Human IgG1 Isotype Research Articles

The Proinflammatory Cytokine Interleukin-32 is expressed in Keratinocytes and Dendritic Cells Obtained from Patients with Chronic Plaque Psoriasis (CPPs) (92.15)

Abstract CPPs is characterized by hyperproliferation of keratinocytes (KC) and T-cell (Tc)/DCs activation. IL-32, which induces TNF and activates NFkB and p38 MAPK, is involved in early inflammation. We investigated the role of IL-32 in the pathogenesis of CPPs by IHC, ECL and qRT-PCR analysis. Biopsy tissues were obtained from CPPs patients with active disease (N=20) and healthy human controls (N=15). A mouse Mab against human IL-32 and mouse IgG1 isotype antibody were used. IHC staining of IL-32 was performed on deparaffinized sections of formalin-fixed tissues by an indirect imunoperoxidase method (EnVision, Dako). IL-32 was expressed in basal KC with cytoplasmic granular staining. IL-32 was also expressed in DCs with strong nuclear and cytoplasmic staining in dermis. IL-32 was weakly expressed in the nucleus of KC in healthy human skin. Induction of IL-32 was detected in human KC lysates after stimulation with TNF and IL-1 or cell contact with activated Tc, but not in KC supernatants, indicating IL-32 is not secreted from KC in vitro. ECL/qRT-PCR results showed IL-32 and other CPPs-relevant cytokines (IL-18, IL-20, 22 and 23) were significantly (P<0.05) increased. Our data support the hypothesis that IL-32 is a novel proinflammatory cytokine which is involved in the pathogenesis of CPPs, especially in epidermal regions of the skin where innate immune mechanisms are central to host defense.

TRIM21 is a trimeric protein that binds IgG Fc via the B30.2 domain

TRIMs comprise a large protein family that include anti-retroviral restriction factors such as TRIM5alpha. Auto-antibodies to TRIM21 (Ro52) are a common serological feature of patients with Sjogren's syndrome and systemic lupus erythematosus (SLE). We show that, in addition to this autoantibody response, TRIM21 binds specifically to the Fc region of human IgG isotypes 1, 2 and 4, via a conformation dependent interaction. The minimal binding epitope was identified as the C-terminal B30.2 domain. The interaction was independent of N-linked glycosylation of the IgG CH2 domain. TRIM21 formed a trimer that competed with protein A for binding to IgG Fc. We conclude that TRIM21 binds to the consensus CH2/CH3 domain interface in the Fc region, overlapping the binding site of several other proteins, including Staphylococcus aureus protein A and Streptococcus spp. protein G. The data suggest that the normal function of TRIM21 involves regulation of IgG functions and that TRIM/B30.2 molecules may have broader and unsuspected roles in innate immunity, beyond that of retroviral restriction.

Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies

Most of the existing therapeutic antibodies that have been licensed and developed as medical agents are of the human IgG1 isotype, the molecular weight of which is ∼ 150 kDa. Human IgG1 is a glycoprotein bearing two N-linked biantennary complex-type oligosaccharides bound to the antibody constant region (Fc), in which the majority of the oligosaccharides are core fucosylated, and it exercises the effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity through the interaction of the Fc with either leukocyte receptors (FcγRs) or complement. Recently, therapeutic antibodies have been shown to improve overall survival as well as time to disease progression in a variety of human malignancies, such as breast, colon and haematological cancers, and genetic analysis of FcγR polymorphisms of cancer patients has demonstrated that ADCC is a major antineoplasm mechanism responsible for clinical efficacy. However, the ADCC of existing licensed therapeutic antibodies has been found to be strongly inhibited by serum due to nonnpecific IgG competing for binding of the therapeutics to FcγRIIIa on natural killer cells, which leads to the requirement of a significant amount of drug and very high costs associated with such therapies. Moreover, enhanced ADCC of non-fucosylated forms of therapeutic antibodies through improved FcγRIIIa binding is shown to be inhibited by the fucosylated counterparts. In fact, non-fucosylated therapeutic antibodies, not including the fucosylated forms, exhibit the strongest and most saturable in vitro and ex vivo ADCC among such antibody variants with improved FcγRIIIa binding as those bearing naturally occurring oligosaccharide heterogeneities and artificial amino acid mutations, even in the presence of plasma IgG. Robust stable production of completely non-fucosylated therapeutic antibodies in a fixed quality has been achieved by the generation of a unique host cell line, in which the endogenous α-1,6-fucosyltransferase (FUT8) gene is knocked out. Thus, the application of non-fucosylated antibodies is expected to be a promising approach as next-generation therapeutic antibodies with improved efficacy, even when administrated at low doses in humans in vivo. Clinical trials using non-fucosylated antibody therapeutics are underway at present.

A Humanized Anti-CD70 Monoclonal Antibody Targets CD70-Expressing Multiple Myeloma.

The tumor necrosis factor family member CD70 (CD27L) is an integral membrane protein transiently expressed on antigen-activated T and B lymphocytes. CD70 regulates lymphocyte survival and differentiation of CD27 positive resting T cells and antigen-primed B cells. Aberrant CD70 expression has been detected on hematologic tumors including diffuse large cell, follicular, and mantle cell lymphomas, chronic lymphocytic leukemia and Reed-Sternberg cells in Hodgkin's disease. Here we report for the first time the expression of CD70 on multiple myeloma (MM). Nine of thirteen MM cell lines examined expressed detectable surface CD70 with receptor copy numbers ranging from 9,000 to 145,000. Furthermore, seven of twelve primary MM isolates and six of six Waldenstrom's macroglobulinemia (WM) isolates tested were positive for CD70. The presence of CD70 on MM and WM, combined with the antigen's limited expression on normal non-hematopoietic tissues makes CD70 an attractive target for antibody-directed therapy of plasma cell disorders. We have engineered chimeric and humanized variants (human IgG1 isotype) of the murine anti-CD70 monoclonal antibody, 1F6. Both chimeric and humanized 1F6 variants retain the binding specificity of the murine parent monoclonal antibody (mAb) and acquired Fc-dependent effector functions including antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytoxicity (CDC) and macrophage-mediated antibody-dependent cellular phagocytic (ADCP) activities against CD70+ tumor cells. Specific lysis (ADCC, CDC) or tumor cell uptake (ADCP) was observed when engineered anti-CD70 mAb were used at ≥10 ng/mL against a panel of CD70-expressing hematologic tumors. The significance of these in vitro observations was further established by the ability of anti-CD70 mAb to significantly improve the survival of mice bearing disseminated CD70-expressing tumors. The in vitro function and in vivo efficacy of anti-CD70 were dependent upon Fc-FcγR interactions, as activity and survival benefit was lost with anti-FcγR blockade or when the targeting antibody was engineered to eliminate FcγR binding. Our data suggest that humanized anti-CD70 mAb possesses potent antitumor activity and has potential utility for therapy of hematologic cancers including MM and WM.

Antiangiogenic chimeric anti-endoglin (CD105) antibody: pharmacokinetics and immunogenicity in nonhuman primates and effects of doxorubicin.

We generated a human/mouse chimeric antibody c-SN6j of human IgG1 isotype from a murine anti-human endoglin (EDG) monoclonal antibody (mAb) SN6j that suppressed angiogenesis, tumor growth and metastasis in mice. We determined pharmacokinetics (PKs) and immunogenicity of c-SN6j in monkeys after multiple i.v. injections. A dose-escalation study was performed by administration of c-SN6j into six monkeys at the dose of 1 mg, 3 mg and 10 mg per kg body weight. In addition, both c-SN6j (3 mg/kg) and doxorubicin (0.275 mg/kg) were injected into two monkeys. c-SN6j and doxorubicin were injected twice a week for 3 weeks. We developed a unique and sensitive ELISA by sequentially targeting the common and idiotypic epitopes of c-SN6j-Fv to quantify plasma c-SN6j. Application of the ELISA showed that increasing the c-SN6j dose resulted in a proportional increase in the circulating c-SN6j after the first injection. In addition, the estimated area under the curve (AUC) for the first injection of c-SN6j is proportional to dose. We carried out detailed analyses of PKs of c-SN6j during and after the repeated injections. Our model of PKs fitted the empirical data well. Addition of doxorubicin modulated the PK parameters. We developed two ELISAs to separately determine the immune responses to the murine part and the human part of c-SN6j in monkeys. Interestingly, the murine part induced a weaker immune response than the human part. Doxorubicin potentiated the immune responses. Increasing the dose of c-SN6j increased plasma levels of c-SN6j but did not increase the immune responses to c-SN6j.

Epithelial tight junction proteins as potential antibody targets for pancarcinoma therapy.

Recombinant monoclonal antibodies are beginning to revolutionize cancer therapy. In combination with standard chemotherapy, high response rates have been reported with antibodies of the human IgG1 isotype for treatment of non-Hodgkin's lymphoma and breast cancer. It is becoming apparent that targets for antibody-based therapies do not necessarily need to be absent from normal tissues but can be present there either in low copy numbers or with binding epitopes shielded from the therapeutic antibody. Here, we studied whether claudin proteins that form tight junctions in normal epithelia are still expressed on carcinoma cells and whether their extracellular domains can be recognized by antibodies. We show that mRNAs of claudins 1, 3, 4, and 7 are all expressed in different human carcinoma cell lines, while claudin 8 was selectively expressed in breast and pancreas cancer lines. Chicken polyclonal antibodies were raised against peptides contained within predicted extracellular domains of claudins 1, 3, and 4. Affinity-purified IgG fractions for claudins 3 and 4 were monospecific and bound to human breast and colon carcinoma lines, but not to a line of monocytic origin. Claudin 3 antibodies also homogeneously stained human renal cell carcinoma tissue and micrometastatic tumor cells as identified by cytokeratin staining in bone marrow biopsies of breast cancer patients. Fluorescence-activated cell sorting and immunocytochemistry indicated that claudin antibodies bound to the surface of tumor cells. By analogy to other tumor-associated antigens that are differentially accessible to antibodies on tumor vs normal tissue, we propose that certain claudin proteins have potential as targets for novel antibody-based therapies of carcinomas.

Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries

IgE antibodies distinctively recognising allergenic epitopes would be ideal reagents in immunodiagnostics to detect and quantify allergens, as well as for the development of allergy diagnostics and therapeutics. We have isolated recombinant human IgE antibodies specific for the major latex allergen, hevein, from antibody phage display libraries using a green fluorescent protein (GFP)–hevein fusion as a selection antigen. Human IgE phage display libraries were constructed by combining the IgE heavy chain genes to kappa and lambda light-chain genes which were isolated from lymphocytes of a latex allergic patient. The screening of antibody libraries resulted in the enrichment of two hevein-binding scFvs designated as 1A4 and 1C2. Both antibodies showed specific binding to the hevein that could be inhibited by both the recombinant GFP–hevein and native hevein isolated from latex examination gloves. The scFvs were prone to aggregate and, thus, for further characterisation, they were converted to Fab fragments with human IgG1 or IgE isotype. Similar hevein-binding properties of the 1A4 and 1C2 Fab fragments and human IgE serum pool, conventionally used in the detection of latex allergens, demonstrate the potential utility of these recombinant antibodies for the analysis of latex allergen.

Influence of the isotype of the light chain on the properties of IgG.

It is widely appreciated that the isotype of the H chain of the Ab molecule influences its functional properties. We have now investigated the contribution of the isotype of the L chain to the structural and functional properties of the Ab molecule. In these studies, the L chain variable region of a murine anti-dansyl Ab was joined to either human kappa or lambda constant region domains and expressed with mouse-human chimeric H chains of the four human IgG isotypes. The resulting Abs were secreted as fully assembled molecules although, as has been previously observed, IgG4 with either kappa or lambda L chains was also secreted as HL half-molecules. However, the isotype of the L chain can influence the kinetics of intracellular assembly with IgG1lambda, IgG2lambda, and IgG4lambda assembling more slowly than their kappa counterparts. The isotype of the L chain also influenced the susceptibility of the interchain disulfide bonds to attack by reducing agents with variable effects, depending on the isotype of the H chains. For IgG2, but not for IgG1, -3, and -4, the isotype of the L chain influenced the rate of clearance in mice, with IgG2lambda having a shorter in vivo half-life than IgG2kappa. Only slight differences were also observed between lambda and kappa molecules in their kinetics of binding to and dissociation from the hapten dansyl. These studies demonstrate that the isotype of the L chain has only a slight impact on the structural and functional properties of variable region identical Abs.

Ligand binding specificities and signal transduction pathways of Fc gamma receptor IIc isoforms: the CD32 isoforms expressed by human NK cells.

We recently reported that human NK cells express, in addition to CD16 [Fcgamma receptor (FcgammaR) IIIA], a second type of FcgammaR, namely CD32 (FcgammaRII). Molecular characterization of CD32 transcripts expressed by highly purified NK cells revealed that they predominantly express products of the FcgammaRIIC gene. Using stable Jurkat transfectants we have analyzed the functional properties of two FcgammaRIIc-specific isoforms isolated from NK cells, namely FcgammaRIIc1 and FcgammaRIIc3, which differ in their cytoplasmic tails. The ligand binding specificity for both murine and human IgG isotypes was found to be similar to that observed for FcgammaRIIb isoforms. Immunoprecipitation studies of FcgammaRIIc isoforms expressed in Jurkat cells revealed a protein of around 40 kDa for FcgammaRIIc1, and a protein of around 32 kDa for FcgammaRIIc3. Signal transduction studies performed on FcgammaRIIc1-expressing Jurkat cells indicated that this molecule is functional, i. e. capable of Ca2+ mobilization and activation of Lck, Zap-70 and Syk protein tyrosine kinases, although the CD3 zeta chain was not found to functionally associate with FcgammaRIIc1. In contrast, FcgammaRIIc3 transfectants showed an impaired ability of this molecule to mobilize Ca2+, but activation of Lck was detected following activation via FcgammaRIIc3. These studies demonstrate the functional activity of FcgammaRIIc isoforms and suggest that the presence of CD32, in addition to CD16, on NK cells may have functional relevance.

A colorimetric–enzymatic microassay for the quantitation of antibody-dependent complement activation

In this report we describe a reliable, sensitive, safe, and easy way to assess antibody-dependent complement-mediated hemolysis. The assay is based on the quantitation of hemoglobin (Hb) released from lysed erythrocytes indirectly, through the generation of fluorene blue, a compound formed from 2-7 diaminofluorene in an enzymatic reaction catalyzed by the Hb molecule. The fact that Hb is the most abundant protein within a mature RBC (∼10 11 molecules per cell) and possesses pseudoperoxidase activity, makes the fluorene blue-coupled assay more sensitive than the simple estimation of Hb adsorption at 410 nm (Soret adsorption maxima of Hb), and as sensitive and reliable as its radioactive equivalent based on the release of 51 Cr from previously loaded RBCs. Using this assay chimeric mouse–human anti-dansyl antibodies, comprising all the human IgG isotypes, were tested for their ability to mediate complement activation. The results obtained agreed with previously reported data, confirming that the fluorene blue-coupled assay is reliable. This assay also has significant advantages over the radioactive-based assay in that the reagents used are inexpensive, and the concerns of using radioactivity and the associated hazards are obviated. Because there is no need for loading the target cells with the analyte to be detected since RBCs are already loaded with Hb, the fluorene blue-coupled assay is simpler and eliminates many steps in comparison to the 51 Cr release based assay.

Amino acid differences in the n-terminus of C H2 influence the relative abilities of IgG2 AND IgG3 to activate complement

The four human IgG isotypes are highly conserved in amino acid sequence, but show differential ability to activate complement (C′): IgG3 and IgG1 are very active, IgG2 is active under certain conditions, and IgG4 is inactive. Although the second constant domain (C H2) is critical for C′ activation, the individual amino acids that confer isotype-specific activity have not been identified. We have generated a series of mutants between IgG2 and IgG3, resulting in the exchange of the four N-terminal and six C-terminal polymorphic residues within C H2. Mutants containing the N-terminus of the C H2 of IgG3 were as effective as wildtype IgG3 in C1q binding, C1 activation and terminal complex (MAC) formation, but had reduced ability to effect C′-mediated lysis. IgG2 and mutants containing the N-terminal portion of the C H2 of IgG2 were reduced compared to IgG3 in activating C1, binding C1q and inducing assembly of the MAC, and were inactive in mediating lysis of target cells. Thus, the amino acid sequence differences in the N-terminus of C H2 play a critical role in determining the relative abilities of IgG2 and IgG3 to bind C1q and activate the C′ cascade although additional residues of C H2 must be involved in mediating optimal target cells lysis. The sequence of the N-terminus of C H2 was less critical in determining C4 and C3 binding. Characterization of domain exchange mutants suggests that intermediate steps may be partly dependent on domains other than C H2. IgGs that do not direct target cell lysis nevertheless activate intermediate steps in the pathway, which may contribute to immune complex-associated disorders.

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.

Production and properties of monoclonal antibodies against human IgG isotypes.

Several monoclonal antibodies (MAbs) against human IgG isotypes were obtained by the fusion of myeloma cells with splenocytes from mice immunized with IgG fractions extracted from human plasma. Four MAbs (F7H7, D4F8, B12A8, and E7E10) were selected by an ELISA technique on the basis of their ability to detect one of the four IgG subclasses. Their specificity was checked using a panel of pure myeloma proteins representative of the main allotypes present on IgG isotypes. In addition, two other MAbs (F3E12 and E6D6) were found able to detect specifically kappa or lambda light chains. The immunochemical properties of these MAbs were analyzed mainly in respect to their capacity to detect and to purify the different human IgG isotypes. The following data were obtained: (1) The ability of the MAbs F7H7, D4F8, B12A8, and E7E10 to measure the concentration of each IgG subclass in serum was estimated by an immunocapture ELISA. Results obtained with the new antibodies were compared with several other MAbs recommended by the IUIS/WHO human Immunoglobulins subcommittee. Similar or better results were obtained with the new anti-IgG1, anti-IgG3, and anti-IgG4, MAbs. (2) The same MAbs were tested for their ability to purify a single IgG subclass from IgG preparations and from normal and pathological sera. Fractions containing about 80% of purified IgG1, IgG3, and IgG4 were obtained after one-step immunoaffinity purification. Consequently, these MAbs proved to be useful to detect, to measure and to purify IgG subclasses.

Anti-renal-cell carcinoma chimeric antibody G250 facilitates antibody-dependent cellular cytotoxicity with in vitro and in vivo interleukin-2-activated effectors.

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy, whereas treatment with biologics has achieved limited success. Although monoclonal antibodies able to recognize human RCC have been identified, most induce little complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC), and thus are of limited potential as therapeutic modalities in their natural conformation. We evaluated a human/ mouse chimeric derivative of the previously described G250 murine monoclonal antibody (mAb), reactive with RCC, to identify a reagent for potential immunotherapy. This chimeric antibody (ch-G250) is composed of the murine variable region from the G250 mAb, which recognizes a tumor-associated antigen expressed on 95% of primary and 86% of metastatic renal cell carcinomas. The constant region of the ch-G250 is comprised of the human IgG1 isotype domains. This chimeric antibody does not bind to normal renal tissue or other normal human tissues, with the exception of gastric mucosal cells and large bile-duct epithelium. Clinical radiolocalization studies have demonstrated the relative tumor-targeting potential of this radiolabeled antibody. This ch-G250 antibody facilitated potent ADCC against several RCC lines when using in vitro and in vivo interleukin-2 (IL-2)-activated peripheral blood mononuclear cells obtained from healthy control donors and patients with cancer, respectively. This lymphocyte-mediated ADCC was specific for RCC cells recognized by the ch-G250 antibody. Using flow cytometry, we found that the level of ADCC was directly related to the degree of binding of ch-G250 to the renal cell target. These in vitro data suggest that this antibody may improve efficacy of IL-2 therapy by targeting cytokine-activated effector cells directly to the tumor and facilitating in vivo ADCC. Clinical studies combining this chimeric antibody with IL-2 treatment will be needed to test the antitumor effects of this ADCC effect in vivo.

A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody

Human immunoglobulin G4 (IgG4) exists in two molecular forms due to the heterogeneity of the inter-heavy chain disulphide bridges in the binge region in a proportion of secreted human IgG4. This heterogeneity is only revealed under denaturing, non-reducing conditions in which an HL “half antibody” is detected, a phenomenon not seen in other human IgG isotypes. In native conditions noncovalent interactions hold the antibody together as the H 2L 2 tetramer. Analysis of the hinge sequences of human IgG heavy chains suggested that the presence of serine at residue 241 might be the cause of this heterogeneity. We therefore changed the serine at 241 to pruline (found at that position in IgG1 and IgG2) in a mouse/human chimeric heavy chain. This single residue substitution leads to the production of a homogeneous antibody. Further, the variant IgG4 has significantly extended serum half-life and shows an improved tissue distribution compared to the original chimeric IgG4.

Interaction of a human Fc gamma RIIb1 (CD32) isoform with murine and human IgG subclasses.

A group of Fc receptor molecules, classified CD32, recognize the Fc moiety of IgG with low affinity. We report the isolation and identification of different hFc gamma RIIb cDNA clones, amongst which are cDNA clones encoding hFc gamma RIIb1 and hFc gamma RIIb2. Two hFc gamma RIIb1 encoding cDNA clones (pIP9 and pIP14) were isolated, which differed by three nucleotides, probably because of allelic variation. The nucleotide differences result in one amino acid change between the allelic hFc gamma RIIb1 variants. This substitution is located at amino acid position 11 of the cytoplasmic tail; a tyrosine in hFc gamma RIIb1 (clone pIP9) was replaced by an aspartic acid in clone pIP14 (encoding hFc gamma RIIb1*). A complication in studying ligand specificity of Fc receptors is the potential co-expression of different classes, subclasses, or polymorphic forms of FcR on the same cell. We therefore used murine fibroblasts transfected with cDNA clone pIP14, encoding a hFc gamma RIIb1* isoform, as our model system. These fibroblasts were found to interact with erythrocytes sensitized with mIgG2a and mIgG2b in rosetting assays performed at 4 and 37 degrees C. Interestingly, hFc gamma RIIb1* transfectants bound mIgG1 sensitized erythrocytes only weakly at 4 degrees C, whereas profound binding was observed at 37 degrees C. The ligand specificity for human (h) IgG isotypes was found to be hlgG3 > or = hlgG1 > hlgG4 > hlgG2, as determined at 4 degrees C with hlgG dimeric complexes. However, when assayed at 37 degrees C, the binding of hlgG2 dimers increased significantly. Next, we evaluated whether these transfectants were capable of supporting anti-CD3 induced T cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Human monoclonal IgG isotypes differ in complement activating function at the level of C4 as well as C1q.

Humanized antibodies are likely to have a major role in therapy and it is important to define their interaction with physiological effectors. By comparing a matched series of chimeric human mAbs we found that igG1 was most efficient in complement lysis, although IgG3 bound more C1q. To resolve this paradox we compared the ability of human IgG1, IgG2, IgG3, IgG4, and IgE and rat IgG2b to cause C1q binding, C1 binding and activation, C4 activation, C4b binding, and C3b binding. Rat IgG2b was included because this isotype has already successfully been used for therapy. Human IgG1 was less efficient than IgG3 and fixing C1q and C1 on the cell surface, but the number of C4 molecules bound per C1 was 10-fold greater for IgG1 than for IgG3. This difference, amplified through later stages of the complement cascade, can account for the superiority of IgG1 for cell lysis. The efficiency of IgG1 in fixing C4 was not due to a favored binding site on the antibody molecule, since virtually all of the bound C4b was attached to the cells. Rather, it appeared that the activation of C4 by C1s was greatly favored by IgG1 compared with IgG3. It should be possible to combine the optimal properties of IgG1 and IgG3 antibodies to produce an improved therapeutic reagent.

A new assay for IgG rheumatoid factor activity and its use to analyse rheumatoid factor reactivity with human IgG isotypes.

A new assay for IgGRF activity is described which employs human IgGFc as the antigen and a radiolabelled monoclonal antibody directed against human IgG (CHI domain) as the developing antibody. Using this assay IgGRF activity against human IgG isotypes was measured and most sera from RA patients were shown to react predominantly with IgG1 and IgG2 but few reacted with IgG3 and IgG4. The same sera were tested for IgMRF to the IgG isotypes. IgG2 was the best antigen, IgG1 and IgG4 were less so and reactivity with IgG3 was the lowest. IgGRF without associated IgMRF was obtained, its specificity compared to that of IgMRF, and found to be broadly similar. With the new assay high levels of serum IgGRF were found in those RA patients with extraarticular disease but not in RA patients with synovitis alone.