Cytotoxic properties of immunoconjugates containing melittin-like peptide 101 against prostate cancer: in vitro and in vivo studies.

BACKGROUND: Monoclonal antibodies (MAbs) can target therapy to tumours while minimising normal tissue exposure. Efficacy of immunoconjugates containing peptide 101, designed around the first 22 amino acids of bee venom, melittin, to maintain the amphipathic helix, to enhance water solubility, and to increase hemolytic activity, was assessed in nude mice bearing subcutaneous human prostate cancer xenografts. METHODS: Mouse MAbs, J591 and BLCA-38, which recognise human prostate cancer cells, were cross-linked to peptide 101 using SPDP. Tumour-bearing mice were used to compare biodistributions of radiolabeled immunoconjugates and MAb, or received multiple sequential injections of immunoconjugates. Therapeutic efficacy was assessed by delay in tumour growth and increased mouse survival. RESULTS: Radiolabeled immunoconjugates and antibodies showed similar xenograft tropism. Systemic or intratumoural injection of immunoconjugates inhibited tumour growth in mice relative to carrier alone, unconjugated antibody and nonspecific antibody-peptide conjugates and improved survival for treated mice. CONCLUSIONS: Immunoconjugates deliver beneficial effects; further peptide modifications may increase cytotoxicity.

Biodistributions of intact monoclonal antibodies and fragments of BLCA-38, a new prostate cancer directed antibody.

BACKGROUND: Monoclonal antibodies (MAbs) are used for targeting agents to tumours while minimizing normal tissue exposure. METHODS: A new anti-prostate cancer MAb, BLCA-38, was radioiodinated (I125) and assessed for its ability to target subcutaneous human prostate cancer (DU-145) xenografts after systemic intraperitoneal administration. For comparison, the profile of J591 MAb (now in clinical trial) against LNCaP-LN3 tumours was examined. Biodistribution profiles were obtained at various times, by assessing injected dose/gram (%ID/g) and xenograft to blood (X/B) ratios. Microautoradiography of xenografts was performed. After conjugation with a melittin peptide toxin, the profiles of BLCA-38 and J591 were compared with that of an irrelevant antibody, DS-1. RESULTS: Xenograft localization by 125I-labeled BLCA-38 and J591 MAbs to their relevant antigen-positive tumors was comparable, and there was no unusual localization in nontumour tissues. F(ab')2 and Fab fragments gave improved X/B ratios, but the %ID/g xenograft was decreased and they accumulated in kidneys, bladder and stomach. In contrast, the conjugates of irrelevant antibody showed no tumour targeting. Microautoradiography showed more tumour accumulation of MAbs than F(ab')2s or Fabs. CONCLUSIONS: BLCA-38 can target prostate cancer in vivo almost as effectively as J591. Given that J591 is used clinically, BLCA-38, which targets a different antigen, has potential for radioimmunoscintigraphy and for therapeutic targeting of prostate cancer.

Noncovalent scFv multimers of tumor-targeting anti-Lewis(y) hu3S193 humanized antibody.

Single-chain variable fragments (scFvs) of anti-Lewis(y) hu3S193 humanized antibody were constructed by joining the V(H) and V(L) domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V(H) domain were removed (-1 residue, -2 residue) and then joined directly to the V(L) domain. An scFv construct in the reverse orientation with the V(L) joined directly to the V(H) domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.

Single-chain Fv multimers of the anti-neuraminidase antibody NC10: the residue at position 15 in the V(L) domain of the scFv-0 (V(L)-V(H)) molecule is primarily responsible for formation of a tetramer-trimer equilibrium.

Single-chain variable fragment of the murine monoclonal antibody NC10 specific to influenza virus N9 neuraminidase, joined directly in the V(L) to V(H) orientation (scFv-0), forms an equilibrium mixture of tetramer and trimer with the tetramer as the preferred multimeric species. In contrast, the V(H)-V(L) isomer was previously shown to exist exclusively as a trimer. Computer-generated trimeric and tetrameric scFv models, based on the refined crystal structure for NC10 Fv domain, were constructed and used to evaluate factors influencing the transition between V(L)-V(H) trimer and tetramer. These model structures indicated that steric restrictions between loops spanning amino acid residues L55-L59 and L13-L17 from the two adjacent V(L) domains within the V(L)-V(H) trimer were responsible for four scFv-0 molecules assembling to form a tetramer. In particular, leucine at position L15 and glutamate at position L57 appeared to interfere significantly with each other. To minimize this steric interference, the site-directed mutagenesis technique was used to construct several NC10 scFv-0 clones with mutations at these positions. Size-exclusion chromatographic analyses revealed that several of these mutations resulted in the production of NC10 scFv-0 proteins with significantly altered tetramer-trimer equilibrium ratios. In particular, introduction of a polar residue, such as asparagine or threonine, at position L15 generated a highly stable NC10 scFv-0 trimer.

Large-scale bacterial fermentation and isolation of scFv multimers using a heat-inducible bacterial expression vector.

This protocol describes optimised large-scale bacterial fermentation conditions for recombinant single-chain Fv molecule (scFv) monomers and multimers (diabodies and triabodies). The heat-inducible bacterial secretion vector, pPOW3, utilising the temperature-regulated tandem lambda promoters is particularly suited to the large-scale fermentation of single-chain antibodies, providing low-cost recombinant protein synthesis. The protein expressed by this vector is secreted into the periplasm where it is found as both the soluble and insoluble protein that is associated with the cell membranes. A protein fractionation method for the rapid extraction and affinity purification of the soluble protein fraction and the urea solubilization and refolding of the insoluble protein fraction expressed from single-chain antibody (Ab) fragment gene constructs is described. This method is simple to perform and utilises inexpensive reagents to provide cost-effective protein synthesis.

A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis.

The new antigen receptor (NAR) from sharks consists of a single immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antibody. Two closely related NARs with affinity for the Kgp (lysine-specific) gingipain protease from Porphyromonas gingivalis were selected by panning an NAR variable domain library. When produced in Escherichia coli, these recombinant NARs were stable, correctly folded, and specifically bound Kgp (K(d)=1.31+/-0.26x10(-7) M). Binding localised to the Kgp adhesin domains, however without inhibiting adhesin activity. These naturally occurring proteins indicate an immune response to pathogenic bacteria and suggest that the NAR is a true antibody-like molecule.