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.

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.

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.

Novel panning and screening methodology was devised to isolate high affinity human recombinant scFv antibody fragments with functionally associated properties in B lymphoma cells. The approach was used to generate a panel of apoptosis-inducing antibodies specific for antigens differentially expressed in B lymphoma vs. T leukaemia cells. The selections resulted in an antibody pool with near perfect selectivity (>99%) for the B lymphoma target cells. Randomly picked clones (72) revealed 7 unique antibody genotypes. Six of these rapidly induced apoptosis in target cells. Following the conversion to full IgGs, the antibodies were shown to be specific for HLA-DR/DP, the B-cell receptor l chain and for CD54/ ICAM-1. The latter receptor was not previously associated with apoptotic properties in B-cell lymphomas. Anti-ICAM-1 IgG induced apoptosis in a broad range of B lymphoma cell lines and were shown by immunohistochemistry to bind strongly to B lymphoma tissue obtained from 5 different B lymphoma patients. The recombinant IgG antibodies had affinities in the subnanomolar (0.3 nM) to nanomolar (3 nM) range. The described technology is generally applicable for the rapid isolation of high affinity human antibodies with specificity for differentially expressed cell surface receptors with intrinsic negative or positive signalling properties from na€ ıve phage libraries. ' 2006 Wiley-Liss, Inc.

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.