Chromosome 2 reciprocal congenic strains to evaluate the effect of the genetic background on blood pressure.

The localisation of quantitative trait loci (QTL) is the first step towards gene identification. This is then verified by the construction of reciprocal congenic strains. The hypertensive SHRSP and normotensive WKY strains were used in a speed congenic approach to confirm the existence of a QTL on rat chromosome 2. Systolic baseline and salt loaded blood pressures were measured by radiotelemetry. Transfer of the chromosome 2 blood pressure QTL region from WKY into an SHRSP background significantly reduced blood pressure, with the increased significance at the salt loaded period, compared to the SHRSP. The reciprocal congenic blood pressure showed a significantly increased baseline systolic pressure compared to the WKY, with no change in significance at the salt loaded period. Thus we have successfully captured a gene(s) which contribute to blood pressure regulation in both congenic strains. This will facilitate further positional cloning of the causative genes first in this model and then in human essential hypertension.

Reciprocal consomic strains to evaluate y chromosome effects.

We have previously demonstrated that the SHRSP Y chromosome contains a locus that contributes to hypertension in SHRSP/WKY F2 hybrids and that SHRSP exhibit an increased vulnerability to focal cerebral ischemia after permanent middle cerebral artery occlusion (MCAO). This increased vulnerability is inherited as a codominant trait, and a putative role for the Y chromosome has been suggested in F1 hybrids. The objective of this study was to investigate further the role of Y chromosome in blood pressure (BP) regulation and in the vulnerability to cerebral ischemia. We have constructed consomic strains by selectively replacing the Y chromosome from WKY rats with that of SHRSP, and vice versa, by using a marker-assisted breeding strategy. Permanent MCAO was carried out by electrocoagulation, with infarct volume expressed as a percentage of the ipsilateral hemisphere. Systolic blood pressure was measured by radiotelemetry during a baseline period of 5 weeks followed by a 3-week period of salt loading. We observed that the transfer of the Y chromosome from WKY onto SHRSP background significantly reduced systolic BP in consomic strains, SP.WKYGlaY(w) (n=6) versus SHRSP (n=6) (209.2+/-10.4 mm Hg versus 241.7+/-7.7 mm Hg, F=5.88, P=0.038) during the salt-loading period. In the reciprocal consomic strain, WKY.SPGlaY(s) (n=5), systolic BP was increased compared with WKY parental strain (n=6) (147.6+/-2.4 mm Hg versus 132.6+/-5.1 mm Hg, F=6.11, P=0.035) during baseline. Infarct volumes in consomic strains were not significantly different from their respective parental strain: WKY.SPGlaY(s) (n=7) versus WKY (n=7), 22.8+/-3.7% versus 22.2+/-8.0%, 95% CI=-12.7, 4.2, P=0.3; SP.WKYGlaY(w) (n=7) versus SHRSP (n=6), 37.7+/-4.4% versus 33.6+/-7.6%, 95% CI=-20.3, 12.1, P=0.5. We conclude that the SHRSP Y chromosome harbors a locus contributing to systolic BP, whereas no contribution to vulnerability to cerebral ischemia can be detected.

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.

Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities.

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.

Enhanced antitumour effect of liposomal daunorubicin using antibody-phospholipase C conjugates or fusion protein.

We have developed a new two-step method for targeting cytotoxic drugs to tumour cells. The method firstly involves the binding to tumour cells of antibody-phospholipase C immunoconjugates or fusion proteins. Further to washing or clearance of the immunoconjugates, liposomes are introduced which are specifically lysed at the tumour site by PLC to release their cytotoxic contents in the vicinity of the tumour cells. For two alternative human cell lines, a synergistic inhibition of cell proliferation was seen for combined treatment with a specific immunoconjugate and daunorubicin encapsulated liposomes. For tumour xenografts in mice, the combined treatment resulted in an inhibition of tumour growth although with no eradication of tumours at the doses used. The two-step antibody-PLC/liposome approach offers broad possibilities for the precise delivery of payloads of cytotoxic drugs to tumour sites.

A humanized antibody against human cytomegalovirus (CMV) gpUL75 (gH) for prophylaxis or treatment of CMV infections.

A humanized monoclonal antibody that binds to the 86-kDa glycoprotein, gpUL75 (gH), of human cytomegalovirus (CMV) has been developed. The six complementarity determining regions of the heavy and light chains of the mouse antibody HCMV16 were transferred to human antibody framework sequences and combined with human antibody constant regions to produce a complete antibody. The reshaped antibody recognized cells infected with a variety of virus strains and neutralized clinical isolates of CMV as efficiently as laboratory strains in a conventional plaque reduction assay. This antibody provides a potential agent for the prevention or treatment of CMV infections in humans.

Identification of framework residues required to restore antigen binding during reshaping of a monoclonal antibody against the glycoprotein gB of human cytomegalovirus.

Introduction of the complementary determining regions (CDRs) from a murine antibody to a human monoclonal antibody is an important technique (humanization) in the development of human immunotherapeutics. We have humanized murine monoclonal antibody HCMV37 which binds to the gB envelope glycoprotein of human cytomegalovirus. Simple transfer of the murine HCMV37 CDRs into heavy- and light-chain framework regions (FRs) based on human NEW and REI, respectively, together with human IgGI and K constant regions, abolished antigen binding because of a suboptimal heavy chain. Replacement of human VH amino acids Leu70, Val71 and Arg94 with murine residues Thr70, Arg71 and Asn94 was insufficient to improve affinity. However, significant restoration of binding was obtained by substitution of human VH amino acids Thr28, Phe29, Ser30 with murine residues Ser28, Ile29, Thr30, in conjunction with the position 94 change. Residue 71, often regarded as critical for antigen binding, was not a major factor.

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.

Efficient generation of a reshaped human mAb specific for the alpha toxin of Clostridium perfringens.

We have used the technique of antibody reshaping to produce a humanized antibody specific for the alpha toxin of Clostridium perfringens. The starting antibody was from a mouse hybridoma from which variable (V) region nucleotide sequences were determined. The complementarity-determining regions (CDRs) from these V regions were then inserted into human heavy and light chain V region genes with human constant region gene fragments subsequently added. The insertion of CDRs alone into human frameworks did not produce a functional reshaped antibody and modifications to the V region framework were required. With minor framework modifications, the affinity of the original murine mAb was restored and even exceeded. Where affinity was increased, an altered binding profile to overlapping peptides was observed. Computer modelling of the reshaped heavy chain V regions suggested that amino acids adjacent to CDRs can either contribute to, or distort, CDR loop conformation and must be adjusted to achieve high binding affinity.

A humanized anti-tumor necrosis factor-alpha monoclonal antibody that acts as a partial, competitive antagonist of the template antibody.

We have constructed several humanized versions of a monoclonal antibody (MAb78) against human tumor necrosis factor-alpha (huTNF-alpha) retaining the complementarity-determining regions (CDR) of the original mouse MAb with or without a variable number of original framework region (FR) residues. All versions, except one, showed a loss of binding affinity and neutralizing potency of at least 10-fold compared to the original mouse MAb or its chimeric equivalent. In some cases, however, the decrease in neutralizing potency was significantly greater than the decrease in binding affinity. Two humanized versions showing the greatest dissociation between these two parameters were studied for their capacity to inhibit the neutralizing activity of chimeric or murine MAb78 when used at concentrations that bound but only partially neutralized huTNF-alpha. One humanized version (MAb78D) was indeed able to do so, whereas the other (MAb78C) was not found to exert any inhibitory activity at all concentrations tested. The antagonistic effect of MAb78D was concentration dependent and could be overcome by increasing the concentrations of chimeric or murine MAb78. Two different models of MAb78-huTNF-alpha interaction that may help explain the antagonist activity of humanized MAb78D are discussed.

Expression of foreign genes in mammalian cells using an antibody fusion system.

An expression system is described whereby a gene product is expressed fused to an antibody Fab fragment to form an antibody-like molecule. The antigen binding function of the original antibody is retained and the foreign gene replaces the CH2 and CH3 regions of the heavy chain. The fusion protein is secreted as if it were an antibody, and can be purified using the antigen-binding function of the Fab-like part of the molecule. In principle, any open reading frame can be expressed and it is not necessary to develop an individual purification scheme, or any analytical reagents such as antibodies, for the expressed protein, as both these functions can be performed by the Fab part of the fusion protein. In practice, the nature of the nonantibody part of the fusion influences the efficiency of expression and secretion, and detailed guidance is given on trouble-shooting and maximizing expression.

Idiotope determining regions of a mouse monoclonal antibody and its humanized versions. Identification of framework residues that affect idiotype expression.

The contribution of framework regions (FRs) of antibody-variable domains to idiotype expression was studied by examining the interaction of various "humanized" versions of a mouse anti-TNF alpha monoclonal antibody (mAb78) with polyclonal and two monoclonal antibodies (mAb1G3 and mAb9F1), generated against the mAb78 idiotype. Humanized mAb78, bearing human constant domains and mouse complementarity-determining regions (CDRs) inserted with human FRs, was found to be five to sevenfold less reactive than mAb78 with polyclonal anti-idiotype antibodies and 200 to 300-fold less active in neutralizing TNF alpha. The substitution of heavy-chain FRs residues of the humanized antibody with original mouse residues 28 to 30, 48 to 49, 67 to 68, 70 to 71, 78, 80 and 82 progressively restored the immunoreactivity with polyclonal immunoglobulin Gs to the level of a version having mouse heavy chain and human light chain FRs, and increased 10 to 20-fold the TNF alpha neutralizing activity. This suggests that at least some of these residues are critical for TNF alpha binding as well as for the expression of idiotopes that are strongly immunogenic in syngeneic animals. All antibody versions with either human or mouse FRs were able to bind to various extents mAb1G3, a gamma-type anti-Id antibody that inhibits mAb78/TNF alpha interaction by paratope blockade. At variance, only the antibody versions containing mouse FRs were able to bind mAb9F1, an alpha-type anti-Id antibody unable to block the access of TNF alpha to mAb78 paratopes. Substitution of heavy chain FR residues 28 to 30 markedly decreased the binding of mAb1G3 (100 to 1000-fold). This suggests that these antibodies recognize CDR and FR idiotopes, respectively, that can be drastically modified by changes in the FRs. In conclusion, the results suggest that CDRs as well as FRs markedly contribute to antibody Id expression. Although strongly immunogenic idiotopes are probably located within the CDRs, the results also suggest that some FR residues are critically involved in shaping antibody Id diversity by affecting the structure of CDR-related idiotopes.

Screening of λgt11 cDNA Libraries Using Monoclonal Antibodies.

The screening of a λgt11 library with monoclonal antibodies described here is a relatively uncomplicated procedure, but it requires a little introduction nevertheless. For simplicity, it is assumed that you have in your possession a library that is ready to screen, i.e., either a library bought from a company or a library that you have made. Construction of a λgt11 library is described in Volume 4, Chapter 19 .

Expression of foreign genes in Mammalian cells using an antibody fusion system.

Whereas the expression of foreign genes in mammalian cells usually proves successful, the purification of gene products is often a difficult and time-consuming process. The availability of monoclonal antibodies to the foreign protein can greatly assist in small scale purification, but where antibodies are not available, alternatives have to be sought One useful approach involves the fusion of the foreign gene adjacent to a gene segment encoding an antibody heavy chain variable region (1). By transfection of this construct into a cell line producing a compatible light chain or by cotransfection of the fusion product with a light chain gene, an antibody-like molecule can be produced and purified using the corresponding antigen.

Screening of λgt11 cDNA Libraries Using Monoclonal Antibodies.

The screening of a λgt11 library with monoclonal antibodies described here is a relatively uncomplicated procedure, but it requires a little introduction nevertheless. For simplicity, it is assumed that you have in your possession a library that is ready to screen, i.e., either a library bought from a company or a library that you have made. Construction of a λgt11 library is described in Volume 4, Chapter 19 .

Expression of foreign genes in Mammalian cells using an antibody fusion system.

Whereas the expression of foreign genes in mammalian cells usually proves successful, the purification of gene products is often a difficult and time-consuming process. The availability of monoclonal antibodies to the foreign protein can greatly assist in small scale purification, but where antibodies are not available, alternatives have to be sought One useful approach involves the fusion of the foreign gene adjacent to a gene segment encoding an antibody heavy chain variable region (1). By transfection of this construct into a cell line producing a compatible light chain or by cotransfection of the fusion product with a light chain gene, an antibody-like molecule can be produced and purified using the corresponding antigen.

Reshaping a human monoclonal antibody to inhibit human respiratory syncytial virus infection in vivo.

We transferred the complementarity determining regions from a murine monoclonal antibody that neutralizes infection by respiratory syncytial virus (RSV) to a human IgG1 monoclonal antibody. The resulting reshaped human antibody lost affinity for RSV, but an additional alteration to one of the framework regions restored binding affinity and specificity. This second generation reshaped human monoclonal antibody cross-reacted with all clinical isolates of RSV tested and both prevented disease and cured mice even when administered four days after infection. We expect the antibody will prove useful in the management of this major childhood disease.