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Biomedical subjects

I Hellström

Publications and source records attributed to I Hellström.

At least 91 records · Page 5Linked to original sources
PubMed1989

Homologous recombination in hybridoma cells: heavy chain chimeric antibody produced by gene targeting.

We demonstrate that murine myeloma cells can efficiently mediate homologous recombination. The murine myeloma cell line J558L was shown to appropriately recombine two transfected DNA molecules in approximately 30% of cells that received and integrated intact copies of both molecules. This activity was then exploited to direct major reconstructions of an endogenous locus within a hybridoma cell line. Production of antigen-specific chimeric heavy chain was achieved by targeting the human IgG1 heavy chain constant region (C gamma 1) exons to the genomic heavy chain locus of a hybridoma cell line secreting antibody specific for a human tumor-associated antigen. The frequency of productive genomic recombinations was approximately 1 in 200 transfectants, with accumulation of the chimeric protein reaching greater than 20 micrograms/ml in culture supernatants.

Animals↗
PubMed1989

Oncogene-associated tumor antigens as targets for immunotherapy.

Cellular antigens encoded by tumor viruses and some antigens encoded by cellular oncogenes offer advantages as targets for immunotherapy by being inextricably associated with the neoplastic phenotype. For example, monoclonal antibodies (MAb) specific for an antigen encoded by the neu oncogene have a direct inhibitory effect on proliferation of antigen-positive tumor cells. Many of the oncogene-encoded cell surface molecules are growth factor receptors, as are some tumor-associated differentiation antigens (TADAs). Therefore, it is not surprising that their level of cancer specificity is similar. There have been some promising findings from using TADAs as targets for various forms of immunotherapy, and one would expect the results to further improve by targeting to molecules that are more directly involved in cell proliferation and/or in maintaining the malignant state.

Animals↗
PubMed1988

Measurement of local Mr 97,000 and 250,000 protein antigen concentration in sections of human melanoma tumor using in vitro quantitative autoradiography.

An assay method that uses 125I-labeled monoclonal antibody (MoAb) and in vitro quantitative autoradiography was developed to determine the local concentration of tumor-associated antigens in tissue sections. Human melanoma biopsy specimens were evaluated for the expression of the Mr 97,000 and 250,000 protein antigens using MoAb-96.5 and MoAb-9.2.27, respectively. Tissue sections were incubated in solutions of increasing concentration of 125I-labeled MoAb with or without an excess of unlabeled antibody. Quantitative autoradiography was performed on the sections and compared with 125I standards to determine tumor-bound radioactivity and calculate bound pmol of MoAb per g of tumor. The total binding, nonsaturable binding, and specific binding of 125I-labeled MoAb to tumor were then computed. Specific binding of MoAbs to tumor tissue was saturable in all antigen-positive tumors. The maximal concentration of specific binding of antibody to tissue (Bmax) represented the tissue antigen concentration. Estimates of the Ka of antigen/antibody binding were also made. The reliability of the measurements was confirmed by testing sections from mixtures of antigen-positive and antigen-negative cells.

Antibodies, Monoclonal↗
PubMed1988

Delivery of melanoma-associated immunoglobulin monoclonal antibody and Fab fragments to normal brain utilizing osmotic blood-brain barrier disruption.

Iodinated monoclonal antibodies (IgG 96.5 and two monomeric Fab fragments 96.5 and 48.7) to melanoma-associated antigens were administered after osmotic blood-brain barrier (BBB) opening in normal rats. Osmotic BBB disruption significantly (P less than 0.0001) increased monoclonal antibody delivery to the brain. Following BBB opening and intracarotid administration, there was no difference in the disrupted brain concentration integral area under the curve between Fab and IgG over the 72-h experimental period. However, Fab concentration in the disrupted brain was initially higher than IgG, and the clearance was more rapid (P less than 0.0001), decreasing 50% by approximately 4.5 h compared to 25.5 h for IgG. Plasma clearance was also more rapid for the Fab than IgG. The levels decreased 50% by 1.5 h for Fab and 15 h for IgG. The route and timing of antibody infusion had a significant effect on delivery to the disrupted brain with the Fab fragments but not with the intact IgG. Antibody recovered from disrupted brain retained its immunological reactivity as measured by a cell binding assay for at least 24 h. IgG and Fab delivery to the ipsilateral brain after BBB disruption increased (P less than 0.001) with increasing dose over a more than 3-log dose range. These data provide information applicable to the therapeutic use of monoclonal antibodies in brain tumor treatment.

Animals↗
PubMed1988

Antibody-mediated killing of human tumor cells by attached effector cells.

Cultured human melanoma, lung carcinoma, and colon carcinoma cells were isotope labeled and incubated with a combination of effector cells and mouse monoclonal antibodies to tumor-associated cell surface antigens. The former were derived from the peritoneal cavity of mice or from peripheral blood of healthy human subjects. Monoclonal antibodies MG-21, 96.5, and L6, which are IgG3, IgG2a, and IgG2a, respectively, were all cytolytic when added in the presence of mouse effector cells to target cells expressing the relevant antigens. MG-21 and L6 were cytolytic also with human effector cells, while monoclonal antibody 96.5 was not. The effector cells attached to plastic surfaces, stained with neutral red, were peroxidase positive and mediated their effect over a 24- to 72-h time period as compared to the 4 h generally sufficient for antibody-dependent cellular cytotoxicity by natural killer cells. In tests on human effector cells with a fluorescence-activated cell sorter, they stained with antibody LCM-3C10 to the CD14 antigen, as well as with antimonocyte antibody 61D3. The cytolytic effect of human effector cells and antitumor antibody was not abolished by incubation with antibodies FC2 or 60.3 to CD16 and CD18, respectively, known to interfere with the antibody-dependent cellular cytotoxicity activity and natural killing of natural killer cells. This suggests, together with the other findings, that the effector cells were macrophages.

Animals↗
PubMed1988

Role of CD2 cross-linking in cytoplasmic calcium responses and T cell activation.

The relationship between the increase of intracellular free Ca2+ concentration ([Ca2+]i) in resting T cells after stimulation with monoclonal antibody (mAb) to CD2 (E rosette receptor) and the subsequent proliferation response was investigated. Although the combination of 9.6 plus 9-1 mAb to CD2 was both mitogenic and induced an increase in [Ca2+]i, cross-linking of an individual CD2 mAb on the cell surface induced an increase in [Ca2+]i without directly stimulating T cell proliferation. The [Ca2+]i response from cross-linking an individual mAb was not epitope dependent, since 21 of 21 mAb to CD2 were effective when cross-linked with a polyclonal goat anti-mouse Ig second step. The kinetics of calcium mobilization was highly dependent upon the procedure for cross-linking, since the binding of biotin-conjugated 9.6 mAb followed by avidin gave a large and rapid response, whereas cross-linking of 9.6 with an anti-kappa mAb, 187.1, caused a minimal response, and the cross-linking of 9.6 followed by a polyclonal goat anti-mouse Ig gave an intermediate response. In addition, ligation of CD2 by rosetting with sheep red blood cells alone was sufficient to cause increased [Ca2+]i. In functional studies only the procedures associated with minimal CD2 cross-linking induced proliferation of resting T cells in combination with interleukin (IL)2. The proliferation also required IL 1 or accessory cells. Cross-linking 9.6 on the cell surface also enhanced proliferation in the presence of phorbol myristate acetate or a CD28 mAb, 9.3, under conditions that were accessory cell independent. In contrast to the proliferation following stimulation with 9.6 plus 9-1, the combination of 9.6 plus 9-1 F(ab')2 fragments lost mitogenic activity. The 9.6 plus 9-1 F(ab')2 combination was similar to 9.6 cross-linking in that either could induce responsiveness to recombinant IL2 or CD28 mAb 9.3. The combination of 9.6 plus 9-1 F(ab')2 fragments was still able to increase [Ca2+]i in T cells with slow kinetics. Together these results suggest that the binding of mAb to CD2 under conditions that cause a slow rather than a rapid increase in [Ca2+]i is associated with T cell activation. Furthermore, they suggest that in studies of T cell activation, sheep erythrocyte rosette formation should not be used to isolate T cells since rosetting may effect [Ca2+]i.

Antibodies, Monoclonal↗
PubMed1988

Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate.

Two anti-tumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), were covalently linked to alkaline phosphatase (AP), forming conjugates that could bind to the surface of antigen-positive tumor cells. The conjugates were capable of converting a relatively noncytotoxic prodrug, etoposide phosphate (EP), into etoposide--a drug with significant antitumor activity. In vitro studies with a human colon carcinoma cell line, H3347, demonstrated that while EP was less toxic than etoposide by a factor of greater than 100, it was equally toxic when the cells were pretreated with L6-AP, a conjugate that bound to the surface of H3347 cells. The L6-AP conjugate localized in H3347 tumor xenografts in nude mice and histological evaluation indicated that the targeted enzyme (AP) was distributed throughout the tumor mass. A strong antitumor response was observed in H3347-bearing mice that were treated with L6-AP followed 18-24 hr later by EP. This response, which included the rejection of established tumors, was superior to that of EP (P less than 0.005) or etoposide (P less than 0.001) given alone. The IF5-AP conjugate did not bind to H3347 cells and did not enhance the toxicity of EP on these cells in vitro. In addition, IF5-AP did not localize to H3347 tumors in nude mice and did not demonstrate enhanced antitumor activity in combination with the prodrug.

Alkaline Phosphatase↗
PubMed1988

Recombinant vaccinia virus vaccine against the human melanoma antigen p97 for use in immunotherapy.

We have constructed a recombinant vaccinia virus, v-p97NY, which expresses the human melanoma-associated glycoprotein p97. Immunization with v-p97NY could induce humoral and cell-mediated immunity to p97, including delayed-type hypersensitivity, in mice and in two of two monkeys (Macaca fascicularis). The fact that an immune response was induced also in monkeys is important because normal cells from monkeys, but not from mice, express a low level of cross-reactive p97. Mice immunized with v-p97NY rejected transplants of syngeneic mouse melanoma expressing p97. A rejection response could be detected also when immunization was started 2 days after tumor transplantation, irrespective of whether the transplanted cells grew subcutaneously or as lung metastases. Evidence was obtained that melanoma cells lacking p97 may be killed as "bystanders" at the site of an immune response to melanoma cells expressing p97.

Animals↗
PubMed1988

Characterization of vaccinia virus growth factor biosynthetic pathway with an antipeptide antiserum.

A synthetic peptide derived from vaccinia virus growth factor (VGF) was used as an immunogen to prepare antiserum able to immunoprecipitate native VGF from both vaccinia virus-infected cell lysate and cell-free medium. Pulse-chase, tunicamycin treatment, and carbohydrate trimming experiments revealed that VGF is synthesized as a 19-kilodalton (kDa) precursor which is rapidly modified to a high-mannose-type 22-kDa protein. This cell-associated form is further processed into a 25-kDa polypeptide which, after proteolytic cleavage, releases the mature VGF into the medium as a 22-kDa glycoprotein.

Amino Acid Sequence↗
PubMed1988

Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody.

We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue.

Animals↗
PubMed1987

Production of a mouse-human chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity.

Mouse monoclonal antibody 2H7 recognizes the CD20 cell surface phosphoprotein that is expressed in normal as well as malignant B cells. CD20 may be a useful target for therapy of B cell lymphomas, since damaged normal B cells can be replaced by their antigen-negative precursors. Monoclonal antibody 2H7 is an IgG2b (kappa) immunoglobulin which cannot mediate antibody-dependent cellular cytotoxicity with human lymphocytes or complement-dependent cytotoxicity with human serum. We have now generated a chimeric 2H7 antibody by substituting the mouse constant domains of 2H7 with the human gamma 1 and kappa domains. This new antibody has the same binding specificities as 2H7 but is highly effective in mediating antibody-dependent cellular cytotoxicity with human effector cells and complement-dependent cytotoxicity with human complement.

Amino Acid Sequence↗
PubMed1987

Tumor-specific idiotopes on suppressor factors and suppressor cells revealed by monoclonal anti-idiotope antibodies.

Two monoclonal anti-idiotope antibodies, previously found to induce tumor-specific cell-mediated immunity in mice, were examined for their relationship to tumor-associated suppressor factors (SF), produced in culture by spleen cells from tumor-bearing mice or present in sera from such mice. A leukocyte adherence inhibition assay was used to detect cellular immunoreactivity to tumor antigens and its inhibition by SF, using peritoneal cells from mice bearing tumor or sensitized with anti-idiotope antibody. The SF were specifically absorbed by the corresponding anti-idiotope antibodies coupled to a solid phase and were recovered by elution. They were also specifically neutralized by the addition of the respective antibodies to the assay system. Anti-idiotope antibody, used with complement to pretreat spleen cells from tumor-bearing mice, prevented these cells from producing SF in culture. Tumor antigen-reactive effector cells, suppressor cells, and SF thus share similar idiotopes, permitting their respective functions to be modulated by appropriate anti-idiotopes.

Animals↗
PubMed1987

Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells.

A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C gamma 2a and C kappa, were substituted by the human C gamma 1 and C kappa by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man.

Amino Acid Sequence↗