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Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications.

Identification of neo-antigenic determinant(s) on parasite infected cell surface is important to control intracellular infections. Such determinant(s) on the surface of intact Plasmodium berghei infected erythrocytes have not been conclusively demonstrated. To generate polyclonal antiserum selectively recognizing the parasite infected cell surface determinant(s), in natural state, we have examined the efficacy of the homologous immunizations, in BALB/c mice, with the membrane rich preparation of: i) erythrocytes in vivo infected with Plasmodium berghei and, ii) macrophages in vitro infected with Leishmania donovani. Anti-infected erythrocyte membrane antiserum specifically recognized, albeit at low level, the infected cell surface as determined by flow cytometry and immunoelectron microscopy. Immunoprecipitation of radiolabeled antigens revealed at least three parasite proteins of > 205 kDa, 160 kDa and 100 kDa specifically present on infected erythrocyte surface. Normal uninfected erythrocytes did not react with the antiserum. Anti-L. donovani-infected macrophage membrane antiserum also recognized only infected macrophage surface and not the normal macrophages. Thus, the approach may find wide application in delineating disease specific determinant(s) on the infected cell surface, particularly to those where animal models are available.

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Many cancer and diseased cells are distinguished from their normal counterparts by an altered expression of cell-surface epitopes. One family of molecules that show altered expression on tumor cells is mucins (MUC). Unlike normal tissue where MUC exists as heavily glycosylated form, the disease- or tumor-associated MUC molecules are underglycosylated. Such underglycosylation of the core protein in cancer tissues exposes new epitopes on the cell surface that are unique to cancer tissues. Several monoclonal antibodies (Mabs) have been generated against these normal and tumor-associated mucins. Enzymatic fragments of Mabs like F(ab')2 and Fab have shown improved clinical utility for diagnosis, imaging, and therapy of cancer. Genetic-engineering methods have been used to design antibody fragments exhibiting high functional affinity, good tumor localization, and rapid clearance from the blood stream thus minimizing radiation exposure to the normal tissues. Such recombinant fragments have shown encouraging results in preclinical studies using xenografted tumor bearing mice and present a whole new avenue for radioimmunotherapy and diagnosis of cancer.