Development of monoclonal antibodies that specifically interact with necrotic lymphoma cells

The immune system has evolved mechanisms to sense not only microbes, but also necrotic cells. The pattern-recognition receptors in macrophages/dendritic cells that stimulate the acquired immune system are closely associated with danger signaling. In this study monoclonal antibodies (mAbs) that specifically interact with necrotic cells were developed. One IgG1 and two IgM mAbs were established, and they recognized a 80 kDa protein expressed in necrotic, but not live or apoptotic, cells. These mAbs, which serve as a probe for necrosis, facilitate analyses of the role of the immune complex that consists of necrotic cells and Ab and contributes to the formation of the inflammatory milieu induced by necrotic cell death.     

T cell-dependent activation of B cell proliferation and differentiation by immobilized monoclonal antibodies to CD3

The capacity of mAb directed at the CD3 molecular complex (64.1) to induce T cell-dependent B cell proliferation and differentiation was examined. Coculture of B cells with mitomycin C-treated T4 cells (T4 mito) stimulated by immobilized 64.1 resulted in marked B cell proliferation and Ig-secreting cells (ISC) generation in the absence of any additional stimulation. The magnitude of the B cell responses induced by immobilized 64.1-stimulated T4 mito was far greater than that induced by other stimuli, such as Staphylococcus aureus plus factors produced by mitogen-activated T cells, PWM, or soluble 64.1. The induction of maximal B cell responsiveness required direct contact between activated T cells and responding B cells. Of note, immobilized 64.1 also induced B cell proliferation and ISC generation in the presence of mitomycin C-treated T8 cells. By contrast, immobilized 64.1 stimulated T4 or T8 cells that had not been treated with mitomycin C induced very modest ISC generation and suppressed B cell responses supported by T4 mito even in the presence of exogenous IL-2 or factors produced by mitogen-activated T cells. The interactions between T and B cells in these cultures not only induced B cell responses, but also enhanced the production of IL-2 by activated T cells. Increased IL-2 production was facilitated when culture conditions afforded the opportunity for contact between B cells and activated T cells. These results indicate that the establishment of interactions between B cells and anti-CD3-stimulated T4 or T8 cells provides all of the signals necessary for proliferation and differentiation of B cells without other stimuli and also augments the production of lymphokines by the activated T cells. The data emphasize the role of Ag-nonspecific interactions between B cells and T cells in promoting polyclonal responses of both cell types.     

Two alcohol binding residues interact across a domain interface of the L1 neural cell adhesion molecule and regulate cell adhesion

Ethanol may cause fetal alcohol spectrum disorders (FASD) in part by inhibiting cell adhesion mediated by the L1 neural cell adhesion molecule. Azialcohols photolabel Glu-33 and Tyr-418, two residues that are predicted by homology modeling to lie within 2.8 Å of each other at the interface between the Ig1 and Ig4 domains of L1 (Arevalo, E., Shanmugasundararaj, S., Wilkemeyer, M. F., Dou, X., Chen, S., Charness, M. E., and Miller, K. W. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 371–375). Using transient transfection of NIH/3T3 cells with wild type (WT-L1) and mutated L1, we found that cysteine substitution of both residues (E33C/Y418C-L1) significantly increased L1 adhesion above levels observed for WT-L1 or the single cysteine substitutions E33C-L1 or Y418C-L1. The reducing agent β-mercaptoethanol (βME) reversibly decreased the adhesion of E33C/Y418C-L1, but had no effect on WT-L1, E33C-L1, or Y418C-L1. Thus, disulfide bond formation occurs between Cys-33 and Cys-418, confirming both the close proximity of these residues and the importance of Ig1-Ig4 interactions in L1 adhesion. Maximal ethanol inhibition of cell adhesion was significantly lower in cells expressing E33C/Y418C-L1 than in those expressing WT-L1, E33C-L1, or Y418C-L1. Moreover, the effects of βME and ethanol on E33C/Y418C-L1 adhesion were non-additive. The cutoff for alcohol inhibition of WT-L1 adhesion was between 1-butanol and 1-pentanol. Increasing the size of the alcohol binding pocket by mutating Glu-33 to Ala-33, increased the alcohol cutoff from 1-butanol to 1-decanol. These findings support the hypothesis that alcohol binding within a pocket bordered by Glu-33 and Tyr-418 inhibits L1 adhesion by disrupting the Ig1-Ig4 interaction.     

The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa

We have identified monoclonal antibodies that inhibit human cell adhesion to collagen (P1H5), fibronectin (P1F8 or P1D6), and collagen and fibronectin (P1B5) that react with a family of structurally similar glycoproteins referred to as extracellular matrix receptors (ECMRs) II, VI, and I, respectively. Each member of this family contains a unique alpha subunit, recognized by the antibodies, and a common beta subunit, each of approximately 140 kD. We show here that ECMR VI is identical to the fibronectin receptor (FNR), very late antigen (VLA) 5, and platelet glycoproteins Ic-IIa and shall be referred to as FNR. Monoclonal antibodies to FNR inhibit lymphocyte, fibroblast, and platelet adhesion to fibronectin-coated surfaces. ECMRs I, II, and FNR were differentially expressed in platelets, resting or activated lymphocytes, and myeloid, epithelial, endothelial, and fibroblast cell populations, suggesting a functional role for the receptors in vascular emigration and selective tissue localization. Tissue staining of human fetal skin localized ECMRs I and II to the basal epidermis primarily, while monoclonal antibodies to the FNR stained both the dermis and epidermis. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to complex extracellular matrix (ECM) produced by cells in culture revealed that complete inhibition of cell adhesion to ECM required antibodies to both the FNR and ECMR II, the collagen adhesion receptor. These results show that multiple ECMRs function in combination to mediate cell adhesion to complex EMC templates and predicts that variation in ECM composition and ECMR expression may direct cell localization to specific tissue domains.     

Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment

Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-alpha-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.     

Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor

Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors.     

Enhancing effect of anti-HLA class I monoclonal antibodies on T cell proliferation induced via CD2 molecule

The mAb 131 to a determinant preferentially expressed on the gene products of the HLA-A locus, the mAb Q6/64 and 4E to determinants preferentially expressed on the gene products of the HLA-B locus, the anti-HLA-A2,A28 mAb CR11-351, HO-2, HO-3, HO-4, and KS1, and the anti-HLA-B7 cross-reacting group mAb KS4 enhanced proliferation of T cells in most, if not all, the PBMC preparations stimulated with the anti-CD2 mAb 9-1 + 9.6. The mAb CR10-215, W6/32, and 6/31 to distinct monomorphic determinants of HLA class I antigens enhanced CD2-induced T cell proliferation in at most 30% of the PBMC preparations tested. The anti human beta 2-microglobulin (beta 2-mu) mAb NAMB-1 displayed no detectable effect on the proliferation of T cells stimulated with the mAb 9-1 + 9.6. The enhancing effect of anti-HLA class I mAb is specific, is dose dependent, is not abrogated by the addition of exogenous IL-1 and IL-2 to the cultures, and reflects the interaction of anti-HLA class I mAb with T cells. Enhancement of CD2 mediated proliferation of T cells is not a unique property of anti-HLA class I mAb, since the anti-HLA class II mAb Q5/6 and Q5/13 also had a similar effect. Analysis of the kinetics of the enhancing effect of anti-HLA class I mAb suggests that they modulate an early event of T cell activation and may affect the interaction of T cells with mAb 9-1. Phenotyping of T lymphocytes activated by mAb 9-1 + 9.6 in the presence of anti-HLA class I mAb suggests that the enhancing effect of anti-HLA class I mAb may reflect the recruitment of a higher percentage of T cells. The present study has shown for the first time that certain, but not all, the determinants of the HLA class I molecular complex are involved in the proliferation of T cells stimulated with the anti-CD2 mAb 9-1 + 9.6. Furthermore, the inhibitory effect of mAb CR11-351, KS1, Q6/64, and W6/32 on the proliferation of T cells stimulated with mAb OKT3 or with mAb BMA 031 indicates that the same determinants of HLA class I antigens play a differential regulatory role in T cell proliferation induced via the CD2 and CD3 pathway.     

Accessory cell independent proliferation of human T4 cells stimulated by immobilized monoclonal antibodies to CD3

The capacity of the monoclonal antibodies (Mab) 64.1 and OKT3 directed at CD3 molecules to induce T4 cell proliferation and interleukin 2 (IL 2) production was examined. Each was tested in soluble form or was immobilized by adhering it to the wells of plastic microtiter wells. Soluble anti-CD3 did not induce proliferation of accessory cell (AC)-depleted T4 cells. In contrast, immobilized anti-CD3 induced T4 cell IL 2 production and proliferation in the complete absence of AC. When T4 cells were stimulated with high density immobilized anti-CD3, responses did not require AC, IL 2, or Mab directed at the Tp44 molecule (9.3). In contrast, responses stimulated by lower densities of immobilized anti-CD3 were enhanced by IL 2, AC, and 9.3, and with even lower densities of immobilized anti-CD3 proliferation, required these additional signals. A variety of other immobilized Mab directed at T cell surface proteins including class I major histocompatibility complex encoded gene products, CD2, CD5, 4F2, and Tp44, did not induce proliferation even in the presence of IL 2. Anti-CD4 Mab (66.1) inhibited immobilized anti-CD3-stimulated T4 cell responses, with a greater degree of inhibition noted when lower densities of immobilized anti-CD3 were used to stimulate T4 cells. The data demonstrate that stimulation of T4 cells by anti-CD3 is completely AC independent when the antibody is immobilized onto a surface. Furthermore, the results indicate that maximal stimulation requires multiple interactions with anti-CD3 without internalization of the CD3 molecule. The observation that additional signals are required to support T4 cell proliferation when the density of immobilized anti-CD3 is diminished suggests that these are necessary only when insufficient interactions with the CD3 molecule have occurred to transmit a maximal activation signal to the cell. Finally, the results indicate that anti-CD4 provides a direct inhibitory signal to the T4 cell, the effect of which is inversely proportional to the intensity of the activation signal.     

Characterization of CD45 and CD45R monoclonal antibodies using transfected mouse cell lines that express individual human leukocyte common antigens

Five cell lines that express one of five isoforms of the human leukocyte common Ag (CD45) were established by transfecting a murine pre-B cell line with leukocyte common Ag cDNA constructs. Using these cell lines, the specificities of CD45 and CD45R mAb were examined. Of the 43 mAb tested, 25 antibodies recognized sequences common to all five isoforms, 16 antibodies recognized isoforms that include exon A encoded sequences, one antibody recognized isoforms that include exon B encoded sequences, and one antibody recognized only the isoform that does not include either exon A, B, or C encoded sequences.     

Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.     

Development and characterization of monoclonal antibodies to a specific domain of human estrogen receptor

We have synthesized three peptides with amino acid sequences identical to those spanning amino acids 201-215, 231-245, and 247-261 of the human estrogen receptor (hER). These peptides were conjugated to keyhole limpet hemocyanin and used as immunogens to develop monoclonal antibodies (MoAbs) to hER. Antibody responses were only elicited by the peptide with amino acid sequence 247-261. Splenocytes from immunized mice were used for hybridoma production. Of the seven MoAbs that recognized the native (functional) form of the ER, four (MoAbs 16, 33, 114, and 213) recognized the ER with high affinity, as demonstrated by the increased sedimentation coefficient of the antibody-complexed ER in sucrose density gradients. Antibodies 318, 35, and 36 bound to ER with low affinity since they immunoprecipitated ER, but the ER-antibody complex appeared to dissociate on sucrose density gradients. The high-affinity MoAbs appear to be site-specific since the peptide competed effectively for binding of the receptor by the antibody. The fact that they reacted with ER from human breast cancer and calf, rat, and mouse uterine tissues suggests that this epitope of the receptor is conserved in these species. Although the DNA-binding region appears to be conserved among the various steroid receptors, these MoAbs did not recognize the native forms of progesterone, androgen, or glucocorticoid receptors. These MoAbs bound to the KCl-activated 4S ER and heat-transformed 5S ER, suggesting that the antibody-binding site is accessible in the monomeric and dimeric forms of ER. The antibodies did not recognize the untransformed 8S ER in the presence of molybdate and without KCl, suggesting that the antibody-binding site in the oligomeric form of ER is inaccessible. The fact that the antibodies did bind to the unoccupied 4S ER was demonstrated by the data obtained with sucrose density gradient analysis followed by postlabeling of ER with [3H]estradiol. The antibodies bound to ERs with high affinity (KD = 0.4 to 1.8 nM). At a fixed concentration of antibody, ERs ranging from 20 to 1,000 fmol were detectable. These MoAbs did not inhibit nuclear or DNA binding of ER in vitro. This can be attributed to the dissociation of the antibodies from ER when the latter interacts with its acceptor site. These results demonstrate the development of site-specific MoAbs to the native form of the hER using synthetic peptides as immunogens.     

Functional glycan-free adhesion domain of human cell surface receptor CD58: design, production and NMR studies.

A general strategy is presented here for producing glycan-free forms of glycoproteins without loss of function by employing apolar-to-polar mutations of surface residues in functionally irrelevant epitopes. The success of this structure-based approach was demonstrated through the expression in Escherichia coli of a soluble 11 kDa adhesion domain extracted from the heavily glycosylated 55 kDa human CD58 ectodomain. The solution structure was subsequently determined and binding to its counter-receptor CD2 studied by NMR. This mutant adhesion domain is functional as determined by several experimental methods, and the size of its binding site has been probed by chemical shift perturbations in NMR titration experiments. The new structural information supports a 'hand-shake' model of CD2-CD58 interaction involving the GFCC'C" faces of both CD2 and CD58 adhesion domains. The region responsible for binding specificity is most likely localized on the C, C' and C" strands and the C-C' and C'-C" loops on CD58.     

Triggering of co-mitogenic signals in T cell proliferation by anti-LFA-1 (CD18, CD11a), LFA-3, and CD7 monoclonal antibodies

Proliferative T cell responses were elicited in a comitogenic assay when purified mAb against CD 18, CD11a, LFA-3, and CD7 were immobilized onto solid plastic surfaces together with submitogenic doses of mAb against the CD3 complex. The proliferative response was associated to the production of IL-2 and to the expression of IL-2R. We explored the possibility that a second signal provided by either PMA or a Ca2+ ionofore could replace the anti-CD3 mAb in the comitogenic assay. Interestingly, our data clearly indicate that PMA but not the ionofore was capable of mediating the co-mitogenic effect in conjunction with solid-bound mAb (CDw18, CD11a, LFA-3, and CD7). We also demonstrate that the mAb (anti-CD4 and anti-CD2) which have been previously described as co-mitogenic in combination with anti-CD3 are capable of eliciting this activating signal in the presence of PMA. These data indicate that mAb to certain cell surface differentiation Ag that in soluble form inhibit T cell function such as LFA-1, LFA-3, and CD2 can under appropriate conditions induce co-mitogenic signals on T cells. Our results support the hypothesis that several cell surface differentiation Ag may participate in conjunction with the T3-Ti complex in the transmembrane signal transduction leading to T cell activation.     

Mapping surface structures of the human insulin receptor with monoclonal antibodies: localization of main immunogenic regions to the receptor kinase domain

A panel of 37 monoclonal antibodies to the human insulin receptor has been used to characterize the receptor's major antigenic regions and their relationship to receptor functions. Three antibodies recognized extracellular surface structures, including the insulin binding site and a region not associated with insulin binding. The remaining 34 monoclonal antibodies were directed against the cytoplasmic domain of the receptor beta subunit. Competitive binding studies demonstrated that four antigenic regions (beta 1, beta 2, beta 3, and beta 4) are found on this domain. Sixteen of the antibodies were found to be directed against beta 1, nine against beta 2, seven against beta 3, and two against beta 4. Antibodies to all four regions inhibited the receptor-associated protein kinase activity to some extent, although antibodies directed against the beta 2 region completely inhibited the kinase activity of the receptor both in the autophosphorylation reaction and in the phosphorylation of an exogenous substrate, histone. Antibodies to the beta 2 region also did not recognize autophosphorylated receptor. In addition, antibodies to this same region recognized the receptor for insulin-like growth factor I (IGF-I) as well as the insulin receptor. In contrast, antibodies to other cytoplasmic regions did not recognize the IGF-I receptor as well as the insulin receptor. These results indicate that the major immunogenic regions of the insulin receptor are located on the cytoplasmic domain of the receptor beta subunit and are associated with the tyrosine-specific kinase activity of the receptor. In addition, these results suggest that a portion of the insulin receptor is highly homologous to that of the IGF-I receptor.     

Gp35/50 mucin molecules of Trypanosoma cruzi metacyclic forms that mediate host cell invasion interact with annexin A2

Host cell invasion is a critical step for infection by Trypanosoma cruzi, the agent of Chagas disease. In natural infection, T. cruzi metacyclic trypomastigote (MT) forms establish the first interaction with host cells. The gp35/50 mucin molecules expressed in MT have been implicated in cell invasion process, but the mechanisms involved are not well understood. We performed a series of experiments to elucidate the mode of gp35/50-mediated MT internalization. Comparing two parasite strains from genetically divergent groups, G strain (TcI) and CL strain (TcVI), expressing variant forms of mucins, we demonstrated that G strain mucins participate in MT invasion. Only G strain-derived mucins bound to HeLa cells in a receptor-dependent manner and significantly inhibited G strain MT invasion. CL strain MT internalization was not affected by mucins from either strain. HeLa cell invasion by G strain MT was associated with actin recruitment and did not rely on lysosome mobilization. To examine the involvement of annexin A2, which plays a role in actin dynamic, annexin A2-depleted HeLa cells were generated. Annexin A2-deficient cell lines were significantly more resistant than wild type controls to G strain MT invasion. In a co-immunoprecipitation assay, to check whether annexin A2 might be the receptor for mucins, protein A/G magnetic beads crosslinked with monoclonal antibody to G strain mucins were incubated with detergent extracts of MT and HeLa cells. Binding of gp35/50 mucins to annexin A2 was detected. Both G strain MT and purified mucins induced focal adhesion kinase activation in HeLa cells. By confocal immunofluorescence microscopy, colocalization of invading G strain MT with clathrin was visualized. Inhibition of clathrin-coated vesicle formation reduced parasite internalization. Taken together, our data indicate that gp35/50-mediated MT invasion is accomplished through interaction with host cell annexin A2 and clathrin-dependent endocytosis.     

Evidence that monoclonal antibodies directed against the integrin beta subunit plexin/semaphorin/integrin domain stimulate function by inducing receptor extension

The overall structure of integrins is that of a ligand-binding head connected to two long legs. The legs can exhibit a pronounced bend at the "knees," and it has been proposed that the legs undergo a dramatic straightening when integrins transit from a low affinity to a high affinity state. The knee region contains domains from both alpha and beta subunits, including the N-terminal plexin/semaphorin/integrin (PSI) domain of the beta subunit. The role played by the knee domains in the regulation of integrin-ligand binding is uncertain. Here we show that: (i) monoclonal antibodies (mAbs) N29 and 8E3 have epitopes in the beta(1) subunit PSI domain and stimulate ligand binding to alpha(5)beta(1); (ii) N29 and 8E3 cause long range conformational changes that alter the ligand binding activity of the head region; (iii) the stimulatory action of these mAbs is dependent on the calf-1 domain, which forms part of the alpha subunit knee; and (iv) the epitopes of 8E3 and N29 map close to the extreme N terminus of the PSI and are likely to lie on the side of this domain that faces the alpha subunit. Taken together, our data suggest that the binding of these mAbs results in a levering apart of the PSI and calf-1 domains, and thereby causes the alpha and beta subunit knees to separate. Several major inferences can be drawn from our findings. First, the PSI domain appears to form part of an interface with the alpha subunit that normally restrains the integrin in a bent state. Second, the PSI domain is important for the transduction of conformational changes from the knee to head. Third, unbending is likely to provide a general mechanism for control of integrin-ligand recognition.     

Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease and are targets for C. difficile-associated disease therapy. Here, recombinant single-domain antibody fragments (V(H)Hs), which specifically target the cell receptor binding domains of TcdA or TcdB, were isolated from an immune llama phage display library and characterized. Four V(H)Hs (A4.2, A5.1, A20.1, and A26.8), all shown to recognize conformational epitopes, were potent neutralizers of the cytopathic effects of toxin A on fibroblast cells in an in vitro assay. The neutralizing potency was further enhanced when V(H)Hs were administered in paired or triplet combinations at the same overall V(H)H concentration, suggesting recognition of nonoverlapping TcdA epitopes. Biacore epitope mapping experiments revealed that some synergistic combinations consisted of V(H)Hs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Further binding assays revealed TcdA-specific V(H)Hs neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin. With favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these V(H)Hs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract.     

Evidence that monoclonal antibodies against CD9 antigen induce specific association between CD9 and the platelet glycoprotein IIb-IIIa complex

Monoclonal antibodies to the CD9 antigen are powerful platelet agonists. We report here the novel finding that the anti-CD9 monoclonal antibodies 50H.19 and ALB6 promote physical association between CD9 antigen and the glycoprotein IIb-IIIa complex (GPIIb-IIIa) component of the platelet fibrinogen receptor. The monoclonal antibodies do not consistently immunoprecipitate proteins other than CD9 from 125I-labeled human platelets even if the platelets are first treated with the homobifunctional cross-linking reagent dithiobis(succinimidyl propionate), indicating that CD9 antigen is not physically associated with other membrane proteins in the resting state. However, the addition of agonistic concentrations of either monoclonal antibody before cross-linking results in the coprecipitation of proteins corresponding in mobility and peptide composition to GPIIb, and GPIIIa. The association of CD9 with the GPIIb-IIIa complex is unaffected by a combination of aspirin and ADP scavengers sufficient to abrogate anti-CD9 monoclonal antibody-induced platelet aggregation, and is therefore not dependent upon thromboxane- and ADP-mediated pathways of intracellular signalling. The specificity of the association is demonstrated by the lack of other coprecipitating major proteins, by the requirement for induction by anti-CD9 monoclonal antibodies, and by the failure to promote reciprocal association with either of the anti-GPIIb-IIIa complex monoclonal antibodies P2 or HuP1-m1a.     

Endocytosis, recycling, and degradation of the insulin receptor. Studies with monoclonal antireceptor antibodies that do not activate receptor kinase

The endocytosis, recycling, and degradation of the insulin receptor were studied in IM-9 cells and U-937 cells by employing two monoclonal antibodies directed at the alpha subunit of the human insulin receptor, antibodies MA-5 and MA-10. Antibody MA-5 is an insulin agonist and MA-10 is an insulin antagonist (Forsayeth, J., Caro, J.F., Sinha, M.K., Maddux, B.A., and Goldfine, I.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3448-3451). Both monoclonal antibodies, like insulin, induced the endocytosis of the insulin receptor within 15 min. Upon removal of extracellular ligand the internalized receptor recycled to the cell surface. At this time there was no degradation of the receptor as measured by a sensitive insulin receptor radioimmunoassay. After 20 h of incubation, insulin and MA-5, but not MA-10, induced significant receptor degradation as measured by both insulin receptor radioimmunoassay and metabolic labeling studies. These studies demonstrated, therefore, that: 1) internalization and recycling of the receptor can be induced by antireceptor monoclonal antibodies that are either insulin agonists or insulin antagonists; 2) enhanced receptor degradation can be induced by monoclonal antibodies that are insulin agonists; and 3) the process of receptor internalization does not necessarily lead to enhanced receptor degradation. Since prior studies have indicated that neither MA-5 nor MA-10 enhance insulin receptor kinase activity, the present studies also suggest that insulin receptor endocytosis and degradation induced by ligands different than insulin can occur without activation of this process.