Retrocyte Display® technology: generation and screening of a high diversity cellular antibody library.

Over the last nearly three decades in vitro display technologies have played an important role in the discovery and optimization of antibodies and other proteins for therapeutic applications. Here we describe the use of retroviral expression technology for the display of full-length IgG on B lineage cells in vitro with a hallmark of a tight and stable genotype to phenotype coupling. We describe the creation of a high-diversity (>1.0E09 different heavy- and light-chain combinations) cell displayed fully human antibody library from healthy donor-derived heavy- and light-chain gene libraries, and demonstrate the recovery of high affinity target-specific antibodies from this library by staining of cells with a labeled target antigen and their magnetic- and flow cytometry-based cell sorting. The present technology represents a further evolution in the discovery of full-length, fully human antibodies using mammalian display, and is termed Retrocyte Display® (Retroviral B lymphocyte Display).

Properties, production, and applications of camelid single-domain antibody fragments.

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications.

Antibody repertoire development in fetal and neonatal piglets: XIX. Undiversified B cells with hydrophobic HCDR3s preferentially proliferate in the porcine reproductive and respiratory syndrome.

Porcine respiratory and reproductive syndrome virus (PRRSV) causes an extraordinary increase in the proportion of B cells resulting in lymphoid hyperplasia, hypergammaglobulinemia, and autoimmunity in neonatal piglets. Spectratypic analysis of B cells from neonatal isolator piglets show a non-Gaussian pattern with preferential expansion of clones bearing certain H chain third complementary region (HCDR3) lengths. However, only in PRRSV-infected isolator piglets was nearly the identical spectratype observed for all lymphoid tissues. This result suggests dissemination of the same dominant B cell clones throughout the body. B cell expansion in PRRS was not associated with preferential VH gene usage or repertoire diversification and these cells appeared to bear a naive phenotype. The B cell population observed during infection comprised those with hydrophobic HCDR3s, especially sequences encoded by reading frame 3 of DHA that generates the AMVLV motif. Thus, the hydropathicity profile of B cells after infection was skewed to favor those with hydrophobic binding sites, whereas the normally dominant region of the hydropathicity profile containing neutral HCDR3s was absent. We believe that the hypergammaglobulinemia results from the products of these cells. We speculate that PRRSV infection generates a product that engages the BCR of naive B cells, displaying the AMVLV and similar motifs in HCDR3 and resulting in their T-independent proliferation without repertoire diversification.

Deletion of the DQ52 element within the Ig heavy chain locus leads to a selective reduction in VDJ recombination and altered D gene usage.

The process of V(D)J recombination that leads to the assembly of Ig gene segments is tightly controlled during B cell differentiation. Two germline transcripts, one of which (mu(0)) originates from the promoter region of DQ52, may control the accessibility of the heavy chain locus. Here, we present the analysis of a mouse line in which the DQ52 gene together with its regulatory sequences is deleted by a Cre/loxP-based strategy. In F(1) (DQ52(+/-)) mice, the use of the JH3 and JH4 elements in DJ or VDJ junctions of the DQ52(-) allele was strongly reduced in both the bone marrow pre-B and spleen cells, while the JH1 and JH2 elements were used with normal frequencies. In addition, IgM(+) B cells of bone marrow and spleen used the DQ52(-) allele less frequently. On DJ joints of the DQ52(-) allele, there was 2 times less processing of JH3 ends, which resulted in clearly increased addition of P nucleotides. Although the use of D elements in DJ joints was quite similar, an altered D repertoire was found in VDJ joints of the DQ52(-) allele. In splenic B cells of the DQ52(-/-) mouse the amino acid distribution of the CDR3 was skewed, probably to compensate for the altered processing of JH3 ends. Thus, we have shown an interesting selective effect of the DQ52 region on controlling accessibility to 3' JH elements on the Ig locus, which also seems to influence the processing of DJ joints. We propose a model in which the DQ52 promoter region enhances the induction of secondary DJ rearrangements.

Expression of IgH chain genes in antibody response to ragweed and purified Amb a 1: evidence for differential regulation of isotype production.

A survey of the work with Ig response to allergens carried out previously reveals an allergen-specific response both by IgE and all of IgG subclasses. Response of non-sensitive people is characterized by the appearance of a variety of the IgG subclasses. We have reexamined ragweed and Amb a 1 specific Ig response in 54 nonsensitive and 147 atopic or atopic-allergic people using a new inverse sandwich immunoassay allowing discrimination based on antibody affinity. We show that non-sensitive people present no, 0 out of 54, Ig response with affinities higher than Ka 10(7) M(-1). The subpopulation of 66 atopics who never have experienced desensitization responds vigorously and solely (56 out of 66) with genes of the sequence gamma2-alpha2. Only ten showed an additional weak response from gamma1-alpha1. This suggests a possible association between the atopic state and selective activation of part of the gene sequence.

Effect of age on the expressed B cell repertoire: role of B cell subsets.

Aged humans and experimental animals are impaired in their responses to most foreign antigens although they produce greater amounts of autoantibodies. We have examined the effect of age on the production of antibodies to a prototypic foreign antigen, sheep erythrocytes (SRBC), and to a prototypic autoantigen, bromelain-treated mouse erythrocytes (BrMRBC), in young and old mice before and after immunization with SRBC. Old mice express more anti-BrMRBC plaque-forming cell (PFC) antibodies before and an even greater number after immunization with SRBC than young mice. Conversely, old mice produce far fewer anti-SRBC PFC than young mice following immunization with SRBC. We hypothesized that the differences in the responses of old mice to BrMRBC and SRBC reflects differences in the activity of CD5+ and CD5- B cells. To test this hypothesis we immunized young and old mice with foreign antigens reported (and confirmed in our studies) to stimulate CD5+ B cells [TNP-ficoll and phosphorylcholine-keyhole limpet hemocyanin (KLH)] or CD5- B cells (SRBC and TNP-KLH). We found that the PFC response of old mice to antigens mediated by CD5+ B cells was equal to or greater than that of young mice. In contrast the PFC response of old mice induced by antigens mediated by CD5- B cells was only 10% that of young mice. Thus it appears that the immune response of old mice is well maintained for antigens which elicit a CD5+ B cell response but not for those which elicit a CD5- B cell response.(ABSTRACT TRUNCATED AT 250 WORDS)

Propagation of a mouse myeloma cell line J558L producing human CD4 immunoglobulin G1.

Transfected mouse myeloma cells are of increasing interest for the production of a wide variety of solubilised recombinant fusion proteins. A stably transfected J558L mouse myeloma subclone (J558L-CD4) secreting human CD4-immunoglobulin type G1 receptor (CD4-H gamma 1) was employed as a model system for cell suspension culture and expression of chimaeric molecules. Cells were grown up to 3-5 x 10(6) cells/ml in serum-free and protein-reduced DHI medium consisting of a mixture of DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL and Pluronic F68. Primatone RL was the essential growth-promoting factor in protein-free medium. The soluble CD4-H gamma 1 receptor, the production of which was not growth-associated, accumulated in the medium to concentrations of 40 micrograms/ml with a specific formation rate of 0.18 micrograms/10(6) cells/h in conventional cultures. The cell density was further increased by growing the cells in dialysis tubing or by using a perfusion system with cell retention. Because of the continuous exchange of nutrients and metabolic end-products average concentrations of 35 x 10(6) cells/ml were achieved. CD4-H gamma 1 accumulated in the dialysis tubing up to 1.3 mg/ml. After an initial rapid growth period, a ten-fold reduction in specific nutrient consumption rates and metabolic end-product formation was observed. Chimaeric proteins purified by protein G chromatography from conventional and perfusion cultures were indistinguishable when compared by SDS-PAGE, limited proteolysis and isoelectric focusing analysis (isoelectric point: 8.5-8.6).

Biased VH gene expression in murine CD5 B cells results from age-dependent cellular selection.

Flow cytometry-purified, peritoneal and splenic CD5+ and CD5- B cells from neonatal and adult C57BL/6 mice were studied for expression of VH and Vx gene families in RNA colony blot assays, and for frequencies of clones secreting antibodies to bromelain-treated mouse red blood cells (BrMRBC), single-stranded DNA, trimethyl ammonium and bovine gamma-globulin, by limiting dilution. The results show few overall differences between the two B cell subsets, which both manifest ontogenic D-proximal VH preferences that are lost with age. Biased VH11 expression in CD5 B cells is high in adult peritoneum and spleen but absent in newborns. It only partly correlates with the selection of anti-BrMRBC reactivity, which is considerably higher in peritoneum than in spleen. No particular Vx bias was observed in any of the populations studied with the possible exception of Vx22 in peritoneal CD5+ B cells. We conclude that the antibody repertoire expressed by peritoneal CD5+ B cells of adult mice is not the result of a genetic program, but rather the consequence of local, age-dependent cellular selection mechanisms.