Conservation of leukocyte cell surface proteins: implications for the generation of monoclonal antibodies against newly identified leukocyte cell surface proteins.

The availability of mouse monoclonal antibodies has been integral to the classification of human leukocyte cell surface proteins under the "Cluster of Differentiation" or "CD" nomenclature system. The sequencing of the human genome has identified many more proteins that have characteristics similar to the known leukocyte cell surface proteins, but which have not so far been identified using monoclonal antibodies. One factor that may have limited the generation of monoclonal antibodies to some of these proteins is the high level of sequence conservation between the mouse and human proteins, in particular in the extracellular regions that are recognized by most of the widely used antibodies. An alternative approach is to use a more distant species, such as chickens, for the generation of antibody reagents. Here we compare the extent of amino acid differences in the protein CD molecules expressed by human leukocytes and their mouse and chicken homologs. The analysis confirms that the human proteins are more similar to the mouse homologs than the chicken homologs. The results indicate that chicken antibodies have the potential to be used as an alternative to mouse reagents where human-mouse sequence conservation is high.

Improved antigen binding by a CD20-specific single-chain antibody fragment with a mutation in CDRH1.

We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.

Penetration of engineered antibody fragments into the eye.

Antibodies are powerful immunotherapeutic agents but their use for treating ocular disorders is limited by their poor penetration into the eye. We hypothesized that antibody fragments of relatively small size might penetrate the cornea more readily. Monovalent single chain variable region (scFv) antibody fragments and divalent miniantibodies were engineered from existing monoclonal antibodies, expressed in a bacterial expression system, and purified by metal ion affinity chromatography. Corneoscleral preparations from normal pig and cat eyes were mounted in a corneal perfusion chamber. Intact antibodies and antibody fragments were applied topically to the anterior corneal surface over 12-h periods, and samples were collected from the artificial anterior chamber. Similar experiments were performed with whole enucleated pig and human eyes. Penetration of antibodies and fragments was quantified by high-sensitivity flow cytometry on appropriate target cells. Both monovalent scFv and divalent miniantibody fragments (but not whole immunoglobulin molecules) passed through de-epithelialized and intact corneas after topical administration, and could be detected by antigen binding. Addition of 0.5% sodium caprate facilitated penetration through intact corneas. Topically-applied scFv was found to penetrate into the anterior chamber fluid of rabbit eyes in vivo. The engineered fragments were stable and resistant to ocular proteases. Monovalent and divalent antibody constructs of molecular weight 28 kD and 67 kD, respectively, can penetrate through intact corneas into the anterior chamber, with retention of appropriate antigen-binding activity. Such constructs may form novel therapeutic agents for topical ophthalmic use.

Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin.

The success of recombinant antibody fragments as diagnostic reagents and therapeutic agents depends on the availability of sufficient functional material. We have produced a bacterial expression vector that combines high-level expression driven by a modified Shine-Dalgarno sequence with the periplasmic chaperonin Skp. Using this vector, we are able to obtain higher yields of soluble antibody fragments from cultures without the need for supplementation of the culture medium during expression. The fragments produced in the presence of the Skp show improved antigen binding activity compared to when the chaperonin is absent.

Identification of additional genes required for O-antigen biosynthesis in Vibrio cholerae O1.

The cloning and expression of the genes encoding the Vibrio cholerae O1 lipopolysaccharide O antigen in a heterologous host have been described previously (P. A. Manning, M. W. Heuzenroeder, J. Yeadon, D. I. Leavesley, P. R. Reeves, and D. Rowley, Infect. Immun. 53:272-277, 1986). It was thus assumed that all the genes required for O-antigen expression were located on a 20-kb SacI restriction fragment. We present evidence for a number of other as yet undescribed genes that are essential for O-antigen biosynthesis in V. cholerae O1 and that these genes are somehow complemented in Escherichia coli K-12. The two genes termed Vibrio cholerae rfbV and rfbU are transcribed in the opposite orientation from the rest of the rfb operon, whereas the galE dehydratase and rfbP (Salmonella enterica) homologs, designated ORF35x7 and rfbW, respectively, are transcribed in the same orientation. The evidence presented here, using chromosomal insertion mutants, clearly shows that the three genes now designated rfbV, rfbU, and rfbW appear to be accessory rfb genes and are essential for O-antigen biosynthesis in V. cholerae but that ORF35x7 is not.

Restricted epitope recognition by precipitin-negative anti-La/SS-B-positive sera.

OBJECTIVE: To determine whether anti-La/SS-B-positive sera that are precipitin negative show a distinct B cell epitope pattern. METHODS: Serum reactivity was tested with recombinant La/SS-B fusion proteins. RESULTS: Among the 18 precipitin-negative anti-La/SS-B-positive sera, reactivity was confined to the full-length recombinant protein (La33.3) in 8 (44%); 5 of 18 (28%) reacted only with La33.3 and with the first 107 N-terminal amino acids (LaA), and 4 (22%) reacted with La33.3, LaA, and the middle region of the La molecule (LaC; amino acids 111-242). One serum reacted with La33.3 and LaC. None of the 18 precipitin-negative sera was positive on a carboxy-terminal fragment (LaL2/3; amino acids 346-408). In contrast, all 26 precipitin-positive anti-La/SS-B-positive sera reacted with La33.3, LaA, and LaC, and 92% reacted with LaL2/3. Rheumatoid factor and serum IgG levels were significantly lower in the precipitin-negative group, providing further evidence of a distinct serologic subset. CONCLUSION: The restricted epitope recognition by these sera may explain the lack of precipitin formation and may represent an early autoantibody response to La/SS-B.