Meredith Mudgett-Hunter

Bio: Meredith Mudgett-Hunter is an academic researcher from Harvard University. The author has contributed to research in topics: Antibody & Monoclonal antibody. The author has an hindex of 12, co-authored 14 publications receiving 3450 citations.

Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.

Abstract: A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.

Variable region framework differences result in decreased or increased affinity of variant anti-digoxin antibodies.

Abstract: Rare spontaneous variants of the anti-digoxin antibody-producing hybridoma 40-150 (Ko = 5.4 x 10(9) M-1) were selected for altered antigen binding by two-color fluorescence-activated cell sorting. The parent antibody binds digoxin 890-fold greater than digitoxin. The variant 40-150 A2.4 has reduced affinity for digoxin (Ko = 9.2 x 10(6) M-1) and binds digoxin 33-fold greater than digitoxin. A second-order variant, derived from 40-150 A2.4 (designated 40-150 A2.4 P.10), demonstrated partial regain of digoxin binding (Ko = 4.4 x 10(8) M-1). The altered binding of the variant 40-150 A2.4 was accounted for by a point mutation resulting in substitution of arginine for serine at position 94 in the heavy chain variable region. Antibody 40-150 A2.4 P.10 also contains this arginine but owes its enhanced antigen binding to deletion of two amino acids from the heavy chain amino terminus. This unusual sequence alteration in an immunoglobulin framework region confers increased affinity for antigen.

Hybridoma proteins expressing the predominant idiotype of the antiazophenylarsonate response of A/J mice.

Abstract: Hybridoma cell lines that secrete monoclonal antiazophenylarsonate antibodies were isolated from the fusion of A/J splenic lymphocytes with a myeloma cell line. A small percentage of these hybridoma proteins were recognized by rabbit antisera that detect the crossreactive idiotype characteristics of the antiazophenylarsonate response of A/J mice. The isotype, pI value, and amino-terminal sequences of four independently derived idiotype-positive hybridoma proteins were determined. These proteins were either of the IgG1 or IgG2a heavy chain class. For two mice tested, the majority of the idiotype in the immune serum was shown to be of the same isotype as the fusion-derived monoclonal antibodies. The pI values of the hybridoma proteins differed from one another and ranged from 6.9 to 7.6. Amino acid sequences of the heavy chains showed a significant degree of homology with each other, but each chain was unique in the framework or the first complementarity determining region (or both). A comparable pattern of sequence variation was evident for the light chains. The azophenylarsonate idiotype, therefore, appears to consist of a family of nonidentical but closely related molecules that are the product of more than one germline gene or the result of somatic mutation of a single germline gene.

Transgenic non-human animals capable of producing heterologous antibodies

Abstract: The invention relates to transgenic non-human animals capable of producing heterologous antibodies and methods for producing human sequence antibodies which bind to human antigens with substantial affinity.

Phage antibodies: filamentous phage displaying antibody variable domains

Abstract: NEW ways of making antibodies have recently been demonstrated using gene technology. Immunoglobulm variable (V) genes are amplified from hybridomas or B cells using the polymerase chain reaction, and cloned into expression vectors. Soluble antibody fragments secreted from bacteria are then screened for binding activities (see ref. 1 for review). Screening of V genes would, however, be revolutionized if they could be expressed on the surface of bacteriophage. Phage carrying V genes that encode binding activities could then be selected directly with antigen. Here we show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage (one in a million) can be isolated after affinity chromatography.

Methods for producing members of specific binding pairs

Abstract: A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.

Abstract: A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.