In the genome of a germ-line cell, the genetic information for an immunoglobulin polypeptide chain is contained in multiple gene segments scattered along a chromosome. During the development of bone marrow-derived lymphocytes, these gene segments are assembled by recombination which leads to the formation of a complete gene. In addition, mutations are somatically introduced at a high rate into the amino-terminal region. Both somatic recombination and mutation contribute greatly to an increase in the diversity of antibody synthesized by a single organism.

Somatic generation of antibody diversity

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.

Methods for producing members of specific binding pairs

The clonal selection principle is used to explain the basic features of an adaptive immune response to an antigenic stimulus. It establishes the idea that only those cells that recognize the antigens (Ag's) are selected to proliferate. The selected cells are subject to an affinity maturation process, which improves their affinity to the selective Ag's. This paper proposes a computational implementation of the clonal selection principle that explicitly takes into account the affinity maturation of the immune response. The general algorithm, named CLONALG, is derived primarily to perform machine learning and pattern recognition tasks, and then it is adapted to solve optimization problems, emphasizing multimodal and combinatorial optimization. Two versions of the algorithm are derived, their computational cost per iteration is presented, and a sensitivity analysis in relation to the user-defined parameters is given. CLONALG is also contrasted with evolutionary algorithms. Several benchmark problems are considered to evaluate the performance of CLONALG and it is also compared to a niching method for multimodal function optimization.

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Learning and optimization using the clonal selection principle

To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.

Making antibody fragments using phage display libraries

Each antibody-producing B cell makes antibodies of unique specificity, reflecting a series of ordered gene rearrangements which must be successfully performed if the cell is to survive. A second selection process occurs during immune responses in which a new antibody repertoire is generated through somatic hypermutation. Here only mutants binding antigen with high affinity survive to become memory cells. Cells expressing autoreactive receptors are counter-selected at both stages. This stringent positive and negative selection allows the generation and diversification of cells while rigorously controlling their specificity.

Clonal selection and learning in the antibody system

https://www.cell.com/cell/pdf/0092-8674(87)90073-0.pdf

Analysis of somatic mutation and class switching in naive and memory B cells generating adoptive primary and secondary responses

While somatic antibody mutants are rare in the preimmune repertoire and in primary immune responses, they dominate secondary and hyperimmune responses. We present evidence that somatic hypermutation is restricted to a particular pathway of B-cell differentiation in which distinct sets of B-cell clones are driven into the memory compartment. In accord with earlier results of McKean et al. (1984) and Rudikoff et al. (1984), somatic mutation occurs stepwise in the course of clonal expansion, before and after isotype switch, presumably at a rate close to 1 X 10(-3) per base pair per generation. At this rate, both selectable and unselectable mutations accumulate in the rearranged V region genes. The distribution of replacement mutations in the V regions shows that a fraction of the mutations in CDRs is positively selected whereas replacement mutations are counterselected in the FRs. By constructing an antibody mutant through site-specific mutagenesis we show that a point mutation in CDR1 of the heavy chain, found in most secondary anti-NP antibodies, is sufficient to increase NP binding affinity to the level typical for the secondary response. Somatic mutation may contribute to the immune repertoire in a more general sense than merely the diversification of a specific response. We have evidence that clones producing antibodies which no longer bind the immunizing antigen can be kept in the system and remain available for stimulation by a different antigen. Somatic mutations are 10 times less frequent in DJH loci than in either expressed or non-expressed rearranged VDJH or VJ loci. We therefore conclude that a V gene has to be brought into the proximity of the DJH segment in order to fully activate the hypermutational mechanism in these loci.

Timing, Genetic Requirements and Functional Consequences of Somatic Hypermutation during B‐Cell Development

A new classification of mouse VH sequences.

VH-gene expression in hybridomas derived from lipopolysaccharide-activated B cells was analyzed. Isolated cytoplasmic RNA was hybridized to probes representing the 9 known VH-gene groups or subjected to mRNA sequencing. In the collection of hybridomas VH genes of all 9 groups are expressed at frequencies which in most correlate reasonably well with the relative complexities of the groups. In 51 out of 54 RNA samples VH-gene transcripts could be identified and corresponded to one of the known VH-gene groups. It therefore appears that the latter essentially represent the VH-gene cluster of the mouse.

VH-gene expression in murine lipopolysaccharide blasts distributes over the nine known VH-gene groups and may be random.

The mouse hybridoma line B1-8.delta 1 secretes a monoclonal IgD, lambda 1 anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody with defined idiotypic determinants. Two spontaneous V-region variants (B1-8.V1/V2) with altered idiotope pattern were selected and the structural variation was located to the variable region of the heavy chain. The amino acid sequences of the B1-8. delta 1 and variant heavy chain V regions were determined. The variant VH regions are identical. Wild-type and variant VH regions differ in 10 positions. Single amino acid exchanges are found in the first and second framework at positions 20 and 43. The majority of replacements (eight substitutions) is clustered in the second complementary-determining region (CDR 2). There are no differences in CDR 1 and CRD 3 and the JH region. The variant, which at first glance appears to have undergone a series of point mutations, arose by recombination, possibly gene conversion, between the rearranged VDJ gene of the wild-type (B1-8.delta 1) and a neighbouring germ line VH gene encoding all of the substitutions.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1982.tb01220.x

Renate Dildrop

Papers

Analysis of somatic mutation and class switching in naive and memory B cells generating adoptive primary and secondary responses

Timing, Genetic Requirements and Functional Consequences of Somatic Hypermutation during B‐Cell Development

A new classification of mouse VH sequences.

VH-gene expression in murine lipopolysaccharide blasts distributes over the nine known VH-gene groups and may be random.

Immunoglobulin V region variants in hybridoma cells. II. Recombination between V genes.