Antibody engineering using very long template-assembled oligonucleotides

Carter, Paul; Garrard, Lisa J.; Henner, Dennis J.

doi:10.1016/s1046-2023(05)80171-9

Cited by 2 publications

(2 citation statements)

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“…Here we report the rapid and simultaneous humanization of heavy-chain (VH) and light-chain (VL) V region genes of mumAb4D5 by using a "gene conversion mutagenesis" strategy (43). Eight humanized variants (humAb4D5) were constructed to probe the importance of several FR residues identified by our molecular modeling or previously by others (22)(23)(24) …”

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Humanization of an anti-p185HER2 antibody for human cancer therapy.

Carter¹,

Presta²,

Gorman³

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

1,672

989

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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 ...

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“…Genes encoding chimeric mAb4D5 light-chain and heavy-chain Fd fragment (VH and CH1 domains) were subcloned together into pUC119 (27) to create pAK1 and were simultaneously humanized in a single step (43). Briefly, sets of six contiguous oligonucleotides were designed to humanize VH and VL (Fig.…”

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confidence: 99%