In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

Concerted evolution is often invoked to explain the diversity and evolution of the multigene families of major histocompatibility complex (MHC) genes and immunoglobulin (Ig) genes. However, this hypothesis has been controversial because the member genes of these families from the same species are not necessarily more closely related to one another than to the genes from different species. To resolve this controversy, we conducted phylogenetic analyses of several multigene families of the MHC and Ig systems. The results show that the evolutionary pattern of these families is quite different from that of concerted evolution but is in agreement with the birth-and-death model of evolution in which new genes are created by repeated gene duplication and some duplicate genes are maintained in the genome for a long time but others are deleted or become nonfunctional by deleterious mutations. We found little evidence that interlocus gene conversion plays an important role in the evolution of MHC and Ig multigene families.

https://www.pnas.org/content/pnas/94/15/7799.full.pdf

Evolution by the birth-and-death process in multigene families of the vertebrate immune system

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 x 10(9) members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody-antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

https://www.pnas.org/content/pnas/95/11/6157.full.pdf

Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

Antibody humanization: a case of the ‘Emperor’s new clothes’?

Sequence and evolution of the human germline V lambda repertoire.

The invention provides a dual-specific ligand comprising a first immunoglobulin variable domain having a first binding specificitiy for a target ligand and a complementary or non-complementary immunoglobulin variable domain having a second binding specificity for a receptor of the target ligand.

Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand

The invention provides a dual-specific ligand comprising a first immunoglobulin variable domain having a first binding specificity and a complementary or non-complementary immunoglobulin variable domain having a second binding specificity.

Dual specific ligands with increased serum half-life

The invention provides a dual-specific ligand comprising a first single immunoglobulin variable domain having a first binding specificity and a complementary immunoglobulin single variable domain having a second binding specificity.

Dual-specific ligand and its use

L'invention concerne un procede simple de production de structures basees sur des anticorps pouvant etre utilisees in vivo. Plus particulierement, cette invention concerne un procede permettant de produire des structures basees sur des anticorps pouvant etre utilisees in vivo comprenant les etapes consistant : (a) a selectionner un seul domaine variable d'anticorps possedant une specificite de liaison a l'epitope; et (b) a fixer le domaine de l'etape (a) a un groupe effecteur. Des utilisations de molecules produites au moyen du procede de l'invention sont egalement decrites.

Greg Winter

Papers

Sequence and evolution of the human germline V lambda repertoire.

Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand

Dual specific ligands with increased serum half-life

Dual-specific ligand and its use

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