell, and suggest a role for MM-302 not only in HER2 3+ cancers, but also in HER2 2+ cancers as well. The ability to treat HER2 2+ tumors offers an exciting opportunity to serve a major unmet clinical need. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3637. doi:10.1158/1538-7445.AM2011-3637

Abstract 3616: Highly potent antibody-amanitin conjugates cause tumor-selective apoptosis

Abstract Alpha-amanitin blocks RNA polymerase II activity and is the most specific and most potent inhibitor of eukaryotic transcription. While widely used as a tool in molecular biology, only a few attempts have been made to employ alpha-amanitin as a therapeutic agent in oncology. The reason for this may be the high liver toxicity caused by the transport of amanitin into hepatocytes by the organic-anion transporter 1B3 (OATP1B3) present on hepatocytes, but not on other cell types. In this study we show that a direct impact on the transcription machinery can provide a novel approach in tumor therapy, provided that the amanitin can be targeted to tumor cells. This was achieved by using tumor-specific antibodies of high affinity armed with amanitin via suitable linker moieties. Using the HER2 specific monoclonal antibody Herceptin (Her) as a carrier, the antibody-drug conjugate (ADC) Her-DSC-30.0134 exhibits antiproliferative activity in HER2-expressing human tumor cell lines like SK-OV-3, SK-BR-3 and NCI-N87 in picomolar concentrations, indicating an up to 100.000-fold higher toxicity of the conjugate over non-conjugated alpha-amanitin. On cells lacking the HER2 receptor, e.g. HEK293 cells, toxicity of Her-DSC-30.0134 was not detected up to micromolar concentrations. This is also observed in HEK293 cells transfected with OATP1B3 (which incorporate amanitin similar as hepatocytes) indicating that amanitin-antibody-conjugates are no longer substrates for OATP1B3. These data suggest that a low hepatotoxicity of amanitin conjugates is expected. Subsequently, antitumoral activity of Her-DSC-30.0134 was evaluated in vivo in a s.c. xenograft mouse model using Herceptin-sensitive SK-OV-3 and Herceptin-resistant JIMT-1 cells. The ADC is well tolerated in nude mice and shows complete tumor remission in 7 out of 7 animals after a single i.v. injection in the SK-OV-3 and JIMT-1 models. By contrast, Herceptin shows only moderate antitumoral activity in the SK-OV-3 and no activity in the resistant JIMT-1 model, even after multiple injections. These data encourages further examination of amanitin-based antibody conjugates as highly potent and selective compounds for the treatment of various maligancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3616. doi:10.1158/1538-7445.AM2011-3616

Antibody-Dependent Natural Killer Cell–Mediated Cytotoxicity Engendered by a Kinase-Inactive Human HER2 Adenovirus-Based Vaccination Mediates Resistance to Breast Tumors

Cancer vaccines may have applications in the therapy and prevention of mammary carcinoma. To investigate such applications, we constructed a recombinant adenoviral vaccine expressing a kinase-inactive mutant form of human HER2 and introduced this into BALB/c wild-type (WT) or HER2 transgenic mice. Here, we report contributions by antibody responses and natural killer (NK) cells in tumor protection in this model. One i.p. vaccination protected WT mice from the HER2-expressing mouse carcinoma D2F2/E2. Half of the HER2 transgenic mice were protected fully and long term after preventive vaccination. Tumor growth in mice that eventually developed neoplastic lesions was delayed. Protection in WT and HER2 transgenic mice was associated with high or low levels of IgG2a antibodies, respectively, whereas CTLs were observed in WT but not in HER2 transgenic mice. Depleting CD4(+) or CD8(+) cells in vaccinated WT mice had limited effects, suggesting that protection was largely independent of CD4(+) or CD8(+) T cells. In contrast, antibody-mediated tumor rejection seemed to contribute significantly based on a loss of protection in mice deficient for Fc-γ RI/III or B cells. Further, a role for antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells was indicated by evidence that vaccine protection could be abolished by in vivo depletion of NK cells. Lastly, NK cells and immune sera purified from WT or HER2 transgenic mice exhibited efficient ADCC of HER2-expressing tumor cells in vitro. Our findings define a critical requirement for NK cells in vaccine-induced protection against HER2-expressing tumors.

Delivery of NKG2D Ligand Using an Anti-HER2 Antibody-NKG2D Ligand Fusion Protein Results in an Enhanced Innate and Adaptive Anti-Tumor Response.

Abstract Background: NKG2D ligands activate via the NKG2D receptor expressed on effector cells belonging to both the innate (NK) and adaptive immune systems (CD8+ T cells). Over-expression of NKG2D ligand on the surface of tumor cells leads to tumor regression in several murine models. We reasoned that antibody targeting of NKG2D ligand to tumor cells could also be used to evoke an enhanced anti-tumor immune response.Methods: We fused the murine NKG2D ligand, Rae-1β, to the 3' end of an anti-HER2 IgG3 containing an intact Fc domain (anti-HER2 IgG3-Rae-1β) to target NKG2D ligand to HER2+ breast tumor cells. The cytotoxicity of the fusion proteins was tested in vitro in direct and indirect assays with the murine NK cell line, KY-2. Anti-tumor efficacy was examined in a murine mammary tumor model.Results: Anti-HER2 IgG3-Rae-1β fusion protein bound to both the HER2 antigen, and to NKG2D as determined by flow cytometry. Anti-HER2 IgG3-Rae-1β fusion induced enhanced cytotoxicity of NK effectors against HER2 expressing tumor cells in vitro. EMT6-HER2 bearing BALB/c mice treated with anti-HER2 IgG3-Rae-1β showed markedly decreased tumor growth relative to anti-HER2 IgG3 or PBS injected control mice. Treatment of mice that were implanted on one flank with EMT6 and contralaterally implanted with EMT6-HER2, resulted in prompt regression of EMT6-HER2, and delayed regression of EMT6, suggesting “epitope spreading.” To examine the involvement of NK, CD4, or CD8 cells in the growth inhibition of the Rae-1β fusion protein targeted tumors, we performed lymphocyte subset depletion studies. Mice depleted of either NK cells or CD8+ T cells showed decreased inhibition of tumor growth following treatment with anti-HER2 IgG3-Rae-1β, compared to control mice. These results suggest that NK cells and CD8+ cells play an important role in the anti-tumor activity of anti-HER2 antibody-Rae-1β fusion. Anti-HER2 IgG3-Rae-1β also inhibited growth of EMT6-HER2 in FcγR-/- mice, indicating activity of the fusion in the absence of FcR interaction. No anti-tumor efficacy of anti-HER2 IgG3-Rae-1β was observed in IFNγ-/- mice.To determine if anti-HER2 antibody-Rae-1β fusion led to priming of an adaptive response, twelve mice that had previously rejected EMT6-HER2 tumors following treatment with anti-HER2 IgG3-Rae-1γ, were rechallenged with either EMT6-HER2, or with parental EMT6. Five of six mice rechallenged with EMT6-HER2 and five of six mice rechallenged with EMT6 showed no evidence of tumor growth at day 25, while both EMT6 and EMT6-HER2 grew rapidly in six of six control mice, respectively.Conclusion: Antibody-NKG2D ligand fusion proteins administration leads to an enhanced anti-tumor innate and adaptive immune response. The immune response evoked by targeting of the fusion to HER2, a single surface antigen, encompassed additional non-targeted antigens. Antibody-NKG2D ligand fusion proteins could potentially be used to treat other malignancies by substituting alternate tumor specificities. Murine NKG2D ligand in the antibody fusion molecules could be replaced with human NKG2D ligands for testing in man. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6102.

Her2-targeted pegylated liposomal doxorubicin: Retention of target-specific binding and cytotoxicity after in vivo passage

Background Receptor-directed targeting of ligand-bearing liposomes to tumor cells may enhance therapeutic efficacy by intracellular delivery of a concentrated payload of liposomal drug. The goal of this study was to assess whether Her2-targeted pegylated liposomal doxorubicin (PLD) retains its binding ability to Her2-expressing target cells through circulation in the blood and extravasation to tumor interstitial fluid. Methods PLD was grafted with a lipophilic conjugate of an anti-Her2 scFv antibody fragment at an approximate ratio of 7.5, 15, or 30 ligands per liposome. BALB/c mice were injected with J6456 lymphoma cells into the peritoneal cavity to generate malignant ascites used as a model for tumor interstitial fluid. When abdominal swelling developed, Her2-targeted (HT-) PLD and non-targeted PLD were injected into the mice i.v. at a dose of 15 mg/kg. The ascitic fluid was collected 48 h later, ascitic tumor cells were removed, and the doxorubicin levels in the cell-free ascitic fluid and plasma were determined. Binding of the cell-free ascitic fluid was tested in vitro against two Her2-expressing human tumor cell lines (N87, SKBR-3) and compared to the binding of shelf formulations (not passaged in vivo) of HT-PLD and PLD, by measuring the amount of cell-associated doxorubicin. Results Plasma and ascitic fluid levels of HT-PLD were only slightly below those of PLD indicating that, the Her2 ligand did not cause any significant change in the clearance rate of PLD. The in vitro binding of HT-PLD containing ascitic fluid to Her2-expressing cells was increased 10 to 20-fold above that of PLD-containing ascitic fluid, similarly to the 20-fold difference in binding between shelf Her2-PLD and PLD. The in vitro cytotoxicity of ascitic fluid containing HT-PLD tested against Her2-expressing tumor cells was far greater than that of PLD, and similar to that of the shelf formulations, indicating that the selective pharmacological activity of HT-PLD is preserved after in vivo passage. Optimal results were obtained with HT-PLD formulated with 15 ligands per liposome. Conclusions HT-PLD retains most of its original binding capacity to Her2-expressing cells after in vivo passage indicating that the ligand is stably maintained in vivo in association with the doxorubicin liposomal carrier, and confirming the validity of the post-formulation ligand grafting approach for liposome targeting. Targeting of PLD using a Her2 antibody fragment provides an important means of in vivo selective drug delivery to tumors expressing the Her2 receptor.

Engineering antigen-specific primary human NK cells against HER-2 positive carcinomas

NK cells are promising effectors for tumor adoptive immunotherapy, particularly when considering the targeting of MHC class I low or negative tumors. Yet, NK cells cannot respond to many tumors, which is particularly the case for nonhematopoietic tumors such as carcinomas or melanoma even when these cells lose MHC class I surface expression. Therefore, we targeted primary human NK cells by gene transfer of an activating chimeric receptor specific for HER-2, which is frequently overexpressed on carcinomas. We found that these targeted NK cells were specifically activated upon recognition of all evaluated HER-2 positive tumor cells, including autologous targets, as indicated by high levels of cytokine secretion as well as degranulation. The magnitude of this specific response correlated with the level of HER-2 expression on the tumor cells. Finally, these receptor transduced NK cells, but not their mock transduced counterpart, efficiently eradicated tumor cells in RAG2 knockout mice as visualized by in vivo imaging. Taken together, these results indicate that the expression of this activating receptor overrides inhibitory signals in primary human NK cells and directs them specifically toward HER-2 expressing tumor cells both in vitro and in vivo.

Construction of a Fully Retargeted Herpes Simplex Virus 1 Recombinant Capable of Entering Cells Solely via Human Epidermal Growth Factor Receptor 2

A novel frontier in the treatment of tumors that are difficult to treat is oncolytic virotherapy, in which a replication-competent virus selectively infects and destroys tumor cells. Herpes simplex virus (HSV) represents a particularly attractive system. Effective retargeting to tumor-specific receptors has been achieved by insertion in gD of heterologous ligands. Previously, our laboratory generated an HSV retargeted to human epidermal growth factor receptor 2 (HER2), a receptor overexpressed in about one-third of mammary tumors and in some ovarian tumors. HER2 overexpression correlates with increased metastaticity and poor prognosis. Because HER2 has no natural ligand, the inserted ligand was a single-chain antibody to HER2. The objective of this work was to genetically engineer an HSV that selectively targets the HER2-expressing tumor cells and that has lost the ability to enter cells through the natural gD receptors, HVEM and nectin1. Detargeting from nectin1 was attempted by two different strategies, point mutations and insertion of the single-chain antibody at a site in gD different from previously described sites of insertion. We report that point mutations at gD amino acids 34, 215, 222, and 223 failed to generate a nectin1-detargeted HSV. An HSV simultaneously detargeted from nectin1 and HVEM and retargeted to HER2 was successfully engineered by moving the site of single-chain antibody insertion at residue 39, i.e., in front of the nectin1-interacting surface and not lateral to it, and by deleting amino acid residues 6 to 38. The resulting recombinant, R-LM113, entered cells and spread from cell to cell solely via HER2.

CTLs Directed against HER2 Specifically Cross-React with HER3 and HER4

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated Ag by T cell-based immunotherapeutical strategies such as cancer vaccines and adoptive T cell transfer. The prerequisite for a successful T cell-based therapy is the induction of T cells capable of recognizing the HER2-expressing tumor cells. In this study, we generated human cytotoxic T cell clones directed against the HER2(369-377) epitope known to be naturally presented with HLA-A*0201. Those HER2-reactive CTLs, which were also tumor lytic, exhibited a similar lysis pattern dividing the targets in lysable and nonlysable tumor cells. Several HER2-expressing tumor cells became susceptible to CTL-mediated lysis after IFN-gamma treatment and, in parallel, up-regulated molecules of the Ag-presenting machinery, indicating that the tumor itself also contributes to the success of CTL-mediated killing. Some of the HER2(369-377)-reactive T cells specifically cross-reacted with the corresponding peptides derived from the family members HER3 and/or HER4 due to a high sequence homology. The epitopes HER3(356-364) and HER4(361-369) were endogenously processed and contributed to the susceptibility of cell lysis by HER cross-reacting CTLs. The principle of "double" or "triple targeting" the HER Ags by cross-reacting T cells will impact the further development of T cell-based therapies.

Her2-targeted pegylated stealth liposomal doxorubicin (PLD) retains its specific targeting ability to Her2-expressing tumor cells after in vivo circulation and extravasation to mouse malignant ascites

13097 Background: Receptor-directed targeting of ligand-bearing liposomes to tumor cells may enhance therapeutic efficacy by intracellular delivery of a concentrated payload of liposomal drug. The goal of this study was to assess whether Her2-targeted PLD retains its binding ability to Her2-expressing target cells through circulation in the blood and extravasation to the ascitic fluid of mice with malignant ascites. Methods: PLD was grafted with a pegylated lipophilic conjugate of an anti-Her2 scFv antibody fragment (F5) at a ratio of 15 ligands per liposome. BALB/c mice were injected with J6456 lymphoma cells into the peritoneal cavity to generate malignant ascites. When abdominal swelling developed, Her2-targeted PLD and nontargeted PLD were injected into the mice i.v. at a dose of 15 mg/kg. The ascitic fluid was collected 48 hr later, ascitic tumor cells were removed, and the doxorubicin levels in the cell-free ascitic fluid and plasma were determined. Binding of the liposome-containing ascitic fluid was tested in vitro against Her2-expressing human tumor cell lines (N87, SKBR-3) and compared to the binding of shelf formulations (not passaged in vivo) of Her2-PLD and PLD, using as parameter the amount of cell-associated doxorubicin. Results: Plasma and ascitic fluid levels of Her2-PLD were only slightly below those of PLD indicating that the Her2 ligand did not cause any significant change in the clearance rate of PLD. Her2-PLD and PLD bound to an equal extent to J6456 cells in vivo. The in vitro binding of Her2-PLD from ascites to Her2- expressing cells was increased 10 to 20-fold above that of PLD from ascites, similarly to the 20-fold difference in binding between shelf Her2-PLD and PLD. Conclusions: Her2-targeted PLD demonstrates similar circulation time to that of nontargeted PLD. After in vivo passage, the targeted liposome retains most of its original binding capacity to Her2 expressing cells indicating that the ligand is stably maintained in association with the doxorubicin liposomal carrier. Targeting of PLD using this Her2 antibody fragment should provide an important means of selective drug delivery to tumors expressing the Her2 receptor. [Table: see text]

Endogenous Anti-HER2 Antibodies Block HER2 Phosphorylation and Signaling through Extracellular Signal-Regulated Kinase

Immunologic targeting of the oncoprotein HER2/neu with monoclonal antibodies is an important component of current therapeutic strategies for patients with locally and systemically advanced breast cancer. Engineered antibodies targeting HER2 may have agonist or antagonist effects on HER2, but little is known about whether endogenous antibodies modulate HER2 activity. Vaccination of patients with HER2 peptides successfully induced antibodies in a minority of patients with HER2-expressing malignancy. A subset of antibodies specifically suppressed phosphorylation of HER2 on tyrosine Y1248, a residue critical for HER2 signaling through extracellular signal-regulated kinase. These antibodies also suppressed extracellular signal-regulated kinase phosphorylation and inhibited colony formation in soft agar. The majority of the antibodies that suppressed HER2 phosphorylation displayed specificity for amino acids 328 to 345 and 369 to 384. The isotype of anti-HER2 antibodies was predominantly IgG3 of low avidity, suggesting a Th1 response to peptide vaccine. Endogenous anti-HER2 antibodies can effectively suppress HER2 kinase activity and downstream signaling to inhibit the transformed phenotype of HER2-expressing tumor cells.

A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells.

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.

A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells.

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.