J. A. Clegg

111In labelling of a branched polypeptide drug carrier with a poly(l-lysine) backbone

A synthetic branched polypeptide drug carrier, based on a polylysine backbone with short side chains of three dl-alanine amino acid residues with a terminal glutamic acid residue, has been labelled with 111In by DTPA chelation. 111In is a tracer suitable for gamma scintigraphy, thus broadening the techniques available for examining the biodistribution of such polymeric drug carriers.

Biodistribution and tumour localization of radiolabelled monoclonal antibody during continuous infusion in nude mice and with human tumour xenografts

The distribution in athymic nude mice of radiolabelled monoclonal antibody 791T/36 has been assessed during continuous infusion from subcutaneously implanted Alzet Osmotic Minipumps. During prolonged administration (up to 15 days) blood levels continued to rise. At 15 days, distribution of radiolabel was virtually identical to that seen after a single parenteral dose. Blood levels were in good agreement to those expected from whole body levels indicating satisfactory entry of antibody into the vascular compartment. Gel filtration chromatography of osmotic minipump contents and circulating radiolabel showed that the antibody had retained its structural integrity. In mice with human tumour xenografts examined a 5-day infusion of a mixture of 131I-791T/36 antibody and 125I-control IgG2b, blood levels of both radiolabels were comparable to those expected from whole body levels and there was effective tumour localization of the antibody to 2.5 times that of control IgG. These studies have demonstrated that prolonged administration of monoclonal antibody is feasible, that antibody enters the vascular compartment satisfactorily and that it can then localize in tumour deposits.

Biodistribution of methotrexate-monoclonal antibody conjugates and complexes: experimental and clinical studies

Hybridoma technology has opened the way to the production of monoclonal antibodies against many antigenic structures including antigens associated with a wide range of common cancers, for example, carcinomas of the colon, lung and breast and against bone tumours and malignant melanoma (3). A promising approach to cancer therapy lies in the use of these monoclonal antibodies as carriers for targeting therapeutic agents such as radionuclides, cytotoxic drugs and plant derived toxins. An additional aspect of monoclonal antibody development has been the production of antibodies against chemotherapeutic agents themselves (12, 24). These antibodies may be useful in drug assay or purification but, in addition, complexing drugs with monoclonal antibodies might allow more prolonged in vivo survival of drugs otherwise rapidly catabolized, with possible concomitant increase in therapeutic efficacy. As a further development, the use of hybrid, bispecific, monoclonal antibodies against drug and a tumour associated antigen may allow site-specific delivery of the drug provided that immune complexes of drug and monoclonal antibody survive satisfactorily in vivo. Clearly an important component of these therapeutic approaches with monoclonal antibodies is an understanding of their biodistribution and tumour localization capacity of conjugates and complexes. Although there is now a growing body of data on the distribution of monoclonal antibodies in experimental systems, and also clinically (3), the biokinetics of conjugated or complexed antibodies may be totally different, and this needs to be investigated. The present studies, carried out with methotrexate (MTX), have examined the biodistribution of drug-ant ibody conjugates and immune complexes in animal models and, to a limited extent, clinically.

Suppression of antibody responses to ricin A chain (RTA) by monoclonal anti-RTA antibodies

Balb/c mice treated with an immunotoxin constructed by conjugation of murine monoclonal antibody 791T/36 via a disulfide linker to ricin A chain generate a pronounced antibody response to peptide epitopes on ricin A chain. Monoclonal anti-RTA antibodies which recognize peptide epitopes have been developed and these have been used to down-regulate anti-RTA antibody responses in 791T/36-RTA immunotoxin-treated Balb/c mice. Of the five MAB tests, two (608/7 and 596/134) proved most effective, inhibiting anti-RTA antibody formation by up to 73%. MAB treatment was effective when initiated up to 3 days after immunotoxin treatment. Pharmacokinetic studies with 791T/36-RTA have shown that the immunotoxin is rapidly eliminated from the circulation, with no more than 4% remaining in blood after 24 hr. It is proposed that the down-regulation of anti-RTA antibodies is effected by MAB interfering with antigen processing.

Comparative biodistribution of methotrexate and monoclonal antibody-methotrexate complexes in mice

The biodistribution of radiolabelled methotrexate and immune complexes of methotrexate and a murine monoclonal anti‐methotrexate antibody has been compared in mice. Complexes formed in‐vitro with the antibody, but not with control immunoglobulin. The complexes were, characteristically, acid labile. In‐vivo, blood levels, organ distribution and whole body catabolism of methotrexate in immune complexes were similar to those of free antibody, and markedly different from those of free drug. These findings suggest the feasibility of prolonging the survival of drugs and altering in‐vivo distribution using complexes with monoclonal antibodies.

Regulation of the contact sensitivity response to urushiol with anti-urushiol monoclonal antibody ALG 991.

Abstract The objective of the studies was to demonstrate that the contact sensitivity (CS) response to poison ivy/oak could be downregulated following treatment with a monoclonal antibody (mAb) reacting with the allergen urushiol. Conjugation of urushiol and its synthetic analogue 3-n-pentadecylcatechol (PDC) to ¶ N -acetylcysteine yielded hydrosoluble derivatives which induced humoral immune responses in BALB/c mice. Hybridomas secreting monoclonal antibodies (mAbs) reacting with urushiol and PDC were generated by fusion of B lymphocytes from immunized mice with mouse myeloma P3NS0 cells. The specificity of mAb ALG 991 (IgM isotype) was defined by inhibition of antibody binding by PDC analogues. This demonstrated that mAb ALG 991 reacted with the catechol moiety of urushiol, the region of the allergen being critically important in the induction of contact dermatitis. The CS response to urushiol in BALB/c mice was suppressed by stimulation with mAb ALG 991 and the role of sensitized T cells, including suppressor T cells, has been considered. Suppression of CS was most effective with low doses (1 μg) of mAb incorporated into a vaccine with Freund’s adjuvant. This treatment suppressed CS responses in BALB/c mice already sensitized to urushiol.

Synthesis and characterization of p-borono-phenylalanine-branched polypeptide-monoclonal antibody ternary systems for potential use in boron neutron capture therapy (BNCT)

The application of the 10B (n,α) 7Li capture reaction to cancer radiotherapy (Boron Neutron Capture Therapy) was studied to avoid the inherent disadvantages of conventional radiation therapy. p-Borono-phenylalanine (Bph) was used as the 10B source and mAb produced against HCMB melanoma cells was applied as targeting device. Since extensive direct boronation of mAb led diminished recognition of antigens, an intermediate carrier was used. Nontoxic, biocompatible, biodegradable and weakly immunogenic branched polypeptides with a polylysine backbone was used to carry a high number of 10B. Protected 10B-Bph was coupled by four different methods to polycationic branched polypeptides. The coupling efficiency varied according to the experimental conditions, with a maximum of 90%. The chiroptical properties of the conjugates indicated an ordered conformation which increased with the number of coupled Bph. The whole body survival (WBS) and tissue distribution profile of mAb (8/6 IgG2a) were markedly altered after conjugation with Bph-branched polypeptide. Decreased WBS and intermediate-carrier-dependent accumulation in the spleen, liver and kidney was observed 24 h after iv. administration. After joining only a few chains of the highly loaded Bph-AK conjugate to mAb, the binding activity of the mAb in the ternary system was preserved compared to control.

Biodistribution and Therapeutic Efficacy of a Monoclonal Antibody-Methotrexate Conjugate in Mice

A human tumour xenograft model has been used to assess the biodistribution and therapeutic potential of a methotrexate human serum albumin monoclon al antibody conjugate. The conjugate significantly retarded growth of a colon carcinoma xenograft whereas the same dose of free methotrexate was ineffective. Biodistribution studies with radiolabelled conjugate showed rapid clearance from plasma, particularly into the liver and spleen, suggesting that alternative conjugates with longer survival might show even greater therapeutic effects.