HER2-overexpressing Tumor Cells Research Articles

Can Trastuzumab Be Effective Against Tumors With Low HER2/Neu (ErbB2) Receptors?

HER2/neu (erbB2) is a member of the erbB family of transmembrane receptor tyrosine kinases, which also includes the epidermal growth factor receptor (EGFR; erbB1), HER3 (erbB3), and HER4 (erbB4). Binding of ligands to the extracellular domain of EGFR, HER3, and HER4 induces the formation of kinase active homoand heterodimers to which HER2 is recruited as a preferred partner. Although HER2 does not bind any of the erbB ligands directly, its catalytic activity can potently amplify signaling by erbB-containing heterodimers via increasing ligand binding affinity and/or receptor recycling and stability. Activation of the erbB network leads to receptor autophosphorylation in C-terminal tyrosines and the recruitment to these sites of cytoplasmic signal transducers that regulate cellular processes such as proliferation, differentiation, motility, adhesion, protection from apoptosis, and transformation. HER2 gene amplification has been reported in approximately 20% of metastatic breast cancers, where it is associated with poor patient outcome. Trastuzumab, a humanized monoclonal immunoglobulin G1 that binds the extracellular domain of HER2, has been shown to induce clinical responses in HER2-overexpressing breast cancers and prolong patient survival when combined with chemotherapy. These studies enrolled either predominantly or exclusively patients with breast cancer that overexpressed HER2 as measured by intense membrane staining with HER2 antibodies in the majority of tumor cells (3 by immunohistochemistry [IHC]) or excess copies of the HER2 gene determined by fluorescent in situ hybridization. Studies with human breast cancer cell lines and primary mammary tumors have shown constitutive phosphorylation of HER2. Thor et al examined the incidence of P-HER2 positivity in 816 primary breast cancers. Only 12% of HER2-positive cancers stained positively with an antibody that recognizes HER2 phosphorylated in Y1248; P-HER2 was not detected in any of the HER2-negative tumors. The molecular basis for this constitutive phosphorylation is not clear but is consistent with the reported ability of wild-type Neu, the mouse/rat homolog of HER2, to multimerize and become activated when present in cells at high density. Thus, it is generally accepted that spontaneous dimerization and activation of HER2 occurs in cancers with HER2 gene amplification. Another potential mechanism of HER2 phosphorylation is transactivation by ligandbound EGFR, HER3, or HER4. Indeed, coexpression of the EGFR ligand transforming growth factor alpha (TGF ) and Neu in the mammary gland of transgenic mice markedly accelerates tumor onset and progression compared with mice expressing Neu or TGF transgenes alone. In this study, TGF Neu bi-transgenic mice exhibited increased tyrosine phosphorylation of both EGFR and Neu. In an analysis of 807 patients with invasive breast cancers, 306 HER2positive tumors also expressed EGFR by IHC. Ninety-seven percent of cancers with phosphorylated HER2 at Y1248 exhibited detectable EGFR and the combination of Y1248 P-HER2 together with cooverexpression of HER2 and EGFR was associated with the shortest patient survival. Finally, HER2 is overexpressed in a cohort of non– small-cell lung cancers and increased HER2 gene copy number has been associated with therapeutic response to EGFR tyrosine kinase inhibitors. These reports provide evidence of EGFR-HER2 cross-talk and of the ability of HER2 to amplify ligand-activated EGFR signals. Immunohistochemical studies have shown HER3 expression and overexpression in over 50% of ductal carcinoma in situ and invasive breast cancers. Although kinase defective, HER3 can be phosphorylated by EGFR or HER2. Phosphorylated HER3 can couple to the phosphatidylinositol-3 kinase (PI3K) pathway directly, whereas EGFR and HER2 cannot. HER2/HER3 heterodimers are potently transforming and both HER2 and HER3 are frequently associated or coexpressed in human breast cancer cell lines, mouse transgenic tumors, and primary breast cancers. In HER2-overexpressing tumor cells, both trastuzumab and EGFR kinase inhibitors inhibit the basal phosphorylation of HER3 and its association with HER2 and with PI3K. Finally, high expression of HER3 predicts early escape from trastuzumab therapy. These data imply that HER3 plays a key role in breast tumor progression mediated by the erbB network, and inhibition of HER3 phosphorylation and/or its cross-talk with HER2 is required for the antitumor effect of HER2 signaling inhibitors. Whether incorporation of markers such as HER3, P-HER2, EGFR, EGFR/HER2 dimers, HER2/HER3 dimers or others that may inform the level of erbB pathway engagement, will allow the identification of patients that respond or escape trastuzumab therapy remains to be investigated. Structural studies using recombinant erbB receptor ectodomains, as well as experiments with cell lines and HER2 monoclonal antibodies that recognize different receptor epitopes, indicate that trastuzumab binds in a region not involved in receptor dimerization and thus is unable to block ligand-induced EGFR/HER2 and HER2/ HER3 heterodimers. These data support the speculation that high levels of HER2-containing heterodimers may be a potential marker of JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 24 NUMBER 23 AUGUST 1

Substitution analog peptide derived from HER-2 can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC50 <1,000 nM) and substituted analog peptides (IC50 <50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.

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.

Future directions of liposome- and immunoliposome-based cancer therapeutics

Nanoscale drug delivery systems including liposomes, polymers, and other nanoparticles provide potential solutions for improved cancer therapeutics. Of these drug delivery systems, liposome-based agents, particularly liposomal anthracyclines, have had the greatest impact in oncology to date. Current liposomal drugs evolved from a number of design strategies for improved biodistribution over free drugs. Reticuloendothelial system-targeted formulations significantly reduce systemic exposure to high peak levels of free drug, but do not facilitate targeting to tumors. Passive or physiologic targeting of drugs to tumors is achievable using long-circulating liposomes, including pure lipid systems as well as surface-modified formulations designed to resist recognition and uptake by reticuloendothelial system cells. The latter, represented by pegylated or STEALTH liposomes, circulate for days as stable constructs and slowly extravasate in neoangiogenic vessels in tumors, providing a degree of passive targeting to tumor tissue. Future liposome therapeutics are building on these validated designs as well as on pharmacologic insights into their mechanisms of delivery. For example, camptothecin analogues, anti-angiogenesis agents, and antisense oligonucleotides each represent rational candidates for delivery in highly stabilized and long-circulating liposomes. For such agents, pegylated liposome delivery offers improved chemical stability of encapsulated drug, enhanced accumulation in tumors, and prolonged drug exposure. True molecular targeting can be achieved using liposomes linked to ligands such as monoclonal antibody fragments directed against cancer-associated antigens. Immunoliposomes combine antibody-mediated tumor recognition with liposomal delivery and, when designed for target cell internalization, provide intracellular drug release. Recent advances in immunoliposome design include rapid selection of phage antibody-derived scFv for targeting, and methods for conjugation of ligands to existing US Food and Drug Administration-approved liposomal drugs such as pegylated liposomal doxorubicin (Doxil/Caelxy [PLD]). An immunoliposome consisting of novel anti-HER2 scFv F5 conjugated to PLD, currently in development, selectively binds to and internalizes in HER2-overexpressing tumor cells. The modular organization of immunoliposome technology enables a combinatorial approach in which a repertoire of monoclonal antibody fragments can be used in conjunction with a series of liposomal drugs to yield a new generation of molecularly targeted agents.

Enhancement of the p27Kip1‐mediated antiproliferative effect of trastuzumab (Herceptin) on HER2‐overexpressing tumor cells

The oncogenic activity of the overexpressed HER2 tyrosine kinase receptor requires its localization in the plasma membrane. The antitumor effect of anti-HER2 antibodies (Abs) is mainly dependent on receptor downregulation and comprises p27Kip1-mediated G1 cell cycle arrest. However, one major limitation of anti-HER2 therapy is the reversibility of tumor growth inhibition after discontinuation of treatment caused by the mitogenic signaling associated with cell surface receptor re-expression. We found that the level of p27Kip1 upregulation, inhibition of Cdk2 activity and magnitude of G1 arrest induced by the humanized Ab trastuzumab (Herceptin, HCT) on BT474 and SKBr3 HER2-overexpressing breast cancer cells correlates with the level of cell surface receptor. Thus, continuous exposure of cells to HCT for 72 hr results in downregulation of the cell surface receptor and a concurrent increase in the level of p27Kip1 protein. Discontinuation of Ab exposure after the first 8 hr results in failure to upregulate p27Kip1 and arrest of cell cycle progression. We show that the lysosomotropic amine chloroquine (CQ) augments receptor internalization in HER2-overexpressing cells either pretreated or continuously treated with HCT and leads to an increased and sustained inhibitory effect. The enhanced CQ-dependent loss of functional HER2 from the cell surface resulted in sustained inactivation of the serine/threonine kinase Akt, upregulation of p27Kip1 protein and inhibition of cyclin E/Cdk2 activity. Potentiation of the inhibitory effect of HCT by CQ was directly related to loss of HER2 from the plasma membrane since prevention of Ab-mediated receptor endocytosis by engagement of the receptor with immobilized HCT abrogated the effect of CQ.

A caspase-6 and anti-human epidermal growth factor receptor-2 (HER2) antibody chimeric molecule suppresses the growth of HER2-overexpressing tumors.

Clinical studies have suggested that human epidermal growth factor receptor-2 (HER2) provide a useful target for antitumor therapy. We previously described the generation of a chimeric HER2-targeted immunocasp-3 protein. In this study, we extend the repertoire of chimeric proapoptotic proteins with immunocasp-6, a construct that comprises a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A, and an active caspase-6, which can directly cleave lamin A leading to nucleus damage and inducing programmed cell death. We demonstrate that the secreted immunocasp-6 molecule selectively recognizes and induces apoptosis in HER2-overexpressing tumor cells in vitro, but not in cells with undetectable HER2. The immunocasp-6 gene was next transferred into BALB/c athymic mice bearing human breast SK-BR-3 tumors by i.m. injection of liposome-encapsulated vectors, by intratumor injection of adenoviral vectors, or by i.v. injection of PBMC modified by retroviral infection. Regardless of the method used, expression of immunocasp-6 suppressed tumor growth and prolonged animal survival significantly. Our data show that the chimeric immunocasp-6 molecule can recognize HER2-positive tumor cells, promptly attack their nucleus, and induce their apoptotic death, suggesting the potential of this strategy for the treatment of human cancers that overexpress HER2.

Secreted Antibody/Granzyme B Fusion Protein Stimulates Selective Killing of HER2-overexpressing Tumor Cells

Targeted cell killing is required for effective treatment of cancers. We previously described the generation of a chimeric immunocasp-3 protein and its potent selective antitumor activity (Jia, L. T., Zhang, L. H., Yu, C. J., Zhao, J., Xu, Y. M., Gui, J. H., Jin, M., Ji, Z. L., Wen, W. H., Wang, C. J., Chen, S. Y., and Yang, A. G. (2003) Cancer Res. 63, 3257-3262). Here we extend the repertoire of another chimeric pro-apoptotic protein immunoGrB, which comprises an anti-HER2 single-chain antibody, a Pseudomonas exotoxin A translocation domain and active granzyme B. Human lymphoma Jurkat cells transfected with the immunoGrB gene expression vector were able to produce and secrete the chimeric protein. The immunoGrB molecule selectively recognized and destroyed HER2-overexpressing tumor cells both in vitro and in nude mouse after intramuscular injection of the immunoGrB expression plasmid. Further in vivo study showed that intravenous administration of immunoGrB gene-modified lymphocytes led to suppression of HER2-overexpressing tumor growth and prolonged animal survival because of continuous secretion of immunoGrB molecules into blood and lymph fluid. These results demonstrate that the chimeric immunoGrB molecule, which is capable of antibody-directed targeting and granzyme B-mediated killing, has therapeutic potential against HER2 tumors, especially in cases in which caspase-dependent apoptosis is inhibited.

Labeling anti-HER2/neu monoclonal antibodies with 111In and 90Y using a bifunctional DTPA chelating agent.

The goal of this investigation was to develop stable radioimmunoconjugates (RICs) of anti-HER2/neu monoclonal antibodies (MoAbs) for imaging and therapy in an animal model bearing human breast tumor xenografts that express normal (MCF-7 cells) and increased amounts of HER2/neu receptors (HCC-1954, BT-474, SKBR-3 cells) on their cell surface membranes. Pharmacy-grade Herceptin, a murine anti-HER2/neu MoAb, and nonspecific mouse IgG protein were conjugated with the recently developed DTPA linker known as CHX-A"-DTPA. These immunoconjugates were labeled with (111)InCl(3) and (90)YCl(3). Using a molar excess of 10:1 CHX-A"-DTPA to immunoglobulin, average specific activities of 1.87 microCi (111)In/microg RIC and 2.71 microCi (90)Y/microg RIC were obtained. The purity of RICs was 96%+ for (111)In and 99%+ for (90)Y. Stability in human plasma at 37 degrees C for both RICs ranged from 98% at 24 h to 85% at 96 h. Binding capacity of the RICs was tested with human cancer cell lines MCF-7, HCC-1954, BT-474, and SKBR-3. Using (111)In-labeled nonspecific IgG protein as a control, (111)In-Herceptin RIC was found to bind to MCF-7 cells with a ratio of 2.5:1 and to SKBR-3 cells with a ratio of 85:1 after 3 h of incubation. (111)In anti-HER2/neu RIC bound to MCF-7 cells with a ratio of 6:1 and to SKBR-3 cells with a ratio of 115:1 after 3 h of incubation. (90)Y-anti-HER2/neu RIC bound 10-times greater to BT-474 cells than to MCF-7 cells. Thus, these MoAbs can be labeled with (111)In and (90)Y using the CHX-A"-DTPA linker. The resulting RICs ((111)In- and (90)Y-anti HER2/neu antibodies) are stable and bind significantly to HER2 overexpressing tumor cell lines.

Mechanism of action of anti-HER2 monoclonal antibodies: scientific update on trastuzumab and 2C4.

The HER family of transmembrane tyrosine kinase receptors is composed of four members, BER1 to HER4. HER2 is a ligand-orphan receptor expressed in many human tumors and overexpressed in 25-30% of breast cancers. HER2 amplifies the signal provided by other receptors of the HER family by forming heterodimers. The essential role of HER2 in the HER signaling network led to the development of anti-HER2 monoclonal antibodies (MAbs) for cancer therapy. In particular, the humanized MAb trastuzumab (Herceptin) has antitumor activity against HER2-overexpressing human breast tumor cells and is widely used for the treatment of women with HER2 overexpressing breast cancers. Trastuzumab induces HER2 receptor downmodulation and, as a result, inhibits critical signalling pathways (i.e. ras-Raf-MAPK and PI3K/Akt) and blocks cell cycle progression by inducing the formation of p27/Cdk2 complexes. Trastuzumab also inhibits HER2 cleavage, preceding antibody-induced receptor downmodulation, and this effect might contribute to its antitumor activity in some cancers. In vivo, trastuzumab inhibits angiogenesis and induces antibody-dependent cellular cytotoxicity. A limitation of trastuzumab is that its activity is largely restricted to breast cancers with the highest level of HER2 overexpression or HER2 gene amplification. However, there is a large population of breast cancers and of many other tumors that have low or moderate HER2 expression. In such tumors, HER2 functions as a preferred coreceptor to form heterodimers with HER1 (EGFR), HER3 or HER4. For this reason, a humanized monoclonal antibody, called 2C4, that targets the role of HER2 as a coreceptor is under active development. 2C4 binds to a different epitope of HER2 ectodomain than trastuzumab and sterically hinders HER2 recruitment in heterodimers with other HER receptors. This results in the inhibition of signalling by HER2-based heterodimers both in cells with low and high HER2 expression. In vitro and in vivo antitumor activity has been reported in a range of breast and prostate tumor models. Therefore, 2C4 may have potential against a wide variety of solid tumors. Phase I trials are underway.

In this issue

Genomic instability is a classic hallmark of tumorigenesis and tumor progression. It is usually thought of as LOH, loss of heterozygosity, most notably at tumor suppressor loci. However, three papers in this issue highlight other chromosomal alterations that occur as different human cancers progress through their respective clinical stages and outline the significance these alterations might play in molecular diagnostics. Ha et al. (pages 615–617) focus on microsatellite instabilities (MSI), represented by insertion or deletion mutations, in head-and-neck squamous cell cancer (HNSCC.) As might be expected, these simple base pair insertions and deletions are dominant features of solid tumors with significant mismatch repair defects. The molecular progression of HNSCC correlates with the phenotypic spectrum of its dysplasia; there is an increase in the prevalence of LOH as the severity of the dysplasia increases. Ha et al. find that MSI become increasingly more common as early dysplastic legions progress to fully malignant HNSCC, and they conclude that these instabilities are probably acquired during this transition. They therefore claim that microsatellite analysis, which can be performed on genetic samples obtained from saliva and serum, can be a supplemental method for early cancer detection as well as for tumor surveillance. In a similar study, Lerebours et al. (pages 618–622) investigate allelic losses associated with primary inflammatory breast cancer (IBC), a rare but highly metastatic form of breast cancer. In contrast to the case for non-IBC, the molecular mechanisms underlying IBC are poorly understood. This group hypothesized that distinct genetic features might account for the unique clinical and histological aggression exhibited by IBC. They report that LOH is more frequent in IBC than in non-IBC; yet the chromosomal regions where heterozygosity is most frequently lost — 8p, 11q, 16q, and 17p — are the same in both cancer types. Likewise, the oncogenes ERBB2, MYC, and CCND1 are amplified with similar frequencies in both IBC and non-IBC. Hypermethylation of CpG islands in the promoter regions of cancer-causing genes is yet another mechanism of gene silencing utilized in human cancers, including gastric cancer. Intestinal metaplasia (IM) is generally considered to be a preneoplastic lesion of the stomach. To et al. (pages 623–628) analyze the presence of promoter hypermethylation in IM of patients with and without gastric cancer in order to correctly place it chronologically in the multistep progression of gastric carcinogenesis. They report that both the prevalence of promoter hypermethylation and the number of hypermethylated promoters are similar in IM in patients with and without gastric cancer, although the promoters of certain of the genes examined — DAP-kinase, p14Arf, p15Ink4b, and p16CDKN2A — were more frequently hypermethylated in IM cells derived from cancer patients than in those derived from healthy patients. They thus conclude that these epigenetic alterations are already present in the preneoplastic stage, and that they may represent an important pathogenetic mechanism in the development of gastric cancers. Cancer cells are usually not targeted by the immune system because they are not recognized as non-self, and thus they are not recognized as dangerous. Current work seeks to use the vaccine model to alert the immune system to the cancer cells' fatal potential and utilize immune-based therapy to attack the transformed cells. Lin et al. (pages 629–637) chose Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, as a vector to orally deliver human papillomavirus type 16 E7 to syngeneic mice injected with TC-1 or B16F1 tumor cells. L. monocytogenes is an attractive vector because it can present secreted proteins to both the MHC class I and class II pathways and thus can stimulate both CD4+ and CD8+ T-cell responses. They observed that oral vaccination with their recombinant L. monocytogenes causes the regression of tumor growth in syngeneic mice, and they suggest that L. monocytogenes encoding HPV-16 E7 may be a useful oral vaccine to combat cervical cancer. This is a particularly appealing idea because cervical carcinoma remains one of the most common malignancies worldwide, and oral vaccines are easier to administer and less expensive to produce than vaccines formulated for injection. Vidovic et al. (pages 660–664) have attempted to develop an effective vaccine against HER-2 overexpressing tumors, such as those in human breast, lung, prostate, ovarian, colorectal, and endometrial carcinomas, by genetically engineering fusion proteins of HER-2 domains with GM-CSF. HER-2 is an attractive target because its overexpression has been correlated with both poor prognosis and shorter survival times. They found that the most potent antigen consists of the hydrophilic intra- and extracellular regions of HER-2 fused to rat GM-CSF; this antigen is best presented by dendritic cells because it is internalized via GM-CSF cell surface receptors. The soluble antigen is then cocultured with autologous APC to trigger the tumor-specific immune response and suppress tumor growth in vaccinated syngeneic mice injected with HER-2 overexpressing tumor cells. In vivotumor elimination assay.

Clonal diversity of the T-cell population responding to a dominant HLA-A2 epitope of HER-2/neu after active immunization in an ovarian cancer patient

Natural antigen processing and presentation of antigen is thought to be important for the generation of a broad functional repertoire of antigen-specific T cells. In this study, the T-cell repertoire to an immunodominant human leukocyte antigen A2 (HLA-A2) binding peptide epitope of HER-2/neu, p369-377, was examined in a patient following immunization with a peptide-based vaccine consisting of helper peptides encompassing HLA-A2 peptide epitopes. The responding T-cell repertoire generated was both phenotypically and functionally diverse. A total of 21 p369-377 clones were generated from this patient. With the exception of two clones, all clones were CD3 +. Sixteen of the clones were CD8 +/CD4 −. Five of the clones were CD4 +/CD8 −, despite being generated with an HLA-A2 binding peptide. Nineteen of 21 of clones expressed the αβ-T-cell receptor (TCR). The remaining two clones expressed the γδ T-cell response (TCR). Selected αβ-TCR clones, both CD8 + and CD4 +, could lyse HLA-A2 transfected HER2 overexpressing tumor cells and p369-377-loaded B-lymphoblastic cell line. In addition to their lytic capabilities these clones could be induced to produce interferon-γ (IFN-γ) specifically in response to p369-377 peptide stimulation. The 2 γδ-TCR clones expressed CD8 and lysed HLA-A2 + HER-2/neu + tumor cells, but not HLA-A2 − HER-2/neu + tumor cells. One of γδ-TCR clones also released IFN-γ directly in response to p369-377 stimulation. These results suggest that a tumor antigen TCR, directed against a specific epitope, can be markedly polyclonal at multiple levels including CD4/CD8 and TCR.

The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells.

Induction of antitumor immunity involves the presence of both CD8(+) CTLs and CD4(+) Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34(+) hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8(+) CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4(+) Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4(+) T cells were cloned that released IFN-gamma upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8(+) and CD4(+) T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.

Herceptin in the treatment of metastatic breast cancer

Amplification of the HER-2/neu (c-erbB-2) gene, resulting in overexpression of the p185HER-2 growth factor, receptor occurs in approximately 25 % of early-stage breast cancers and is associated with a poor clinical outcome. Antibodies to the HER2 receptor have a cytostatic effect by suppressing growth of HER2 overexpressing tumor cells. The humanized antibody rhuMAb4D5 (Herceptin), which has recently been approved by the U. S. Food and Drug Administration for the treatment of metastatic breast cancer, has been shown to improve the response rate, response duration to chemotherapy and to extend 12-month overall survival in HER2-amplified breast cancers. The response rate to Herceptin given as a single agent is a modest 23 %. Preclinical data demonstrate a therapeutic advantage in the administration of Herceptin in combination with chemotherapeutic agents. In a multicenter clinical trial for patients with HER2/neu-overexpressing metastatic breast cancer the use of Herceptin in combination with doxorubicin/cyclophosphamide or paclitaxel resulted in significantly improved objective clinical responses and prolongation of survival. We report on recent studies with Herceptin as a single agent and in combination with chemotherapy. Mechanisms of action of Herceptin are presented. Diagnosic issues in determining HER2 overexpression are discussed. At present Herceptin has only been approved in North America. In conclusion, guidelines for Germany for the treatment of HER2-positive metastatic breast cancer are given.

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.