Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies.

Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.

Mining the Plasma Cell Transcriptome for Novel Cell Surface Proteins.

Antibody Secreting Cells (ASCs) are a fundamental component of humoral immunity, however, deregulated or excessive antibody production contributes to the pathology of autoimmune diseases, while transformation of ASCs results in the malignancy Multiple Myeloma (MM). Despite substantial recent improvements in treating these conditions, there is as yet no widely used ASC-specific therapeutic approach, highlighting a critical need to identify novel methods of targeting normal and malignant ASCs. Surface molecules specifically expressed by the target cell population represent ideal candidates for a monoclonal antibody-based therapy. By interrogating the ASC gene signature that we previously defined we identified three surface proteins, Plpp5, Clptm1l and Itm2c, which represent potential targets for novel MM treatments. Plpp5, Clptm1l and Itm2c are highly and selectively expressed by mouse and human ASCs as well as MM cells. To investigate the function of these proteins within the humoral immune system we have generated three novel mouse strains, each carrying a loss-of-function mutation in either Plpp5, Clptm1l or Itm2c. Through analysis of these novel strains, we have shown that Plpp5, Clptm1l and Itm2c are dispensable for the development, maturation and differentiation of B-lymphocytes, and for the production of antibodies by ASCs. As adult mice lacking either protein showed no apparent disease phenotypes, it is likely that targeting these molecules on ASCs will have minimal on-target adverse effects.

The BTB-ZF transcription factor Zbtb20 is driven by Irf4 to promote plasma cell differentiation and longevity.

The transcriptional network regulating antibody-secreting cell (ASC) differentiation has been extensively studied, but our current understanding is limited. The mechanisms of action of known "master" regulators are still unclear, while the participation of new factors is being revealed. Here, we identify Zbtb20, a Bcl6 homologue, as a novel regulator of late B cell development. Within the B cell lineage, Zbtb20 is specifically expressed in B1 and germinal center B cells and peaks in long-lived bone marrow (BM) ASCs. Unlike Bcl6, an inhibitor of ASC differentiation, ectopic Zbtb20 expression in primary B cells facilitates terminal B cell differentiation to ASCs. In plasma cell lines, Zbtb20 induces cell survival and blocks cell cycle progression. Immunized Zbtb20-deficient mice exhibit curtailed humoral responses and accelerated loss of antigen-specific plasma cells, specifically from the BM pool. Strikingly, Zbtb20 induction does not require Blimp1 but depends directly on Irf4, acting at a newly identified Zbtb20 promoter in ASCs. These results identify Zbtb20 as an important player in late B cell differentiation and provide new insights into this complex process.

Germinal center-independent, IgM-mediated autoimmunity in sanroque mice lacking Obf1.

Mice homozygous for a point mutation in the Rc3h1 gene encoding Roquin1, designated sanroque mice, develop a severe antibody-mediated autoimmune condition. The disease is T-cell intrinsic, exacerbated by macrophage-intrinsic defects and driven by excessive T follicular helper cell generation and spontaneous germinal centre (GC) formation. This culminates in abnormally high numbers of plasma cells secreting high-affinity autoreactive immunoglobulin G (IgG). Obf1 is a transcriptional co-activator required for normal T-cell-dependent antibody responses, and it is essential for GC formation under all circumstances so far tested. We crossed sanroque mice with Obf1-null mice to determine whether the hyperactivity of sanroque T cells could drive Obf1(-/-) B cells to differentiate to GC B cells, or conversely, if Obf1 loss would prevent sanroque-mediated autoimmune disease. Surprisingly, while sanroque/Obf1(-/-) mice did not form GC, they still developed autoimmune disease and succumbed even more rapidly than did sanroque mice. The disease was mediated by autoreactive IgM, which may have been derived from a pre-existing population of autoreactive B cells in the Obf1(-/-) mice responding to the over-exuberant activity of sanroque CD4 cells.

Circulating precursor CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure.

Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory.

Blimp1 is limiting for transformation in a mouse plasmacytoma model.

Multiple myeloma (MM) and plasmacytomas are cancers of antibody-secreting cells (ASCs). PRDM1/BLIMP1 is an essential regulator of ASC development. Histologic evidence shows that 100% of MM expresses PRDM1/BLIMP1, indicating that PRDM1/BLIMP1 is important for the development or persistence of MM. In contrast, some diffuse large B-cell lymphomas (DLBCLs) lose PRDM1 expression, suggesting that PRDM1 may act as a tumor suppressor in DLBCL. Thus, the role of PRDM1/BLIMP1 in transformation of mature B cells is unclear. We have used a plasmacytoma-prone transgenic mouse model to study the effect of Blimp1 loss on plasmacytoma prevalence, latency, and phenotype. Two possible outcomes could be envisaged: loss of Blimp1 might decrease plasmacytoma prevalence, through reduction of plasma cells, and so the number of susceptible transformation targets. Alternatively, Blimp1 may participate in the transformation process itself. Our results support the latter scenario, showing that decreasing Blimp1 dosage does not change plasma cell number in nontransgenic mice in vivo, but it significantly reduces plasmacytoma prevalence in transgenic mice. Loss of functional Blimp1 completely prevents plasmacytoma formation in this tumor model. These observations suggest that Blimp1 is limiting for plasma cell transformation and thus has potential as a target for new therapies to combat MM.

CD93 is required for maintenance of antibody secretion and persistence of plasma cells in the bone marrow niche.

Plasma cells represent the end stage of B-cell development and play a key role in providing an efficient antibody response, but they are also involved in numerous pathologies. Here we show that CD93, a receptor expressed during early B-cell development, is reinduced during plasma-cell differentiation. High CD93/CD138 expression was restricted to antibody-secreting cells both in T-dependent and T-independent responses as naive, memory, and germinal-center B cells remained CD93-negative. CD93 was expressed on (pre)plasmablasts/plasma cells, including long-lived plasma cells that showed decreased cell cycle activity, high levels of isotype-switched Ig secretion, and modification of the transcriptional network. T-independent and T-dependent stimuli led to re-expression of CD93 via 2 pathways, either before or after CD138 or Blimp-1 expression. Strikingly, while humoral immune responses initially proceeded normally, CD93-deficient mice were unable to maintain antibody secretion and bone-marrow plasma-cell numbers, demonstrating that CD93 is important for the maintenance of plasma cells in bone marrow niches.

Silencing and nuclear repositioning of the lambda5 gene locus at the pre-B cell stage requires Aiolos and OBF-1.

The chromatin regulator Aiolos and the transcriptional coactivator OBF-1 have been implicated in regulating aspects of B cell maturation and activation. Mice lacking either of these factors have a largely normal early B cell development. However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes. In mice deficient for Aiolos and OBF-1, the numbers of immature B cells are reduced, small pre-BII cells are increased and a significant impairment in immunoglobulin light chain DNA rearrangement is observed. We identified genes whose expression is deregulated in the pre-B cell compartment of these mice. In particular, we found that components of the pre-BCR, such as the surrogate light chain genes lambda5 and VpreB, fail to be efficiently silenced in double-mutant mice. Strikingly, developmentally regulated nuclear repositioning of the lambda5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos. These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function.

Targeting the extracellular membrane-proximal domain of membrane-bound IgE by passive immunization blocks IgE synthesis in vivo.

The classical allergic reaction starts seconds or minutes after Ag contact and is committed by Abs produced by a special subset of B lymphocytes. These Abs belong to the IgE subclass and are responsible for Type I hyperreactivity reactions. Treatment of allergic diseases with humanized anti-IgE Abs leads primarily to a decrease of serum IgE levels. As a consequence, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells. The biological mechanism behind anti-IgE therapy remains partly speculative; however, it is likely that these Abs also interact with membrane IgE (mIgE) on B cells and possibly interfere with IgE production. In the present work, we raised a mouse mAb directed exclusively against the extracellular membrane-proximal domain of mIgE. The interaction between the monoclonal anti-mIgE Ab and mIgE induces receptor-mediated apoptosis in vitro. Passive immunization experiments lead to a block of newly synthesized specific IgEs during a parallel application of recombinant Bet v1a, the major birch pollen allergen. The decrease of allergen-specific serum IgE might be related to tolerance-inducing mechanisms stopping mIgE-displaying B cells in their proliferation and differentiation.

Inefficient processing of mRNA for the membrane form of IgE is a genetic mechanism to limit recruitment of IgE-secreting cells.

Immunoglobulin E (IgE) is the key effector element in allergic diseases ranging from innocuous hay fever to life-threatening anaphylactic shock. Compared to other Ig classes, IgE serum levels are very low. In its membrane-bound form (mIgE), IgE behaves as a classical antigen receptor on B lymphocytes. Expression of mIgE is essential for subsequent recruitment of IgE-secreting cells. We show that in activated, mIgE-bearing B cells, mRNA for the membrane forms of both murine and human epsilon (epsilon) heavy chains (HC) are poorly expressed compared to mRNA for the secreted forms. In contrast, in mIgG-bearing B cells, mRNA for the membrane forms of murine gamma-1 (gamma1) and the corresponding human gamma4 HC are expressed at a much higher level than mRNA for the respective secreted forms. We show that these findings correlate with the presence of deviant polyadenylation signal hexamers in the 3' untranslated region (UTR) of both murine and human epsilon genes, causing inefficient processing of primary transcripts and thus poor expression of the proteins and poor recruitment of IgE-producing cells in the immune response. Thus, we have identified a genetic steering mechanism in the regulation of IgE synthesis that represents a further means to restrain potentially dangerous, high serum IgE levels.