Tarun Ghose

[20] Preparation of antibody-linked cytotoxic agents☆

Publisher Summary This chapter presents the preparation of antibody-linked cytotoxic agents. It discusses the strategy of linking cytotoxic agents to immunoglobulins so that biologically active conjugates retaining agent and antibody activities are produced. Immunoglobulins share a number of distinctive structural features, while exhibiting enormous structural diversity compared to other biologically active proteins. Distinctive features include (a) a tetrameric arrangement of two light (L) chains (Mr 23,000) and two heavy (H) chains (Mr 50,000-70,000) linked by disulfide bridges and (b) the division of each dimer made up of one L and one H chain into variable (V) (antigen binding) and constant (C) regions. The V region occurs near the amino terminal end of the immunoglobulin chain. Immunoglobulins of different classes differ considerably in the amino acid sequence of the C segment of their H chains, in chain length, in the number of homologous domains in the chain, in the disulfide bridge pattern, in the degree of polymerization, and in the position, numbers, and kind of oligosaccharides. When L or H chains from the same class and species are compared, the C region is found to contain an invariant sequence characteristic of the class and species, whereas the V region shows multiple substitutions ranging from 10 to 60 in the first 110 residues.

Immunochemotherapy of human malignant melanoma with chlorambucil-carrying antibody

Abstract Heterologous anti-melanoma sera produced against any one patients tumour cross-react, to varying degrees, with both the cytoplasmic and surface antigens of melanoma cells from other patients. Intravenous injections of cross-reacting antimelanoma antibodies bound to chlorambucil were followed by the regression of a considerable number of skin metastases in a patient with disseminated malignant melanoma. When immunochemotherapy had to be discontinued because of anaphylaxis, many of the metastases which had almost completely regressed, reappeared and grew progressively in spite of chemotherapy. One metastatic nodule which had not regressed and another which appeared during immunochemotherapy contained a much larger proportion of tumour cells lacking the cross-reacting surface antigen, when compared to tumour cells in metastatic nodules excised before immunochemotherapy.

Antibody as carrier of 131I in cancer diagnosis and treatment

Cell‐surface localizing heterologous antibodies against mouse EL4 lymphoma, Ehrlich ascites carcinoma, and several human malignant tumors could be bound to varying amounts of131I without interfering with the reactivity of these antibodies with their respective tumor cells. Exposure of the mouse tumor cells to radio‐iodinated antitumor antibodies in vitro, or the injection of radioiodinated antitumor antibodies into mice preinoculated with tumor cells resulted in either partial or complete tumor inhibition depending upon the amount of 131I activity carried by the antibodies. Injecten of comparable amounts of the immunoglobulin alone or of 131I bound to normal globulin did not cause any tumor inhibition. Intraperitoneally injected radio‐iodinated anti‐EL4 antibody was found to localize preferentially in the subcutaneous transplants of EL4 lymphoma. Similar localization of intravenously injected radio‐iodinated antibodies was observed in the metastases of two cancer patients.

Radionuclide imaging of metastases from renal-cell carcinoma by 131I-labeled antitumor antibody.

On immunofluorescence assay, goat antisera reacted with renal-cell carcinomas from 20 patients, but not with adult normal human tissues (including patients normal kidney). After in travenous injection, the 131I-labeled antibody localized preferentially in metastatic renal carcinoma in 6 consecutive patients. Labeled antitumor antibodies may have the specificity for tumor imagining which current radiopharmaceuticals lack.

Tumor Localization of 131I-Labeled Antibodies by Radionuclide Imaging

Intravenous injections of 131I-Iabeled anti-EL4 lymphoma antibodies showed progressive localization of radioactivity in EL4 transplants but not in B16 melanoma in mice carrying both tumors. Normal rabbit globulin labeled with 131I did not localize in either tumor and cleared more slowly from the internal organs. Metastatic localization of intravenous 131I-labeled anti-tumor antibodies was also observed in 2 cancer patients.

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

SummaryIntravenous 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 × 106 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 isotypematched 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.

Induction of MHC class II expression in recipient tissues caused by allograft rejection

MHC class II antigens (DR) are not commonly expressed on parenchymal cells of kidney and liver except when they are allografts undergoing rejection. The objective of this study was to determine whether allograft rejection can also induce DR upregulation in parenchymal cells of autologous recipient organs. Dogs had unilateral renal autografts to facilitate kidney sampling. All kidneys were tubular cell DR-negative. After 8-14 days each dog received a tubular cell DR-negative allograft. Tubular cell DR became positive in both allograft and autograft simultaneously, its onset and intensity correlating with blast cell infiltration and rejection in the allograft. Blast cells were first detected in the autograft after allograft nephrectomy, and then disappeared as autograft tubular cell DR diminished over the next 6-8 days. This was reproduced on repeat allografting. In 2 untreated dogs hepatocytes became positive on day 4, with no hepatic blast infiltrate. Four other dogs received cyclosporine immunosuppression. Allograft and autograft tubular cell DR, and hepatocyte DR, increased in all dogs, but were delayed while on CsA until onset of rejection despite transient earlier allograft blast infiltration. Downregulation in autograft and liver occurred together after allograft nephrectomy. An interferon-like substance appeared in plasma after allografting in association with the DR changes in native kidney and liver. Renal allorejection therefore induces upregulation of parenchymal DR expression in autologous liver and kidney of the recipient. It is probably mediated by an interferon-like substance derived from cells infiltrating the allograft. The effect is modified by CsA.

Covalent binding of antibodies to liposomes using a novel lipid derivative

N‐[3−(2−Pyridyldithio) propionyl] stearylamine (PDP‐SA) was synthesized from a reaction between stearylamine and the heterobifunctional reagent N‐succinimidyl−3−(2−pyridyldithio) propionate. Use of this PDP‐SA to covalently couple antibodies to liposomes was investigated. The binding efficiency was found to be 24–32%. The antibodies bound to liposomes were shown to retain the specific antibody activity. This new procedure of coupling antibodies to lipsosomes could be an efficient means to deliver drugs to selected target organs, especially in cancer chemotherapy.

Inhibition of human renal cancer by methotrexate linked to a monoclonal antibody.

The monoclonal antibody DAL K29 against a human renal cell carcinoma associated cell surface antigen was covalently linked to the antifolate methotrexate with full retention of antibody reactivity and partial retention of drug activity. In a colony inhibition assay, antibody-conjugated methotrexate was 400% more potent in inhibiting the growth of the human kidney cancer line Caki-1 than equimolar amounts of the free drug. Comparable amounts of the antifolate linked to normal mouse IgG did not inhibit the growth of Caki-1 cells. Furthermore, the methotrexate-DAL K29 conjugate had no effect on the two nontarget human cell lines tested, melanoma M21 and B cell leukemia D10-1 cells, even when the conjugate contained amounts of methotrexate equivalent to the 50% inhibitory concentration of the free drug for the nontarget cell lines or an amount equivalent to the 50% inhibitory concentration of the conjugated drug for Caki-1 cells.

Antibody directed targeting of methotrexate-containing small unilamellar vesicles

SummaryThe potential of antibody-linked SUVs containing MTX in anticancer therapy was investigated. The SUVs, mean diameter 50±20 nm, were prepared by probe sonication of MTX-containing MLVs and were covalently linked either to a RAMG or NRG. After incubation with M21 melanoma cells for 2 h, RAMG-linked SUVs showed 2 and 4 times more binding than NRG-linked MTX-containing SUVs or MTX-containing SUVs unlinked to any Ig. Furthermore, on incubating M21 melanoma cells with RAMG-linked 3H MTX-containing SUVs for 2, 4, and 8 h at 4° C or 37° C, a higher radioactivity was associated with cells at 37° C than at 4° C. Membrane immunofluorescence revealed aggregation of and cap formation by RAMG-linked SUVs after 2 h (37° C) and endocytosis at 4 and 8 h at 37° C. Electron microscopic and autoradiographic studies confirmed aggregation of 3H MTX-containing SUVs around and on the surface of M21 cells. Electron microscopy also revealed these SUVs inside invaginations of and under the plasma membrane of melanoma cells. A colony inhibition assay showed that RAMG-linked, MTX-containing SUVs were 60 times, 8 times, and 4.5 times more growth inhibitory than free MTX, NRG-linked MTX-containing SUV, and MTX-containing SUVs unlinked to any Ig, but not toxic to a human kidney cancer line (that did not react with RAMG).