Decreased expression of natriuretic peptide A receptors and decreased cGMP production in the choroid plexus of spontaneously hypertensive rats

CD44 isoforms, such as CD44s (the standard form), contain at least one ankyrin-binding site within the 70-amino acid (aa) cytoplasmic domain and several hyaluronic acid (HA)-binding sites within the extracellular domain. To study the role of CD44s-ankyrin interaction in regulating human prostate tumor cells, we have constructed several CD44s cytoplasmic deletion mutants that lack the ankyrin-binding site(s). These truncated cDNAs were stably transfected into CD44-negative human prostate tumor cells (LNCaP). Our results indicate that a critical region of 15-amino acids (aa) between aa 304 and aa 318 of CD44s is required for ankyrin binding. Biochemical analyses, using competition binding assays with a synthetic peptide containing the 15 aa between aa 304 and aa 318 (NSGNGAVEDRKPSGL), further support the conclusion that this region contains the ankyrin-binding domain of CD44s. Deletion of this 15-aa ankyrin-binding sequence from CD44s results in a drastic reduction of HA-mediated binding/cell adhesion, Src p60 kinase(s) interaction and anchorage-independent growth in soft agar. These findings suggest that the binding of cytoskeletal proteins, such as ankyrin, to the cytoplasmic domain of CD44s plays a pivotal role in regulating HA-mediated functions as well as Src kinase activity and prostate tumor cell transformation.     

A blood group-related polymorphism of CD44 abolishes a hyaluronan-binding consensus sequence without preventing hyaluronan binding

CD44 is a widely expressed integral membrane protein that acts as a receptor for hyaluronan (HA) and is proposed to be important to cell-extracellular matrix interaction. The Indian (In) blood group antigens reside on CD44, and most individuals express the Inb antigen. Homozygosity for the Ina allele occurs as a rare event and is associated with production of alloantibody to the common Inb antigen after transfusion or pregnancy. The present study demonstrates that a single point mutation (G252 --> C) causes an Arg46 --> Pro substitution, which is responsible for the Inb/Ina polymorphism. Additional mutations were found in In(a+b-) cDNA but were not necessary to the antigenic phenotype as determined in site-directed mutagenesis studies. In studies using CD44 chimeric constructs, Arg46 has previously been shown to be crucial for maintenance of HA-binding ability to a CD44 peptide. However, the present study demonstrates that the Arg46 --> Pro substitution does not reduce HA binding to the intact CD44 protein, which contains two proposed extracellular HA-binding motifs. Down-regulation of HA binding to In(a+b-) CD44 by anti-CD44 monoclonal antibody (mAb) ligands, however, was weakened, although all mAbs tested bound In(a+b-) and In(a-b+) CD44 equally well. Competitive inhibition studies using human anti-Inb also showed that some mAbs that inhibit HA binding to CD44 may do so by interacting with a domain separate from, but affecting the structure of, the Inb epitope.     

Monoclonal antibodies to the extracellular domain of prostate-specific membrane antigen also react with tumor vascular endothelium

Prostate-specific membrane antigen (PSMA), initially defined by monoclonal antibody (mAb) 7E11, is a now well-characterized type 2 integral membrane glycoprotein expressed in a highly restricted manner by prostate epithelial cells. 7E11 has been shown to bind an intracellular epitope of PSMA that, in viable cells, is not available for binding. Herein, we report the initial characterization of the first four reported IgG mAbs that bind the external domain of PSMA. Competitive binding studies indicate these antibodies define two distinct, noncompeting epitopes on the extracellular domain of PSMA. In contrast to 7E11, these mAbs bind to viable LNCaP cells in vitro. In addition, they show strong immunohistochemical reactivity to tissue sections of prostate epithelia, including prostate cancer. These mAbs were also strongly reactive with vascular endothelium within a wide variety of carcinomas (including lung, colon, breast, and others) but not with normal vascular endothelium. These antibodies should prove useful for in vivo targeting to prostate cancer, as well as to the vascular compartment of a wide variety of carcinomas.     

Structure-function study of the extracellular domain of the human IFN-alpha receptor (hIFNAR1) using blocking monoclonal antibodies: the role of domains 1 and 2

We have performed a structure-function analysis of extracellular domain regions of the human IFN-alpha receptor (hIFNAR1) using mAbs generated by immunizing mice with a soluble hIFNAR1-IgG. Five mAbs described in this study recognize different epitopes as determined by a competitive binding ELISA and by alanine substitution mutant analyses of the hIFNAR1-IgG. Two mAbs, 2E1 and 4A7, are able to block IFN-stimulated gene factor 3 (ISGF3) formation and inhibit the antiviral cytopathic effect induced by several IFN-alpha (IFN-alpha 2/1, -alpha 1, -alpha 2, -alpha 5, and -alpha 8). None of these anti-IFNAR1 mAbs were able to block activity of IFN-beta. mAb 4A7 binds to a domain 1-hIFNAR1-IgG but not to a domain 2-hIFNAR1-IgG, which suggests that its binding region is located in domain 1. The binding of the most potent blocking mAb, 2E1, requires the presence of domain 1 and domain 2. The most critical residue for 2E1 binding is a lysine residue at position 249, which is in domain 2. These findings suggest that both domain 1 and domain 2 are necessary to form a functional receptor and that a region in domain 2 is important. IFN-beta recognizes regions of the hIFNAR complex that are distinct from those important for the IFN-alpha.     

Naturally-occurring anti-thymocyte autoantibody which identifies a restricted CD4+CD8+CD3-/lo/int thymocyte subpopulation exhibits extensive polyspecificity

In this study, naturally-occurring, monoclonal IgM kappa anti-thymocyte autoantibodies from the neonatal inbred Balb/c mouse-derived hybridoma NMT-1 (NMT-1 mAb), previously reported to identify a restricted CD4+CD8+CD3/lo/int thymocyte subpopulation, have been shown to exhibit extensive polyspecificity. Using immunofluorescence, immunoblotting and antibody titration and competition ELISAs, NMT-1 mAbs exhibited polyspecific binding to 12 apparently structurally unrelated self and non-self antigens. The autoreactive component of the polyspecificity profile of NMT-1 mAbs encompassed reactivity to developmentally-related 14.5 and 18.3 kDa Thy-1 glycoforms expressed on a CD4+CD8+CD3-/lo/int thymocyte subpopulation. The autoreactivity profile of NMT-1 mAbs also included recognition of the heavy and light chains of mouse IgG1 and mouse cytokeratins within thymic medullary epithelium and basal epithelial cells of stratified squamous epithelium of mouse tongue, oesophagus, stomach, skin and vagina. Examination of the polyspecificity profile of NMT-1 mAbs was also undertaken using a panel of 23 antigens including heterologous proteins, phospholipids, haptens and bacterial antigens by antibody titration and competition ELISAs. Antibody titration ELISAs demonstrated that NMT-1 mAbs bound nine antigens including bovine carbonic anhydrase, ovalbumin, cardiolipin, phosphatidylserine, the haptens, DNP and FITC and the bacterial antigens including Escherichia coli beta-galactosidase and the toxoids from Corynebacterium tetani and Clostridium diphtheria. Competition ELISAs, based on the inhibition of NMT-1 mAb binding to antigens adsorbed to ELISA plate surfaces by inhibitor antigens in solution, demonstrated that NMT-1 mAb interactions were not dependent on multivalent binding. In these assays, NMT-1 mAbs recognized unmodified (native) epitopes on the solution phase forms of the protein antigens, including E. coli beta-galactosidase and toxoids from Corynebacterium tetani and Clostridium diphtheria, providing further evidence for the hypothesis that the binding of multiple, apparently unrelated, antigens by NMT-1 mAbs occurs via unique polyspecific antigen combining sites.     

Structural variability of CD44v molecules and reliability of immunodetection of CD44 isoforms using mAbs specific for CD44 variant exon products

CD44 can be considered structurally and functionally one of the most variable surface molecules. Alternative splicing of variant exons as well as posttranslational modifications of the molecule (differences in glycosylation) generate a rich repertoire of CD44 isoforms (CD44v), some of which seem to play a key role in tumor growth and progression. Immunodetection of CD44 isoforms in vivo, using mAbs specific for CD44 variant exon products, is largely used to identify those CD44 molecules involved in tumor growth and progression and to interfere with CD44-mediated processes. In the present work we demonstrate that the immunoreactivity of some mAbs directed to CD44 exon-specific epitopes can be impaired by the structural variability of the molecule. Our findings demonstrate that (1) specific exon assortment and/or posttranslational modifications of CD44v molecules can mask CD44 exon-specific epitopes; (2) glycosaminoglycan side chains, carried by some CD44v isoforms of high molecular weight, may play a critical role in determining the exact conformation of the molecule, which is necessary for the detection of CD44 variant epitopes by specific mAbs; and (3) in a panel of stable transfectants expressing CD44 N-glycosylation site-specific mutants, generated in the constant region of CD44 extracellular domain, asparagine-isoleucine substitution is sufficient per se to impair the immunoreactivity of several mAbs to pan-CD44. Thus, conformational changes due to the alternative splicing of CD44 variant exons and/or posttranslational modifications of the molecule (different degree of glycosylation), which are cell type-specific, are likely to generate CD44 variants that elude immunodetection. These findings strongly suggest that immunohistochemical analysis of CD44 expression in vitro and in vivo, using mAbs specific for CD44 variant exon epitopes, can potentially be impaired by a large number of false negative results.     

Definition of the specificity of monoclonal antibodies against porcine CD45 and CD45R: report from the CD45/CD45R and CD44 subgroup of the Second International Swine CD Workshop

Swine cell binding analyses of a set of 48 monoclonal antibodies (mAbs), including eleven standards, assigned to the CD44 and CD45 subset group of the Second International Swine CD Workshop yielded 13 clusters. Although none of these corresponded to CD44, seven mAbs formed a cluster which was identified as being specific for restricted epitopes of CD45 (CD45R). In addition, a T-cell subset specific cluster comprised of four mAbs was also identified. Two mAbs (STH106 and SwNL 554.1) reacted exclusively with CD8 bright lymphocytes, the other two (2B11 and F01G9) with a subset of CD4 lymphocytes. The other 10 clusters were either specific for MHC-class I like molecules or overlapped with clusters identified by the adhesion molecule subgroup and are therefore just briefly discussed in this report. The specificity of all the mAbs in the CD45R cluster was verified by their ability to immunoprecipitate distinct proteins and to react with CHO cells expressing individual isoforms of CD45. Three CD45R mAbs (3a56, MIL5, -a2) did react with a 210 kDa isoform(s), while another three (STH267, FG2F9, 6E3/7) only recognized a 226 kDa isoform(s). The remaining one (MAC326) precipitated both a 210 and 226 kDa protein. The specificity of all the mAbs in the CD45R cluster, and of the CD45 common mAbs, was confirmed by their reactivity with CHO cells transfected with cDNAs encoding the extracellular and transmembrane portions of distinct CD45R isoforms. Those mAbs recognizing a 210 kDa protein reacted with CHO cells expressing the CD45RC isoform, while those capable of precipitating a 226 kDa, but not the 210 kDa, polypeptide recognized CHO cells expressing either the CD45RAC and the relatively rare CD45RA isoform. MAC326 was unique in its inability to react with CHO cells engineered to produce the CD45RC and CD45RAC isoforms. Thus, three mAbs (6E3/7, STH267, and FG2F9) appear to be specific for an epitope(s) encoded by the A exon, while one (MAC326) recognizes a determinant encoded by the C exon. The remaining three mAbs (3a56, -a2, MIL5) are apparently specific for an epitope(s) which results from the fusion of the C exon to the invariant leader sequence and is destroyed by inclusion of the A exon. All three CD45 common mAbs, K252.1E4, MAC323 and 74.9.3, did react with the CHO cells lines expressing either the CD45RA, CD45RC, CD45RAC or CD45RO isoforms, but not with untransfected CHO cells. When the natural expression of CD45 isoforms was examined by reacting lymphocytes with CD45R mAbs, a high level expression of isoforms containing the A exon-generated domain was detected in all B cells while the majority of CD4+ T cells had undetectable or lower expression density of this protein than B cells. In contrast, the density of expression of the CD45 isoform(s) containing the C exon-generated domain ranged from undetectable to high in CD4+ T cells whereas the amounts were approximately ten-fold lower in B cells. Overall this panel of CD45 mAbs will be very useful in analyzing the maturation and differentiation of swine lymphoid cells subsets.     

Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens

The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix 'w' which will lead to 'wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.     

Expression of the sialosyl-Tn epitope on CD45 derived from activated peripheral blood T cells

The cell surface protein tyrosine phosphatase CD45 is a major target of IgM anti-T cell autoantibodies in systemic lupus erythematosus (SLE). The autoreactive determinants on CD45 are O-linked glycans expressed on activated T cells and certain T cell lines, rather than linear or conformational polypeptide epitopes or N-linked glycans. To identify oligosaccharide structures that may play a role in the functional interactions of CD45 or are candidate target epitopes of SLE anti-CD45 autoantibodies, autoreactive CD45 purified from Jurkat T cells and non-autoreactive CD45 purified from CLL B cells were tested by ELISA for expression of mucin-type O-glycan structures. Monoclonal antibodies (mAbs) directed against blood group A, type 1 H chains, type 2 H chains, T, Le(a), sialylated-Le(a), Le(b), sialylated-Le(c), Le(x), sialylated-Le(x), multi-fucosylated Le(x), Le(y), and sialylated-extended Le(v) failed to react with CD45 from either B cells or T cells. However, mAbs directed against Tn (galNAcalpha1-->O-ser/thr) or sialosyl-Tn (neuNAcalpha2-6gaINAcalpha1-->O-ser/thr) structures reacted with CD45 derived from Jurkat T cells, but not from CLL B cells. Anti-Tn mAbs also reacted in western blotting procedures with CD45 isolated from Jurkat T cells, but did not react with CD45 isolated from CEM, MOLT-3, or PEER T cells; Daudi, Raji, or CLL B cells; or resting or Con A-activated PBL. However, anti-sialosyl-Tn mAbs stained blots of CD45 isolated from Jurkat and CEM T cells and Con A-activated PBL, a pattern of reactivity similar to that of the anti-CD45 autoantibodies. Flow cytometric analyses demonstrated that the sialosyl-Tn epitopes are expressed on a subpopulation of CD4 +/CD8- T cells.     

Site-specific de-N-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding

CD44 is a cell surface receptor for the glycosaminoglycan hyaluronan (HA). Not all CD44-positive cells bind HA, and binding ability is strictly regulated. Three different HA binding states have been defined: inactive, inducible (by certain CD44-specific monoclonal antibodies), and constitutively active. The observation that sets of genetically related cell lines representing different HA binding states showed correlated differences in N-glycosylation of CD44, and that inhibition of N-glycosylation enhanced HA binding (Lesley et al., J. Exp. Med., 182: 431-437, 1995) led us to examine directly whether specific N-glycosylation site modifications were involved in regulating the HA binding function. CD44-negative, -active, and inducible cell lines were stably transfected with mutant constructs in which each of the five N-glycosylation sites of murine CD44 had been separately inactivated. Ability to bind soluble HA was examined over a range of CD44 expression levels. For the active cell line, AKR1, transfectants for all N-glycosylation mutants bound HA as well as did transfectants for wild type CD44. No inhibitory effects of inactivating specific N-glycosylation sites were observed. HA binding was activated when two of the mutant constructs were transfected into a novel CD44-negative inducible cell line. Inactivation of N-glycosylation sites at residues 25 or 120 converted the inducible cell line to constitutively active, whereas inactivation of other sites had little or no effect. Fusion proteins secreted from inactive, inducible, or active cell lines were purified, bound to beads, and assayed for HA binding activity by flow cytometric analysis. Fusion proteins derived from inactive, inducible, and constitutively active cells exhibited three distinguishable "threshold" densities required for HA binding ability. The results imply that the CD44 molecules produced in cells in these three activation states have intrinsic differences in HA binding function. Treatment of the fusion proteins with neuraminidase altered the HA binding state, and glycosylation mutations that affected the phenotype of the inducible cell line lowered the threshold required for HA binding of CD44-immunoglobulin fusion proteins derived from the inducible cell line. Thus, alterations of glycosylation of CD44 itself can affect HA binding ability as manifested by a change in HA binding state.     

L-Selectin ligands that are O-glycoprotease resistant and distinct from MECA-79 antigen are sufficient for tethering and rolling of lymphocytes on human high endothelial venules

During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)-containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLex on HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of different sLex-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLex, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLex on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease-resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLex mAb 2H5 blocks binding by approximately 60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.     

Identification of functional domains in murine granulocyte-macrophage colony-stimulating factor using monoclonal antibodies to synthetic peptides

Granulocyte-macrophage (GM)-CSF is an important hematopoietic cytokine that regulates proliferation and differentiation of macrophages, neutrophils, and eosinophils. In this study, we generated mAb to five synthetic peptides that correspond to regions along the murine GM-CSF molecule. The ability of anti-peptide mAb to bind to and inhibit biologic activity of murine (m) GM-CSF was determined. mAb with the highest neutralization titers were derived from mice immunized with peptide II, which correspond to amino acids 27 to 38 of mGM-CSF. Immunochemical studies showed that peptide II specifically blocked binding of anti-peptide II mAb to GM-CSF. mAb to two other peptides in the N-terminal half corresponding to residues 7 to 17 and 47 to 58, respectively, of mGM-CSF also inhibited GM-CSF-dependent proliferation and differentiation of murine bone marrow precursors for macrophages and granulocytes. Anti-peptide mAb also inhibited growth of a murine hematopoietic cell line FDCP1 and a murine T cell line HT-2, which was shown to be dependent on GM-CSF for growth in vitro. Biologic activity of both natural and recombinant mGM-CSF was neutralized by anti-peptide mAb. These findings indicate that epitopes in the N-terminal region of mGM-CSF are important for biologic activity, and the epitope defined by peptide II (residues 27 to 38) lies within a particularly important functional domain of the mGM-CSF molecule.     

Fibrinogen coating density affects the conformation of immobilized fibrinogen: implications for platelet adhesion and spreading

Adhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 microg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 microg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for gamma 1-78 and 95-265, respectively, suggesting that the fibrinogen gamma-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 microg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at > 30 microg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.     

Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes

The combination of anti-CD2 mAb 9.6 and 9-1, specific for distinct epitopes, induces proliferation of resting human T cells. The mitogenic activity of this mAb mixture depends upon accessory cells and the 9-1 mAb Fc domain. To further study the functional properties of these mAb, their variable regions were cloned and expressed as monospecific single-chain Fv (scFv) proteins fused to the human IgG1 Fc domain (scFvIg). A novel bispecific scFvIg was constructed by cloning the two monospecific scFv binding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1 scFvIg. Monospecific scFvIg binding to CD2 was comparable to that of the corresponding parental mAb, while the bispecific scFvIg exhibited binding activity similar to that of the 9-1 scFvIg. The combination of 9.6 scFvIg and 9-1 mAb was mitogenic, whereas mixtures including the 9-1 scFvIg were non-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc. Without the IgG3 tail, the bispecific 9.6/9-1 scFvIg was directly mitogenic and was a more potent mitogen than the mAb mixture, but was accessory cell dependent. Unlike the combination of mAb, the bispecific reagent did not directly mobilize calcium in T cells. In comparison to the mAb mixture, bispecific 9.6/9-1 scFvIg-mediated stimulation of a mixed lymphocyte reaction was significantly more resistant to inhibition of the CD28 co-stimulatory pathway by the inhibitor CTLA-4-Ig. These results show that expression of the 9.6 and 9-1 binding sites together on a bispecific scFvIg increased the mitogenic properties of the mAb and altered the degree of accessory cell signals required for T cell activation.     

The development of anti-CD79 monoclonal antibodies for treatment of B-cell neoplastic disease

The B-cell antigen receptor (BCR) consists of cell surface IgM associated with the CD79 alpha/beta heterodimer. In this paper we describe a panel of monoclonal antibodies (mAbs) recognising the extracellular regions of human CD79 alpha and beta. FACS analysis demonstrated that the mAbs bind to a range of Burkitt's lymphoma lines, a mouse B-cell line (JO-72) transfected with human CD79 alpha and beta, and tumour biopsies from NHL patients. The specificity of the mAbs was confirmed by immunoprecipitation. The Ka for the binding of IgG from the anti-CD79 alpha mAbs to cell surface CD79 alpha on Ramos cells was 3 x 10(8) M-1, and their maximum level of binding, 1.7-2 x 10(5) molecules/cell, matched that obtained with anti-Fc mu and anti-Fd mu mAbs. All four anti-CD79 beta mAbs were of lower affinity. Interestingly, in growth arrest studies, we found that while all anti-Fc mu mAbs caused profound inhibition of proliferation of Ramos cells, a range of other anti-BCR mAbs, which included the anti-CD79, anti-Fab mu, anti-gamma and anti-idiotype reagents, all performed poorly giving a maximum of 25% inhibition. These differences in performance are believed to relate to the ability of anti-BCR mAbs to cross-link neighbouring surface BCR and suggest that, unlike anti-Fc mu which favours cross-linking, most of these mAbs are binding in a monogamous, non-cross-linking, union with the BCR.     

Interaction between CD44 and hyaluronic acid regulates human prostate cancer development

PURPOSE: This study was designed to investigate the significance of the interaction between CD44 and hyaluronic acid in the development of human prostate cancer. MATERIALS AND METHODS: We transfected the standard CD44 isoform (CD44s) cDNA into PC3, a human prostate cancer cell line that barely expresses CD44s protein. The effects of the reintroduction of CD44s into PC3 cells on the ability to bind hyaluronic acid (HA) were analyzed by the cell adhesion assay and by the cell migration assay. The in vitro growth rate of CD44s transfected PC3 was measured by using the MTT assay. We then evaluated the in vivo tumor development of CD44s transfected PC3 cells by subcutaneous, intravenous, and intraperitoneal injection models in athymic nude mice. RESULTS: The introduction of CD44s in PC3 cells markedly enhanced the binding and migration of these cells to HA, but not to other extracellular matrix molecules. In vitro growth of CD44s-transfected PC3 was found to be significantly decreased. In addition, the CD44s-transfected PC3 cells also demonstrated reduced tumorigenicity and metastatic potential in in vivo experimental models. CONCLUSIONS: These findings suggest that the CD44s downregulation plays an important role in the development of human prostate cancer, in part through reduction of the ability to bind HA.     

N-terminal EFRH sequence of Alzheimer's beta-amyloid peptide represents the epitope of its anti-aggregating antibodies

Monoclonal antibodies 6C6 and 10D5 raised against the N-terminal of beta-amyloid peptide interfere with the formation of beta-amyloid and trigger reversal to its non-toxic components. The epitopes of these antibodies were localized employing a library composed of filamentous phage displaying random combinatorial hexapeptides. Among 44 positive phage-clones, selected from the library by both antibodies, 40 clones carried the consensus sequence EFRH. These EFRH phage-clones bind specifically mAbs 6C6 or 10D5 with an apparent binding constant of approximately 10(-9) M. The peptide EFRH inhibits binding of mAbs 6C6 or 10D5 to beta-amyloid peptide in affinities identical to those obtained. with the peptides corresponding to positions 1-9, 1-16 and 1-40 of beta-peptide. These findings confirm that the peptide EFRH which is located at positions 3-6 within beta-amyloid peptide represents the sequential epitope of mAbs 6C6 and 10D5.     

Evolution of mammalian apolipoprotein A-I and conservation of antigenicity: correlation with primary and secondary structure

We have evaluated the immunoreactivity of 20 monoclonal antibodies (mAbs) directed against human apolipoprotein (apo)A-I with a panel of high density lipoproteins (HDL) from 13 mammalian species. The pattern of cross-reactivity showed that 20 mAbs had different specificity. While not all mAbs recognized apoA-I from all of the different species, the antigenicity of some sequences was well conserved. Thus, mAb A05 cross-reacted with all species except guinea pig and rat. In contrast, the mAb 4H1, which recognized residues 2-8, required a specific proline in position 3, as no immunoreactivity was found in the species missing this amino acid. Furthermore, the presence of a threonine residue in place of serine (in position 6) in the cynomolgus monkey was associated with a 20-fold loss of immunoreactivity in radioimmunometric assay with 4H1. As most of the epitopes were found in CNBr fragments 2 and 3, we sequenced these regions in four species (horse, goat, sheep, and cat) and analyzed the alignment of most known sequences to evaluate their consensus. Except for the rat and the chicken, considerable identity was observed. This permitted us to deduce the involvement of the residues in some antigenic epitopes. In the middle of apoA-I, a conservative mutation Asp103-->Glu was found sufficient to eliminate all reactivity of this epitope for A11 (residues 99-105 ... 12l6-132) in five species (rabbit, cow, goat, sheep, and rat). The residues essential to the expression of two other epitopes overlapping with A11 were also characterized. Edmundson-wheel representation of 18-residue repeated sequences of the different apoA-I species (for the eight amphipatic helices of residues 46-63, 68-85, 101-118, 123-140, 143-160, 167-184, 189-206, and 222-239) showed that secondary structure of apoA-I was more conserved than the antigenic epitopes. The N-terminal region, residues 1 to about 98, is rich in both strictly preserved sequences and epitope expression in most of the species surveyed. This evolutionary conservation of the N-terminal domain suggests an important yet unknown function.