Role of the B-cell receptor in chronic lymphocytic leukemia: where do we stand?

The past 15 years have witnessed an enormous effort in studying B-cell Chronic Lymphocytic Leukemia. A great number of researches brought significant novel information and a better understanding of the natural history of this disease. This mini review will focus on the studies related to the Immunoglobulin variable (IgV) genes rearrangements that compose the B-cell receptor (BcR) of the leukemic clones. These studies have defined a role for the antigen(s) in the paths that lead to leukemic clone generation/expansion and underscore the informative value represented by BcR analyses.

A recombinant bispecific single-chain fragment variable specific for HLA class II and Fc alpha RI (CD89) recruits polymorphonuclear neutrophils for efficient lysis of malignant B lymphoid cells.

Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against Fc alpha RI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. Fc alpha RI and HLA class II-directed single-chain fragment variable (scFv) fragments were isolated from phage display libraries, established from the hybridomas A77 and F3.3, respectively. The two scFv molecules were connected with a 20 aa flexible linker sequence. After expression in SF21 insect cells and chromatographic purification, the bispecific molecule showed specific binding to both Ags at K(D) values of 148 +/- 42 nM and 113 +/- 25 nM for the anti-Fc alpha RI and anti-HLA class II scFv components in the bsscFv, respectively. In Ab-dependent cytotoxicity assays with PMNs as effectors and a series of lymphoma-derived cell lines (ARH-77, RAJI, REH, NALM-6, RS4;11), the bsscFv was significantly more cytotoxic than the parental murine IgG1 and its chimeric IgG1 derivative. When targeting primary tumor cell isolates from six patients with B cell malignancies, the killing capacity of the (Fc alphaRI x HLA class II) bsscFv compared favorably to conventional HLA class II mAb. Importantly, the cell lines NALM-6 and RS411, as well as two primary tumor cell isolates, were exclusively lysed by the bsscFv. To our knowledge, this is the first report of an Fc alpha RI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (Fc alpha RI x HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.

Transgenic expression of single-chain anti-CTLA-4 Fv on beta cells protects nonobese diabetic mice from autoimmune diabetes.

T cell-mediated immunodestruction of pancreatic beta cells is the key process responsible for both the development of autoimmune diabetes and the induction of rejection during islet transplantation. In this study, we investigate the hypothesis that transgenic expression of an agonistic, membrane-bound single-chain anti-CTLA-4 Fv (anti-CTLA-4 scFv) on pancreatic beta cells can inhibit autoimmune processes by selectively targeting CTLA-4 on pathogenic T cells. Strikingly, transgenic expression of anti-CTLA-4 scFv on pancreatic beta cells significantly protected NOD mice from spontaneous autoimmune diabetes. Interestingly, local expression of this CTLA-4 agonist did not alter the diabetogenic properties of systemic lymphocytes, because splenocytes from transgenic mice or their nontransgenic littermates equally transferred diabetes in NOD/SCID recipients. By analyzing the T cell development in anti-CTLA-4 scFv/Th1/Th2 triple transgenic mice, we found that beta cell-specific expression of CTLA-4 agonist did not affect the development of Th1/Th2 or CD4(+)CD25(+) regulatory T cells. Most strikingly, islets from transgenic mice inhibited T cell response to immobilized anti-CD3 in a T cell-islet coculture system, suggesting a trans-mediated inhibition provided by transgenic islets. Finally, transgenic islets implanted in diabetic recipients survived much longer than did wild-type islets, indicating a therapeutic potential of this genetically modified islet graft in autoimmune diabetes.

Tumor targeting carboxylesterase fused with anti-CEA scFv improve the anticancer effect with a less toxic dose of irinotecan.

Irinotecan (CPT-11) is a key drug for the treatment of various cancers. CPT-11 can be considered to be a prodrug, since it needs to be activated into the toxic drug SN-38 by the enzyme carboxylesterase. However, CPT-11 may induce severe diarrhea and bone marrow suppression as adverse effects, thus leading to treatment interruption. The tumor-specific activation of CPT-11 is a possible strategy to avoid the severe toxicities by reducing the serum concentration of CPT-11. In this study, we constructed human liver carboxylesterase-2 fused with anticarcinoembryonic antigen (CEA) scFv as a targeting molecule. The recombinant enzyme anchors onto the tumor cell surface CEA, and thus metabolize CPT-11 extracellularly. In addition a secreted tumor-targeted form of carboxylesterase should help prevent the leakage of the enzyme from the site of the tumor into the circulation. This fusion protein showed CPT-11 activation to SN-38 and specific binding to CEA-expressing cells. In combination with CPT-11, the recombinant carboxylesterase protein exerted antiproliferative effects on human cancer cells. This recombinant enzyme is, therefore, a promising new tool in enzyme prodrug therapy for the treatment of carcinoma with CPT-11.

Structure-function relationships of the variable domains of monoclonal antibodies approved for cancer treatment.

Due to their exquisite specificity for a given epitope on the target antigen, recombinant monoclonal antibodies (rmAb) can deliver "targeted therapy" in oncology. This review focuses on the structural bases of "antigen specificity" to aid clinical researchers and pharmacologists in managing these new drugs. The fine structure of the Fv (Fragment variable) module (combination of VH and VL domains) from the five unconjugated antibodies currently approved for cancer treatment, namely rituximab, cetuximab, alemtuzumab, trastuzumab and bevacizumab, is presented and analysed. Co-crystal and functional studies are reviewed to define rmAb residues contributing to antigen binding site (paratope)-epitope interfaces. The genetic origin of these recombinant monoclonal antibodies, determined through the IMGT/3Dstructure-DB database and IMGT/V-QUEST (http://imgt.cines.fr), is presented, allowing the evaluation of homologies between antibodies and their closest germline human counterparts and hence their possible immunogenicity. Overall, the IMGT standards appear as a first and crucial step in the evaluation of recombinant antibodies.

Peptidomimics of antigen are present in variable region of heavy and light chains of anti-idiotypic antibody and function as surrogate antigen for perpetuation of immunological memory.

Peptide antigens composed of relevant B cell and T cell epitopes, capable of inducing protective immune response against the whole pathogen, are potentially safe, alternative vaccine antigens for prevention of wide range of diseases. Here, we show that short peptides derived from internal image sequences of anti-idiotypic antibody (peptidomimics) can function as both B and T cell epitopes and perpetuate antigen specific immunological memory. We have sequenced the variable regions of heavy and light chains of the anti-idiotypic antibody specific to rinderpest virus hemagglutinin protein and predicted T cell epitopes in these sequences by an immuno-informatics approach. We have studied the interaction of these epitopes with MHC class I by in vitro assays and in silico analysis by molecular modeling of the idiopeptide-MHC complexes as well as antigen-derived peptide-MHC complexes. The functional capacity of anti-idiotypic antibody derived peptides to stimulate antigen specific T cells in vitro was tested. The ability of peptidomimics to proliferate the immune splenocytes in vitro was 10 times more when compared with that of a control peptide taken from the constant region of immunoglobulin. Similarly three- to fivefold more amounts of IL-2 and IFN-gamma were secreted by immune splenocytes in response to in vitro re-stimulation with peptidomimics. Further, we have provided evidence for the generation of antibodies against peptidomimics in memory response generated on antigen or anti-idiotypic antibody immunizations. In summary, our experiments suggest that peptidomimics are generated in the body after antigen immunization and may have important roles in vivo in regulating antigen specific immunological memory.

Functional mapping and single chain construction of the anti-cytokeratin 8 monoclonal antibody TS1.

The anti-cytokeratin (CK) 8 monoclonal antibody (mab) TS1 has been shown to efficiently bind to CK8 expressed in carcinomas in vivo. The anti-idiotypic antibody of TS1, alphaTS1, can be used to regulate the tumor:non-tumor ratio of TS1 by clearing non-tumor binding TS1 from the circulation. If the interaction of TS1 to CK8 and alphaTS1 is fully understood, mutations can be used to improve the tumor:non-tumor ratio. A scFv was made of the mab TS1 and residues earlier identified by Erlandsson et al. as important for the interaction with both its antigen CK8 and its anti-idiotype alphaTS1, were mutated to alanine or amides and expressed in E. coli. The effects of the mutations were studied by ELISA and residues important for the interactions to both CK8 and alphaTS1 were identified as mainly tyrosines, charged residues, a serine and a tryptophan. Altogether, nine amino acid residues in TS1 were found to be important in the interaction to alphaTS1 and six residues for the interaction to CK8. Important residues, clustered together in the modelled protein, were identified as residues from CDR 3 of the heavy chain and the unexpected participation of a residue in CDR 2 of the light chain. Some of the important residues are likely to be hotspots. Hotspots constitute a few residues in an interaction that contribute most to the binding, energetically. Amino acid residues in hotspots often cluster together in the center of the interaction interface, but can also be spread out to the periphery. The hotspots are often surrounded by hydrophobic patches, which are seen in the modelled TS1 protein used in this study. Amino acid residues that increased the affinity when mutated were also identified for both interactions. These residues are likely to be located outside the interacting interface. It can from this study be concluded that it is wise to precede the mutational procedure with experiments that can give guidelines for the selection of which amino acid residues to mutate. If the guidelines from the chemical modifications from Erlandsson et al. not had been used, this study would have left some residues unmutated and thereby missed important information.

[Inhibitory effect of anti-type IV collagenase intrabody on invasiveness of human pulmonary giant cell carcinoma PG cells in vitro].

OBJECTIVE: To explore the inhibitory effects of endoplasmic reticulum-retained intrabody on the secretion of type IV collagenase and the invasion of human pulmonary giant cell carcinoma PG cells in vitro. METHODS: Two expression plasmids were constructed, pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv encoding cytoplasm-retained and endoplasmic reticulum-retained single chain antibodies against the type IV collagenase, respectively. The intracellular antibody genes were transfected into the human pulmonary giant cell carcinoma PG cells. Western blot was performed to detect the expression of pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv. Gelatin zymography was performed to detect seretion of type IV collagenase in PG cells and Matrigel assay was employed for determination of the cell invasiveness. RESULTS: Both of cytoplasm-retained and endoplasmic reticulum-retained introbodies, CP.scFv and ER.scFv, were expressed in PG cells. ER.scFv, significantly inhibited the secretion of type IV collegenase. As shown, matrix metalloproteinase 9 and matrix metalloproteinase 2 were inhibited by 85.7% and by 51.2%, respectively. However, CP.scFv did not show such inhibitory effect. The ER.scFv encoding gene-transfected PG cells were much less invasive than parental or vector control cells, the inhibition rate was 76.3% (P < 0.05), whereas CP.scFv encoding gene-transfected PG cells showed no reduction in invasiveness. CONCLUSION: Those findings demonstrate that endoplasmic reticulum (ER)-retained intracellular antibody technology may selectively abrogate the activity of type IV collagenase in the protein trafficking and secretory pathway and effectively inhibit tumor cell invasion in vitro. Anti-type IV collagenase intrabody may be further used in cancer gene therapy.

The role of interface framework residues in determining antibody V(H)/V(L) interaction strength and antigen-binding affinity.

While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region fragment (V(H))/antibody light chain variable region fragment (V(L)) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak V(H)/V(L) interaction strength, in the absence of antigen. To investigate the possible relationship between antigen-binding affinity and V(H)/V(L) interaction strength, a novel phage display system that can switch two display modes was employed. We focused on the two framework region 2 regions of the HyHEL-10 V(H) and V(L), facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.3, which has a far stronger V(H)/V(L) interaction. The phagemid library, encoding V(H) gene 7 and V(L) amber codon gene 9, was used to transform TG-1 (sup+), and the phages displaying functional variable regions were selected. The selected phages were then used to infect a nonsuppressing strain, and the culture supernatant containing V(H)-displaying phages and soluble V(L) fragment was used to evaluate the V(H)/V(L) interaction strength. The results clearly showed the existence of a key framework region 2 residue (H39) that strongly affects V(H)/V(L) interaction strength, and a marked positive correlation between the antigen-binding affinity and the V(H)/V(L) interaction, especially in the presence of a set of particular V(L) residues. The effect of the H39 mutation on the wild-type variable region was also confirmed by a SPR biosensor as a several-fold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region.

Antibody-mediated Hsp70 protein therapy.

Intracellular Hsp70 provides cytoprotection against a variety of stressful stimuli, and an effective means of increasing intracellular Hsp70 levels could prove beneficial in the prevention and treatment of a variety of human diseases. A novel protein transduction domain consisting of the single chain Fv fragment of an anti-DNA antibody known to penetrate into living cells and tissues, mAb 3E10, has recently been used to deliver functional proteins to cells. The ability of the single chain Fv fragment to deliver Hsp70 into living cells was tested by generating an Fv-Hsp70 fusion protein. Fv-Hsp70 was produced as a secreted protein in both COS-7 cells and the methylotropic yeast strain Pichia pastoris and was shown capable of penetrating into COS-7 cells and primary rat cortical neurons. Pre-treatment with Fv-Hsp70 protected both COS-7 cells and primary rat cortical neurons against subsequent exposure to hydrogen peroxide. These results provide the first evidence that the Fv fragment of mAb 3E10 is capable of delivering proteins to neurons and indicate its potential in the development of Hsp70 protein therapy.

Targeted apoptosis activation with GrB/scFvMEL modulates melanoma growth, metastatic spread, chemosensitivity, and radiosensitivity.

GrB/scFvMEL, a fusion protein composed of human granzyme B (GrB) and the single-chain antibody scFvMEL, targets melanoma gp240 antigen and exerts impressive cytotoxic effects by inducing apoptosis. We evaluated the effects of GrB/scFvMEL on chemotherapy, radiation therapy, metastasis in vitro, and the growth of human melanoma A375 xenograft tumors in nude mice. GrB/scFvMEL showed synergistic cytotoxicity when coadministered with doxorubicin, vincristine or cisplatin, and additive effects, in combination with etoposide or cytarabine. Optimal cytotoxic effects were obtained when cells were treated first with GrB/scFvMEL followed by exposure to the agent (rather than the reverse). Pretreatment of A375 cells with GrB/scFvMEL significantly sensitized melanoma cells to ionizing radiation assessed using a clonogenic survival assay. Subtoxic doses of GrB/scFvMEL inhibited the invasion of A375 cells into Matrigel. GrB/scFvMEL (37.5 mg/kg) was administered intravenously to nude mice bearing A375 tumors. Saline-treated tumors increased 24-fold, whereas tumors treated with GrB/scFvMEL showed a significant tumor growth delay increasing four-fold. Tumor tissue displayed an increase in apoptotic nuclei compared to control. Thus, the targeted delivery of GrB to tumors may have a significant potential for cancer treatment. Targeted therapeutic agents specifically designed to impact cellular apoptotic pathways may represent a novel class of therapeutic agents.

Role of the monoclonal kappa chain V domain and reversibility of renal damage in a transgenic model of acquired Fanconi syndrome.

Acquired Fanconi syndrome (FS) is a complication of monoclonal gammopathies featuring a generalized dysfunction of the proximal tubule of the kidney, due to the storage within proximal tubular cells of a monoclonal immunoglobulin light chain. We engineered transgenic mice in which the endogenous mouse Jkappa cluster was replaced by a human VkappaJkappa rearranged gene cloned from a patient with smoldering myeloma-associated FS. The V region belonged to the VkappaI subgroup and was related to the O2-O12 germ-line gene, a V segment previously found associated with FS and light-chain crystallization in several patients with myeloma. Association of the human VkappaI domain with a mouse kappa constant domain in transgenic animals yielded a nephrotoxicity pattern similar to that observed in patients, strongly suggesting that the whole pathogenic effect of FS light chains can be ascribed to a peculiar structure of the V domain. Morphologic alterations of the kidney tubular cells, which contained rhomboid-shape crystals, were observed in mice, together with alterations of the proximal tubule reabsorption function. Moreover, the number of renal crystalline inclusions was dramatically reduced after conditional deletion of the human VkappaI transgene, showing that proximal tubular lesions are reversible upon suppression of the nephrotoxic light chain secretion.

Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli.

c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus, single-chain antibody fragment (scFv) specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. Here, we constructed the expression system for anti-c-Met scFv fused with T7 tag at its N-terminus using pET vector and investigated the expression conditions to achieve a functional and soluble expression of the scFv in the cytoplasm of recombinant Escherichia coli. The redox potential of E. coli cytoplasm was the most critical factor for the functional expression of anti-c-Met scFv. The employment of a host with oxidizing cytoplasm, E. coli trxB/gor double mutant, improved the productivity of functional anti-c-Met scFv by approximately 10-fold compared to the production of anti-c-Met scFv in the reducing cytoplasm of wild type E. coli. Productivity of functional anti-c-Met scFv could be further enhanced by co-expressing molecular chaperones such as GroELS, trigger factor, and DsbC with the scFv. Coexpression of DsbC increased the yield of functional anti-c-Met scFv about 2.5-fold in the cytoplasm of E. coli trxB/gor mutant compared to the production of scFv without DsbC coexpression. Lowering the IPTG concentration from 1 to 0.05 mM led to the slight enhancement, approximately 1.6-fold, of productivity of functional scFv. Although the use of low temperature for anti-c-Met scFv expression increased the ratio of soluble scFv fraction to insoluble fraction, productivity of soluble scFv decreased owing to the significant reduction of expression rate. The addition of 0.5 M sucrose in the medium inhibited the formation of intracellular insoluble anti-c-Met scFv. To purify the anti-c-Met scFv simply, we fused hexahistidine at the C-terminus of scFv and purified the scFv showing 98% of purity through the interaction between Ni2+ and histidine.

Systemic autoimmune disease caused by autoreactive B cells that receive chronic help from Ig V region-specific T cells.

B cells present BCR V region-derived Id-peptides on their MHC class II molecules to Id-specific CD4+ T cells. Prolonged Id-driven T-B collaboration could cause autoimmune disease, but this possibility is difficult to test in normal individuals. We have investigated whether mice doubly transgenic for an Id+ Ig L chain and an Id-specific TCR develop autoimmune disease. Surprisingly, T cell tolerance was not complete in these mice because a low frequency of weakly Id-reactive CD4+ T cells accumulated with age. These escapee Id-specific T cells provided chronic help for Id+ B cells, resulting in a lethal systemic autoimmune disease including germinal center reactions, hypergammaglobulinemia, IgG autoantibodies, glomerulonephritis, arthritis, skin affection, and inflammatory bowel disease. Inflamed tissues contained foci of Id-driven T-B collaboration, with deposition of IgG and complement. The disease could be transferred with B and T cells. The results demonstrate a novel mechanism for development of autoimmune disease in which self-reactive Id+ B cells receive prolonged help from Id-specific T cells, thus bypassing the need for help from T cells recognizing conventional Ag.

Anti-beta 2-glycoprotein-I antibodies in scFv format.

Phage display was introduced almost 20 years ago. It has been used to produce large amounts of diverse proteins, to analyze protein-ligand interactions, to improve the affinity of proteins for their binding receptors, and to characterize antibody binding sites. The recombinant version of the antibody Fv is termed single-chain variable fragment (scFv). Many large phage libraries have been developed that have yielded antibodies to several hundred antigens, but only 5 human anti-beta2-glycoprotein-I and three anti-prothrombin antibodies in scFV have been so far characterized. Antibodies to beta2GP-I thus generated show 92-94% homology with their nearest germ line genes. Their mutations frequently appear to be independent of antigen. Two anti-prothrombin antibodies show strong crossreactivity with beta2GP-I. Four mouse anti-beta2GP-I scFV show less binding properties than their original counterparts, but had the same capacity of inducing experimental antiphospholipid syndrome. This pathogenicity appears to reside in the V(H)DJ(H)C(H) region of the scFv since the V(H)DJ(H)C(H) regions of pathogenic scFV combined with irrelevant V(L) J(L)C(L) regions retained their pathogenicity while the opposite failed to do so.

Neutralizing human anti-B-cell-activating factor of the TNF family (BAFF) scFv selected from phage antibody library.

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-BAFF. After four rounds of panning against BAFF, thirty-two out of 92 phage clones displayed BAFF binding activity. One of the positive clones, designated F8, bound to BAFF with relatively high affinity and neutralized BAFF bioactivity in vitro. F8 clone was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC). The purified scFv recognized BAFF with the affinity constant (K(aff)) of 2.5 x 10(7)M(-1) without cross-reaction to APRIL. In addition to binding, the purified scFv could does-dependently inhibit BAFF-induced mouse spleen B lymphocyte proliferation. Together with its fully human mature, F8 scFv may have therapeutic implications in therapy of autoimmune disorders mediated by BAFF.

Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells.

In the past decade, intracellular antibodies have proven to be a useful tool in obtaining the phenotypic knock-out of selected gene function in different animal and plant systems. This strategy is based on the ectopic expression of recombinant forms of antibodies targeted towards different intracellular compartments, exploiting specific targeting signals to confer the new intracellular location. The functional basis of this technology is closely linked to the ability of intracellular antibodies to interact with their target antigens in vivo. This interaction allows either a direct neutralising effect or the dislodgement of the target protein from its normal intracellular location and, by this mechanism, the inactivation of its function. By using this approach, the function of several antigens has been inhibited in the cytoplasm, the nucleus, and the secretory compartments. In this article, we shall describe all the steps required for expressing single-chain Fv fragments in different subcellular compartments of mammalian cells and their subsequent use in knock-out experiments, starting from a cloned single-chain Fv fragment. This will include the analysis of the solubility properties of the new scFv fragment in transfected mammalian cells, the intracellular distribution of the antigen-antibody complex, and the resulting phenotype.

The function of conserved amino acids in or near the complementarity determining regions for related antibodies to Cryptococcus neoformans glucuronoxylomannan.

Most monoclonal antibodies (mAbs) to Cryptococcus neoformans glucuronoxylomannan (GXM) antigen (Ag) are grouped as Class II based on usage of V(H)7183, Vkappa5.1, J(H)2, and Jkappa1 gene elements. Comparative analysis of 43 Class II mAbs revealed conservation of I51, G54, and D61 in heavy-chain variable region (V(H)) complementarity determining region 2 (CDR2), and R95 and D96 in CDR3. Furthermore, position 100b (Kabat numbering scheme) in CDR3 always had an aromatic amino acid (aa) and F was found at position 100c in 96% of mAbs. The function of these conserved residues for binding to GXM and peptide mimetics, and idiotype (Id) structure, was investigated using site-directed mutagenesis. In addition, we mutated W36 and V37 in the second framework. Mutations W36A, Y100bA, and F100cA interfered with antibody (Ab) secretion, but not assembly, and cytoplasmic Ab bound to GXM and Id mAbs. In contrast, mutations V37A, I51A, G54A, and D61A did not affect assembly, secretion, or binding to GXM. Mutating the R95-D96 motif in CDR3 to DR, DD, RR, RA, AD, KD, HD, RE, RN or AA revealed that the positive charge at position 95 was essential for binding GXM, whereas the negative charge at position 96 could be substituted for a non-charged aa. Our results: (1) extend the concept that CDR3 diversity is essential for Ag and Id specificity to a polysaccharide-binding Ab; (2) show that aa conservation in CDRs does not imply a requirement for Ag binding; (3) establish a role for W36 in secretion; and (4) demonstrate that aa motifs used for binding GXM and peptide mimetics can differ.

Intracellularly expressed single-domain antibody against p15 matrix protein prevents the production of porcine retroviruses.

The presence of porcine endogenous retroviruses presents a potential risk of transmission of infectious diseases (xenozoonosis) if tissues and organs from genetically modified pigs are to be used in xenotransplantation. Here, we report that intracellular expression of a llama single-domain antibody against p15, the matrix domain protein of the porcine endogenous retrovirus Gag polyprotein, blocks retrovirus production, providing the possibility of eliminating the risk of infection in xenotransplantation.

Interaction of murine precursor B cell receptor with stroma cells is controlled by the unique tail of lambda 5 and stroma cell-associated heparan sulfate.

Efficient clonal expansion of early precursor B (pre-B) cells requires signals delivered by an Ig-like integral membrane complex, the so-called pre-B cell receptor (pre-BCR). A pre-BCR consists of two membrane micro H chains, two covalently associated surrogate L chains, and the heterodimeric signaling transducer Igalphabeta. In contrast to a conventional Ig L chain, the surrogate L chain is a heterodimer composed of the invariant polypeptides VpreB and lambda5. Although it is still unclear how pre-BCR signals are initiated, two recent findings support a ligand-dependent initiation of pre-BCR signals: 1) a pre-BCR/galectin-1 interaction is required to induce phosphorylation of Igalphabeta in a human precursor B line, and 2) soluble murine as well as human pre-BCR molecules bind to stroma and other adherent cells. In this study, we show that efficient binding of a soluble murine pre-BCR to stroma cells requires the non-Ig-like unique tail of lambda5. Surprisingly however, a murine pre-BCR, in contrast to its human counterpart, does not interact with galectin-1, as revealed by lactose blocking, RNA interference, and immunoprecipitation assays. Finally, the binding of a murine pre-BCR to stroma cells can be blocked either with heparin or by pretreatment of stroma cells with heparitinase or a sulfation inhibitor. Hence, efficient binding of a murine pre-BCR to stroma cells requires the unique tail of lambda5 and stroma cell-associated heparan sulfate. These findings not only identified heparan sulfate as potential pre-BCR ligands, but will also facilitate the development of appropriate animal models to determine whether a pre-BCR/heparan sulfate interaction is involved in early B cell maturation.