A platform for phenotypic discovery of therapeutic antibodies and targets applied on Chronic Lymphocytic Leukemia.

Development of antibody drugs against novel targets and pathways offers great opportunities to improve current cancer treatment. We here describe a phenotypic discovery platform enabling efficient identification of therapeutic antibody-target combinations. The platform utilizes primary patient cells throughout the discovery process and includes methods for differential phage display cell panning, high-throughput cell-based specificity screening, phenotypic in vitro screening, target deconvolution, and confirmatory in vivo screening. In this study the platform was applied on cancer cells from patients with Chronic Lymphocytic Leukemia resulting in discovery of antibodies with improved cytotoxicity in vitro compared to the standard of care, the CD20-specific monoclonal antibody rituximab. Isolated antibodies were found to target six different receptors on Chronic Lymphocytic Leukemia cells; CD21, CD23, CD32, CD72, CD200, and HLA-DR of which CD32, CD200, and HLA-DR appeared as the most potent targets for antibody-based cytotoxicity treatment. Enhanced antibody efficacy was confirmed in vivo using a patient-derived xenograft model.

Regulation of B lymphocyte development by the truncated immunoglobulin heavy chain protein Dmu.

The development of B lymphocytes from progenitor cells is dependent on the expression of a pre-B cell-specific receptor made up by a mu heavy chain associated with the surrogate light chains, immunoglobulin (Ig)alpha, and Igbeta. A variant pre-B cell receptor can be formed in which the mu heavy chain is exchanged for a truncated mu chain denoted Dmu. To investigate the role of this receptor in the development of B cells, we have generated transgenic mice that express the Dmu protein in cells of the B lineage. Analysis of these mice reveal that Dmu expression leads to a partial block in B cell development at the early pre-B cell stage, probably by inhibiting VH to DHJH rearrangement. Furthermore, we provide evidence that Dmu induces VL to JL rearrangements.

Targeted disruption of the V(H) 81X gene: influence on the B cell repertoire.

We have generated a mutant mouse in which the most D-proximal V(H) gene (V(H)81X) has been disrupted by introducing a neomycin-resistance gene into the V(H)81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the V(H)81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of V(H) gene rearrangements in the V(H)7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of V(H)7183 genes is concerned.

B-1a, B-1b and B-2 B cells display unique VHDJH repertoires formed at different stages of ontogeny and under different selection pressures.

Analyses of VHDJH rearrangements isolated from murine peritoneal B-1a cells (CD5+, IgMhi, B220lo), peritoneal B-1b cells (CD5-, IgMhi, B220lo), and conventional splenic B cells provide evidence that a unique repertoire of VH regions is displayed by each of these B-cell subsets. The B-1a subset is characterized by a low N-region diversity, by a high frequency of sequence homologies in the VH-D and D-JH junctions, and by a limited exonuclease nibbling of the terminals of the joining gene segments. Through expansion in ageing mice, B-1a clones with these properties are favoured. B-1b cells are similar to conventional B-2 cells with respect to N-region diversity, but are unique in terms of D gene expression. Thus, while most murine pre-B and B cells preferentially use DSP and DFL gene segments in a given reading frame (RF1), B-1b cells frequently express D genes in another reading frame (RF2). Together, these findings provide structural evidence for a model where B-1a, B-1b and B-2 cells are produced by separate progenitors that are active at different stages of ontogeny.

V-region directed selection in differentiating B lymphocytes.

We here analyse the repertoire of VH7183 rearrangements isolated from different stages of B cell differentiation in adult mice. The nucleotide sequence analyses of VH7183-D-JH rearrangements derived from large pre-B cells (B220+, mu-), small pre-B cells (B220+, mu-) and mature B cells (B220+, mu+) isolated from adult bone marrow revealed a sequential accumulation, among functional rearrangements, of D segments of the FL16 family and a depletion of D segments using the second and the third reading frame (RF). One member (VH7183.1) of the VH7183 gene family was utilized in 60-80% of the rearrangements of all populations analysed. In neonates the majority of the rearrangements utilizing this gene was found to be functional. In contrast, > 96% of the VH7183 rearrangements isolated from adult spleen were non-functional. These data provide evidence for cellular selection of VH regions acting at different points of the B cell differentiation pathway and at the transition of B cells from the bone marrow to the periphery.