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The Biology Behind |
Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
In the early 1980s, monoclonal antibodies were prepared that reacted with a surface membrane nonhematopoietic differentiation antigen overexpressed on human 
25% of human breast carcinomas (1)
. Similar reactivity was seen with other carcinomas, but expression in normal tissues was found to be limited (2)
. Within several years, the antigen was recognized as the HER2 or ErbB-2 transmembrane tyrosine kinase receptor encoded by the neu proto-oncogene (3
, 4)
. HER2 heterodimerizes with HER1, HER3, or HER4, and transmits signals that lead to cell proliferation and survival (5)
. Point mutation or overexpression of HER2 can lead to cell transformation (6
, 7)
, and overexpression of HER2 in breast and ovarian carcinomas correlates with an unfavorable patient prognosis (8)
. Thus, therapeutics directed at HER2 may be useful for a significant number of cancer patients.
In 1986, a monoclonal antibody to rat HER2 was prepared and found to inhibit the growth of a rat neuroblastoma cell line both in vitro and in vivo (9) . Subsequently, a number of murine monoclonal antibodies to the extracellular domain of human HER2 were found to inhibit tumor cell growth (10, 11, 12) . One of these antibodies (4D5) was humanized by grafting murine complementary-determining regions onto a human IgG1 framework (13) . The humanized antibody, trastuzumab, retained antitumor activity against HER2-overexpressing tumor cells in tissue culture (13) , in animal models (14) , and in Phase I-III clinical trials in patients with HER2-positive breast cancers (15) . Trastuzumab was approved by the FDA in 1998 for clinical use for HER2-overexpressing metastatic breast cancer.
Further development of anti-HER2 antibodies for the treatment of HER2-overexpressing malignancies will be facilitated by better understanding of how such antibodies inhibit cancer growth. Seminal observations include the findings: (a) that only a fraction of anti-HER2 antibodies inhibit tumor growth (10 , 12 , 16) ; (b) that combinations of anti-HER2 antibodies work better than individual antibodies (11 , 17 , 18) ; and (c) anti-HER2 antibody plus chemotherapy agents yield synergistic tumor cell kill both in animal models and patients (14 , 19) . Observations by Spiridon et al. (20) in this issue of Clinical Cancer Research add evidence that combinations of anti-HER2 antibodies are more active than single antibodies and suggest one approach to improve their therapeutic potential. Can we explain these preclinical and clinical findings with the proposed mechanisms for anti-HER2 antibodies? Will hypotheses for the molecular mechanism of action of anti-HER2 antibodies lead to 2additional improvements in therapy of HER2-positive cancer patients?
At least four different hypotheses have been advanced to explain the antitumor activity of anti-HER2 antibodies. Hurwitz et al. (16) propose that bivalent antibody binding cross-links HER2 on the cell surface and triggers endocytosis. The endocytosed HER2 is routed to the lysosomes where it is degraded. Reduced HER2 at the cell surface allows less HER2 heterodimer formation, resulting in reduced growth factor-induced signaling and proliferation. Both unlabeled and [35S]methionine-labeled HER2 were more rapidly degraded when cells were exposed to inhibitory anti-HER2 antibodies (12 , 21) , but Fab' fragments of inhibitory antibodies, which lack cross-linking functions, were unable to induce HER2 degradation or produce tumor inhibition (10 , 16 , 18) . Inhibitory anti-HER2 antibodies became inaccessible to acid treatment after cell incubation and colloidal gold conjugates of these antibodies were observed in endosomes by electron microscopy. In contrast, stimulatory anti-HER2 antibodies were removed by acid treatment (thus remained on the cell surface), and their gold conjugates were not seen by electron microscopy in endosomes (16) . A similar correlation between tumor growth inhibition and HER2 down-regulation was observed measuring the half-life of [35S]methionine-labeled HER2 in NIH3T3 mouse cells transfected with human HER2 and treated with a panel of inhibitory antibodies (11) . Antibodies producing greater tumor growth inhibition were associated with greater HER2 degradation. Interestingly, in the study by Harweth et al. (11) , the greatest HER2 degradation was seen with an antibody combination. In a similar study by Hurwitz et al. (16) , [35S]cysteine-labeled HER2 was degraded extremely rapidly in cells exposed to a combination of two anti-HER2 antibodies, but significant degradation or tumor growth inhibition was not observed when the cells were treated with each antibody alone. A novel approach was used to confirm the effects of HER2 down-regulation. Transfection of tumor cells with single chain anti-HER2 antibodies containing the COOH-terminal endoplasmic reticulum retention sequence KDEL yielded intracellular sequestration of HER2, loss of surface HER2, and tumor growth inhibition (22 , 23) . How do the anti-HER2 antibodies trigger endocytosis and degradation? After incubation of tumor cells at 37°C with 125I-labeled anti-HER2 antibodies, the radiolabeled antibodies became resistant to proteases added to the medium (24) . Using immunoelectron microscopy and colloidal gold-conjugated anti-HER2 antibodies, the antibody-HER2 complexes were found to traffic to clathrin-coated pits, coated vesicles, endosomes, and then multivesicular bodies (24) . Once internalized, the complexes recruited c-Cbl ubiquitin ligase and were ubiquitinated (25) . When the HER2 c-Cbl docking site at Tyr-1112 was mutated, both ubiquitination and degradation were blocked (25) . In contrast to the preceding observations, no correlation was found when Neve et al. compared the rates of internalization for several 125I-labeled single chain and bivalent anti-HER2 antibodies with their growth inhibitory activity (26) . However, the fate of HER2 was not examined in the latter study. The HER2 may have recycled to the cell surface after single chain antibody binding and not undergone ubiquitination or degradation.
Codony-Servat et al. (27) have described that HER2 undergoes a proteolytic cleavage by a metalloprotease that results in the release of the extracellular domain and the production of a truncated membrane-bound fragment (p95). The truncated, membrane-bound p95 has constitutive kinase activity and is found on many HER2-overexpressing tumor cells (28 , 29) . The same investigators in Barcelona have shown that trastuzumab but not the less tumor-inhibitory antibody 2C4 inhibited the proteolytic cleavage that produces p95 (29) . Trastuzumab reacts with an epitope between amino acid residues 529 and 627 in the cysteine-rich domain II (CRD2) close to the transmembrane region. This is the same region of HER2 that is cleaved to generate p95. The antibody may sterically block the protease cleavage site in HER2, thereby preventing generation of p95. Less p95, may yield reduced intracellular signaling and cell proliferation. However, the biological role of HER2 p95 in oncogenesis remains poorly characterized although recent data by Molina et al. (28) have shown that breast cancers expressing high levels of p95 have worse prognostic features than those with full-length HER2. It will be important to determine the phenotype of mutant HER2 transfectants that lack the p95 cleavage site. Additional anti-HER2 antibodies that inhibit tumor cell growth but react with epitopes on CRD-1 should be examined.
Anti-HER2 antibodies may directly interfere with ligand-HER receptor binding and receptor dimerization. Growth factor ligands contain a low affinity site for interaction with HER2 (30) . Antibodies (10) , antibody fragments (26) , or peptidomimetics (31) may compete with this interaction reducing dimerization and tyrosine kinase activation. The peptide inhibitor, FCGDGFYACYMDV, bound tightly to HER2 and inhibited cell proliferation and signaling (31) . This peptide was reportedly less efficient than trastuzumab at down-modulating HER2 from the cell surface, but the data were not shown. Klapper et al. (10) described a group of anti-HER2 antibodies (class II), which inhibited tumor cell growth but had reduced ability to down-modulate HER2 from the cell surface. These antibodies were able to block ligand binding at 4°C and thus prevented initial ligand-receptor interactions.
After tumor cell binding, the Fc portions of anti-HER2 antibodies can engage Fc
receptors on effector cells leading to tumor cell killing. Anti-HER2 antibodies administered to tumor-bearing nude mice were less effective when the mice lacked FcR receptors or the antibodies were mutated (D265A) to reduce Fc binding (31)
. In contrast the antibodies were more effective in mice lacking the inhibitory FcRIIB receptor (32)
. The most important effector cells appear to be CD16 positive natural killer cells. Incubation of 51Cr-labeled tumor cells with trastuzumab showed maximal cytotoxicity with natural killer cells (33)
. The antitumor efficacy does not depend on complement. Depletion of complement by cobra venom factor had no effect on in vivo antitumor efficacy (34)
.
In addition to these major molecular pathways of tumor cell injury, anti-HER2 antibodies have been shown to inhibit a number of downstream signaling events associated with HER family receptor activation. Thus, antibody-treated tumor cells have reduced expression of vascular endothelial growth factor (35)
, and phosphatidylinositol 3'-kinase and Akt kinase activity (33)
, and increased expression of p27Kip1 (36)
, E-cadherin, and 
2-integrins (37)
. These changes are likely secondary to reduced upstream HER2 signaling. Similarly, chemosensitization by anti-HER2 antibodies may be because of reduced HER receptor signaling. HER2 overexpression is associated with resistance to cisplatin and taxanes secondary to increased DNA repair, impaired cell cycle checkpoints, and increased apoptotic thresholds (38)
. Anti-HER2 antibodies reverse this chemoresistant state.
A reasonable assessment is that multiple mechanisms are involved in the antitumor efficacy in animals and patients of anti-HER2 antibodies. These pathways provide opportunities such as those proposed in this issue of Clinical Cancer Research by Spiridon et al. (20) to improve on the current single anti-HER2 therapeutic. The hyper-cross-linking mixture may increase HER2 down-modulation, additionally diminish ligand-receptor interactions or metalloprotease cleavage, or improve effector cell recruitment. Other approaches to modify HER2 cellular trafficking, processing, or signaling, or to increase anti-HER2 immune function may be tested soon including use of anti-HER2 antibody-geldanamycin conjugates to accelerate HER2 internalization and ubiquitination (39) , combinations of anti-HER2 antibody with matrix metalloprotease inhibitors to reduce p95 generation (36) , combinations of anti-HER2 and anti-HER1 antibodies or combinations of anti-HER2 antibody with HER1 tyrosine kinase inhibitor to inhibit signaling (40 , 41) , or combinations of anti-HER2 antibody with low dose interleukin 2 to improve effector cell function (42) . The chemosensitizing effect of anti-HER2 antibodies is being exploited in clinical trials testing new combinations of trastuzumab with cytotoxic chemotherapies (43) . These various studies may additionally increase the therapeutic index of this new and exciting class of anticancer agents, and, hopefully, improve the quality and duration of life for patients with HER2-positive cancers.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be addressed, at Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27157. 
Received 2/25/02; accepted 5/25/02.
REFERENCES
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