Fine epitope mapping of the human SS-B/La protein. Identification of a distinct autoepitope homologous to a viral gag polyprotein.

To analyze the autoepitopes on the SS-B/La protein, a cDNA covering the entire region coding the protein was isolated from a human cDNA library. The cDNA was subcloned into an expression plasmid vector, pEX, to express its protein product as a fusion protein with cro-beta-galactosidase in Escherichia coli. A recombinant pEX plasmid expressing three-fourths of the protein (amino acid 112-408) was also constructed. The antigenicities of these recombinant proteins were confirmed with a patient's serum. Their various deletion mutants were produced with exonuclease III treatment from the 3' ends of the cDNAs without changing the proper translational frame. Immunoblot analysis and enzyme-linked immunosorbent assay were used to evaluate the reactivities of the recombinant proteins with patients' sera to determine the autoepitopes. A narrow segment (amino acid 88-101) and the region where several epitopes were located (amino acid 283-338) on the SS-B/La protein were universally recognized by all the sera with anti-SS-B/La antibodies examined. An additional epitope region (amino acid 179-220) was recognized by some patients' sera. Computer analysis revealed that the most distinct autoepitope, amino acid 88-101, had a striking homology to a retroviral gag polyprotein. These findings indicate that exogenous or endogenous retroviruses may play a role in initiation of the anti-SS-B/La autoimmunity.     

Definition of a discontinuous immunodominant epitope at the NH2 terminus of the La/SS-B ribonucleoprotein autoantigen.

High-titer IgG autoantibodies to the La/SS-B ribonucleoprotein (RNP) are a hallmark of patients with primary Sjogren's syndrome. Anti-La/SS-B-positive human sera bind to multiple epitopes on recombinant La/SS-B, although the initial response is against an immunodominant epitope within the first 107 NH2-terminal amino acids (aa). Sequence analysis has identified a striking homology between aa 88-101 in this NH2-terminal region of La/SS-B and a feline retroviral gag polypeptide suggesting the anti-La/SS-B response may be initiated by cross-reactivity with an exogenous agent. In the present study, detailed mapping of this NH2-terminal epitope, using recombinant La/SS-B purified from the expression of overlapping DNA fragments spanning aa 1-107, has shown that this immunodominant epitope is a complex conformational or discontinuous epitope dependent upon both aa 12-28 and 82-99 for expression, even though these regions share no homology with each other. This requirement questions the significance of the homology between La/SS-B and a retroviral gag polypeptide in the generation of the B cell response to La/SS-B and is in accord with the general concept that B cells recognize conformational epitopes on antigens rather than small linear peptide sequences. The finding also reinforces the notion that native autoantigen could be the initiator of the autoimmune response.     

Targeting von Willebrand factor and platelet glycoprotein Ib receptor

Atherothrombotic events, such as acute coronary syndrome or stroke, are the result of platelet activation. Von Willebrand factor (vWF), a multimeric glycoprotein, plays a key role in aggregation of platelets, especially under high-shear conditions. Acting as bridging element or ligand between damaged endothelial sites and the glycoprotein Ib (GPIb) receptor on platelets, vWF is responsible for platelet adhesion and aggregation. This vWF activation and further platelet aggregation mainly occurs under high shear stress present in small arterioles or during deficiency of the vWF-cleaving protease ADAMTS13. There are several substances targeting vWF itself or its binding receptor GPIb on platelets. Two antibodies are directed against vWF: AJW200, an IgG4 humanized monoclonal antibody, and 82D6A3, a monoclonal antibody of the collagen-binding A-3 domain of vWF. ALX-0081 and ALX-0681 are bivalent humanized nanobodies targeting the GPIb binding site of vWF. Aptamers are oligonucleotides with drug-like properties that share some of the attributes of monoclonal antibodies. ARC1779 is a second-generation, nuclease-resistant aptamer, binding to the activated vWF A1 domain and ARC15105 is a chemically advanced follower with an assumed higher affinity to vWF. Antibodies targeting GPIbα are h6B4-Fab, a murine monoclonal antibody; GPG-290, a recombinant, chimeric protein containing the amino-terminal 290 amino acids of GPIbα linked to human IgG1 Fc; and the monoclonal antibody SZ2. There are a number of promising preclinical results and development of some agents (AJW 200, ARC1779 and ALX-0081) has already reached Phase II trials.     

Targeting von Willebrand factor and platelet glycoprotein Ib receptor

Atherothrombotic events, such as acute coronary syndrome or stroke, are the result of platelet activation. Von Willebrand factor (vWF), a multimeric glycoprotein, plays a key role in aggregation of platelets, especially under high-shear conditions. Acting as bridging element or ligand between damaged endothelial sites and the glycoprotein Ib (GPIb) receptor on platelets, vWF is responsible for platelet adhesion and aggregation. This vWF activation and further platelet aggregation mainly occurs under high shear stress present in small arterioles or during deficiency of the vWF-cleaving protease ADAMTS13. There are several substances targeting vWF itself or its binding receptor GPIb on platelets. Two antibodies are directed against vWF: AJW200, an IgG4 humanized monoclonal antibody, and 82D6A3, a monoclonal antibody of the collagen-binding A-3 domain of vWF. ALX-0081 and ALX-0681 are bivalent humanized nanobodies targeting the GPIb binding site of vWF. Aptamers are oligonucleotides with drug-like properties that share some of the attributes of monoclonal antibodies. ARC1779 is a second-generation, nuclease-resistant aptamer, binding to the activated vWF A1 domain and ARC15105 is a chemically advanced follower with an assumed higher affinity to vWF. Antibodies targeting GPIbα are h6B4-Fab, a murine monoclonal antibody; GPG-290, a recombinant, chimeric protein containing the amino-terminal 290 amino acids of GPIbα linked to human IgG1 Fc; and the monoclonal antibody SZ2. There are a number of promising preclinical results and development of some agents (AJW 200, ARC1779 and ALX-0081) has already reached Phase II trials.     

Complete congenital heart block is associated with increased autoantibody titers against calreticulin

Complete congenital heart block (CCHB) is associated with anti-Ro/SS-A and anti-La/SS-B antibodies. Calreticulin, a calcium-binding, multifunctional protein of the endoplasmic reticulum with C-terminal KDEL-sequence, is not part of the Ro/SS-A ribonucleoprotein complex. In this study anti-calreticulin autoantibody responses in serum samples from 18 infants with CCHB, their mothers and in a control group of 11 anti-Ro/SS-A or anti-La/SS-B positive infants without heart block and their mothers were analysed. Specific enzyme-linked immunosorbent assays were performed. Nine out of 18 sera with CCHB contained IgG anti-calreticulin antibodies. Four sera of those with IgG antibodies also had IgM antibodies. One serum contained anti-calreticulin IgM antibodies only. In the non-CCHB group two sera were positive for IgG and one serum was positive for IgM anti-calreticulin antibodies. Sera of healthy infants were negative both for anti-IgG and anti-IgM calreticulin antibodies. Calreticulin is involved in calcium storage and therefore anti-calreticulin antibodies might influence the development of CCHB. The new finding of IgM autoantibodies and the observed differences in antibody response in infants and mothers support the hypothesis of a fetally mediated and passively acquired autoimmune disease.     

Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity

IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.     

Demonstration of antibody for glutamic pyruvic transaminase (GPT) in chronic hepatic disorders.

The present paper describes the detection of an autoantibody for glutamic pyruvic transaminase (GPT) in sera of patients with chronic hepatic disorders. In 16 out of 500 patients, the existence of an antibody for pig GPT was demonstrated by the double antibody method, gel filtration and radioimmunoelectrophoresis. The antibody was demonstrated as an immunoglobulin G (IgG) with either polyclonal or monoclonal type (kappa or lambda). The binding portion of IgG with GPT was determined as the fragment Fab, but not Fc of IgG. Because the binding of 125I-pig GPT with the patient's antibody was displaced by human GPT, this antibody may have the characteristic of cross reacting with both pig and human GPT. Although the mechanism of production of the antibody for GPT and the pathological significance of the antibody in chronic hepatic disorders remained obscure, possible inhibition of GPT activity in serum is suggested in the presence of this antibody.     

抗卵巢上皮癌单克隆抗体的制备和鉴定

以人的浆液性卵巢上皮癌细胞系ＨＯ－８９１０为抗原，制备分泌抗卵巢癌单克隆抗体的杂交瘤，经间接免疫荧光组化鉴定证实。制备的单克隆抗体ＯＣ１Ｃ３与ＨＯ－８９１０呈强阳性反应，与其他肿细胞系则无反应或反应极弱。在冰冻切片中，单克隆抗体ＯＣ１Ｃ３与卵巢恶性肿瘤呈强阳性反应（７／８），与绒癌有部分交叉反应（１／２），与正常卵巢和胚胎组织无反应。结果表明ＯＣ１Ｃ３是卵巢恶性肿瘤的一种特异性抗原的单克隆抗体。为     

Identification of a human-specific epitope in a conserved region of the La/SS-B autoantigen.

Human anti-La/SS-B autoantibodies are known to react with highly conserved epitopes suggested to be functional or active sites on the La/SS-B polypeptide. This study was designed to determine whether the autoantibodies also react with poorly conserved regions of La/SS-B as predicted by an antigen-driven autoimmune response. Binding of human autoantibodies to purified human, mouse, and bovine recombinant fragments representing immunodominant regions of the La/SS-B polypeptide was compared using Western blotting and ELISA. A cross-reactive epitope was located in the highly conserved NH2-terminal region of La/SS-B. Significantly, human-specific epitopes were identified in both the conserved RNA-recognition motif and a poorly conserved COOH-terminal fragment, providing direct evidence for an autoantigen-driven response. The lack of autoantibody cross-reactivity with a conserved domain of mouse and bovine La/SS-B implies that a small number of residues in human autoepitopes may be critical for autoimmunogenicity.     

Studies on a monoclonal antibody to human factor viii coagulant activity, with a description of a facile two-site factor VIII coagulant antigen assay

IgG was purified from the ascites tumor fluid obtained from mice injected with a monoclonal cell line secreting antibody that inhibited VIII:C. With a modified Bethesda assay method (18 hr, 4 degrees C), the titer of the purified IgG was 14,000 U/mg. In a fluid-phase IRMA for VIII:CAg utilizing the Fab fragment prepared from the monoclonal IgG, two high-titer human anti-VIII:C inhibitors (IgG fractions) showed no demonstrable competition for the monoclonal VIII:CAg binding site. Conversely, neither human antibody (125I-Fab fragment) was displaced from its VIII:CAg binding site by the monoclonal IgG molecule. When the monoclonal antibody was used in a fluid-phase IRMA, slightly decreased VIII:CAg levels were found in serum. A facile one-step, two-site IRMA using Sepharose-bound human anti-VIII:C and labeled monoclonal IgG was designed. With this assay, in contrast to the finding with the fluid-phase IRMA, both the rate and apparent level of binding of VIII:CAg "sandwiched" between the two antibodies were increased approximately twofold in serum compared to the native plasma. A similar increase in rate and apparent level of binding was also found after thrombin treatment of VIII:C/vWf relative to the untreated control preparation.     

Suppression of NZB/NZW murine nephritis by administration of a syngeneic monoclonal antibody to DNA. Possible role of anti-idiotypic antibodies.

Suppression of circulating antibodies to double-stranded DNA was achieved in NZB/NZW f1 female mice by repeated administration of an IgG2a monoclonal antibody to DNA. Deaths from nephritis were delayed; glomerular deposition of IgG and of the cationic IgG DNA antibodies characteristic of murine lupus nephritis were diminished. Quantities of circulating antibodies to single-stranded DNA were not reduced compared with untreated or IgG myeloma-treated control mice. Antibodies directed against the monoclonal anti-DNA appeared in the circulation of treated mice after three inoculations of the idiotype. Those antibodies did not react with another monoclonal anti-DNA of the same allotype. One monoclonal anti-idiotypic antibody was obtained in hybridoma cultures derived from a spleen of a treated mouse. Cross-reactive or common idiotypes were found in 30-50% of NZB/NZW f1 sera and monoclonal DNA antibodies. Deletions of portions of the spectrotype of antibodies to DNA were found in sera containing anti-idiotypic antibodies, suggesting suppression of clones producing antibodies with isoelectric points similar to that of the immunizing idiotype. Deletions of some of the anti-idiotypic antibodies also occurred as the mice aged. Rheumatoid factors were not detectable in any sera. Therefore, administration of an antibody to DNA bearing an idiotype occurring with high frequency in NZB/NZW f1 females resulted in relatively specific suppression of the antibody response to double-stranded DNA, as well as suppression of nephritis. Reduction of anti-DNA synthesis by anti-idiotypic antibodies may have been an important suppressive mechanism. Experiments are in progress to test this hypothesis.     

Mechanism of first-dose cytokine-release syndrome by CAMPATH 1-H: involvement of CD16 (FcgammaRIII) and CD11a/CD18 (LFA-1) on NK cells.

The administration of the immunosuppressive humanized monoclonal antibody CAMPATH 1-H, which recognizes CD52 on lymphocytes and monocytes, is associated with a first-dose cytokine-release syndrome involving TNFalpha, IFNgamma, and IL-6 clinically. In vitro models have been used to establish the cellular source and mechanism responsible for cytokine release, demonstrating that cytokine release is isotype dependent, with the rat IgG2b and human IgG1 isotype inducing the highest levels of cytokine release, which was inhibited with antibody to CD16, the low affinity Fc-receptor for IgG (FcgammaR). Cross-linking antibody opsonized CD4 T lymphocytes failed to stimulate TNFalpha release, which together with the observation that TNFalpha release by purified natural killer (NK) cells stimulated by fixed autologous CAMPATH 1-H-opsonized targets was inhibited with anti-CD16, indicates that cytokine release results from ligation of CD16 on the NK cells, rather than Fc-receptor (FcR)-dependent cross-linking of CD52 on the targeted cell. Since the hierarchy of isotypes inducing cytokine release in these cultures matches that seen clinically, we conclude that ligation of CD16 on NK cells is also responsible for cytokine release after injection of CAMPATH 1-H in vivo.     

Mouse-human immunoglobulin G1 chimeric antibodies with activities against Cryptococcus neoformans.

Passive antibody administration is a potentially useful approach for the therapy of human Cryptococcus neoformans infections. To evaluate the efficacy of the human immunoglobulin G1 (IgG1) constant region against C. neoformans and to construct murine antibody derivatives with reduced immunogenicities and longer half-lives in humans, two mouse-human IgG1 chimeric antibodies were generated from the protective murine monoclonal antibodies 2D10 (IgM) and 18B7 (IgG1). The 2D10 mouse-human IgG1 chimeric antibody (ch2D10) had significantly lower binding affinity than its parent murine antibody (m2D10), presumably because of a loss of avidity contribution on switching from IgM to IgG. The 18B7 mouse-human IgG1 chimeric antibody (ch18B7) had higher affinity for cryptococcal polysaccharide antigen than its parent murine antibody (m18B7). ch18B7 and ch2D10 promoted phagocytosis of C. neoformans by primary human microglial cells and the murine J774.16 macrophage-like cell line. ch18B7 and m18B7 enhanced fungistatic or fungicidal activity of J774.16 cells and prolonged the survival of lethally infected mice. We conclude that the human IgG1 constant chain can be effective in mediating antifungal activity against C. neoformans. ch18B7 or similar antibodies are potential candidates for passive antibody therapy of human cryptococcosis.     

Genetic delivery of the murine equivalent of bevacizumab (avastin), an anti-vascular endothelial growth factor monoclonal antibody, to suppress growth of human tumors in immunodeficient mice

Vascular endothelial growth factor (VEGF) produced by tumor cells plays a central role in stimulating angiogenesis required for solid tumor growth. VEGF-specific antibodies inhibit tumor cell line growth in animal models and a humanized monoclonal anti-VEGF antibody (bevacizumab [Avastin]) is approved as a treatment for metastatic cancer. We hypothesized that administration of an adenoviral (Ad) vector expressing the murine monoclonal antibody equivalent of bevacizumab would suppress human tumor growth in vivo. The Ad vector (AdalphaVEGF) encodes the light chain and heavy chain cDNAs of monoclonal antibody A.4.6.1, a murine antibody that specifically recognizes human VEGF with the same antigen-binding site as bevacizumab. AdalphaVEGF efficacy in vivo was evaluated with A-673 rhabdomyosarcoma and DU 145 prostate carcinoma cells in human tumor cell xenografts in SCID mice. For both tumor models, AdalphaVEGF directed the expression of high anti-human VEGF IgG antibody titers in vivo, the numbers of mitotic nuclei and blood vessels in the tumor were significantly decreased (p < 0.05), tumor growth was suppressed (p < 0.05), and there was increased survival (p < 0.005). Thus, AdalphaVEGF, encoding a murine monoclonal antibody that is the equivalent of bevacizumab, effectively suppresses the growth of human tumors, suggesting gene therapy as an alternative to bevacizumab monoclonal antibody therapy.     

The potency of immunoglobulin G fragments for inhibition of interference caused by anti-immunoglobulin antibodies in a monoclonal immunoradiometric assay for thyrotropin

Heterophile antibodies in patients' serum may produce false increases in apparent analyte concentrations in "sandwich"-type immunoassays. Using three patients with endogenous anti-mouse IgG antibodies and a two-site mouse monoclonal assay for thyrotropin, we studied the ability of IgG fragments to block this positive interference. Mouse whole IgG and IgG Fc fragment blocked the interference virtually completely; IgG F(ab')2 and Fab fragments did not. Rat and horse immunoglobulin fragments gave variable results. We suggest that sandwich assays formulated with IgG Fab or F(ab')2 fragments may be less susceptible to positive interference by heterophile antibodies. Unlike sera from the three patients, simulated human specimens containing heterologous anti-IgG antibodies showed little or no selectivity in inhibition by IgG fragments, and therefore are not useful to study this phenomenon.     

Evaluation of the RheumaStrip ANA profile test: a rapid test strip procedure for simultaneously determining antibodies to autoantigens U1-ribonucleoprotein (U1-RNP), Sm, SS-A/Ro, SS-B/La, and to native DNA

The "LipoGen RheumaStrip ANA Profile" test method (LipoGen, Inc.) is a new assay format for autoantibody detection in which recombinant autoantigens are used. This enzyme immunoassay, in test-strip format, detects antibodies to autoantigens U1-ribonucleoprotein (U1-RNP), Sm, SS-A/Ro, SS-B/La, and to native DNA (nDNA). We evaluated 200 antinuclear antibody (ANA)-positive and 100 ANA-negative sera for the presence of antibodies to U1-RNP, Sm, SS-A/Ro, SS-B/La, and nDNA by the new test-strip procedure. These data correlated well with those obtained with either Ouchterlony double immunodiffusion for U1-RNP, Sm, SS-A/Ro, and SS-B/La or with Crithidia luciliae indirect immunofluorescence for anti-nDNA. Assay sensitivity and assay specificity of the ANA Profile method as compared with those of established procedures were respectively as follows: 89.8% and 98.8% for U1-RNP, 86.4% and 95.3% for Sm, 97.9% and 89.3% for SS-A/Ro, 98.3% and 86.3% for SS-B/La, and 97.5% and 93.1% for nDNA. Agreement between the ANA Profile test and these other test methodologies ranged from 88.7% for the SS-B/La test to 97.3% for the U1-RNP test. This new test procedure substantially decreases the time and effort required to perform these assays. Total hands-on time and overall assay time were decreased by 72% and 97%, respectively.     

Development of a sandwich ELISA to detect IgG and IgG sub-class antibodies specific for a major antigen (Ag 7) of Aspergillus fumigatus

A sandwich ELISA has been developed, using an affinity purified monospecific antiserum as a capture antibody, to detect specific IgG and IgG sub-classes to a major antigen (Ag 7) of Aspergillus fumigatus in the sera of patients with allergic bronchopulmonary aspergillosis (ABPA). Significantly elevated levels of specific IgG to Ag 7 were detected in 97% of ABPA sera tested, as compared to control sera and to sera from A. fumigatus skin-prick test positive individuals. IgG sub-class antibody levels to Ag 7 were also determined in a similar sandwich ELISA, but using specific monoclonal antisera instead of the polyclonal anti-IgG. Both Ag 7 specific IgG1 and IgG4 levels were found to be significantly raised in the ABPA sera compared to controls. It is proposed that this antigen-specific ELISA may provide a more specific diagnostic test for IgG antibody detection in sera of ABPA patients.     

Inhibition of phagocytosis of complement C3- or immunoglobulin G-coated particles and of C3bi binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol).

Events that lead to phagocytosis of complement (C3)- or IgG-coated particles after their interaction with specific cell surface receptors are poorly understood. Two mouse monoclonal antibodies (an IgM and an IgG2a) to a human granulocyte-monocyte surface membrane differentiation antigen (Mol) inhibited ingestion by granulocytes both of oil Red O particles opsonized with normal human serum or with IgG and of sheep erythrocytes sensitized with IgG. In addition, they specifically inhibited rosetting between phagocytes and sheep erythrocytes coated with C3bi, a fragment of the complement component C3, generated by cleaving C3b with C3b inactivator and beta IH protein. These monoclonal anti-Mol antibodies did not inhibit IgG Fc, C3b or C3d receptor-mediated binding of erythrocytes coated with the respective proteins. The Fab fragment of the IgG2a monoclonal antibody inhibited noncytotoxic enzyme release from granulocytes when these cells were stimulated with zymosan coated with C3bi. Electrophoretic transfer of polymorphonuclear leukocyte detergent lysates to nitrocellulose, followed by immunofixation with monoclonal antibody, showed that these antibodies were directed to a 155,000-mol wt glycoprotein. This surface membrane structure appears to be involved in Fc and C3 receptor-dependent phagocytosis and closely associated with the C3bi receptor.     

Human heterophilic antibodies display specificity for murine IgG subclasses

OBJECTIVES: The study investigated heterophilic antibodies: the human immunoglobulin classes involved and their specificity for different murine IgG subclasses. DESIGN AND METHODS: Using immunofluorometric assays for human IgA, IgM and IgG binding murine IgG1, we analyzed 173 samples displaying positive interference and 97 negative control samples from a previous study. We also set up assays for heterophilic antibody interference using Mabs from different murine IgG subclasses. Three Mabs each of murine IgG1, IgG2a and IgG2b subclasses, one murine IgG3 Mab and one rat Mab were used. RESULTS: Elevated levels of human murine IgG1-binding immunoglobulins of IgM class only were found in 40% of interference-positive samples, human IgG only in 1.7%, and human IgA only in 2.3% of the samples. Both elevated human IgG and IgM classes were found in 3.5% of the samples, IgA and IgM in 4.0%, and finally, all three immunoglobulin classes in 1.7% of the samples. Eighty percent of interference positive samples showed heterophilic assay interference for at least one murine IgG1 Mab, 35% for IgG2a, 66% for IgG2b, 52% for IgG3a and 17% for the rat Mab. CONCLUSIONS: Heterophilic antibody interference is mainly caused by IgM class human antibodies with a marked murine IgG subclass specificity. Combining assay antibodies from different murine IgG subclasses may reduce interference in immunoassays.