The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (pl85"m), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "hum " antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant do was constructed. Light-and heavy-chain variable regions were simultaneously humned in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonudleotides, respectively. The protooncogene HER2 encodes a protein tyrosine kinase (pl85HER2) that is homologous to the human epidermal growth factor receptor (1-3). Amplification and/or overexpression of HER2 is associated with multiple human malignancies and appears to be integrally involved in progression of 25-30%o of human breast and ovarian cancers (4, 5).Furthermore, the extent of amplification is inversely correlated with the observed median patient survival time (5). The murine monoclonal antibody mumAb4D5 (6), directed against the extracellular domain (ECD) of p185HER2, specifically inhibits the growth of tumor cell lines overexpressing p185HER2 in monolayer culture or in soft agar (7,8).mumAb4D5 also has the potential of enhancing tumor cell sensitivity to tumor necrosis factor (7,9). Thus, mumAb4D5 has potential for clinical intervention in carcinomas involving the overexpression of p185HER2.A major limitation in the clinical use of rodent mAbs is an anti-globulin response during therapy (10,11). A partial solution to this problem is to construct chimeric antibodies by coupling the rodent antigen-binding variable (V) domains to human constant (C) domains (12)(13)(14). The isotype of the human C domains may be varied to tailor the chimeric antibody for participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (15). Such chimeric antibody molecules are still =30% rodent in sequence and are capable of eliciting a significant anti-globulin response.Winter and coworkers (16-18) pioneered the "humanization" of antibody V domains by transplanting the complementarity determining regions (CDRs), which are the hypervariable loops involved in antigen binding, from rodent antibodies into human V domains. The validity of this approach is supported by the clinical efficacy of a humanized antibody specific for the CAMPATH-1 antigen with two non-Hodgkin lymphoma patients, one of whom had previously developed an anti-globulin response to the parental rat antibody (17,19). In some cases, transplanting hypervariable loops from rodent antibodies into human frameworks is sufficient to transfer high antigen binding affinity (16, 18), whereas in other cases it has been necessary to also replace one (17) or several (20) framework region (FR) residues. For a given antibody, a small number of FR residues are ...

The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-alpha (HRG-alpha) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 +/- 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

Modulation of the growth of human and murine cell lines in vitro by recombinant human tumor necrosis factor-α (rTNF-α) and recombinant human interferon-γ (rIFN-γ) was investigated. rTNF-α had cytostatic or cytolytic effects on only some tumor cell lines. When administered together with rIFN-γ, rTNF-α showed enhanced antiproliferative effects on a subset of the cell lines tested. In contrast to its effects on sensitive tumor cells, rTNF-α augmented the growth of normal diploid fibroblasts. Variations in the proliferative response induced by rTNF-α were apparently not due to differences in either the number of binding sites per cell or their affinity for rTNF-α. These observations indicate that the effects of rTNF-α on cell growth are not limited to tumor cells, but rather that this protein may have a broad spectrum of activities in vivo.

Macrophages are important in the induction of new blood vessel growth during wound repair, inflammation and tumour growth. We show here that tumour necrosis factor-alpha (TNF-alpha), a secretory product of activated macrophages that is believed to mediate tumour cytotoxicity, is a potent inducer of new blood vessel growth (angiogenesis). In vivo, TNF-alpha induces capillary blood vessel formation in the rat cornea and the developing chick chorioallantoic membrane at very low doses. In vitro, TNF-alpha stimulates chemotaxis of bovine adrenal capillary endothelial cells and induces cultures of these cells grown on type-1 collagen gels to form capillary-tube-like structures. The angiogenic activity produced by activated murine peritoneal macrophages is completely neutralized by a polyclonal antibody to TNF-alpha, suggesting immunological features are common to TNF-alpha and the protein responsible for macrophage-derived angiogenic activity. In inflammation and wound repair, TNF-alpha could augment repair by stimulating new blood vessel growth; in tumours, TNF-alpha might both stimulate tumour development by promoting vessel growth and participate in tumour destruction by direct cytotoxicity.

Since the poor prognosis associated with HER2 amplified breast cancers might be explained by a mechanistic association between p185HER2 overexpression and therapeutic resistance, we assessed the chemo-endocrine sensitivity of estrogen receptor (ER) containing MCF-7 breast cancer cells transfected with full-length HER2 cDNA. Of the 36 isolated MCF/HER2 subclones, 7 were found to overexpress p185HER2 surface receptor at levels 3 to 45-fold greater than parental or control transfected cells (MCF/neo). The overexpressing transfectants possessed increased inositol-1,4,5-triphosphate-3'-kinase activity comparable to enzyme activity in the endogenously HER2 amplified breast cancer cell lines SK-Br-3 and BT-474. The anti-p185HER2 monoclonal antibody and receptor-specific partial agonist, muMAb4D5 (4D5), known to inhibit growth of SK-Br-3 and BT-474 cells, produced no significant growth inhibitory effect on any of the transfectants including the 45-fold overexpressing MCF/HER2-18 cells which were studied in greater detail. MCF/HER2-18 cells contained at least partially functioning exogenous receptor since 4D5 (3 micrograms/ml) specifically stimulated phosphorylation of p185HER2 and its co-precipitating ptyr56 substrate within 5 min, and this was followed at 1 h by a transient induction of c-myc but not c-fos mRNA. ER content and the in vitro sensitivity of MCF/HER2-18 cells to 5-fluorouracil and adriamycin were identical to those of control transfectants and parental cells. However, these highly overexpressing transfectants had acquired low level (2 to 4-fold) resistance to cisplatin and were no longer sensitive to the antiestrogen tamoxifen (TAM). To compare the hormone-dependent tumorigenicity of the HER2 transfectants, MCF/HER2-18 and control cells (MCF, MCF/neo-3) were implanted into ovariectomized athymic nude mice. No tumors were produced in the absence of estradiol (E2) administration. In E2 supplemented mice, MCF/HER2-18 tumors grew most rapidly. When E2 treatment was stopped and daily TAM injections were initiated, MCF-7 and MCF/neo-3 tumor growth ceased immediately, while MCF/HER2-18 tumors continued to show an accelerated growth rate lasting weeks. This pattern of hormone-dependent, TAM-resistant growth exhibited by the MCF/HER2-18 tumors in nude mice supports the possibility that p185HER2 overexpression in human breast cancers may be linked to therapeutic resistance.