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Biomedical subjects

A H Blair

Publications and source records attributed to A H Blair.

At least 19 recordsLinked to original sources
PubMed1991

Tumor localization of monoclonal antibodies against human renal carcinoma in a xenograft model.

We investigated the localization of intravenously injected DAL K45 and DAL K29, two monoclonal antibodies (MABs) against human renal cell carcinoma (RCC), and their F(ab)2 fragments in nude mice bearing intrarenal transplants of the RCC line Caki-1. More of the MABs or their F(ab)2s specifically localized in the tumor than in any normal tissue with the exception of blood. Compared to parent MABs, F(ab)2s were cleared faster from all tissues. In serum, the MABs and F(ab)2s showed a single radioactive peak retaining partial immunoreactivity. DAL K45-F(ab)2 showed the highest tumor:normal tissue localization ratios and the most distinct gamma-camera image at 24 h.

Adult↗
PubMed1991

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen.

Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 x 10(6) cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotype-matched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-Mr MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.

Animals↗
PubMed1991

Inhibition of human renal cancer by monoclonal-anti-body-linked methotrexate in an ascites tumor model.

The monoclonal antibody DAL K29 against a cell-surface antigen associated with a human renal cell carcinoma was covalently linked to the antifolate methotrexate with full retention of antibody activity and partial retention of drug activity. Using an ascites tumor model, developed after intraperitoneal (i.p.) inoculation of 5 x 10(6) cells of the human kidney cancer line Caki-1 per pristane-primed nude mouse, we showed that the methotrexate-Dal-K29 conjugate was a more potent tumor inhibitor (P less than 0.0005) of human renal cell carcinoma (which is resistant to currently available modalities including chemotherapy) than the drug or mAb alone, the drug linked to an isotype-matched nontumor-specific IgG or a mixture of the drug and the mAb. Only the conjugate could produce tumor-free survival in a proportion of the mice during the period of observation (i.e. 150 days after tumor inoculation).

Animals↗
PubMed1989

Synthesis of site-specific methotrexate-IgG conjugates. Comparison of stability and antitumor activity with active-ester-based conjugates.

With a view to increasing drug incorporation without loss of antibody activity, tritium-labeled methotrexate (MTX) was covalently linked to a polyclonal rabbit IgG antibody against bovine serum albumin and a monoclonal mouse IgG antibody against human renal cancer (Dal K20) by a site-specific method based on hydrazone bond formation between MTX hydrazide and the aldehyde groups generated by periodate oxidation of carbohydrate moieties in IgG (which are uncommon in the antigen-binding region). These conjugates were compared with the corresponding non-site-specific MTX-IgG conjugates produced by the N-hydroxysuccinimide active-ester method with regard to synthesis, stability, retention of antibody activity, inhibition of the target enzyme dihydrofolate reductase and antitumor effect. Incorporation levels achieved with the hydrazide method were no greater than with the active-ester method, typically 6-7 mol MTX/mol IgG. Approximately the same dihydrofolate-reductase-inhibitory capacity was observed for MTX bound by either method. Hydrazide conjugates lost bound drug more rapidly than active-ester conjugates on freezing and thawing, on incubation at 37 degrees C and 51 degrees C, and in the presence of serum or rat liver homogenates. Exposure to rat liver homogenates at 37 degrees C, pH 4.6, for 24 h led to the loss of 50%-60% of the bound drug from hydrazide conjugates compared to 20%-30% from the active ester conjugates. Bio-Gel P-2 chromatography of low-molecular-mass fractions, obtained after exposure of each of the conjugates to liver homogenates, revealed the presence of a compound that had the same elution volume and RF on thin-layer chromatography as free MTX. Enzyme-linked immunosorbent assay showed loss of antibody activity of both types of conjugates at 51 degrees C and on freezing and thawing. In a clonogenic assay, the active-ester conjugate of Dal K20 appeared to be equally effective or slightly better as a tumor inhibitor than the corresponding hydrazide conjugate. The hydrazide method may be useful in linking MTX to those monoclonal antibodies that tend to denature when subjected to the active-ester method of linkage.

Antibodies, Monoclonal↗
PubMed1989

Synthesis of methotrexate-antibody conjugates by regiospecific coupling and assessment of drug and antitumor activities.

In order to increase the retention of drug activity, regiospecific coupling has been used to synthesize conjugates of methotrexate (MTX, 1) with normal rabbit IgG (NRG) and a mouse anti-human renal cancer monoclonal IgG (Dal K-20). MTX gamma-methyl ester (4) was produced either by selective esterification of MTX or by coupling of 4-amino-4-deoxy-N10-methylpteroic acid (2) with suitable glutamic acid derivatives. The MTX gamma-methyl ester (4) was then converted to the corresponding hydrazide 6. An amide-linked conjugate was formed when the MTX gamma-hydrazide (6) was converted to reactive acylating species 7 by using tert-butyl nitrite or trifluoroacetaldehyde, which were reacted with nucleophilic centers, presumably epsilon-amino groups, in native IgG. A hydrazone-linked conjugate was formed when MTX gamma-hydrazide (6) was reacted directly with IgG that had first been oxidized with periodate to form polyaldehyde IgG. The regiospecifically synthesized conjugates were somewhat more effective inhibitors in vitro of dihydrofolate reductase and of colony formation by human renal cancer (Caki-1) cells than were control nonregiospecific conjugates.

Antibodies, Monoclonal↗
PubMed1988

Hydrolysis of methotrexate-immunoglobulin conjugates by liver homogenates and characterization of catabolites.

Methotrexate (MTX) linked to antitumor antibodies inhibits tumor growth better than free MTX, free antibody, or MTX linked to normal rabbit IgG (NRG), in spite of the less effective inhibition of the target enzyme dihydrofolate reductase (DHFR) by conjugated MTX. In addition to the demonstrated higher uptake of MTX linked to antitumor antibodies (compared with the uptake of free MTX or nonspecific IgG conjugates), a contributory factor to the superior tumor inhibitory action of MTX-IgG conjugates may be the prolonged release of active drug from the internalized conjugate. Therefore, we have investigated whether an MTX-IgG conjugate could be hydrolyzed to release free MTX or fully active MTX-containing fragments after incubation with liver homogenates and have characterized the catabolites according to the presence of free MTX and their capacity to inhibit DHFR. Catabolism was optimal at pH 4.6, activated by dithiothreitol, and inhibited by antipain and N-alpha-p-tosyl-L-lysine chloromethyl ketone, thus implicating lysosomal enzymes. Liver homogenates produced an MTX-containing, low-molecular-weight fraction that was isolated by gel filtration. Further purification of this fraction by DEAE-cellulose chromatography gave two MTX-containing peaks, neither of which migrated as free MTX on thin-layer chromatography or inhibited DHFR more effectively than the parent conjugate. However, the presence of amino acid residues in these catabolites could contribute to their observed prolonged intracellular retention and superior antitumor action.

Antipain↗
PubMed1987

The design of cytotoxic-agent-antibody conjugates.

The rationale for the use of antibodies as carriers of cancer chemotherapeutic agents is based upon: the presence on cells of tumor-associated cell surface antigens (TAA); the ability to obtain specific polyclonal or monoclonal antibodies against them; and the availability of methods for binding appropriate toxic agents or radionuclides with retention of activity of both antibody and agents. The general finding so far has been that both conventional polyclonal and hybridoma-derived monoclonal antibodies can deliver cytocidal amounts of toxic agents to target tumor cells both in vitro and in vivo. Moreover, various agents with different modes of antitumor activity (e.g., DNA intercalation or alkylation, enzyme inhibition, and cell surface modification) have all produced superior tumor inhibition in conjugate form compared to the individual or synergistic inhibition produced by agent and antibody. Recent studies are contributing to the understanding of the mechanism of action of drug-antibody conjugates and are thus establishing guidelines for this approach to cancer therapy.

Antibiotics, Antineoplastic↗
PubMed1985

Conjugation of methotrexate to IgG antibodies and their F(ab)2 fragments and the effect of conjugated methotrexate on tumor growth in vivo.

Methotrexate (MTX) was first conjugated to antibovine serum albumin IgG (antiBSA) or its F(ab)2 fragment to define conditions for retention of drug and antibody activity. With identical drug: protein molar ratios, incorporation in the F(ab)2 fragment was lower than in intact antiBSA, an observation consistent with analysis of the number of lysine residues (22 in F(ab)2 compared to 40 in antiBSA). In either case, up to approximately 10 mol MTX could be incorporated per mol protein, with recovery of 70% of the protein. At an incorporation ratio of 6 mol MTX per mol protein, MTX-antiBSA retained 100% of antibody activity and MTX-F(ab)2-antiBSA retained 75%. MTX-antiBSA and MTX-F(ab)2antiBSA were equally potent in vitro inhibitors of dihydrofolate reductase. Conjugates prepared from antiEL4 IgG (AELG) and from F(ab)2AELG significantly increased survival in EL4 lymphoma-bearing mice compared with mice receiving equal amounts (5 mg MTX/kg) of free MTX, MTX linked to the F(ab)2 fragment of normal rabbit IgG, or a simple mixture of MTX and F(ab)2AELG. MTX-AELG at this dose level produced longer survival than MTX-F(ab)2AELG (0.005 less than P less than 0.01).

Animals↗
PubMed1985

Uptake of methotrexate linked to polyclonal and monoclonal antimelanoma antibodies by a human melanoma cell line.

[3H] Methotrexate [( 3H]MTX) was covalently linked to monoclonal antibody (MoAb) 225.28S against human melanoma, to a rabbit anti-human melanoma IgG absorbed either with human red blood cells (AHMGR) or with red blood cells and a variety of normal human tissues (AHMGR + T), or to normal rabbit IgG (NRG). Human melanoma M21 cells were incubated at 0 degrees C or 37 degrees C with 10 microM free MTX or 10 microM MTX linked to one of the above carriers. The order of net uptake of MTX during 6 hours was MTX-MoAb 225.28S greater than MTX-AHMGR greater than MTX-AHMGR + T greater than MTX-NRG greater than or equal to MTX. This order of uptake by the three antibody conjugates corresponded to the amount of conjugate bound at equilibrium at 0 degrees C and to the immunofluorescence titers. Binding sites for MoAb 225.28S were more efficient for internalization of MTX than were those for the two polyclonal antibody preparations. When M21 cells preloaded with MTX by incubation at a drug concentration of 1.0 or 10 microM were incubated in drug-free medium, the amount of cell-associated MTX rapidly declined to 1.8 pmol/mg protein, i.e., the level of intracellular dihydrofolate reductase (DHFR). However, when cells preloaded to a drug content of 112 pmol/mg protein by incubation with 10 microM MTX linked to AHMGR were transferred to conjugate-free medium, 65 pmol MTX/mg remained cell associated after 12 hours. The efflux was inhibited by chloroquine. Both the efflux medium and M21 cells after a 9.5-hour incubation period had MTX-containing catabolic fragments that inhibited DHFR.

Absorption↗
PubMed1984

Tumor and tissue distribution of a methotrexate-anti-EL4 immunoglobulin conjugate in EL4 lymphoma-bearing mice.

The uptake and tissue distribution of [3H]methotrexate [( 3H]MTX) at doses of 5 mg/kg i.p. either free or linked to anti-EL4 immunoglobulin G (AELG) or normal rabbit globulin (NRG) was studied in EL4 lymphoma-bearing C57BL/6J mice. When the uptakes of MTX-AELG, MTX-NRG, and free MTX were assayed as cell-associated 3H activity, comparison 3 hr after administration showed that uptake of MTX administered as the AELG conjugate was 2.5 times the uptake of MTX administered as the NRG conjugate and 6 times the uptake of MTX administered free. In contrast to the difference in the uptake of MTX-AELG and MTX-NRG by tumor cells, the pattern of uptake in all the other tissues studied was generally similar for the two conjugates. Conjugated MTX persisted in all tissues and serum and ascites fluid, whereas free MTX declined rapidly after reaching peak levels around 1 hr, except in EL4 cells where 45% was retained at 24 hr. The levels of intracellular MTX after administration of these three agents exceeded the intracellular dihydrofolate reductase level and correlated with the relative tumor-inhibitory effect in vivo of the agent (MTX-AELG greater than MTX-NRG greater than MTX).

Animals↗
PubMed1983

Uptake of methotrexate linked to an anti-EL4-lymphoma antibody by EL4 cells.

To study the mechanism of tumor inhibition, the uptake of methotrexate (MTX) covalently linked to a rabbit IgG antibody against a tumor-associated antigen on the surface of mouse EL4 lymphoma cells (AELG) has been compared with the uptake of free MTX and of MTX covalently linked to normal rabbit IgG (NRG). When EL4 cells were incubated at 37 degrees C with 10 microM free MTX uptake leveled off after 30 min, at 30 pmol/mg protein. In contrast, uptake of both conjugates under these conditions continued throughout an observation period of 6 h. At 6 h the net uptake of MTX bound to AELG was 40 pmol/mg protein and that of MTX bound to NRG was 24 pmol/mg protein. These results show that both MTX-AELG and MTX-NRG conjugates are taken up by EL4 cells. The rate at which EL4 cells took up bound MTX was much slower than that of free MTX but, at 6 h, the net uptake of MTX-AELG exceeded that of the free drug.

Animals↗
PubMed1982

Inhibition of a mouse hepatoma by the alkylating agent Trenimon linked to immunoglobulins.

Trenimon was conjugated in active alkylating form to rabbit anti-mouse H6 hepatoma globulin (AHG) with retention of antibody activity. H6 hepatoma-inoculated mice were given various combinations of conjugates, free Trenimon, and unconjugated immunoglobulins in daily injections for 5 days. Linkage of Trenimon to immunoglobulins reduced systemic toxicity of the drug, with comparative retention of its antitumor activity. The antitumor action of Trenimon was potentiated by AHG irrespective of whether the drug was directly linked to AHG or free AHG was administered along with Trenimon linked to normal rabbit globulin (NRG). In vitro, Trenimon bound to AHG was less inhibitory to hepatoma cells than free Trenimon, but more inhibitory than Trenimon-NRG conjugates. There was no significant endocytosis of conjugates by the hepatoma cells. This suggests that unlike free Trenimon, the target molecules of Trenimon-immunoglobulin conjugates are not intracellular DNA but are located on the surface of the hepatoma cells.

Alkylating Agents↗
PubMed1982

Imaging of human melanoma xenografts in nude mice with a radiolabeled monoclonal antibody.

External photoscanning with display of radioactivity data as a color-scaled image detected xenografts of human melanoma in male nude inbred mice of BALB/c background 48 hours after injection of 131I-labeled monoclonal IgG 225.28S that is specific for human melanoma. A 131I-labeled polyclonal goat IgG against human melanoma-associated antigens could also image the tumor, but with this preparation there was considerable localization of radioactivity in normal tissues, resulting in less satisfactory tumor definition. Labeled normal mouse IgG did not image the melanoma grafts. Assay of radioactivity in tissues of melanoma-grafted mice confirmed tumor-specific localization of the antimelanoma antibodies. The tumor:blood ratio of radioactivity was 6.55 with the monoclonal antimelanoma IgG and 0.45 with the polyclonal IgG.

Animals↗