T. Ghose

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

SummaryMethotrexate (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)2antiBSA 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

Inhibition of human renal cancer by monoclonal antibody targeted methotrexate-containing liposomes in an ascites tumor model

The monoclonal antibody DAL K29 against a human renal cell carcinoma associated cell surface antigen was covalently linked to small unilamellar lipid vesicles (SUV) containing the antifolate, methotrexate (MTX), with full retention of antibody activity. In an ascites tumor model developed after intraperitoneal inoculation of 5 x 10(6) cells of the human kidney cancer line Caki-1 per pristane primed nude mouse, the DAL K29 linked MTX-containing SUV was a more potent tumor inhibitor (P less than 0.0005) than the drug or MAB alone, MTX-containing SUV, a mixture of DAL K29 and MTX-containing SUV or MTX-containing SUV linked to an isotype matched nontumor specific IgG.

Synthesis of site-specific methotrexate-IgG conjugates

SummaryWith 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° C and 51° C, and in the presence of serum or rat liver homogenates. Exposure to rat liver homogenates at 37° 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° 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.

Uptake of methotrexate linked to an anti-el4-lymphoma antibody by el4 cells.

SummaryTo 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°C with 10 μM 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.

Binding of Chlorambucil with Antitumor Globulins and its Effect on Drug and Antibody Activities

Abstract A method for noncovalently binding chlorambucil to antitumor globulins (ATG) at a pH of about 2·0 is described. Binding of chlorambucil to ATG by this method did not impair either the alkylating activity of chlorambucil or the immunological reactivity of ATG and led to more consistent binding and better retention of both drug and antibody activities than binding at alkaline pH. Variations of temperature between 0°–37° C had no effect on the binding. Gammaglobulins from different species showed different patterns of binding with chlorambucil. Chlorambucil to y-globulin ratio and the concentration of chlorambucil in the reaction mixture were important factors in determining the amount of drug which bound to ATG. Up to 3000 moles of chlorambucil could be bound to each mole of a goat anti-EL 4 IgG, however, there was substantial reduction of antibody activity when 20 moles of chlorambucil bound to a mole of the ATG. Binding of chlorambucil also changed the immunoelectrophoretic mobility of the ATG. Chlorambucil bound anti-EL 4 globulin carried more chlorambucil to target EL 4 cells in vitro than equivalent amounts of chlorambucil bound to normal goat globulin.

Immunoprophylaxis and immunotherapy of EL4 lymphoma

Abstract Injections of appropriate numbers of irradiated, but not iodoacetate treated, EL4 lymphoma cells into C57BL/6J mice were effective in immunoprophylaxis against 106 EL4 cells and in immunotherapy against 102 EL4 cells per mouse. The addition of BCG injections made immunotherapy with irradiated EL4 cells effective against a load of 104 EL4 cells/mouse. Though inoculation of BCG into BALB/c mice inhibited the growth of the allogeneic Ehrlich carcinoma, immunoprophylaxis with BCG only was ineffective against EL4 in C57BL/6J mice. Coating irradiated EL4 cells with concanavalin A did not increase their immunogenicity. Immunotherapy with vibrio cholera neuraminidase and mitomycin treated EL4 cells or with sensitized syngeneic lymphoid cells did not protect EL4 inoculated C57BL/6J mice. However, injections of several rabbit anti-EL4 globulins completely suppressed EL4 lymphoma in a proportion of mice even when treatment was started 120 hr after i.p. inoculation of 104 EL4 cells per mouse. When administered within 72 hr of tumour inoculation 131I or chlorambucil noncovalently bound to nontumour inhibitory anti-EL4 globulins were at least as effective tumour inhibitors as the inhibitory anti-EL4 globulins.

Tumor inhibition by chlorambucil covalently linked to antitumor globulin

Abstract The alkylating agent chlorambucil has been covalently bound to antitumor globulins directed against cell surface antigen(s) of the mouse EL 4 lymphoma. The resulting conjugates retained alkylating and antibody activities and were used to treat C 57 BL 6 j mice after prior inoculation with the syngeneic EL 4 lymphoma. Forty per cent of mice treated with the covalent conjugate, 78 % of mice treated with non-covalent conjugate and 50 % of mice treated with chlorambucil followed by antitumor globulin survived tumor-free for more than 100 days . Untreated mice had a mean survival of 18 ± 1.3 days and the groups that received chlorambucil or antitumor globulin only, or chlorambucil covalently bound to normal rabbit globulin died within 30 days of tumor inoculation.

Inhibition of Epstein-Barr-virus-transformed human chronic lymphocytic leukaemic B cells with monoclonal-antibody-adriamycin (doxorubicin) conjugates

The anthracyclin antineoplastic agent doxorubicin (Adriamycin) was linked by four different methods of linkage to DalB02, an IgG1κ murine monoclonal antibody (mAb) against surface-associated antigens on human chronic lymphocytic leukaemia (CLL) B cells. All the four conjugates fully retained the immunoreactivity of the parent DalB02. When the inhibitory effect of these conjugates was evaluated in vitro against the target D10–1 cells (a clone derived from an Epstein-Barr-virus-transformed human CLL B cell line that binds DalB02) it was observed that one conjugate was more potent than the free drug but the others were not. When131I-labelled unmodified DalB02 and the131I-labelled DalB02-containing conjugate that was found to be potent were injected i.v. into nude mice bearing a subcutaneous D10–1 xenograft, the percentages of the injected dose (%ID) of both131I-DalB02 and the131I-DalB02-containing conjugate that localized in the tumour were much higher than the %ID of the respective preparations that localized in normal tissues of D10–1-xenografted mice. The systemic toxicity of the conjugate was less than that of the free drug. At an equitoxic dose level, this conjugate was a more effective inhibitor of established D10–1 xenografts than the free drug.

Tumor localization and therapeutic potential of an antitumor-anti-CD3 heteroconjugate antibody in human renal cell carcinoma xenograft models

A heteroconjugate (HC) antibody, constructed with the monoclonal antibody (MoAB) Dal K29 to human renal cell carcinoma (RCC) and an anti-CD3 MoAb, could induce a very high level of lysis of human RCC cells when incubated with human peripheral blood lymphocytes (PBL) in vitro (Kerr et al., 1990, J. Immunol., 144, 4060-4067). We now report that this HC antibody selectively localizes in RCC xenografts in nude mice and could inhibit RCC in an ascites tumor xenograft model when administered intraperitoneally together with PBL.

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

SummaryThe 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 × 106 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 <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).