An immunoglobulin E-reactive chimeric human immunoglobulin G1 anti-idiotype inhibits basophil degranulation through cross-linking of FcepsilonRI with FcgammaRIIb.

BACKGROUND: IgE binds to mast cells and basophils via its high-affinity receptor, FcepsilonRI, and cross-linking of FcepsilonRI-bound IgE molecules by allergen leads to the release of allergic mediators characteristic of type I hypersensitivity reactions. Previous work has shown that cross-linking of FcepsilonRI with FcgammaRIIb, an ITIM-containing IgG receptor, leads to inhibition of basophil triggering. 2G10, a chimeric human IgG1 anti-idiotype, has broad reactivity with human IgE and as such has the potential to bind simultaneously to FcepsilonRI-bound IgE, via its Fab regions, and the negative regulatory receptor, FcgammaRIIb, via its Fc region. OBJECTIVE: To assess the ability of human 2G10 to inhibit anti-IgE and allergen-driven basophil degranulation through cross-linking of FcepsilonRI-bound IgE with FcgammaRIIb. METHODS: 2G10 was assessed for its ability to bind to FcgammaRIIb on transfected cells and on purified basophils. In the basophil degranulation assay, basophils were purified from peripheral blood of atopic individuals and activated with either anti-IgE or the house dust mite allergen Der p 1, in the presence or absence of human 2G10. Basophil activation was quantified by analysis of CD63 and CD203c expression on the cell surface, and IL-4 expression intracellularly, using flow cytometery. RESULTS: Human 2G10 was able to bind to FcgammaRIIb on transfected cells and on purified basophils, and induce a dose-dependent inhibition of both anti-IgE and Der p 1-driven degranulation of basophils. CONCLUSION: The inhibition of basophil degranulation by the human IgG1 anti-idiotype 2G10 highlights the therapeutic potential of IgE-reactive IgG antibodies in restoring basophil integrity through recruitment of the inhibitory receptor FcgammaRIIb.

Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors.

The Lewis Y (Ley) antigen is a blood group-related antigen that is expressed in a high proportion of epithelial cancers (including breast, colon, ovary, and lung cancer) and is an attractive target for monoclonal antibody-directed therapy. The murine monoclonal 3S193 (IgG3) was generated in BALB/c mice by immunization with Ley-expressing cells of the MCF-7 breast carcinoma cell-line. The murine 3S193 showed high specificity for Ley in ELISA tests with synthetic Ley and Ley-containing glycoproteins and glycolipids and also reacted strongly in rosetting assays and cytotoxic tests with Ley-expressing cells. We generated a humanized form of the murine 3S193 antibody by linking cDNA sequences encoding the variable region of murine 3S913 with frameworks of the human KOL heavy chain and REI K chain. The genes for the humanized 3S193 monoclonal antibody IgG1 were transfected into mouse myeloma NS0 cells and cloned for the establishment of high antibody-producing colonies. Humanized 3S193 antibody was subsequently produced through in vitro culture and under good manufacturing practice conditions using hollow-fiber bioreactors. The purified humanized 3S193 (hu3S193) was subsequently characterized and validated for use in preliminary immunotherapy investigations. hu3S193 reacted specifically with Ley antigen, with similar avidity to the murine form. hu3S193 demonstrated potent immune effector function, with higher antibody-dependent cell-mediated cytotoxicity than its murine counterpart and potent complement-dependent cytotoxicity (ED50, 1.0 microg/ml). The in vivo immunotherapeutic potential of hu3S193 was assessed in a human breast xenograft model using MCF-7, Ley-positive cells. Six i.v. doses of up to 1 mg of hu3S193 were administered to animals bearing established tumors (120-130 mm3) with no significant effect on tumor growth. In contrast, in an MCF-7 xenograft preventive model, a 1-mg hu3S193 dosage schedule was able to significantly slow tumor growth compared with placebo and isotype-matched control IgG1 antibody. hu3S193 has promise for immunotherapy of Ley-positive tumors and is currently entering Phase I clinical trials.

Sequences of heavy and light chain variable regions from four bovine immunoglobulins.

Oligodeoxyribonucleotide primers based on the 5' ends of bovine IgG1/2 and lambda constant (C) region genes, together with primers encoding conserved amino acids at the N-terminus of mature variable (V) regions from other species, have been used in cDNA and polymerase chain reactions (PCRs) to amplify heavy and light chain V region cDNA from bovine heterohybridomas. The amino acid sequences of VH and V lambda from four bovine immunoglobulins of different specificities are presented.

Cloning and expression in Escherichia coli of the cDNA encoding human cardiac troponin I.

We have cloned and sequenced the human cardiac troponin I (cTnI)-encoding cDNA with the aim of expressing the cDNA in Escherichia coli. The cDNA was successfully expressed as a fusion product with beta-galactosidase and as an unfused protein. Both polypeptides were recognised by an anti-human cTnI antibody.

The contrasting IgG-binding interactions of human and herpes simplex virus Fc receptors.

A virally encoded, high-affinity Fc receptor (FcR) is found on herpes simplex virus type 1 (HSV-1) particles and infected cells where its binding of non-immune IgG protects cells from host-mediated lysis. Whilst mutation or aglycosylation of the IgG CH2 domain reduced binding to human FcR, the interaction with HSV-1 FcR was not affected. However, the HSV-1 FcR, unlike human FcR, discriminates between human IgG1 allotypes, being sensitive to changes at positions 214 (CH1) and 356/358 (CH3), away from its proposed binding site at the CH2-CH3 interface. The biological consequences are not known but this is the first evidence of a major functional difference between IgG1 allotypes.

Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities.

Subclasses of human IgG have a range of activity levels with different effector systems but each triggers at least one mechanism of cell destruction. We are aiming to engineer non-destructive human IgG constant regions for therapeutic applications where depletion of cells bearing the target antigen is undesirable. The attributes required are a lack of killing via Fcgamma receptors (R) and complement but retention of neonatal FcR binding to maintain placental transport and the prolonged half-life of IgG. Eight variants of human IgG constant regions were made with anti-RhD and CD52 specificities. The mutations, in one or two key regions of the CH2 domain, were restricted to incorporation of motifs from other subclasses to minimize potential immunogenicity. IgG2 residues at positions 233 - 236, substituted into IgG1 and IgG4, reduced binding to FcgammaRI by 10(4)-fold and eliminated the human monocyte response to antibody-sensitized red blood cells, resulting in antibodies which blocked the functions of active antibodies. If glycine 236, which is deleted in IgG2, was restored to the IgG1 and IgG4 mutants, low levels of activity were observed. Introduction of the IgG4 residues at positions 327, 330 and 331 of IgG1 and IgG2 had no effect on FcgammaRI binding but caused a small decrease in monocyte triggering.

The herpes simplex virus type 1 Fc receptor discriminates between IgG1 allotypes.

Herpes simplex virus type 1 (HSV-1) expresses a complex of two virally encoded glycoproteins, gE and gl, which is capable of binding nonimmune human IgG. The gE-gl complex has thus become known as an Fc receptor (FcR), which reportedly binds human IgG subclasses in the order IgG4 > IgG1 > or = IgG2 and does not bind IgG3 from many individuals. There is, however, allelic variation in the genes encoding the human IgG1 heavy chain constant region and this gives rise to allotypes of IgG1. Using recombinant monoclonal IgG molecules of known isotype and mutants thereof we have unexpectedly discovered that the HSV-1 FcR discriminates between IgG1 allotypes. This is evidence of functional differences between IgG1 allotypes that may account for their distribution in populations. Furthermore, these findings suggest HSV-1 FcR binding sites on the IgG molecule some distance from the proposed binding site in the CH2-CH3 domain interface.

The production and characterisation of a chimaeric human IgE antibody, recognising the major mite allergen Der p 1, and its chimaeric human IgG1 anti-idiotype.

BACKGROUND: Two mouse monoclonal antibodies have been described, namely: mAb 2C7 (IgG2bkappa), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1kappa), which is an anti-idiotypic antibody raised against mAb 2C7. Given its broad IgE specificity, anti-idiotype mAb 2G10 could potentially have immunomodulatory applications. For example, a chimaeric human IgG version of mAb 2G10 could prove to be a useful molecule for binding to mast cell and basophil FcepsilonRI bound IgE, and in doing so co-ligating FcepsilonRI with FcgammaRIIB, which has been reported to have downregulatory effects. AIMS: To produce a chimaeric human IgE version of mAb 2C7 (mAb 2C7huE) and a chimaeric human IgG1 version of its anti-idiotype mAb 2G10 (mAb 2G10huG1). METHODS: The Vkappa and VH regions of mAb 2C7 and its anti-idiotype mAb 2G10 were engineered into human constant regions of the IgE and IgG1 isotypes, respectively. RESULTS: The production of chimaeric mAb 2C7huE and its anti-idiotype mAb 2G10huG1 confirmed that the respective mouse antibody V regions were successfully engineered into human constant regions and still retained the specificity of the original murine V regions. CONCLUSION: The newly constructed chimaeric antibodies will be useful to investigate the downregulation of IgE mediated hypersensitivity by the crosslinking of FcepsilonRI with FcgammaRIIB.

A rapid one-stage whole-blood HPA-1a phenotyping assay using a recombinant monoclonal IgG1 anti-HPA-1a.

Severe neonatal alloimmune thrombocytopenia and patients with HPA-1a-specific antibodies require transfusion of HPA-1a-negative platelets. Identifying HPA-1a-negative donors requires simple and reliable typing methods. Most existing techniques use polyclonal antibodies, are time consuming and involve platelet isolation. We have used a horseradish peroxidase (HRP)-conjugated recombinant IgG1 anti-HPA-1a (CAMTRAN007) to develop a rapid and reliable enzyme-linked immunosorbent assay (ELISA), which eliminates sample preparation and reduces the incubation and wash steps associated with traditional sandwich ELISAs. The assay uses simultaneous incubation of the monoclonal antibody RFGP56 to capture GPIIbIIIa from whole blood and the recombinant IgG1 antibody to detect captured HPA-1a antigen. It allows 96 samples to be typed in less than 1 h and can be used on stored samples. Initial testing of 85 samples of known HPA-1a genotype demonstrated that HPA-1a-negative samples had OD values of < 0.266, whereas HPA-1a-positive samples had OD values of > 0.6. Testing of 1862 random donor samples in two blood centres confirmed these OD cut-off values and identified 45 HPA-1a-negative samples (2.4%), all except one giving OD values of < 0.2. The remaining HPA-1a-negative sample had an OD value of 0.303. The HPA-1a status on all the negative samples and an equivalent number of randomly selected positive samples was confirmed by flow cytometry and polymerase chain reaction with sequence-specific primers (PCR- SSP).

Specificity analysis of blood group Lewis-y (Le(y)) antibodies generatedagainst synthetic and natural Le(y) determinants.

Le(y)-reactive monoclonal antibodies (mAbs) were generated in mice by immunization with synthetic Le(y) neoglycoproteins or with Le(y)-expressing cells. Serological analysis indicated that mAbs raised against synthetic Le(y) (i) reacted strongly with synthetic Le(y) but poorly with natural Le(y), (ii) cross-reacted with Le(x) or H-type 2 structures, and (iii) were IgG1, IgG2a, or IgG2b. mAbs raised against Le(y)-expressing cells (i) reacted with both synthetic Le(y) and natural Le(y), (ii) were of two types: cross-reactive with Le(x) or H-type 2 structures or specific for Le(y), and (iii) were IgM or IgG3. One of the mAbs raised against natural Le(y), mAb 3S193 (IgG3), showed high specificity for Le(y) in ELISA tests with synthetic Le(y) and Le(y) containing glycoproteins and glycolipids; it also reacted strongly in rosetting assays and cytotoxic tests with Le(y)-expressing cells. mAb 3S193 did not lyse O, A, AB, and B human erythrocytes in the presence of human complement. In flow cytometry, there was weak reactivity with granulocytes, a reactivity also observed with two previously described highly specific Le(y) mouse mAbs--BR55-2 (IgG3) and B3 (IgG1). A humanized version of mAb 3S193 has been constructed, and the specificity pattern and reactivity for Le(y) remain very similar to mouse mAb 3S193.

Rapid phenotyping of HPA-1a using either diabody-based hemagglutination or recombinant IgG1-based assays.

BACKGROUND: The HPA-1 system is carried on the beta3 integrin. HPA-1a (Zw(a), Pl(A1)) is immunogenic in an HPA-1b homozygote (HPA-1b1b). In pregnancy, 1 of 365 women forms anti-HPA-1a, which causes severe thrombocytopenia in 1 in 1100 neonates. Identification of women at risk of forming anti-HPA-1a and the screening of donors to obtain HPA-1a-negative platelets for therapy need reliable, low-cost, automated assays. STUDY DESIGN AND METHODS: A diabody with dual specificity for HPA-1a x D and an IgG1 anti-HPA-1a have been constructed by the use of the genes encoding the first anti-HPA-1a fragment. With these reagents, two complementary HPA-1a phenotyping assays have been developed. RESULTS: This diabody was used in a simple hemagglutination technique to perform HPA-1a phenotyping on soluble glycoprotein IIb/IIIa from EDTA plasma samples. Over 1000 unselected donors have been correctly HPA-1a-phenotyped by use of the diabody. The human recombinant IgG1 anti-HPA-1a was produced in a rat myeloma cell line and was fluorescein labeled for use in a whole-blood flow cytometric HPA-1a phenotyping assay. This IgG1 anti-HPA-1a shows a clear differential between HPA-1a-positive and HPA-1a-negative platelets at nM antibody concentrations. CONCLUSIONS: The two recombinant reagents described are highly suitable for screening and confirmatory HPA-1a phenotyping. They permit rapid determination of the HPA-1a phenotype and are amenable to automation.