An in vivo model for screening peptidomimetic inhibitors of gelatinase A.

Gelatinase A, a matrix metalloproteinase, is frequently associated with human solid tumors, and its secretion and activation in the tumor milieu is considered important in the process of angiogenesis, invasion, and metastasis. Consequently, metalloproteinase inhibitors may be of value in the therapy of cancer as well as other disease states involving tissue remodeling and release of biologically active peptide/protein by proteolytic cleavage. Here we describe the development of a rapid screening assay for in vivo activity of peptidomimetic inhibitors of gelatinase A that involves assessment of inhibition of an enzyme-substrate reaction in a circumscribed body compartment, the mouse pleural cavity. As examples of the utility of this assay, in vivo activity of the aryl sulfonamide, sulfamyl urea, morpholino and carboxylic acid functionality at the P3' position of a series of hydroxamic acid inhibitors was examined after administration both intraperitoneally (ip) (to approximate systemic administration) and orally. For up to 2 h after ip administration, all inhibitors tested showed marked activity (> 90% inhibition) at 17 mumol/kg (approximately 10 mg/kg). This activity declined in a dose-responsive manner to insignificant levels at 0.67 mumol/kg (approximately 0.4 mg/kg). Aryl sulfonamides showed significant inhibition (> 50%) for up to 7 h after administration. A higher dosage (136 mumol/kg, approximately 80 mg/kg) was required to reveal oral activity, which was observed only with morpholino compounds (> 50% inhibition). Thus, the model described may be of value in the identification of orally active gelatinase A inhibitors.

Improved tumor targeting with chemically cross-linked recombinant antibody fragments.

The construction and use of recombinant chimeric and later fully humanized (CDR-grafted) antibodies to tumor-associated antigens has reduced the immune response generated to these antibodies in clinical studies. However, their long circulating half-life is a disadvantage for tumor imaging and therapy. Fragments such as F(ab')2, Fab', Fv and single chain Fv (scFv) offer faster blood clearance but also lower overall tumor doses. We have examined the tumor targeting of several novel fragments produced by chemical cross-linking of Fab' or scFv to dimeric and trimeric species. To facilitate cross-linking of Fab' fragments, a chimeric B72.3 Fab' fragment has been expressed with a hinge sequence containing a single cysteine residue. B72.3 scFv was also produced with a similar hinge region peptide attached to the COOH terminus to allow cross-linking. These fragments, Fab' delta Cys and scFv' delta Cys were cross-linked with linkers containing two or three maleimide groups to produce dimeric and trimeric molecules with increased avidity for antigen. Cross-linkers were also designed to contain a 12-N-4 macrocycle capable of stable radiolabeling with 90Y. This allowed the production of site-specifically-labeled, fully immunoreactive proteins. Biodistribution studies in the nude mouse LS174T xenograft model with scFv, di-scFv, and tri-scFv demonstrated that these fragments clear extremely rapidly from the circulation and give rise to only low levels of activity accumulated at the tumor. Di-Fab (DFM) and tri-Fab (TFM) however, accumulated relatively high levels of activity at the tumor with high tumor:blood ratios generated, demonstrating improved targeting compared to IgG. cB72.3 90Y-labeled tri-Fab was found not to accumulate in the kidney or the bone, resulting in an attractive antibody fragment for tumor therapy.

Structural studies on the [Bu(t)-Cys18](19-37)-fragment of human beta-calcitonin-gene-related peptide.

High-field n.m.r. studies were undertaken upon a peptide fragment of the C-terminal region of human beta-calcitonin-gene-related peptide (beta-hCGRP). Studies on the antigenic [Bu(t)-Cys18]beta-hCGRP-(19-37)-fragment revealed that several elements of secondary structure were present when the peptide was dissolved in [2H6]dimethyl sulphoxide. In particular an unspecified turn in the region of Ser19-Gly20 and a type I beta-turn in the region of Asn31-Val32-Gly33 were identified. Through-space connections between the terminal Phe37 amide group and the beta-protons of Thr50 suggest that the peptide may be folded into a loop-type conformation. These structural elements appear to overlap with the epitopes of a number of monoclonal antibodies and provide a molecular basis for understanding the role of the terminal Phe37 amide residue in the immune recognition of beta-hCGRP.

The in vivo release of 90Y from cyclic and acyclic ligand-antibody conjugates.

Yttrium binding ligands DOTA, caDTPA and CT-DTPA were each conjugated to monoclonal antibody B72.3, labelled with 90Y and injected into mice in order to assess the in vivo inertness of the antibody-linked 90Y-ligand complexes. Levels of 90Y in femur shafts of the DOTA-B72.3 mice were low, being approximately 7 and 44%, respectively, of levels in the femur shafts of the caDTPA-B72.3 and CT-DTPA-B72.3 treated mice. This finding demonstrates the greater inertness and by implication the greater suitability for immunotherapy of the DOTA-90Y complex.