Methods and Compositions Related to Soluble Monoclonal Variable Lymphocyte Receptors of Defined Antigen Specificity

a monoclonal variable and lymphocyte receptor technology, applied in the field of antigen specific polypeptides, can solve the problem of not having a large-scale vlr cell line availabl

Inactive Publication Date: 2012-07-26
UAB RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no cell lines were available for large scale VLR production because of these characteristics.

Method used

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  • Methods and Compositions Related to Soluble Monoclonal Variable Lymphocyte Receptors of Defined Antigen Specificity
  • Methods and Compositions Related to Soluble Monoclonal Variable Lymphocyte Receptors of Defined Antigen Specificity
  • Methods and Compositions Related to Soluble Monoclonal Variable Lymphocyte Receptors of Defined Antigen Specificity

Examples

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example 1

VLRs that Specifically Bind Bacillus anthracis

[0103]VLR-positive lymphocytes from Bacillus anthracis exosporium immunized lamprey were harvested and their RNA isolated. Primers were used to amplify VLR cDNAs which were cloned into an expression vector and transformed into bacteria. Selected colonies were screened by PCR with VLR specific primers. The heterogeneous size of the PCR products indicated the diversity of the VLR cDNA library. Plasmids were purified from individual colonies and transfected into HEK-293 cells, which were tested for VLR expression by Western blotting of detergent-soluble cell lysates with anti-VLR mAb under reducing conditions. The first six VLRs expressed were composed of monomeric VLR units of different sizes (FIGS. 3A, 3B and 3C) due to variable numbers of their constituent LRR modules. When the culture supernatants of the transfected HEK-293 cells were examined for the presence of secreted VLRs, the products of three of these six VLR clones were secrete...

example 2

VLRs that Specifically Bind H Blood Group Determinant

[0106]Lampreys were immunized with 1×107 type O human erythrocytes once a week for four weeks. One week following the last immunization, lamprey plasma was collected. Two CHO cell lines were also employed, one transfected with α1,2-fucosyltransferase to produce the H antigen on the surface of CHO cells and the other transfected with the vector alone (Prieto et al., J Biol. Chem. 1997 Jan. 24; 272(4):2089-97.) Cells were first incubated in 1:10 dilution of lamprey plasma or 1:50 of the monoclonal antibody 92 FR A2, which is specific for the H antigen. All cells were washed and those cells incubated with lamprey plasma were then incubated in mAb 4C4 which recognized VLR molecules and then washed. All cells were stained with a goat anti mouse-RPE secondary antibody and then washed twice. FACS histogram shows that only plasma from lamprey immunized with human erythrocytes stained CHO cells transfected with the enzymes to produce the H...

example 3

Production and Characterization of Lamprey Monoclonal VLR-B Antibodies of Defined Antigen Specificity

[0107]Isolation of antigen specific VLR-B clones. To surmount the constraints for culturing VLR-producing lamprey cells or hybridomas, a heterologous expression system was developed utilizing HEK-293T cells transfected with full-length VLR-B cDNAs, which spontaneously secrete recombinant oligomeric VLR-B antibodies into the tissue culture supernatant. The secretion of VLR-B clones by HEK-293T cells provided the means to screen a large number of clones for antigen binding using a methodology similar to hybridoma screening. The procedure enables antigen specific VLR-B clones to be isolated utilizing techniques accessible to biological laboratories and requires a time investment comparable to monoclonal antibody production (FIGS. 8A and 8B). Lamprey larvae were immunized every two weeks for a total of eight weeks before FACS isolation of VLR-B+ lymphocytes from blood samples by FACS. RN...

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Abstract

Disclosed are compositions and methods related to variable lymphocyte receptors (VLRs). More particularly, disclosed are a variety of antigen specific polypeptides, including soluble, monoclonal, and multivalent forms, as well as methods of using the polypeptides, antibodies that bind the antigen specific polypeptides, and nucleic acids, vectors and expression systems that encode the polypeptides. Antigen specific polypeptides that selectively bind pathogens, like anthrax, and carbohydrates, like blood group determinants, are specifically disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Ser. No. 60 / 835,033 filed Aug. 2, 2006, which is incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION[0002]Jawless vertebrates were recently demonstrated to have antigen specific receptors called variable lymphocyte receptors (VLRs). These VLRs play a role in adaptive immunity but are distinctly different from immunoglobulin-type antigen receptors found in jawed vertebrates. VLRs are clonally diverse and comprise leucine-rich repeat (LRR) modules. VLRs were previously isolated from lampreys or hagfish and are known to have a GPI anchor and be membrane bound. However, no cell lines were available for large scale VLR production because of these characteristics.SUMMARY OF THE INVENTION[0003]As embodied and broadly described herein, the present application relates to antigen specific polypeptides and methods and compositions related thereto. The present application further relat...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K16/00C12Q1/04C07H21/04C12N15/63C12N5/10C12P21/02G01N33/53
CPCC07K16/1018C07K16/1063C07K2317/20C07K16/34C07K16/1278A61P31/12A61P31/16A61P31/18C07K2317/14C07K2317/33C07K2317/35C07K2317/76
Inventor COOPER, MAX D.HERRIN, BRANTLEY R.ALDER, MATTHEW N.
Owner UAB RES FOUND
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