Cornelius B. Alexander

Rabbit Immune Repertoires as Sources for Therapeutic Monoclonal Antibodies: The Impact of Kappa Allotype-correlated Variation in Cysteine Content on Antibody Libraries Selected by Phage Display

The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. On the basis of phage display technology, we recently reported the first humanization of a rabbit monoclonal antibody. The allotypic diversity of rabbit immunoglobulins prompted us to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. In particular, we evaluated the diversity of unselected and selected chimeric rabbit/human Fab libraries that were derived from different kappa light chain allotypes. Most rabbit light chains have an extra disulfide bridge that links the variable and constant domains in addition to the two intrachain disulfide bridges shared with mouse and human kappa light chains. Here we evaluate the impact of this increased disulfide bridge complexity on the generation and selection of chimeric rabbit/human Fab libraries. We demonstrate that rabbits with mutant bas and wild-type parental b9 allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones with b9 allotype is a rabbit/human Fab that binds with a dissociation constant of 1nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer. Examination of 228 new rabbit antibody sequences allowed for a comprehensive comparison of the LCDR3 and HCDR3 length diversity in rabbits. This study revealed that rabbits exhibit an HCDR3 length distribution more closely related to human antibodies than mouse antibodies.

The structural and genetic basis for expression of normal and latent VHa allotypes of the rabbit

The immunoglobulin heavy chain variable regions of the rabbit are unusual in having genetically controlled, serologically detectable alternative forms, the VHa allotypes, as well as minor VH allotypes of the x, y and w groups. New insights into the probable structural basis for the VHa allotypes have come from re-examination of earlier protein sequence data in the light of newly deduced protein sequences derived from sequencing cloned cDNAs and genomic DNAs encoding VH regions. Here we review this sequence information, and define the allotype-correlated differences at seven positions in framework region 1 and 10 positions in framework region 3 that may lead to the serologically detectable allotypic determinants (allotopes). Most alternative amino acids at allotype-correlated positions can be derived from each other by single-base changes. Thus somatic mutations and/or gene conversion-like events must be considered along with other serological and genetic explanations for various reported observations of the production of latent VHa allotypes. The proximity of rabbit VH genes (approximately 3 kb apart) might enhance the likelihood of conversion-like events in both germline and somatic cells.

Tritium (3H) radiolabeling of protein A and antibody to high specific activity: Application to cell surface antigen radioimmunoassays

Staphylococcal protein A and several different immunoglobulins have been radiolabeled to high specific activities (greater than 10(6) cpm/microgram) by reductive methylation with tritiated (3H) sodium borohydride. The proteins retain excellent functional and antigenic properties. The utility of these reagents in a variety of assays for cell surface antigens is illustrated. The results indicate that this radiolabeling procedure may become the method of choice for many cell surface and solution immunoassays.

Nucleotide sequence of a rabbit IgG heavy chain from the recombinant F-I haplotype

We report the sequence of a cDNA encoding a rabbit immunoglobulin γ heavy chain of d12 and e14 allotypes with high homology to partial cDNA sequences from rabbits of d11 and e15 allotypes. The encoded rabbit protein shows homologies with human (68–70%) and mouse (60–63%) γ chains. The nucleotide sequence homologies of the CH domains range from 76–84% with human and 64–76% with mouse sequences. Comparison of the portion of VH encoding amino acid positions 34–112 with a previously determined VH sequence of the same allotype shows high conservation of sequences in the second and third framework segments but more marked differences both in length and encoded amino acids of the second and third complementarity-determining regions (CDRs). We also found a high degree of homology with a human genomic V-region, VH26 (77%) and a remarkable similarity between rabbit and human second CDR sequences and human genomic D minigenes. These results provide additional evidence that D minigene sequences share information with the CDR2 portion of VH regions.

Genetics and expression of kappa-type light chains in Basilea rabbits.

In contrast to rabbits of b4, b5, b6, and b9 allotypes whose serum immunoglobulins (Igs) are predominantly composed of kappa-type light chains, rabbits of the mutant Basilea strain have serum Igs that are largely of lambda type. We prepared several antisera that recognized a minor K2 (bas) light chain that is produced by Basilea rabbits. With these antisera we identified the K2 (bas) isotype in the serum of the original b9/b9 male rabbit whose offspring displayed the Basilea mutant phenotype. It was present in one half of his nonmutant offspring which inherited b9 from him and another b allotype from their mothers. Breeding was conducted both in Basel and at the NIH to develop and maintain colonies of mutant Basilea strain rabbits. The data obtained during colony development confirm that the trait of expression of the bas allotype maps to the same genetic region (b locus) that is known to control the allelic b allotypes b4, b5, b6 and b9. Homozygotes or heterozygotes of b4, b5 or b6 allotype (bb/bb) were mated with homozygous bbas/bbas rabbits to produce F1s, and then F2s as well as progeny of backcrosses to both homozygous parental types (bb/bb and bbas/bbas) were produced. The bas allotype segregates as an allele (or pseudoallele) at the b locus although there was a deficiency in recovery of homozygous bas offspring in both the F2 and backcross matings to bbas/bbas parental type in the NIH colony. This selective deficiency may reflect a deleterious effect on survival of homozygous bas progeny.

Partial primary structure of the immunoglobulin light chain constant region of a single rabbit of b5 allotype

Abstract The partial amino acid sequence of the constant region of the b5 light chain from the normal IgG of a single rabbit is reported. For structure determination, the IgG light chain was fully reduced and carboxymethylated, then digested with chymotrypsin or trypsin. All chymotryptic peptides covering the constant region from positions 116–210 (117–214 in the standardized numbering system of Kabat et al. ∗ ) were isolated and purified by column and paper chromatography. Sequences were then determined using traditional sequencing methods. Overlapping was obtained by use of large tryptic peptides, covering positions 138–210 (139–214 ∗ ). The chymotryptic peptide ending with Leu 115(116 ∗ ) could not be obtained in a pure state owing to insolubility and perhaps heterogeneity. When the sequence of this light chain is compared to those of light chains of b4 and b9 allotypes, about 77% homology is found with b4 and 62% with b9. This confirms serological data which would indicate that b4 and b5 allotypes are more similar to each other in structure than they are to b9. The allotypic differences are distributed throughout the whole constant portion of the light chain.

Genetic analyses of restriction fragment length polymorphisms using high molecular weight DNA from sperm or lymphocytes embedded in agarose

A simple and efficient method for determining restriction fragment length polymorphism types on large numbers of individuals using small samples of peripheral blood or sperm cells is described. Whole cells embedded in low gelling/melting temperature agarose were treated with a series of enzyme, detergent, and washing steps to release high molecular weight DNA that was then digested with standard restriction enzymes such as EcoRI and PstI, electrophoresed, blotted, and probed as in normal Southern analyses. The technique should be readily adaptable to any application requiring DNA from small numbers of cells for Southern analyses or pulsed field gel electrophoresis.

Expression of K2 ISOTYPE mRNA in normal and basilea rabbits

Molecular genetic techniques were used to study the regulated expression of the kappa light chains in the rabbit. Two isotypic kappa genes, kappa 1 and kappa 2, have been identified in the genome of all rabbits; however, the majority of secreted immunoglobulins produced by most domestic rabbits bear only K1 light chains. S1 nuclease protection experiments utilizing a single-stranded cDNA probe encoding the K2 constant region were performed to identify K2 mRNA in normal rabbits and in the mutant Basilea rabbit strain in which K2 light chains were first described. Varying amounts of K2 message were observed in the non-Basilea samples, between 0.05-1% of the K2 RNA found in a comparable preparation of Basilea RNA. Evidence for alternatively spliced messages was also noted. In addition, a K2 oligonucleotide probe is described which will distinguish between the K2 allotypic forms, bas1 and bas2.

Molecular analysis of recombination sites within the immunoglobulin heavy chain locus of the rabbit.

Previously, recombinations involving genes of the rabbit immunoglobulin heavy chain locus have been documented serologically. These data indicated that the sites at which the causative recombination events occurred could have been anywhere from within the VH gene cluster up to, or 3′ of, Cμ. Since these sites could not be localized further by serological methods, we attempted to do this using techniques of molecular biology. DNAs from homozygous recombinant rabbits and from the appropriate non-recombinant parental haplotypes were characterized using Southern blots hybridized with a panel of probes derived from cloned regions of the rabbit immunoglobulin heavy chain gene complex. In all three recombinants, the site was downstream of the entireVH cluster and upstream of the JHcluster within an ∼50 kilobase (kb) egion containing expanses of repetitive-sequence DNA as well as DH genes. DH-specific probes further showed that in two of the recombinants, the recombination appears to have occurred within or 5′ of DH1 and 5′ of DH2 genes; in the third it occurred 3′ of the DH2 genes but at least ∼5 kb 5′ of the JH region.

Allotype-specific probes: A molecular approach to the study of serologically defined determinants

A new approach to the study of serologically defined immunoglobulin determinants is described. We designed DNA probes which distinguished between the rabbit kappa light chain allotypic sequences in Northern analyses of mRNAs and Southern analyses of genomic DNAs. S1 nuclease protection experiments are described which detect allotype-specific sequences in as little as 100 pg of total RNA. The use of molecular biological techniques overcomes many of the problems inherent in using serological reagents and techniques. In addition, the sensitivity of the assays described here allows the detection of low level expression of the allotypic genes. This work was extended to include the discrimination of the VHa allotypes.