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Amino acid sequence of the N-terminal sixty-nine residues of heavy chain derived from a homogeneous rabbit antibody.

ABSTRACT: The sequence of the N-terminal 69 residues of heavy chain from a homogeneous rabbit antibody to type III pneumococcal polysaccharide was determined. The sequence is similar to that found in heavy chains of normal pooled rabbit immunoglobulins of the same allotype Aa1. Two regions of the homogeneous heavy chain (residues 35-46 and 62-69) are very similar to corresponding regions of heavy chains from rabbit Aa2 immunoglobulin, as well as from mouse, guinea-pig and human immunoglobulins. In contrast, residues 47-62 appear to be variable. Comparison in this section with another homogeneous anti-pneumococcal antibody (Strosberg et al., 1972) of related specificity and of the same allotype indicates sequence variation in at least three positions. An antibody to group C streptococcal carbohydrate of allotype Aa2 (Fleischman, 1971) differs by five amino acids in the same region of the heavy chain. Sequence variability between these three antibodies does not occur in homologous positions within this variable section. Allotype-related sequences could not be identified in section 34-65.

SUBMITTER: Jaton JC

PROVIDER: S-EPMC1174434 | biostudies-other | 1972 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundMonoclonal antibodies have been used to effectively treat various tumors. We previously established a unique strategy to identify tumor specific antibodies by capturing B-cell response against breast tumor antigens from patient-derived sentinel lymph nodes. Initial application of this approach led to identification of a tumor specific single domain antibody. In this paper we optimized our previous strategy by generating heavy chain antibodies (HCAbs) to overcome the deficiencies of single domain antibodies. Here we identified and characterized a heavy chain antibody (HCAb2) that targets cell surface HSP90 antigen on breast tumor cells but not normal cells.MethodsEight HCAbs derived from 4 breast cancer patients were generated using an in vitro expression system. HCAbs were screened against normal breast cells (MCF10A, HMEC) and tumor cell lines (MCF7, MDA-MB-231) to identify cell surface targeting and tumor specific antibodies using flow cytometry and immunofluorescence. Results observed with cell lines were validated by screening a cohort of primary human breast normal and tumor tissues using immunofluorescence. Respective antigens for two HCAbs (HCAb1 and HCAb2) were identified using immunoprecipitation followed by mass spectrometry. Finally, we generated MDA-MB-231 xenograft tumors in NOD scid gamma mice and performed in vivo tumor targeting analysis of HCAb1 and HCAb2.ResultsFlow cytometry screen revealed that HCAb2 selectively bound to the surface of MDA-MB-231 cells in comparison to MCF10A and MCF7 cells. HCAb2 showed punctate membrane staining on MDA-MB-231 cells and preferential binding to human breast tumor tissues in comparison to normal breast tissues. In primary breast tumor tissues, HCAb2 showed positive binding to both E-cadherin positive and negative tumor cells. We identified and validated the target antigen of HCAb2 as Heat shock protein 90 (HSP90). HCAb2 also selectively targeted MDA-MB-231 xenograft tumor cells in vivo with little targeting to mouse normal tissues. Finally, HCAb2 specifically targeted calnexin negative xenograft tumor cells.ConclusionsFrom our screening methodology, we identified HCAb2 as a breast tumor specific heavy chain antibody targeting cell surface HSP90. HCAb2 also targeted MDA-MB-231 tumor cells in vivo suggesting that HCAb2 could be an ideal tumor targeting antibody.

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Amino acid sequence of the N-terminal sixty-nine residues of heavy chain derived from a homogeneous rabbit antibody.

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