ErbB-2 is an orphan receptor that belongs to a family of tyrosine kinase receptors for either epidermal growth factor (EGF) or Neu di erentiation factor (NDF/ neuregulin). Because overexpression of the erbB-2 proto-oncogene is frequently associated with an aggressive clinical course of certain human adenocarcinomas, the encoded protein is an attractive target for immunotherapy. Indeed, certain monoclonal antibodies (mAbs) to ErbB-2 e ectively inhibit tumor growth in animal models and in clinical trials, but the underlying mechanism is incompletely understood. To study this question, we generated a large battery of mAbs to ErbB-2, that were classi®ed epitopically. Whereas most antibodies stimulated tyrosine phosphorylation of ErbB-2, their anti-tumor e ect correlated with its accelerated endocytic degradation. One group of tumor-inhibitory mAbs (Class II mAbs) was elicited by the most antigenic site of ErbB-2, and inhibited in trans binding of NDF and EGF to their direct receptors. The inhibitory e ect was due to acceleration of ligand dissociation, and it resulted in the reduction of the ability of ErbB-2 to transactivate the mitogenic signals of NDF and EGF. These results identify two potential mechanisms of antibody-induced therapy: acceleration of ErbB-2 endocytosis by homodimerization and blocking of heterodimerization between ErbB-2 and growth factor receptors.

ABSTRACT

Immunization of mice with acetaldehyde conjugated to human plasma proteins resulted in the production of polyclonal antibodies that reacted with erythrocyte protein-acetaldehyde conjugates, but not with control erythrocyte proteins. Such antibodies recognized erythrocyte protein-acetaldehyde conjugates prepared with 20-100 microM acetaldehyde, concentrations that exist in the blood of alcoholics. The antibodies also recognized acetaldehyde condensation products with synthetic poly-(L-lysine). Immunization with keyhole limpet hemocyanin-acetaldehyde conjugates resulted in antibodies against both plasma protein-acetaldehyde and erythrocyte protein-acetaldehyde conjugates, which did not cross-react with the respective unmodified carrier proteins. Immunization with human erythrocyte protein-acetaldehyde condensates led to the production of antibodies against both the protein moiety as well as the condensate. Monoclonal antibodies with affinities 50 times greater for the condensate than for the carrier protein were produced by hybridization of spleen cells from the immunized mice. Chronic alcohol administration to mice for 45-50 days led to the generation of antibodies that reacted against protein-acetaldehyde conjugates, suggesting that such adducts are formed in vivo and can act as neoantigens. Antibodies against acetaldehyde adducts should be of value in the identification of alcohol consumption and in the study of the biology of the adducts in relation to organ pathology.

The ERBB2 (also called HER2, neu, and c-erbB-2) gene product, which encodes a growth factor receptor, was implicated in the malignancy of human adenocarcinomas. An antibody directed to the rat oncogenic receptor has been previously shown to have an antitumor effect in model systems. In an attempt to extend this observation to the protooncogenic human receptor and also to understand the underlying mechanism, we generated a panel of monoclonal antibodies specific to the extracellular portion of the ERBB2 protein. The effects of the antibodies on tumor growth were compared with their cellular and biochemical actions in vitro. Surprisingly, opposing in vivo effects were observed: although some antibodies almost completely inhibited the growth in athymic mice of transfected murine fibroblasts that overexpress Erbb-2, other antibodies either accelerated tumor growth or resulted in intermediate responses. When tested on cultured human breast carcinoma cells or ERBB2 transfectants, the tumor-stimulatory antibody was found to induce significant elevation of tyrosine phosphorylation of the ERBB2 protein. In contrast, only partial correlation was observed between the capacity to restrict tumor growth and the effects of the antibodies on receptor degradation and cellular proliferation in vitro. This suggests that the antitumor antibodies affect both receptor function and host-tumor interactions. Our results may help establish experimental criteria for the selection of specific antibodies for use either alone or in cogiunction with other molecules as pharmacological antitumor agents.Evidence has been accumulated in recent years for the involvement of growth factors and their receptors in the process of malignant transformation. The ERBB2 protein is a receptor tyrosine kinase (1), homologous to the epidermal growth factor (EGF) receptor (2, 3). The rat homologue ofthe gene undergoes oncogenic activation through a single point mutation (4). The ERBB2 protein was found to be overexpressed in several types of human adenocarcinomas, especially in tumors of the breast and the ovary (5-7), and the overexpression was correlated with short time to relapse and poor survival of breast cancer patients (5).The potential use of monoclonal antibodies (mAbs) in diagnosis and treatment of cancer has been studied extensively (8 (20), and the hybridomas were selected with hypoxanthine/aminopterin/thymidine medium. Supernatants of the growing cells were screened by using an indirect binding assay. CHO cells transfected with the ERBB2 gene (HCC cell line) were plated on a flexible 94-well plate, previously coated with polylysine (1 mg/ml). The cells were fixed with 3% (wt/vol) paraformaldehyde, and supernatants of hybridomas were incubated for 1 hr at 22°C with the fixed cells. The bound Abbreviation: mAb, monoclonal antibody. 8691The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Iodine-125-labeled monoclonal antibody 108.4 (108.4 mAb), raised against the extracellular domain of the epidermal growth factor (EGF) receptor, was shown to visualize sc xenografts of human oral epidermoid carcinoma (KB) cells in nude mice. In vitro, although EGF caused an increase in the number of KB cell colonies (150% at a concentration of 160 mM), the anti-EGF receptor antibodies reduced clone formation. At a concentration at which EGF caused a 50% increase in colony number, the addition of a 100-fold molar excess of 108.4 mAb resulted in a decrease in the number of cell colonies to 20% of the original value. Therefore, the effect of antibody on the KB tumor was studied in vivo in three different modes of tumor transplantation. Antitumor activity was demonstrated first by retardation (versus controls) of the growth of tumor cells as sc xenografts (P greater than .017), then by prolongation of the life span of animals with the ip form of the tumor (P less than .001), and finally on an experimental lung metastasis by a reduction in the number and size of tumors (P less than .05). When the anti-EGF receptor antibodies were added together with cisplatin, the antitumor effect was greatly enhanced, suggesting that the toxic activity of these agents is synergistic (P less than .007). The antitumor effect persisted when animals were treated with the F(ab)'2 fragment of the antibody, although it was less efficient. The Fab fragment of the antibody, whose ability to bind to the cell-associated receptor was completely conserved, did not affect the growth of the tumor. The activity manifested by the F(ab)'2 fragment of the anti-EGF receptor antibodies suggested that the antitumor effect was not due to immune mechanisms requiring the Fc portion of the antibody.

Daunomycin was coupled to dextrans of various molecular sizes. The binding to the dextran carriers augmented the therapeutic efficacy of the antitumor agent in a murine lymphoma line (YAC). When the treatment with the drug or its conjugates was given concomitantly with the tumor cells at separate sites, the unbound drug was able, at its optimally effective doses, to prevent tumors in 40% of the mice, whereas the drug-dextran was efficient in 80% of the mice. The advantage of the drug-dextran over the free drug was also manifested when the treatment was given 6 days after tumor transplantation. However, a further delay of the treatment resulted in a decrease in the potency of the drug-dextran. Similar behavior was observed when increasing tumor loads were transplanted (10(5)-10(8) cells) and when the treatment was administered immediately. The most favorable effect of the drug-dextran was obtained with 10(7) cells, but against 10(8) cells neither the free drug nor the bound one was effective.

Daunomycin was covalently attached via a dextran bridge to specific antibodies against rat a-fetoprotein produced in a horse. The effect of this conjugate on an ei-fetoproteinproducing tumor was investigated in terms of cytotoxicity and inhibition or retardation of tumor development. Under the experimental conditions used, the covalent conjugate was by both criteria more efficient than either daunomycin alone or a mixture of daunomycin and specific antibodies or a conjugate ofdaunomycin with horse immunoglobulin. These results show that the conjugate may be useful as a specific cytotoxic agent against at-fetoprotein-producing tumors.The cytotoxic activity of a specific antiserum to a-fetoprotein (AFP) on AFP-producing tumors was determined both in vitro (1, 2, t) and in vivo (3, 4). The observation that tumor cells that produce very low levels of AFP are able to survive selectively on treatment with the antiserum in vitro (5) suggests that the cytotoxic activity of the antiserum may depend on the level of AFP production by the tumor cells. Recent studies (1, 6, 7) have shown that AFP is detectable on the cell surface of AFP-producing tumor cells and suggest that the binding of antibody to AFP may have a fundamental role in its cytotoxic effect on such cells.Daunomycin (8, 9) is a well-known antitumor drug, widely used in cancer therapy. The major drawback, however, is that daunomycin, like many other drugs effective in killing tumor cells, also has detrimental effects on rapidly proliferating normal cells. This toxicity, which limits the effective use of chemotherapy in treatment of neoplastic diseases, may perhaps be overcome or reduced by binding the drug to a carrier that has specific affinity to the tumor cells (10,11). In previous studies, we have tested the effects of daunomycin-antitumor immunoglobulin conjugates on various tumors (12). These conjugates retained most of their original drug and antibody activities (13,14). Conjugates with specific antibodies were more effective than drug conjugates with normal immunoglobulin and, under certain conditions, were an improvement over the free drug (15).The aim ofthis study was to test specific antibodies produced in a horse against rat AFP as carriers of daunomycin. Daunomycin-anti-AFP conjugates were tested for their effect on rat hepatoma and compared with free daunomycin, anti-AFP, and a mixture of anti-AFP and daunomycin linked to normal horse immunoglobulin. A preliminary report ofthese studies has been presented. § A report describing the in vitro effect of daunomycin attached by a direct linkage to anti-mouse a-fetoprotein has also appeared (16). MATERIALS AND METHODSSpecific Antibodies to AFP. Specific antiserum to rat AFP was produced in a horse by weekly subcutaneous injections of 1 mg of purified AFP (17) emulsified in Freund's complete adjuvant. Specific antibodies to rat AFP were purified by affinity chromatography on activated Sepharose 4B coupled to rat AFP (18). (Fab')2 fragment from the specific antibody was prepared by pepsin digesti...

Monoclonal antibodies to rat a-fetoprotein (AFP) were produced by hybridization of mouse myeloma cells with spleen cells from mice immunized with rat AFP. The monoclonal antibodies as well as horse anti-rat AFP were coupled via a dextran bridge to daunomycin. Both types of conjugates were tested in vitro and in vivo for their anti-tumor activity. They were equally cytotoxic to rat AH66 hepatoma cell line in culture. Rats challenged with hepatoma cells were treated with the conjugates either by intraperitoneal or intravenous injections. Daunomycin conjugates with horse anti-AFP and monoclonal mouse anti-AFP were capable of delaying the tumor development more efficiently than the controls of antibodies or free drug, mixtures of drug with antibodies, and a conjugate ofdrug and normal immunoglobulin. The specific conjugates were considerably more effective when the treatments were given intravenously. The specific conjugates produced 60% long-term survival, whereas the controls delayed only slightly tumor development. Chemotherapeutic studies were performed with both types of conjugates in the treatment of rat hepatoma. Although previously the tumor and the treatments were delivered by the same route, intraperitoneally (i.p.) (1), treatments now also were given intravenously (i.v.). The systemic treatment with the specific carriers ofthe i.p. tumor produced 60% long-term survival, suggesting high efficacy and successful targeting at the tumor sites. MATERIALS AND METHODSSpecific Antibodies to AFP. The preparation ofhorse anti-rat AFP and the purification of the specific antibody by affinity chromatography have been described (10, 11). Monoclonal antirat AFP was prepared by hybridization (2) according to a procedure described by Eshhar et al. (12). Spleen cells were taken from mice that were immunized with rat AFP according to the following schedule. The mice were injected into the footpad twice (10 days apart) with 10 ,Ag of rat hepatomal AFP (13) in complete Freund adjuvant. Six and 5 days prior to the hybridization they received 10 ,g ofAFP, iLv., and i.p., respectively. Positive cultures were detected by solid-phase indirect radioimmunoassay (14), by using as a second antibody "2I-labeled goat anti-mouse F(ab')2 (specific activity, 2 X 10 cpm/mg). They were cloned subsequently in agar or by limiting dilutions. The clones were grown in vitro in culture or in CD2 mice.Tumor Cells. The rat ascites cell line AH66 was used throughout the experiments. It was maintained by i.p. passage in syngeneic Donryu rats or cultivation in vitro (15,16 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.