Prolonged in vivo residence times of antibody fragments associated with albumin

Antibody fragments can be expressed at a high level in microbial systems, but they may have limited therapeutic value because they are rapidly eliminated from the body. We demonstrate here that site-specific conjugation or binding of bacterially derived Fab' to the long-lived protein serum albumin allows full retention of the antibody's binding characteristics while imparting the albumin's longevity in vivo. In rats the area under the curve for Fab' conjugated to rat serum albumin was 17-fold greater than for the control of Fab' conjugated to cysteine. Again, a bispecific F(ab')(2) with specificity for rat serum albumin showed an area under the curve about 8-fold greater than did a F(ab')(2) without specificity to albumin. Genetic fusions of scFv to albumin were similarly long-lived and could be expressed in yeast to provide the basis of a cost-effective production system.     

The prolonged half-lives of new erythropoietin derivatives via peptide addition

Erythropoietin, or Epo, is a hematopoietic cytokine that promotes erythropoiesis, and recombinant human Epo has been used in the treatment of anemia in various chronic diseases. Here, we have constructed novel Epo derivatives with prolonged half-lives by adding peptides to the carboxy terminus of Epo without using linkers. The fused peptides were selected from the carboxy terminal region of human chorionic gonadotropin (hCG) or human thrombopoietin (hTpo), which promote the proper folding, secretion, and stabilization of bioactive glycoproteins. Addition of these peptides did not interfere with secretion or receptor binding, and significantly increased the in vivo half-life of human Epo, as measured by intravenous administration in rats. The plasma half-life of the Epo constructs was longest when the carboxy terminal 28 aa of the beta subunit of hCG was added (Epo-CGC), a half-life that was slightly longer than NESP (Aranesp), which is the most effective Epo product in current clinical use. The transformation of four Ser glycosylation sites to Ala on the CGC sequence also lengthened the plasma half-life of Epo, indicating that the in vivo stabilizing effect of the hCG peptide was due to both structures within the peptide itself and its O-glycosylations. The application of the carboxy terminal half of hTpo also resulted in remarkably reduced elimination of the Epo chimera (Epo-TpC), possibly due to protection by the TpC sequence. The in vivo hematopoietic activity of Epo derivatives in mice was consistent with their pharmacokinetic profiles. Therefore, these derivatives with prolonged half-lives may provide opportunities for developing new Epo therapeutics with less frequent administration.     

Retroviral vectors displaying functional antibody fragments.

We have made retrovirus particles displaying a functional antibody fragment. We fused the gene encoding an antibody fragment directed against a hapten with that encoding the viral envelope protein (Pr80env) of the ecotropic Moloney murine leukemia virus. The fusion gene was co-expressed in ecotropic retroviral packaging cells with a retroviral plasmid carrying the neomycin phosphotransferase gene (neo), and retroviral particles with specific hapten binding activities were recovered. Furthermore the hapten-binding particles were able to transfer the neo gene and the antibody-envelope fusion gene to mouse fibroblasts. In principle, the display of antibody fragments on the surface of recombinant retroviral particles could be used to target virus to cells for gene delivery, or to retain the virus in target tissues.     

Specific labeling of protein domains with antibody fragments

Monovalent antibody Fab fragments, prepared from antisera raised against two different types of crystalline arrays made of either intact, or a proteolytic fragment of bacteriophage T4 major capsid protein, gp23*, were employed to stoichiometrically label different gp23* protein domains on the outer surface of a tubular variant (i.e., "polyheads") of bacteriophage T4 capsids. Computer filtrations of both negatively stained and freeze-dried/metal-shadowed specimens permitted approximate mapping of the Fab binding sites within the capsomere of the polyheads.     

Therapeutic antibody engineering

It is an important event in any knowledge area when an authority in the field decides that it is time to share all accumulated knowledge and learnings by writing a text book. This does not occur often in the biopharmaceutical industry, likely due to both the highly dynamic environment with tight timelines and policies and procedures at many pharmaceutical companies that hamper knowledge sharing. To take on a task like this successfully, a strong drive combined with a desire and talent to teach, but also an accommodating and stimulating environment is required. Luckily for those interested in therapeutic monoclonal antibodies, Dr. William R. Strohl decided about two years ago that the time was right to write a book about the past, present and future of these fascinating molecules. Dr. Strohl’s great expertise and passion for biotechnology is evident from his life story and his strong academic and industry track record. Dr. Strohl pioneered natural product biotechnology, first in academia as a full professor of microbiology and biochemistry at Ohio State University in Columbus, Ohio and later in industry while at Merck. Despite his notable advances in recombinant natural products, industry interest in this area waned and in 2001 Dr. Strohl sought new opportunities by entering the field of antibody therapeutics. He initiated antibody discovery through phage display at Merck, and then moved to Centocor Research and Development Inc. (now Janssen Biotech, Inc.) in 2008 to head Biologics Research, where he now directs the discovery of innovative therapeutic antibody candidates.     

Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody.

We have determined the structure of a human rhinovirus (HRV)-Fab complex by using cryoelectron microscopy and image reconstruction techniques. This is the first view of an intact human virus complexed with a monoclonal Fab (Fab17-IA) for which both atomic structures are known. The surface area on HRV type 14 (HRV14) in contact with Fab17-IA was approximately 500 A2 (5 nm2), which is much larger than the area that constitutes the NIm-IA epitope (on viral protein VP1) defined by natural escape mutants. From modeling studies and electrostatic potential calculations, charged residues outside the neutralizing immunogenic site IA (NIm-IA) were also predicted to be involved in antibody recognition. These predictions were confirmed by site-specific mutations and analysis of the Fab17-IA-HRV14 complex, along with knowledge of the crystallographic structures of HRV14 and Fab17-IA. The bound Fab17-IA reaches across a surface depression (the canyon) and meets a related Fab at the nearest icosahedral twofold axis. By adjusting the elbow angles of the bound Fab fragments from 162 degrees to 198 degrees, an intact antibody molecule can be easily modeled. This, along with aggregation and binding stoichiometry results, supports the earlier proposal that this antibody binds bivalently to the surface of HRV14 across icosahedral twofold axes. One prediction of this model, that the intact canyon-spanning immunoglobulin G molecule would block attachment of the virus to HeLa cells, was confirmed experimentally.     

Immunocytochemical localization of simian virus 40 T antigen with peroxidase-labeled antibody fragments.

T antigen was localized in simian virus 40 lytically infected and transformed cells by Fab' antibody conjugates at the ultrastructural level. This virus-specific protein appeared in the cytoplasm of permissive cells as early as 3 h after infection. At later time intervals, the T antigen was localized in both the cytoplasm and nucleus and finally (24 h) in the nucleus. These results suggest a synthesis of T antigen on cytoplasmic ribosomes, with subsequent transfer to the nucleus.     

Tumor necrosis therapy antibody interleukin-2 fusion protein elicits prolonged and targeted antitumor effects in vivo

Interleukin-2 (IL-2) is one of the most successful cytokines applied in tumor immunotherapy because of its ability to stimulate potent cellular immune response. The life-threatening toxicity of vascular leak syndrome (VLS) associated with the high-dose IL-2 treatment regimen has limited its use in tumor immunotherapy. To reverse this situation, a tumor-targeted fusion protein, recombinant human TNT-IL2 (rhTNT-IL2), was generated with both the cytokine activity of IL-2 and the tumor-targeting ability of TNT antibody. TNT is a human tumor necrosis therapy monoclonal antibody capable of binding intracellular antigens which are accessible and abundant in necrotic regions of tumors. The immunotherapeutic potential of this fusion protein was tested in murine melanoma and lung cancer models, and tumor-bearing mice showed satisfied tumor regressions after rhTNT-IL2 immunotherapy. Immunohistochemical study showed a distinct penetration of IL-2 in tumors in mice treated with rhTNT-IL2, indicating its evident tumor-targeting activity. Moreover, the rhTNT-IL2 was well tolerated in cynomolgus monkeys in a 12-week long-term repeated toxicity study. These studies indicate that the targeting of IL-2 to necrotic areas of tumors might be a new approach for the immunotherapy of solid tumors.     

Calculation of half-lives of proteins in vivo. Heterogeneity in the rate of degradation of yeast proteins

Summary A method is given for the calculation of half-lives of proteins in vivo from the measurement of the decrease of radioactivity in pulse-labelled proteins with time. This method could be particularly useful for the study of the degradation of proteins in cells that have a low growth rate.The method applied to growing yeast indicates that there are two major classes of protein. The class with low turnover constitutes the bulk of yeast protein and has a half-life of 160 h in a medium with glucose or galactose and of 50 h in a medium with ethanol. The class of proteins with high turnover (half-life between 0.8 and 2.4 hours) represents from 1% of total protein in yeast growing on glucose to 7% in yeast growing on ethanol.It is shown that some proteins which are derepressed during growth on ethanol or induced during growth on galactose are particularly susceptible to degradation in a medium which contains glucose.It is proposed that protein degradation is regulated by a coarse control at the level of protease activity and a fine control on the susceptibility of individual proteins to proteases.     

Complement-mediated alteration of antibody specificity in vivo.

The simultaneous injection of heterologous anti-EL4 lymphoma serum and complement results in the rapid disappearance of such antibody from the periphery of non-tumor bearing mice. However, this phenomenon is only observed when a complement source capable of mediating the lysis of EL4 cells sensitized with such heterologous antibody is used. This complement mediated enhancement of anti-tumor antibody absorption was observed in vivo for three strains of mice. Omission of complement or the use of genetically deficient complement sources resulted in no effect on circulating antibody titer when compared to the titer of heterologous anti-tumor antibody observed in the periphery when injected alone. Exogenous complement did not enhance the clearance of heterologous anti-tetanus toxin serum, thereby suggesting that the increased absorption of anti-EL4 in vivo is not related simply to the enhanced clearance of foreign gamma-globulin. Confirmatory evidence of the role of complement in altering anti-tumor antibody specificity in vivo was obtained in a guinea pig tumor model as well. The data suggest that anti-tumor serum shown to be relatively specific for the tumor cell gains additional specificity in the presence of functional complement and consequently manifests avidity for cross-reactive determinants previously thought to be unrelated.     

Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.     

Polymerizable Fab' antibody fragments for targeting of anticancer drugs.

We have designed a new pathway for the synthesis of targeted polymeric drug delivery systems, using polymerizable antibody Fab' fragments (MA-Fab'). The targeted systems can be directly prepared by copolymerization of the MA-Fab', N-(2-hydroxypropyl)methacrylamide (HPMA) and drug-containing monomers. Both MA-Fab' and the Fab'-targeted copolymers can effectively bind to target cells. An MA-Fab' (from OV-TL 16 Ab) targeted HPMA copolymer containing mesochlorin e6 (Mce6) was synthesized by copolymerization of MA-Fab', HPMA, and MA-GFLG-Mce6. The targeted copolymer exhibited a higher cytotoxicity toward OVCAR-3 human ovarian carcinoma cells than the nontargeted Mce6-containing copolymer or free Mce6. The targeted copolymer was internalized more efficiently by OVCAR-3 cells than the nontargeted copolymer.