Induction of sterile transcription from the kL chain gene locus in V(D)J recombinasedeficient progenitor B cells

B cell development in RAG-2-deficient (RAG-2T) mice is impeded at an early stage, due to the inability of these animals to rearrange their endogenous lg gene loci. Expression of an Eμ-bci-2 transgene in these mice did not change this phenotype. However, stromal cell/IL-7-reactive B cell progenitors (pro-B cells) were found in fetal liver and bone marrow of RAG-2T and RAG-2T/Eμ-bci-2 transgenic mice in numbers comparable to normal mice. Like cells from normal mice they are c-kit+, surrogate L chain+ and CD25−, and can proliferate in vitro for long periods of time. Upon IL-7 deprivation, they can be induced to differentiate into c-kit−, surrogate L chain+ and CD25− cells that are no longer clonable on stromal cells and IL-7. Furthermore, sterile transcription from the kL.chain gene loci is induced. The latter was also observed with pro-B cells directly isolated ex vivo from the bone marrow of RAG-2-deficient animals. The results suggest that progenitor B celldifferentiation can occur in cells from V(D)J recombinase-deficient mice to the stage where KL chain gene rearrangements would normally be initiated. It further indicates that some molecular programs of early B cell differentiation can take place in the absence of lg gene rearrangement

Rearrangement and expression of χ light chain genes can occur without μ heavy chain expression during differentiation of pre-B cells

The kinetics of χ light (χL) chain gene rearrangement and expression on mRNA and protein level has been studied with four stromal cell/IL-7 reactive, long-term in vitro proliferating pre-B cell lines and clones, two from fetal liver of normal mice and two from fetal liver of EμH-bcl-2 transgenic (bcl-2-tg) mice. These pre-B cell lines and clones are DJH-rearranged on both H chain alleles. Two of the clones harbor H chain rearrangements which do not allow the expression of VHDJH rearranged H chain genes as μH chain proteins. Upon removal of IL-7 from the pre-B cell cultures all four cell lines rearrange VH-DJH and VL-JL gene segments, loose the surface expression of c-kit, CD43, and surrogate light chain, as well as the capacity to be clonable on stromal cells in the presence of IL-7. Pre-B cells from normal mice die by apoptosis during differentiation, while those from bcl-2-tg mice do not. All four lines and clones express comparable levels of mRNA for μH and μL chains with the same time kinetics during 3 days of differentiation. However, only two of the four pre-B cell lines and clones express μH chain protein, whereas all four pre-B cell lines and clones express μL chain protein at comparable levels between 2×105 and 1.40×106 μL chain molecules per cell. These results suggest that μH chain expression is not mandatory for rearrangement and normal expression of μL chain genes when pre-B cells differentiate to B cell

A Novel Molecular Complex Expressed on Immature B Cells: A Possible Role in T Cell-Independent B Cell Development

To identify surface molecules that may play a role in regulating ileal Peyer's patch (PP) B cell growth, we generated monoclonal antibodies (mAbs) and then selected them for a unique reactivity with ileal PP B cells. Flow cytometric analysis identified a mAb (SIC4.8R) that labeled 97% of ileal and 50–60% of jejunal PP sIgM+B cells. SIC4.8R also labeled a subpopulation of cortical thymocytes but few B or T cells in other lymphoid tissues, including bone marrow. Immunohistochemistry revealed intense SIC4.8R staining of B cells in the cortex of ileal PP follicles. SIC4.8R also labeled bovine PP B cells, a murine pro-B cell line, and pre-B cells in human bone marrow. Protein chemistry revealed that a structurally similar molecular complex was expressed on sheep ileal PP B cells and thymocytes and murine pro-B cells. Addition of soluble SIC4.8R to cultured ileal PP B cells reduced apoptotic cell death, elevated proliferative responses, partially inhibited anti-Ig-induced cell death, and induced IL-4 responsiveness. In contrast, soluble SIC4.8R had an antiproliferative effect on a mouse pro-B cell line. Finally, SIC4.8R labeling declined following the stimulation of ileal PP B cells with CD40 ligand. In conclusion, the present investigation determined that SIC4.8R identified a novel molecular complex that is expressed at several stages of T cell-independent B cell development in a variety of mammalian species. This observation confirmed that PP B cells are developmentally distinct from other B cell populations in sheep and suggested that the bone marrow may not be a site of B lymphopoiesis in young lambs

IL-2 receptor α chain (CD25JAC) expression defines a crucial stage in pre-B cell development

The analysis of the expression of the a chain of the IL-2 receptor (CD25.TAC) on the surface of B lineage cells In mouse bone marrow reveals that it is a useful marker to distinguish pre-B-I from pre-B-II cells. CD25 Is not expressed on CD45R(B220)+ c-kit+ CD43+ TdT+ λ5+ Cμ− slg− lgH chain locus DJH-rearranged pre-B-I cells of mouse bone marrow. It is expressed on large cycling CD45R(B220)+ c-kit+ CD43+ TdT+ λ5+ Cμ− sig− and on small resting CD45R(B220)+ c-kit+ CD43− TdT+ λ5+ Cμ− sig− sig- IgH chain locus VHDJH-rearranged pre-B-II cells. Therefore, the transition from pre-B-I to large pre-B-II cells is marked by the downregulation of c-kit and terminal deoxynucleotldyl transferase (TdT), and by the upregulatton of CD25. SCID, RAG-2T, μMT and γ6T mutant mice do have normal, If not elevated numbers of pre-B-I cells but lack all CD25+ pre-B-II cells in their bone marrow. The expression of a transgenic H chain under control of the μH chain enhancer in RAG-2T bone marrow B lineage precursors allows the development of large and small CD25+ pre-B-II cells. The results suggest that the differentiation of pre-B-I to pre-B-II cells in mouse bone marrow requires the expression of μH chains and surrogate L chains in membranes, probably on the surface of precursor B cell

Novel Antibody Drug Conjugates Targeting Tumor-Associated Receptor Tyrosine Kinase ROR2 by Functional Screening of Fully Human Antibody Libraries Using Transpo-mAb Display on Progenitor B Cells

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes (“Transpo-mAb Display”) for ROR2 binding. Individual cellular “Transpo-mAb” clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs

<i>In vitro</i> killing of HER-2-overexpressing breast cancer cells by sortase A-conjugated anti-HER-2 ADCs.

<p>HER-2-overexpressing SKBR3 cells (A) and HER-2-non-overexpressing T47D cells (B) were grown in the presence of serial dilutions of the indicated ADCs. Viable cells were quantified using a Luminescent Cell Viability Assay. Datapoints represent mean of two replicates and error bars represent SD.</p

Analytical summary of conjugates manufactured in this study.

<p>DAR, drug-to-antibody ratio, determined by hydrophobic interaction and/or reverse phase chromatography; Mono (start/conj), % momomer content before/after conjugation, determined by size exclusion chromatography.</p><p>Analytical summary of conjugates manufactured in this study.</p

Sortase A-mediated conjugation of Gly₅-modified FITC to C-terminally tagged mAb Ac10 IgH and IgL chains.

<p>The tag sequences are shown on top, with the sortase A recognition motif in bold print, and the Strep II tag underlined. (A) Spacer-free design; (B) Design using a 5 amino acid (GGGGS) spacer on the IgL chain. Antibody was incubated with 2-fold serial dilutions of sortase A in the presence of a 20-fold excess of Gly₅-modified FITC. The crude reaction products were then separated by SDS-PAGE, and the FITC conjugates visualized by placing the gel on a UV box. IgH, heavy chain; IgL, light chain; GF, Gly₅-FITC.</p

Down-regulation of RAG1 and RAG2 gene expression in PreB cells after functional immunoglobulin heavy chain rearrangement

AbstractTwo waves of immunoglobulin gene rearrangements, first of the heavy, then of the light chain gene loci form functional immunoglobulin genes during B cell development. In mouse bone marrow the differential surface expression of B220 (CD45R), c-kit, CD25, and surrogate light chain as well as the cell cycle status allows FACS separation of the cells in which these two waves of rearrangements occur. The gene products of two recombination activating genes, RAG1 and RAG2 are crucial for this rearrangement process. Here, we show that the expression of the RAG genes is twice up- and down-regulated, at the transcriptional level for RAGI and RAG2, and at the postranscriptional level for RAG2 protein. Expression levels are high in D → JH and VH → DJH rearranging proB and preB-I cells, low in preB cells expressing the preB cell receptor on the cell surface, and high again in VL → JL rearranging small preB-II cells. In immature B cells expressing on the cell surface RAGI and RAG2 mRNA is down-regulated, whereas RAG2 protein levels are maintained. Down-regulation of RAGI and RAG2 gene expression after productive rearrangement at one heavy chain allele might be part of the mechanisms that prevent further rearrangements at the other allele

Generation of an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins

The antigen-specific targeting of autoreactive B cells via their unique B cell receptors (BCRs) is a novel and promising alternative to the systemic suppression of humoral immunity. We generated and characterized cytolytic fusion proteins based on an existing immunotoxin comprising tetanus toxoid fragment C (TTC) as the targeting component and the modified Pseudomonas aeruginosa exotoxin A (ETA') as the cytotoxic component. The immunotoxin was reconfigured to replace ETA' with either the granzyme B mutant R201K or MAPTau as human effector domains. The novel cytolytic fusion proteins were characterized with a recombinant human lymphocytic cell line developed using Transpo-mAb (TM) technology. Genes encoding a chimeric TTC-reactive immunoglobulin G were successfully integrated into the genome of the precursor B cell line REH so that the cells could present TTC-reactive BCRs on their surface. These cells were used to investigate the specific cytotoxicity of GrB(R201K)-TTC and TTC-MAPTau, revealing that the serpin proteinase inhibitor 9-resistant granzyme B R201K mutant induced apoptosis specifically in the lymphocytic cell line. Our data confirm that antigen-based fusion proteins containing granzyme B (R201K) are suitable candidates for the depletion of autoreactive B cells