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 HER2 protooncogene encodes a 185-kDa transmembrane protein (p185HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. These data also support the hypothesis that p185HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics. The anti-p185HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185HER2. The monoclonal antibody is directed at the extracellular (ligand binding) domain of this receptor tyrosine kinase and presumably has its effect as a result of modulating receptor function. In vitro assays have shown that muMAb 4D5 can specifically inhibit the growth of tumor cells only when they overexpress the HER2 protooncogene. MuMAb 4D5 has also been shown to enhance the TNF-alpha sensitivity of breast tumor cells that overexpress this protooncogene. Relevant to its clinical application, muMAb 4D5 may enhance the sensitivity of p185HER2-overexpressing tumor cells to cisplatin, a chemotherapeutic drug often used in the treatment of ovarian cancer. In vivo assays with a nude mouse model have shown that the monoclonal antibody can localize at the tumor site and can inhibit the growth of human tumor xenografts which overexpress p185HER2. Modulation of p185HER2 activity by muMAb 4D5 can therefore reverse many of the properties associated with tumor progression mediated by this putative growth factor receptor. Together with the demonstrated activity of muMAb 4D5 in nude mouse models, these results support the clinical application of muMAb 4D5 for therapy of human cancers characterized by the overexpression of p185HER2.

The product of the HER2 protooncogene, p185HEP2, represents an attractive target for cancer immunotherapies. We The HER2 (c-erbB2, neu) protooncogene and pl85HER2, the growth factor receptor-tyrosine kinase it encodes, appear to play a central role in the pathogenesis of many human cancers.

Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.

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