Efficient generation of antibodies to oncoproteins by using synthetic peptide antigens.

Tanaka, Takeo; Slamon, Dennis J.; Cline, Martin J.

doi:10.1073/pnas.82.10.3400

Cited by 66 publications

(36 citation statements)

References 27 publications

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“…The basis for this peptide design was that the 13-amino acid peptide should have been of a sufficient length to generate an antibody response, while it was hypothesized that the two linear stretches of 5 and 7 amino acid peptides of normal sequence may not have been of sufficient length to generate an immune response. In general, synthetic peptides with <10 amino acid residues fail to elicit an effective antibody response (19). The 5-amino acid sequence on the aminoterminal side of the fusion point amino acid glycine also represents the 5 amino-terminal residues in the normal EGFR (20), and thus the fusion junction synthetic peptide has the same sequence as the new amino terminus of the mutant EGFR.…”

Section: Discussionmentioning

confidence: 99%

Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.

Humphrey

Wong

Vogelstein

et al. 1990

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

344

188

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We have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate sitespecific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.

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Section: Discussionmentioning

confidence: 99%

Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.

Humphrey

Wong

Vogelstein

et al. 1990

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

344

188

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“…First, the length of the immunizing peptide is critical . To raise antibodies to a peptide, a minimum length of six amino acids is required, and peptides of >10 amino acids generally induce antibodies that may bind to the native protein (10). Equally important is the part of the protein from which the sequence is derived.…”

Section: Discussionmentioning

confidence: 99%

“…Not all attempts to raise protein-reactive peptide antibodies have been successful (10,11), however, and many failures occur despite attention to selecting amino acid sequences of appropriate size, charge, and helix formation . While investigating the sharing of amino acid sequences between virus proteins and a defined region of the a chain of the human acetylcholine receptor (HuAChR), we noted (T .…”

mentioning

confidence: 99%

Peptides as antigens. Importance of orientation.

Dyrberg¹,

Oldstone²

1986

The Journal of Experimental Medicine

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Factors known to be important in producing protein-reactive peptide antibodies include the accessibility of the region from which the peptide sequence is derived, the hydrophilic-phobic character of the sequence, and the length of the peptide. The data presented here indicate that the orientation of the peptide coupled to a carrier protein also influences the binding pattern of peptide antibodies. An octapeptide, representing a sequence from the alpha chain of the human acetylcholine receptor, was coupled either through an N- or C-terminal cysteine-glycine-glycine linker to a carrier protein and used to immunize rabbits. The resulting antisera reacted at comparable titers to the uncoupled immunizing peptides, but did not crossreact with the identical but opposite-linked peptide. Characterization of the binding to other homologous peptides showed that immunization with the N-terminal-linked peptide induced antibodies reactive specifically with the C-terminal amino acid(s). Immunization with the C-linked peptide resulted in antibodies reactive with a site of the peptide near the C-terminus.

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“…Materials-Anti-Ras antiserum Has6 (Tanaka et al, 1985) is a generous gift of Takeo Tanaka (Kure National Hospital, Kure). Antibodies against MAPK (05-157), phosphotyrosine (05-321), and Shc (06 -203) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).…”

Section: Methodsmentioning

confidence: 99%

Ras Is Not Required for the Interleukin 3-induced Proliferation of a Mouse Pro-B Cell Line, BaF3

Terada

Kaziro

Satoh

1995

Journal of Biological Chemistry

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It has been demonstrated that Ras is involved in in-. Furthermore, the activation of fos gene promoter following IL-3 stimulation was almost completely abolished when Ras(S17N) was induced. Under these conditions, Ras(S17N) exhibited no inhibitory effect on IL-3-dependent proliferation assessed by the increase of cell numbers and a mitochondrial enzyme activity. The results indicate that Ras-dependent pathways, including the Raf/MAPK/Fos pathway, are dispensable for IL-3-induced growth stimulation. When BaF3 cells were treated with a tyrosine kinase inhibitor, herbimycin A, IL-3-dependent proliferation of the cells was impaired, suggesting that tyrosine kinase-mediated pathways are critical for growth promotion. On the other hand, apoptotic cell death caused by deprivation of IL-3 was prevented by the induction of the activated mutant Ras(G12V), although the rate of cell number increase was markedly reduced. Thus, it is likely that Rasindependent pathways play important roles to facilitate the proliferation although they may not be essential for IL-3-stimulated antiapoptotic signal transduction.In various types of cells, Ras functions as a molecular switch that regulates intracellular signaling pathways for proliferation, differentiation, and other physiological responses. Tyrosine kinase receptors, such as epidermal growth factor receptor and platelet-derived growth factor receptor, when stimulated by their specific ligands, form a complex with a variety of signal-transducing molecules including phosphatidylinositol 3-kinase, Ras-GTPase activating protein (Ras-GAP), 1 phospholipase C-␥1, and adaptor proteins (e.g. Grb-2, Nck, and Shc) through the interaction between specific phosphotyrosines on the receptor and Src homology 2 (SH2) domains of the binding molecules Schlessinger, 1993). Among the above components of the signal-transducing complex, Shc and Grb-2 are well characterized as elements that link the receptor and a Ras-guanine nucleotide exchange factors (Ras-GEFs), such as mSos-1. Adaptors and Ras-GEFs are known to participate also in Ras regulation through non-tyrosine kinase-type receptors including interleukin 2 (IL-2), IL-3, and T cell antigen receptors (Burns et al., 1993;Cutler et al., 1993;Ravichandran et al., 1993;Sato et al., 1993;Buday et al., 1994;Reif et al., 1994;Ravichandran and Burakoff, 1994;Welham et al., 1994). The active GTP-bound form of Ras specifically binds RasGAPs (Boguski and McCormick, 1993), c-Raf-1 (Avruch et al., 1994), B-Raf (Moodie et al., 1994;Vaillancourt et al., 1994), phosphatidylinositol 3-kinase (Rodriguez-Viciana et al., 1994), Ral-guanine nucleotide dissociation stimulator (Hofer et al., 1994;Kikuchi et al., 1994;Spaargaren and Bischoff, 1994), mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase (ERK) kinase (MEK) kinase (MEKK) (LangeCarter and Johnson, 1994), and Rin1 (Han and Colicelli, 1995), among which Raf proteins are best characterized as direct targets of Ras (Daum et al., 1994;Marshall, 1995). After the binding of Ras, the serine/t...

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Efficient generation of antibodies to oncoproteins by using synthetic peptide antigens.

Cited by 66 publications

References 27 publications

Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.

Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.

Peptides as antigens. Importance of orientation.

Ras Is Not Required for the Interleukin 3-induced Proliferation of a Mouse Pro-B Cell Line, BaF3

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