Secretory immunoglobulin produced by single cells and methods for making and using same

a technology of secretory immunoglobulin and cultured cells, which is applied in the direction of antibody medical ingredients, immunological disorders, peptide/protein ingredients, etc., can solve the problems of inefficient formation of disulfide bonds between diga and sc in vitro, unstable non-secretory forms with relatively short half-lives, and limited therapeutic use, so as to avoid sig alterations, reduce hospitability, and be more efficient

Inactive Publication Date: 2002-09-12
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently available monoclonal IgA is of limited therapeutic use since stable, secretory forms can only be produced in limited amounts and the non-secretory forms are unstable with relatively short half-lives in vivo.
However, the formation of disulfide bonds between dIgA and SC in vitro was inefficient.

Method used

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  • Secretory immunoglobulin produced by single cells and methods for making and using same
  • Secretory immunoglobulin produced by single cells and methods for making and using same
  • Secretory immunoglobulin produced by single cells and methods for making and using same

Examples

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example 1

Cloning Human Ectoplasmic Domain

[0061] To produce sIgA a gene coding for human pIgR was obtained from Dr. Charlotte S. Kaetzel (University of Kentucky, Lexington, Ky.). A fragment from pIgR containing only the ectoplasmic domain (SC) and lacking the transmembrane and the cytoplasmic domains was generated. A 1402 bp PCR fragment was generated using the complete human pIgR cDNA in pBluescript (Tamer et al., Mol. Immunol. 30 413-421, 1993, this article and all other articles cited herein are incorporated herein by reference) as template and the primers:

1 1. 5'-GGGCAGAACGGTGACCATCAACTGCCCTTT-3' (SEQ ID NO:1) and 2. 5'-AAGGAATTCCTACTCTGCAAAAAGCCTGGGGTCCTGAATGGC-3' (SEQ ID NO:2)

[0062] The second primer included a silent base change upstream of Glu589 to delete a BamHI site in the SC coding region to facilitate cloning. A stop codon, shown by underlining, followed by an EcoRI site downstream of Glu589 were also included. A stop codon was introduced at amino acid 590, the position of normal...

example 2

Expression of Cloned Human Ectoplasmic Domain in Cells Secreting Mouse-Human Chimeric IgA1

[0063] Sp2 / 0 transfectants secreting monomeric and dimeric forms of mouse-human chimeric IgA1 have been previously reported (Chintalacharuvu et al., J. Immunol. 157 3443, 1996). Cells secreting mouse-human chimeric IgA1 were transfected with the SC expression vector by electroporation. Sp2 / 0 cells were plated in 96-well tissue culture plates in presence of Histidinol. The surviving colonies were screened for SC secretion by ELISA using goat anti-.kappa. as the trapping antibodies and rabbit anti-human SC as the detecting antibody. The clone producing the highest quantity of sIgA was expanded and adapted to growth in serum free medium.

[0064] Since SC is a cleavage product of the pIgR a stop codon was introduced at the site of cleavage (FIG. 1A). Murine transfectomas secreting mouse-human chimeric IgA1 specific for the hapten dansyl (Chintalacharuvu et al., J. Immunol 157 3443, 1996) were transfe...

example 3

Analysis of Culture Supernatants

[0065] The levels of sIgA in culture supernatants were determined by ELISA as described previously (Chintalacharuvu et al., J. Immunol 157 3443, 1996). Microtiter plates coated with dansylated BSA was used to capture sIgA. Bound sIgA was detected by incubation with rabbit antiserum to human SC (Chintalacharuvu et al., J. Cell. Physiol. 148 35, 1991) diluted 1:2000 in phosphate buffered saline (PBS) containing 1% (w / v) BSA (PBS-1% BSA). Bound rabbit antibody was detected using an alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma Imm. Chem.) diluted 1:10,000 in PBS-1% BSA. Color was developed by adding 5 mg / ml of disodium p-nitrophenyl phosphate (Sigma Imm. Chem.).

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Abstract

Disclosed is a method of producing secretory Ig molecules. The method comprises transfecting a cell producing an Ig with a polynucleotide encoding an SC to form SC transfected Ig producing cells. Secretory Ig molecules, such as secretory IgA, can be used to treat or prevent infection.

Description

[0001] This application is based on United States provisional application serial No. 60 / 050,969, filed Jun. 19, 1997, the contents of which are incorporated herein by reference. Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.FIELD OF THE INVENTION[0003] The present invention relates to secretory Ig molecules produced by a single cultured cell. The invention also relates to methods of producing and using the secretory Ig molecules.BACKGROUND OF THE INVENTION[0004] Secretory IgA (sIga) is found in external secretions such as colostrum, respiratory and intestinal mucin, saliva, tears and genitournary tract mucin and is often the first line of defense against infectious agents.[0005] Monoclonal antibodies, specific for different diseases are available to combat infectio...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00A61K39/395A61P37/04C07K16/00C12P21/04
CPCA61K38/00A61K39/39591C07K16/00C07K2317/24Y10S530/808A61P37/04
Inventor MORRISON, SHERIE L.CHINTALACHARUVU, KOTE R.
Owner RGT UNIV OF CALIFORNIA
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