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Background Expressing afucosylated human IgG1 antibodies with Chinese hamster ovary (CHO) cells deficient of α-(1,6)-fucosyltransferase (FUT8) is being more and more accepted as a routine method to enhance antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies, especially for anti-cancer regimens. However, in pre-clinical studies relying on disease models other than mice and primates, e.g., those underrepresented species for infectious diseases, it is less clear whether such afucosylated antibodies can demonstrate enhanced therapeutic index. This is because the orthologues of human FcγRIIIA or mouse FcγRIV from those species have not been well characterized. Methods We set up a luciferase-based ADCC assay with Jurkat reporter cells expressing FcγRIIIA/FcγRIV from human, mouse, rat, hamster, guinea pig, ferret, rabbit, cat, dog, pig and monkey, and also produced human, mouse, hamster, rabbit and pig IgG from wild type and Fut8−/− CHO cells or hybridomas. Results We confirmed that enhanced stimulation through FcγRIIIA/FcγRIV by afucosylated IgG, as compared with wild type IgG, is a cross-species phenomenon. Conclusions Thus, efficacy and toxicology studies of the next generation afucosylated therapeutic IgG and Fc fusion proteins in these underrepresented animal models should be expected to generate translatable data for treating human diseases, leading to the expanded applications of this new class of glycoengineered biologics.

Most pathogens establish infection through mucosa, where secretory immunoglobulin A (sIgA) plays an ‘immune exclusion’ role in humoral defense. Extravasation of intravenously (i.v.) administrated therapeutic immunoglobulin G (IgG) mainly relies on convection and/or neonatal Fc receptor-mediated transcytosis from circulation into interstitial space. Active transport of interstitial IgG further across epithelium into mucosa, like sIgA, is a much desired feature for the next generation of therapeutic antibodies, especially for anti-infection purposes. For the first time, we report the engineering of an IgA mimicry of IgG, with its Fc portion in fusion with the 18-aa tail piece (tp) of sIgA and the J chain, possessing sIgA’s full binding activity towards polymeric immunoglobulin receptor that mediates mucosa transcytosis. In a diphtheria toxin receptor (DTR) knockin mouse model, i.v. injected anti-diphtheria toxin (DT) IgG(tp)J protected DTR+ cells from deletion upon DT injection. The compact design of IgG(tp)J opens new revenues for more effective therapeutic IgG mimicking some of the important biological functions of IgA.

Glyco-engineered recombinant antibodies are currently being developed as the next generation therapeutics to treat human diseases, including cancer, autoimmunity and infection. Antibodies lacking core fucosylation show great increase in affinity for FcγRIIIA, leading to an improved receptor-mediated effector function. While afucosyl human IgG1 exhibits 50-100-fold increase in antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism underlying the anti-cancer effect of some approved therapeutic antibodies, it is not clear whether such glyco-engineered antibodies would find similar use for infectious disease. Due to the species difference, human antibodies may have different binding properties towards corresponding IgG receptors from animals used for modeling infection and intoxication. During the course of studying a recombinant human IgG1 in neutralizing diphtheria toxin (DT) in Guinea pigs (Cavia porcellus), we identified a previously uncharacterized Guinea pig protein H0VDZ8 from UNIPROT database that shows high sequence homologies to human FcγRIIIA and mouse FcγRIV. This Fcγ receptor, which we named as gpFcγRIV, also demonstrates functional similarity although not to the same extent as the human and mouse counterparts, in that it binds to afucosyl human and mouse IgG much stronger than to the wild type antibodies. Thus, Guinea pigs can be used to compare the efficacies of wild type vs. afucosyl anti-DT human IgG1 in toxin removal and animal protection. Molecular and functional characterization of human FcγRIIIA and mouse FcγRIV equivalents in other species could expand the list of preclinical animal models for testing afucosyl human antibodies in treating various human diseases.

BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas(-/-) murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance.

Numerous studies demonstrate that polycyclic aromatic hydrocarbons (PAH) suppress immunity by modifying the function of both B and T cells. Relatively few studies have assessed the effects of these common environmental chemicals on immature lymphocytes. In the present study, long-term primary bone marrow cultures were employed to investigate the effects of a prototypic PAH and aryl hydrocarbon receptor (AhR) agonist, 7,12-dimethylbenz[ a ]anthracene (DMBA), on immature B lymphocytes. In this system, immature preB cells are maintained in a supportive microenvironment provided by bone marrow stromal cells. Results presented here demonstrate that (1) exposure of primary bone marrow cultures to DMBA results in preB cell death by apoptosis; (2) notably low doses of DMBA (≥10 −8 m ) induce preB cell apoptosis; (3) in long-term cultures, bone marrow stromal cells, but not preB cells, express AhR mRNA and protein as determined by in situ hybridization, RT-PCR, and immunoblotting; (4) freshly isolated unfractionated bone marrow cells, but not purified bone marrow B cells, express AhR protein as assessed by immunohistochemistry; (5) α-naphthoflavone, a competitive AhR inhibitor and cytochrome P450 antagonist, completely blocks DMBA-induced preB cell apoptosis in primary bone marrow cultures; and (6) DMBA or benzo[ a ]pyrene injection in vivo results in bone marrow cell apoptosis consistent with the death of hematopoietic cells clustered around stromal elements. The results implicate programmed cell death as a mechanism underlying DMBA-mediated immunosuppression and suggest that preB cell death is influenced by local interactions with AhR + bone marrow stromal cells.

Recent studies suggest that environmental chemicals such as polycyclic aromatic hydrocarbons (PAH) compromise the immune system in part through the induction of programmed cell death (apoptosis). Nevertheless, mechanisms through which PAH induce apoptosis remain elusive. In particular, the role of the 8S AhR remains controversial and the nature of intracellular signal transduction in PAH-induced apoptosis remains largely undefined. To extend previous studies to the T cell compartment and to develop a clonal system in which intracellular signals leading to PAH-induced apoptosis can be dissected, the ability of fluoranthene, a ubiquitous, but less well-studied PAH, to induce apoptosis in murine T cell hybridomas was evaluated. Particular emphasis was placed on the role of the 8S AhR. The data indicate that (1) three of four hybridomas studied undergo apoptosis within 8 hr of fluoranthene exposure; (2) fluoranthene induces growth arrest concurrent with apoptosis; (3) at doses sufficient to induce lymphocyte apoptosis, fluoranthene does not induce AhR nuclear translocation in cells expressing high AhR levels; (4) fluoranthene-responsive hybridomas do not express AhR mRNA or protein; (5) the Ca 2+ chelating agent EGTA partially inhibits fluoranthene-induced apoptosis. These results (1) indicate the immunosuppressive potential of fluoranthene; (2) support a role for apoptosis in PAH immunotoxicity; (3) demonstrate that fluoranthene-mediated T cell death and growth arrest are AhR independent; and (4) illustrate similarities between PAH- and antigen-specific receptor-mediated apoptosis. These findings encourage consideration of AhR-independent events in PAH risk assessment.

The focal adhesion adapter protein p130(cas) regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130(cas). AND-34/BCAR3, one of three NSP family members, binds the p130(cas) carboxyl terminus, adjacent to a bipartite p130(cas) Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130(cas). Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130(cas) complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130(cas) to bind the Src SH3 domain through an RPLPSPP motif in the p130(cas) SBD. Although our prior work identified phosphorylation of the serine within the p130(cas) RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130(cas). The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130(cas) complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130(cas) substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130(cas). Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130(cas) and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130(cas) complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130(cas) SBD.

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.

Antibody produced by a variant of the murine antidigoxin hybridoma 26-10 has reduced affinity for digoxin but enhanced recognition of the digoxin 12-hydroxyl due to a Tyr to His substitution at heavy chain position 50 (Schildbach, J. F., Panka, D. J., Parks, D. R., Jager, G. C., Novotny, J., Herzenberg, L. A., Mudgett-Hunter, M., Bruccoleri, R. E., Haber, E., and Margolies, M. N. (1991) J. Biol. Chem. 266, 4640-4647). Consistent with these data, the 26-10 Fab-digoxin x-ray crystal structure (Jeffrey, P. D., Strong, R. K., Sieker, L. C., Chang, C. Y., Campbell, R. L., Petsko, G. A., Haber, E., Margolies, M. N., and Sheriff, S. (1993) Proc. Natl. Acad. Sci. U. S. A., in press) reveals that Tyr-50 contacts a region of digoxin that includes the hapten-12 carbon. To determine the effects of other heavy chain position 50 substitutions, mutant antibodies were engineered, and their affinities for digoxin and digoxin analogues were measured. The affinity of the mutant antibodies for digoxin roughly correlates with the size of the position 50 side chain. Substitutions of Trp or Phe have no effect on affinity, whereas substitutions of Asn, His, Leu, Ala, Gly, and Asp confer progressively lower affinities. Although Trp and Phe mutants exhibit wild-type specificity, Asn and Asp mutants have improved affinity for digoxin relative to digitoxin (12-deshydroxydigoxin). Leu, Ala, and Gly mutants have improved affinity for 12-acetyldigoxin relative to digoxin as compared with 26-10. These results indicate that position 50 is a determinant of both antibody affinity and fine specificity for antibody 26-10 and that single-amino acid substitutions can alter antibody fine specificity. Models of the mutants were computationally constructed, and haptens were docked into the modeled binding sites. The results suggest that 12-acetyldigoxigenin occupies different orientations in the 26-10 and in the Ala mutant binding sites, resulting in altered binding.

Antibody LB4, produced by a spontaneous variant of the murine anti-digoxin monoclonal antibody 26-10, has an affinity for digoxin two orders of magnitude lower than that of the parent antibody due to replacement of serine with phenylalanine at position 52 of the heavy chain variable region (Schildbach, J.F., Panka, D.J., Parks, D.R., et al., 1991, J. Biol. Chem. 266, 4640-4647). To examine the basis for the decreased affinity, a panel of engineered antibodies with substitutions at position 52 was created, and their affinities for digoxin were measured. The antibody affinities decreased concomitantly with increasing size of the substituted side chains, although the shape of the side chains also influenced affinity. The crystal structure of the 26-10 Fab complexed with digoxin (P.D.J., R.K. Strong, L.C. Sieker, C. Chang, R.L. Campbell, G.A. Petsko, E.H., M.N.M., & S.S., submitted for publication) shows that the serine at heavy chain position 52 is not in contact with hapten, but is adjacent to a tyrosine at heavy chain position 33 that is a contact residue. The mutant antibodies were modeled by applying a conformational search procedure to position side chains, using the 26-10 Fab crystal structure as a starting point. The results suggest that each of the substituted side chains may be accommodated within the antibody without substantial structural rearrangement, and that none of these substituted side chains are able to contact hapten. These modeling results are consistent with the substituents at position 52 having only an indirect influence upon antibody affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS PAGE while NSP-1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype.

Purpose AND-34/BCAR3 (Breast Cancer Anti-Estrogen Resistance 3) associates with the focal adhesion adaptor protein, p130CAS/BCAR1. Expression of AND-34 regulates epithelial cell growth pattern, motility, and growth factor dependence. We sought to establish the effects of the loss of AND-34 expression in a mammalian organism. Methods AND-34−/− mice were generated by homologous recombination. Histopathology, in situ hybridization, and western blotting were performed on murine tissues. Results Western analyses confirmed total loss of expression in AND-34−/− splenic lymphocytes. Mice lacking AND-34 are fertile and have normal longevity. While AND-34 is widely expressed in wild type mice, histologic analysis of multiple organs in AND-34−/− mice is unremarkable and analyses of lymphocyte development show no overt changes. A small percentage of AND-34−/− mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber. Following initial vacuolization and liquefaction of the lens cortex first observed at postnatal day three, posterior lens rupture occurs in all AND-34−/− mice, beginning as early as three weeks and seen in all mice at three months. Western blot analysis and in situ hybridization confirmed the presence of AND-34 RNA and protein in lens epithelial cells, particularly at the lens equator. Prior data link AND-34 expression to the activation of Akt signaling. While Akt Ser 473 phosphorylation was readily detectable in AND-34+/+ lens epithelial cells, it was markedly reduced in the AND-34−/− lens epithelium. Basal levels of p130Cas phosphorylation were higher in AND-34+/+ than in AND-34−/− lens epithelium. Conclusions These results demonstrate the loss of AND-34 dysregulates focal adhesion complex signaling in lens epithelial cells and suggest that AND-34-mediated signaling is required for maintenance of the structural integrity of the adult ocular lens.

Over-expression of AND-34/BCAR3/NSP2 (BCAR3) or its binding-partner p130Cas/BCAR1 generates anti-estrogen resistance in human breast cancer lines. Here, we have compared BCAR3 to two related homologs, NSP1 and NSP3/CHAT/SHEP, with regards to expression, anti-estrogen resistance, and signaling. BCAR3 is expressed at higher levels in ERalpha-negative, mesenchymal, than in ERalpha-positive, epithelial, breast cancer cell lines. Characterization of "intermediate" epithelial-like cell lines with variable ER-alpha expression reveals that BCAR3 expression correlates with both mesenchymal and ERalpha-negative phenotypes. Levels of the BCAR3/p130Cas complex correlate more strongly with the ERalpha-negative, mesenchymal phenotype than levels of either protein alone. NSP1 and NSP3 are expressed at lower levels than BCAR3 and without correlation to ERalpha/mesenchymal status. Among NSP-transfectants, only BCAR3 transfectants induce anti-estrogen resistance and augment transcription of cyclin D1 promoter constructs. Over-expression of all homologs results in activation of Rac, Cdc42 and Akt, suggesting that these signals are insufficient to induce anti-estrogen resistance. BCAR3 but not NSP1 nor NSP3 transfectants show altered morphology, transitioning from polygonal cell groups to rounded, single cells with numerous blebs. Whereas stable over-expression of BCAR3 in MCF-7 cells does not lead to classic epithelial-to-mesenchymal transition, it does result in down-regulation of cadherin-mediated adhesion and augmentation of fibronectin expression. These studies suggest that BCAR's ability to induce anti-estrogen resistance is greater than that of other NSP homologs and may result from altered interaction of breast cancer cells with each other and the extracellular matrix.

AND-34 is a murine protein that binds by a cdc25-like GDP exchange factor domain to the focal adhesion docking protein p130Cas. Overexpression of either of the human homologues of AND-34 and p130Cas, BCAR3 and BCAR1, respectively, has been reported to induce resistance to antiestrogens in breast cancer cell lines. Here we show that overexpression of AND-34 leads to activation of the Rho family GTPases Cdc42 and Rac. Consistent with these findings, BCAR3 overexpression induced alterations in F-actin distribution and augmented both autophosphorylation and kinase activity of the Cdc42/Rac-responsive serine/threonine kinase PAK1. p130Cas-associated BCAR3 protein was detected in the estrogen-independent breast cancer cell line 578-T, but not in estrogen-dependent MCF7 or ZR-75-1 cells. Stable ZR-75-1 transfectants overexpressing BCAR3, but not vector-only transfectants, grew in the presence of the pure antiestrogen ICI 182,780. Stable transfection with RacV12, a constitutively active form of Rac1, also induced antiestrogen resistance in ZR-75-1 cells. Transient transfection of BCAR3 in estrogen-dependent MCF7 cells induced activation of luciferase constructs containing the proximal 1745 or 163 bp but not 66 bp of the cyclin D1 promoter. Such cyclin D1 promoter activation was inhibited by dominant negative forms of Rac1 and PAK1. Overexpression of the PAK1 autoinhibitory domain (residues 83-149) but not an inactive PAK1 autoinhibitory domain point mutant (L107F) also blocked BCAR3-mediated cyclin D1 activation. These studies suggest that AND-34/BCAR3 induces antiestrogen resistance in breast cancer cell lines by a Rac1- and PAK1-dependent pathway.

AND-34, a novel GDP exchange factor, is expressed constitutively at significant levels in murine splenic B cells, but not in murine splenic T cells or thymocytes. In B cell lines, anti-IgM treatment up-regulates AND-34 transcript levels. B cell AND-34 associates with both the docking molecules p130Cas and HEF1. AND-34 binds by its GDP exchange factor domain to the C terminus of HEF1, a region of HEF1 previously implicated in apoptotic, adhesion, and cell cycle-regulated signaling. Overexpression of AND-34 in murine B cell lines activates the Rho family GTPase Cdc42, but not Rac, Rho, RalA, or Rap1. Consistent with this, a subpopulation of AND-34 overexpressing B cells have long filamentous actin-containing cellular extensions. AND-34 overexpression augments both autophosphorylation and kinase activity of the Cdc42/Rac-responsive serine/threonine kinase PAK1. As previously reported for lymphoid cells transfected with constitutively active Cdc42, AND-34 overexpression inhibits SDF-1α-induced B cell polarization. These studies suggest that p130Cas and HEF1-associated AND-34 may regulate B cell adhesion and motility through a Cdc42-mediated signaling pathway.

A family of murine anti-p-azophenylarsonate (Ars) antibodies share a variable (V) region serologically defined marker, the 36-60 idiotype (Id36-60). Most mouse strains possess five genes highly homologous to the gene encoding the heavy (H) chain V region of antibodies bearing Id36-60 (VH36-60); however, only one of these genes is ever utilized by hybridomas whose antibodies bind Ars and bear Id36-60. The relevant VH genes were cloned from A/J and BALB/c mouse DNA libraries. Their DNA sequences were found to differ at only two positions. Southern blot analysis, protein sequence determination, and nucleic acid sequence determination indicate that the above hybridomas utilize the same joining (JH3), diversity (D), and VH gene segments regardless of BALB/c or A/J strain origin. Despite this virtual identity, BALB/c and A/J mouse strains express quite different serum levels of Id36-60-bearing antibodies when immunized with Ars. The basis of this regulatory process is discussed.