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Biomedical subjects

A J George

Publications and source records attributed to A J George.

At least 19 recordsLinked to original sources
PubMed1995

Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.

Amino Acid Sequence↗
PubMed1995

Measurement of kinetic binding constants of a panel of anti-saporin antibodies using a resonant mirror biosensor.

We have used a resonant mirror biosensor to determine the kinetics of binding of four antibodies, and their Fab' fragments, to their antigen, the plant-derived ribosome-inactivating protein (RIP) saporin. The analysis of the affinity of the antibodies was in reasonable agreement with values obtained by conventional techniques. However, the kinetic data showed that all four antibodies have a high dissociation rate constant (kdiss). These antibodies have been used in the construction of bispecific antibodies used to deliver saporin to tumour cells, and it is highly probable that the in vivo efficacy of the bispecific antibodies is limited by the high rate of dissociation of antibody-toxin complexes.

Allergy and Immunology↗
PubMed1995

Delivery of the ribosome-inactivating protein, gelonin, to lymphoma cells via CD22 and CD38 using bispecific antibodies.

It is well established that bispecific antibodies (BsAbs) can be used effectively in targeting the ribosome-inactivating protein (RIP), saporin, against neoplastic B cells. We have now extended this delivery system for use with gelonin. By measuring antigen-binding characteristics and epitope mapping a panel of anti-gelonin MAbs using the IAsys resonant mirror bisensor, we were able to rapidly select the most suitable for making BaAbs. The Fab' fragments from these MAbs were chemically conjugated with Fab' from either anti-CD22 or anti-CD38. Cytotoxicity assays showed that BsAbs were highly efficient at delivering gelonin to cultured Daudi cells and achieved levels of toxicity which correlated closely with the affinity of the BsAbs. Using pairs of anti-CD22 BsAbs we were able to generate bivalent BsAb-gelonin complexes which achieved IC50 values of 2 x 10(-11) M gelonin, a potency which is equivalent to that reached by saporin in this targeting system. However, because gelonin is 5-10 times less toxic than saporin, the therapeutic ratio for gelonin is superior, making it potentially a more useful agent for human treatment. Cytotoxicity assays and kinetic analysis showed that targeting gelonin via CD38 was 2-5 times less effective than delivery through CD22. However, with a pair of BsAbs designed to co-target gelonin via CD22 and CD38, the cytotoxicity achieved equalled that obtained with a pair of anti-CD22 BsAbs (IC50 = 1 x 10(-11) M). This important result suggests that the anti-CD38 helps bind the gelonin to the cell and is then 'dragged' or 'piggy-backed' into the cell by the anti-CD22 BsAb. The implication of these findings for cancer therapy is discussed.

ADP-ribosyl Cyclase↗
PubMed1995

The glycophosphatidylinositol anchor affects the conformation of Thy-1 protein.

Thy-1 has the structure of a single variable-type immunoglobulin domain anchored to the external face of the plasma membrane via a glycophosphatidylinositol moiety. When the lipid is removed from this anchor by either phospholipase C or D, the reactivity of the delipidated Thy-1 for a range of antibodies, including those known to be determined by amino acid residues, is impaired. We have investigated in detail the effect of delipidation on the reaction with the OX7 monoclonal antibody, determined by the allelic variant residue Arg 89. Analysis of the kinetics of OX7 binding shows that delipidation affects primarily the dissociation of antibody, increasing the dissociation rate constant kdiss from 0.27 x 10(-3) s-1 to 2.39 x 10(-3) s-1. Addition of phospholipase to preformed antibody-antigen complex causes an immediate change from the slow to the faster dissociation rate, implying that delipidation induces a conformational change in the Thy-1 protein that is sufficiently strong to dissociate bound antibody. This conformational change can be demonstrated directly by the circular dichroism spectrum of human Thy-1 that detects changes in the environment of Tyr residues located near the antigenic epitopes. Molecular dynamics studies suggest that, on delipidation, a conformational change occurs in the glycan chain that affects the protein in the region of the antigenic epitopes. This study thus demonstrates that the glycophosphatidylinositol anchor strongly influences the conformation of Thy-1 protein by a mechanism that could occur generally with membrane proteins of this class.

Amino Acid Sequence↗
PubMed1994

Mammalian expression and secretion of functional single-chain Fv molecules.

Single-chain Fv (sFv) proteins are genetically engineered molecules that consist of the two variable domains of an antibody connected by a polypeptide linker; they contain the antigen binding function of the parental protein in a single 30-kDa polypeptide chain. sFvs are usually produced in bacteria where they are insoluble and therefore require extensive refolding in vitro. In this report we followed the processing of three antibody sFvs (145-2C11 directed against murine CD3 epsilon chain, OKT9 against the human transferrin receptor, and U7.6 against dinitrophenyl groups) by transfected mammalian (COS-7) cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv with high efficiency. The sFvs contained an immunoglobulin light chain leader sequence, which directed them to the endoplasmic reticulum and allowed secretion into the medium. We found that the sFvs were secreted at different rates, with the rate-limiting step of secretion being their exit from the endoplasmic reticulum. We increased the secretion rate of one of the sFvs by introducing an asparagine-linked glycosylation site in FR1 of the heavy chain, and by using tunicamycin (an inhibitor of glycosylation) we found that glycosylated antibody sFvs were secreted faster than their nonglycosylated counterparts. All secreted sFvs specifically bound their antigens; where tested, at least 90% of the secreted sFv was functional. Therefore, mammalian cells can effectively fold and secrete sFv antibody and can provide a convenient system for testing and producing sFv proteins.

Animals↗
PubMed1994

Antibody engineering.

A century ago, in his private laboratory in Berlin, Paul Ehrlich conceived and developed the idea of using antibodies to target toxic molecules. Recent advances in genetic engineering have enormously extended the potential of this concept for the treatment of malignant diseases, by making it possible to isolate genes encoded for the antibody variable regions and to manipulate them to create new molecules.

Animals↗
PubMed1994

Applications of monoclonal antibodies in clinical oncology.

Application of monoclonal antibodies (mAbs) to clinical settings has proved to be slower than originally hoped. However, a recent conference provided evidence that the field is coming of age, and that antibodies, and their recombinant derivatives, will continue to find a role in the clinic.

Antibodies, Monoclonal↗
PubMed1992

Targeting of anti-tumor responses with bispecific antibodies.

T cells can be induced to specifically lyse tumor cells with bispecific antibodies containing anti-T cell receptor mAbs crosslinked to anti-tumor mAbs. Such "targeted cytolysis" requires that the target cell be bound directly to the cytotoxic cell. In addition, targeted T cells mediate a second activity, the secretion of factors that can block the growth of both tumor target cells and bystander tumor cells. When given to nude mice bearing intraperitoneal human ovarian carcinoma, targeted human T cells cause the rapid removal of most tumor cells from the peritoneum, and markedly prolong the times of survival of treated mice. The efficacy of targeted T cells for treating human cancer is currently being tested in clinical trials.

Animals↗
PubMed1992

Targeted cytokine production.

It has been well established that bispecific antibodies containing anti-T-cell receptor MAbs crosslinked to anti-tumor MAbs induce T cells to lyse tumor cells, as measured in a 51Cr-release assay. Such lysis requires direct attachment between target and cytotoxic cells and most probably involves the exocytosis of cytolytic substances into the cell:cell interface. In addition, targeted T cells mediate a second activity, the secretion into the medium of factors that can block the growth of bound tumor cells and unbound bystander cells. In order to test how targeted effector cells mediate anti-tumor effects in vivo, we are currently developing a totally syngeneic murine system in which murine T cells are targeted against mouse mammary tumors. The system allows us to treat both primary tumors and tumor transplants, using a mammary-tumor-virus antigen as the entity that is specifically recognized on the tumor cells.

Animals↗
PubMed1991

Intra-thecal treatment of leptomeningeal lymphoma with immunotoxin.

An animal model has been established to investigate the effect of intra-thecal (i.t.) immunotoxins in the treatment of leptomeningeal metastases of acute lymphoblastic leukaemia (ALL). Direct inoculation of L2C lymphoma cells into the cisterna magna of guinea-pigs gives rise to a leptomeningeal pattern of growth, similar to that of human ALL, and to a systemic leukaemia which develops in approximately 14 days. In our model the systemic disease could be controlled with cyclophosphamide while the meningeal disease progressed and provided a target for i.t. immunotoxin. The immunotoxin used consisted of an anti-idiotypic antibody disulphide-bonded to the ribosome-inactivating protein saporin. It was highly cytotoxic to L2C cells in vitro, being around 30,000 times more potent than a control immunotoxin at inhibiting protein synthesis. In vivo, the maximum tolerated dose of i.t. immunotoxin was 10 micrograms. From the rate at which radiolabelled immunotoxins appeared in the plasma following i.t. injection, we were able to estimate that its half-life in the cerebrospinal fluid (CSF) was between 1 1/2 and 2 hr. Intrathecal treatment of guinea-pigs with immunotoxin 1 day after inoculation of L2C cells into the cisterna magna had a remarkable therapeutic effect. All guinea-pigs treated with 0.5 or 5 micrograms of immunotoxin survived, and remained tumour-free for more than 100 days after treatment, while control animals given cyclophosphamide alone or an irrelevant immunotoxin had a mean survival time of 28 days. Provided concerns over toxicity can be overcome, these results indicate that i.t. immunotoxins offer an alternative, highly specific form of treatment in leptomeningeal neoplasia.

Animals↗
PubMed1991

Idiotypic vaccination against B-cell lymphoma leads to dormant tumour.

Idiotypic immunoglobulin, which can be considered to bear tumour-associated antigens in the context of B-cell lymphoma, has been obtained from the splenic A31 tumour, purified, and used to immunise syngeneic mice. On subsequent exposure to a lethal challenge of lymphoma cells, the mice showed no overt tumour development over an observation period of 6 months, whereas mice immunised with an unrelated idiotypic immunoglobulin succumbed to lymphoma after about 20 days. Anti-idiotypic immunity persisted in protected mice, since a second exposure to a lethal tumour dose 4 months after the first challenge also failed to induce lymphoma. Anti-idiotypic antibody appeared to have a major role in protection when analysed by passive transfer experiments, with no contribution from transferred cells. Protected mice were investigated for the presence of lymphoma cells 4-8 months following exposure to tumour, but the spleens, which were of normal weight and appearance, contained few or no tumour cells by phenotypic analysis. However, passage of cells dispersed from these spleens led, in 60% of cases, to tumour development in unimmunised recipients. The emergent tumours were indistinguishable from the original A31 lymphoma, with no evidence for variants, indicating that the cells were unable to grow in the immune mice, but that this dormant state could be disrupted by transfer.

Animals↗
PubMed1991

Characterization of a new monoclonal anti-Fc gamma RII antibody, AT10, and its incorporation into a bispecific F(ab')2 derivative for recruitment of cytotoxic effectors.

Fc gamma RII (CDw32) on monocytes is capable of triggering both phagocytosis and lysis of chick red blood cells (CRBC) coated with antibody of the appropriate isotype. In this report we describe the production and characterization of a mouse monoclonal IgG1 antibody specific for Fc gamma RII and compare its activity in binding studies, tissue distribution and redirected cellular cytotoxicity (RCC), with the previously identified anti-Fc gamma RII antibodies KB61 and IV.3. Immunohistochemical and flow cytometry analyses demonstrated that AT10 binds very strongly to Fc gamma RII on normal monocytes, but only weakly to that expressed on lymphocytes. This pattern does not correspond to the staining seen with either KB61 or IV.3, and appears to give an intermediate profile. The binding constant (Ka) for the Fab' fragment of AT10 was calculated at 5.3 x 10(8) M-1, four times higher than that for KB61 (1.4 x 10(8) M-1). Bispecific F(ab')2 antibodies were constructed from Fab' fragments of AT10 or KB61 thioether-linked to Fab' from an anti-CRBC monoclonal antibody. These bispecific derivatives directed monocyte cytotoxicity against CRBC as efficiently as either a monoclonal or polyclonal anti-chick erythrocyte antibody. The bispecific F(ab')2 antibodies had a distinct advantage over the conventional reagents, in that they were not blocked in the presence of human Fc gamma at 3.5 mg/ml (a concentration comparable with that provided by IgG in serum). Therefore, bispecific derivatives constructed with the high affinity anti-Fc gamma RII antibody, AT10, may be used as therapeutic reagents for targeting tumour cell lysis in vivo.

Animals↗
PubMed1991

Characterisation of a light chain loss variant of the BCL1 lymphoma.

Vaccination of BALB/c mice with idiotypic (id) IgM derived from the murine B cell lymphoma BCL1, protects the animals from challenge with tumour cells. Escape of the tumour cells from immune control is associated with the selection of variant cells which fail to express significant levels of id IgM on their cell surface. We have previously isolated one such variant, SNAG 1, and shown that, while it expresses less than 10% of the levels of surface IgM of the parental BCL1 lymphoma, it continues to synthesise id material which can be detected within the cell. In this report we present a detailed characterisation of this variant and show that the tumour cells no longer synthesise the lambda light chain. This failure to produce the light chain causes the mu heavy chains in SNAG 1 to remain marooned in the endoplasmic reticulum. The mu heavy chains in SNAG 1 have a normal mol. wt and isoelectric point, and so appear not to be mutated. This is unlike the vast majority of light chain loss variants, in which the heavy chains have been shown to contain deletions. Investigation of the mechanisms responsible for the loss of light chain synthesis demonstrated that, while mRNA for the light chain is present, and of a normal size, there was no production of light chain protein in a cell free system. This indicates that the failure to express light chain by SNAG 1 cells is due to an inability to translate the light chain mRNA into the detectable levels of lambda light chain protein.

Animals↗
PubMed1991

Monoclonal antibodies raised against the idiotype of the murine B cell lymphoma, BCL1 act primarily with heavy chain determinants.

Anti-idiotypic antibodies can be used as probes to distinguish neoplastic cells from their normal counterparts. In addition they have been used in the passive therapy of B cell tumors. In this report we describe a panel of 7 rat monoclonal antibodies raised against idiotypic determinants carried by the IgM molecule of the BCL1 lymphoma. The majority (6/7) of these antibodies recognize private idiotypic determinants that are carried on the isolated mu heavy chain of the molecule, and do not require the lambda chain for reactivity. This is unusual for antibodies raised against the idiotype of the whole immunoglobulin molecule, which normally require both chains for reactivity. The antibodies do not, however, bind peptides corresponding to the complementarity determining regions of the mu heavy chain of BCL1. The antibodies perform well in complement mediated cytotoxicity, and, in at least one case, are effective in the passive immunotherapy of BCL1 lymphoma.

Animals↗
PubMed1990

Recognition of a B cell lymphoma by anti-idiotypic T cells.

Idiotypic IgM derived from a B cell lymphoma can act as a tumor-associated Ag, in that immunization with this purified protein generates an anti-idiotypic immune response that specifically suppresses tumor development. Spleens of immune mice contain T cells that proliferate in response to idiotypic IgM. However, idiotypic Ag is presented to the T cells most efficiently in its natural form at the surface of the lymphoma cells, than as soluble IgM plus presenting cells. Variant tumors that display either little or no idiotypic IgM at the cell surface, but which are otherwise indistinguishable from parental tumor, induce a weak response or fail to stimulate the T cells, respectively. Anti-idiotypic lines and clones have been derived from the splenic T cells by growth in the presence of irradiated tumor cells. Phenotypic analysis revealed that cells from both lines and clones express CD3 and CD4 Ag, but not CD8. Recognition of tumor Id, which required no added presenting cells, was inhibited by antibody against MHC class II Ag, and variably by anti-CD4. Proliferative responses were inhibited by anti-idiotypic antibodies, but also by antibodies against the constant region of the mu H chain, indicating that perturbation of the surface IgM abrogates availability of idiotypic determinants to the T cells.

Animals↗
PubMed1990

Anti-idiotypic therapy of leukemias and lymphomas.

Idiotypic determinants of the Ig expressed by the majority of B cell tumors present an attractive target for immunotherapeutic manipulations. The idiotypic Ig is molecularly defined and the behavior of the target cells placed under anti-idiotypic attack might have broader implications for cancer immunotherapy. Simple administration of monoclonal antibody reactive with these determinants clearly has only a limited effect on tumor load, due largely to the multiplicity of strategies by which the tumor cell can avoid such attack. These include modulation, change in idiotypic determinants due to somatic mutation, and complete loss of expression at the cell surface. If the first antibody treatment can be made more effective, for example by tailoring molecules to recruit available effector mechanisms more efficiently, or by the use of antibodies capable of delivering a lethal hit via a toxin, the tumor cell will presumably have less opportunity to escape. A second strategy is to immunize the tumor-bearing host with idiotypic Ig obtained from tumor cells. Once in place, such an immune response should suppress tumor growth on a continuing basis. In animal lymphoma, this approach appears to prolong survival although some of the escape mechanisms encountered for passive antibody therapy are apparently operative for active therapy. Study of these mechanisms should allow insight into how cells control expression of idiotypic determinants at the cell surface and open the possibility of further therapeutic intervention. A combined approach to the treatment of human lymphoma might be envisaged whereby tumor load is reduced by passive antibody, thus leaving the immune system relatively unscathed, and even perhaps releasing natural antitumor responses which could be further stimulated by immunization.

Antibodies, Anti-Idiotypic↗