Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease.

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein-protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.

Engineered antibodies take center stage.

The start of the post-genomic era provides a useful juncture for reflection on the state of antibody engineering, which will be a critical technology for relating function and pathology to genomic sequence in biology and medicine. The phenomenal progress in deciphering the human genome has given significant impetus to the application of engineered antibodies in proteomics. Thus, advances in phage display antibody libraries can now help to define novel gene function and the measurement of abnormal protein expression in pathological states. Furthermore, intrabody and antibody engineering provide vehicles for the development of molecular medicines of the future. In addition to these new directions, antibody engineering has begun to show concrete success in its long-term efforts to develop targeted immunotherapies for cancer and other diseases. The cornerstones of clinical development are the detailed academic clinical trials that continue to push the boundaries of engineered antibodies into the real world. The field displays a healthy impatience for practical results, as research accelerates with concerted efforts to transfer preclinical insights into clinical trials. Growing private and governmental expenditures will lead to the rapid expansion of life-saving immunotherapeutic agents. The present review developed from our effort to report on the 11th Annual International Conference on Antibody Engineering (3-6 December 2000). This annual meeting is a forum for discussions on the latest advances in antibody engineering groups from around the world, and now includes the broader agenda of engineering in molecular immunology. In bringing scientists together to exchange ideas at this open forum, new collaborations and the threads of new discoveries are woven. For example, Professors Gerhard Wagner (Harvard Medical School), Dennis Burton (Scripps Research Institute), and Peter Hudson (CSIRO, Melbourne, Australia) gave exciting insights on structural immunobiology that had implications across many disciplines. The growth in antibody engineering was highlighted by the attendance of some 600 participants at the meeting, doubling that of the 1999 meeting. Dramatic clinical acceptance of monoclonal antibodies during the past two years has fostered this growth, with sales in 2000 of 1.8 billion dollars and projections for 2001 of 3 billion dollars. However, economic measures cannot begin to convey the medical revolution that is being effected by these first humanized and chimerized monoclonal antibodies. At this juncture, the 10 monoclonal antibody therapeutics in clinical use are of murine origin, of which 3 are entirely murine (OKT3, Mylotarg, 90Y-labeled Bexxar), 4 have been chimerized (human constant domains replacing murine) (ReoPro, Rituxan and its 131I-labeled analogue (Zevalin), Simulect, Remicade) and 3 were chimerized and humanized (human residues being substituted for at least some mouse-specific framework residues in VH and VL) (Zenapax, Herceptin, Synagis). Fully humanized anti-CD52 (CAMPATH-1H) has also been approved by the FDA for the treatment of B-cell chronic lymphocytic leukemia and should become available in late 2001. Humanization was initially developed by Dr. Greg Winter at the MRC Laboratory of Molecular Biology (Cambridge, UK), who presented the meeting's keynote address, "Antibodies as a Paradigm for Molecular Evolution". His pioneering work in antibody phage display libraries has been reformulated into a daring approach to develop truly novel proteins with genetically paired structural elements. He described studies in combinatorial protein engineering with enormous implications for both industrial and therapeutic applications of macromolecules.

Extended half-life and elevated steady-state level of a single-chain Fv intrabody are critical for specific intracellular retargeting of its antigen, caspase-7.

8 h) and high steady-state levels of protein accumulation, while the H2 intrabodies had a half-life of 2 h and less protein at steady state. These results suggest that the choice of sFv as an intrabody depends critically on the intracellular sFv protein having an extended half-life and elevated steady-state level. Thus, extended half-life must be considered together with sFv antibody specificity and affinity when choosing an optimal sFv intrabody for functional studies of cellular proteins.

Symmetry of Fv architecture is conducive to grafting a second antibody binding site in the Fv region.

Molecular modeling studies on antibody Fv regions have been pursued to design a second antigen-binding site (chi-site) in a chimeric single-chain Fv (chi sFv) species of about 30 kDa. This analysis has uncovered an architectural basis common to many Fv regions that permits grafting a chi-site onto the Fv surface that diametrically opposes the normal combining site. By using molecular graphics analysis, chimeric complementarity-determining regions (chi CDRs) were defined that comprised most of the CDRs from an antibody binding site of interest. The chain directionality of chi CDRs was consistent with that of specific bottom loops of the sFv, which allowed for grafting of chi CDRs with an overall geometry approximating CDRs in the parent combining site. Analysis of 10 different Fv crystal structures indicates that the positions for inserting chi CDRs are very highly conserved, as are the corresponding chi CDR boundaries in the parent binding site. The results of this investigation suggest that it should be possible to generally apply this approach to the development of chimeric bispecific antibody binding site (chi BABS) proteins.

Single-chain Fv radioimmunotargeting.

The availability of engineered antibody species has catalyzed new developments in radioimmunotargeting. This chapter summarized recent studies of single-chain Fv (sFv) proteins, which are minimal antibody binding sites engineered as single polypeptide chains. The single-chain Fv can be as small as 26 kDa monomers or may be engineered as larger fusion proteins designed to self-associate into dimeric or multimeric species. They typically exhibit rapid clearance that results in high targeting specificity within a matter of hours. We have compared different modes of administration to allow further manipulation of their biodistribution and targeting properties. Results of the present study comparing intravenous (i.v.) and intraperitoneal (i.p.) administration show comparable long-term retention in circulation, but the i.v. route showed an initially high peak blood level while i.p. injection did not. As with a single sFv dose, repeated bolus injections of sFv attained high target-to-background ratios, whereas continuous sFv infusion reached a steady state level of free sFv in blood and kidney that exceeded that in tumor xenografts. We observed improved localization of radioiodinated sFv in tumor xenografts if the radioiodine label resisted dehalogenation from the protein, which was accomplished, for example, through conjugation of a para-131I-benzoyl group to Iysyl epsilon-amino groups of the protein. Modification of the sFv by genetic incorporation of a cysteinyl peptide (to form sFv') provided a chelation site for radiometals that simplified incorporation of 99mTc with the opportunity for improved diagnostic imaging in cancer and other diseases. Therapeutic applications of sFv radioimmunotargeting could rely on sFv' complexed to 186Re or 188Re. Engineering sFv of sFv' with increased antigen-binding affinity and appropriately manipulating their mode of administration should promote sustained tumor retention conducive to clinically useful therapeutic indices.

Enhanced tumor specificity of 741F8-1 (sFv')2, an anti-c-erbB-2 single-chain Fv dimer, mediated by stable radioiodine conjugation.

UNLABELLED: The goal of this study was to determine if the stabilization of the radioiodine-protein bond by the N-succinimidyl p-iodobenzoate (PIB) method improved the degree and specificity of tumor localization of 125I-741F8-1 (sFv')2, an anti-c-erbB-2 sFv dimer, in an immunodeficient murine model. METHODS: Gamma camera images were acquired 21 hr after intravenous administration of 131I-741F8-1 (sFv')2 labeled by the p-iodobenzoate or chloramine T methods. The stability of the radioiodine-protein bond also was assessed in plasma samples after intravenous injection of 125I-741F8-1 (sFv')2 labeled by either the chloramine T or p-iodobenzoate methods. RESULTS: By 6 hr postinjection, 97% of the activity associated with the 125I-741F8-1 (sFv')2 labeled by the p-iodobenzoate method was protein bound compared with 61% after labeling with the chloramine-T method. These observations indicate that increasing the stability of the conjugation between the radioiodine and the sFv molecule can significantly increase the degree and specificity of tumor targeting. Significantly greater tumor retention (p < 0.005) and lower blood (p < 0.001), spleen (p < 0.001) and stomach (p < 0.005) retention were observed in biodistribution studies when the p-iodobenzoate conjugate was used. This resulted in superior tumor-to-organ ratios for all tissue samples studied. CONCLUSION: These observations may have clinical relevance for the use of radiolabeled sFv as imaging agents.

Redirection of cellular cytotoxicity. A two-step approach using recombinant single-chain Fv molecules.

In this article the authors discuss an indirect system for redirecting cellular cytotoxicity, which utilizes a "universal" bispecific antibody to redirect T-cells to kill cells targeted with single-chain Fv (sFv) fusion proteins that carry a peptide tag recognized by the bispecific antibody. This approach has a number of theoretical advantages in the immunotherapy of cancer.

Optimization of in vivo tumor targeting in SCID mice with divalent forms of 741F8 anti-c-erbB-2 single-chain Fv: effects of dose escalation and repeated i.v. administration.

Single-chain Fv molecules in monovalent (sFv) and divalent [(sFv')2] forms exhibit highly specific tumor targeting in mice as a result of their small size and rapid systemic clearance. As a consequence, there is a rapid reversal of the sFv blood/tumor gradient, resulting in diminished retention of sFv species in tumors. In this report we investigate two distinct strategies, dose escalation and repetitive intravenous (i.v.) dosing, aiming to increase the absolute selective retention of radiolabeled anti-c-erbB-2 125I-741F8 (sFv')2 in c-erbB-2-overexpressing SK-OV-3 tumors in mice with severe combined immunodeficiency (SCID). A dose-escalation strategy was applied to single i.v. injections of 125I-741F8 (sFv')2. Doses from 50 micrograms to 1000 micrograms were administered without a significant decrease in tumor targeting or specificity. High doses resulted in large increases in the absolute retention of 125I-741F8 (sFv')2. For example, raising the administered dose from 50 micrograms to 1000 micrograms increased the tumor retention 24 h after injection from 0.46 microgram/g to 9.5 micrograms/g, and resulted in a net increase of greater than 9 micrograms/g. Over the same dose range, the liver retention rose from 0.06 microgram/g to 1 microgram/g, and resulted in a net increase of less than 1 microgram/g. The retention of 9.5 micrograms/g in tumor 24 h following the 1000-micrograms dose of (sFv')2 was comparable to that seen 24 h after a 50-micrograms dose of 125I-741F8 IgG, indicating that the use of large doses of (sFv')2 may partially offset their rapid clearance. When two doses were administered by i.v. injection 24 h apart, the specificity of delivery to tumor observed after the first dose was maintained following the second injection. Tumor retention of 125I-741F8 (sFv')2 was 0.32 microgram/g at 24 h and 0.22 micrograms/g at 48 h following a single injection of 20 micrograms, while 0.04 microgram/ml and 0.03 microgram/ml were retained in blood at the same assay times. After a second 20-micrograms injection at the 24-h assay time, tumor retention increased to 0.49 micrograms/g, and blood retention was 0.06 microgram/ml, at the 48-h point. These results suggest that multiple high-dose administrations of radiolabeled 741F8 (sFv')2 may lead to the selective tumor localization of therapeutic radiation doses.

In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library.

BACKGROUND: Antibody-based reagents have failed to live up to their anticipated role as highly specific targeting agents for cancer therapy. Targeting with human single-chain Fv (sFv) molecules may overcome some of the limitations of murine IgG, but are difficult to produce with conventional hybridoma technology. Alternatively, phage display of antibody gene repertoires can be used to produce human sFv. OBJECTIVES: To isolate and characterize human single chain Fvs which bind to c-erbB-2, an oncogene product overexpressed by 30-50% of breast carcinomas and other adenocarcinomas. STUDY DESIGN: A non-immune human single-chain Fv phage antibody library was selected on human c-erbB extracellular domain and sFv characterized with respect to affinity, binding kinetics, and in vivo pharmacokinetics in tumor-bearing scid mice. RESULTS: A human single-chain Fv (C6.5) was isolated which binds specifically to c-erbB-2. C6.5 is entirely human in sequence, expresses at high level as native protein in E. coli, and is easily purified in high yield in two steps. C6.5 binds to immobilized c-erbB-2 extracellular domain with a Kd of 1.6 x 10(-8) M and to c-erbB-2 on SK-OV-3 cells with a Kd of 2.0 x 10(-8) M, an affinity that is similar to sFv produced against the same antigen from hybridomas. Biodistribution studies demonstrate 1.47% injected dose/g tumor 24 h after injection of 125I-C6.5 into scid mice bearing SK-OV-3 tumors. Tumor:normal organ ratios range from 8.9:1 for kidney to 283:1 for muscle. CONCLUSIONS: These results are the first in vivo biodistribution studies using an sFv isolated from a non-immune human repertoire and confirm the specificity of sFv produced in this manner. The use of phage display to produce C6.5 mutants with higher affinity and slower k(off) would permit rigorous evaluation of the role of antibody affinity and binding kinetics in tumor targeting, and could result in the production of a therapeutically useful targeting protein for radioimmunotherapy and other applications.

Mammalian cell expression of single-chain Fv (sFv) antibody proteins and their C-terminal fusions with interleukin-2 and other effector domains.

The production of several single-chain Fv (sFv) antibody proteins was examined by three modes of mammalian cell expression. Our primary model was the 741F8 anti-c-erbB-2 sFv, assembled as either the VH-VL or VL-VH, and expressed alone, with C-terminal cysteine for dimerization, or as fusion proteins with carboxyl-terminal effector domains, including interleukin-2, the B domain of staphylococcal protein A, the S-peptide of ribonuclease S, or hexa-histidine metal chelate peptide. Constructs were expressed and secreted transiently in 293 cells and stably in CHO or Sp2/0 cell lines, the latter yielding up to 10 mg per liter. Single-chain constructs of MOPC 315 myeloma and 26-10 monoclonal antibodies were also expressed, as were hybrids comprising unrelated VH and VL regions. Our results suggest that mammalian expression is a practical and valuable complement to the bacterial expression of single-chain antibodies.

Redirection of T cell-mediated cytotoxicity by a recombinant single-chain Fv molecule.

We have produced two single-chain Fv (sFv) proteins by bacterial periplasmic secretion, one sFv with specificity for the hapten DNP, and the other for the human transferrin receptor. After solubilization and refolding, we recovered several mg of active sFv per liter of bacterial culture. Each sFv bound to cells bearing the appropriate Ag and could be used to direct targeted cellular cytotoxicity. Targeting relied on a universal bispecific antibody designed to cross-link CD3 on the cytotoxic T cell with a peptide fused to the sFv carboxyl-terminus. The universal bispecific antibody was used in combination with the Ag-specific sFv to redirect human cytotoxic T cells to kill a variety of target cells. Such an approach has a number of advantages that may make it useful for the immunotherapy of cancer and other diseases.

Tumor targeting in a murine tumor xenograft model with the (sFv')2 divalent form of anti-c-erbB-2 single-chain Fv.

This investigation has utilized novel forms of the single-chain Fv (sFv), wherein a cysteine-containing peptide has been fused to the sFv carboxyl terminus to facilitate disulfide bonding or specific cross-linking of this sFv' to make divalent (sFv')2. The 741F8 anti-c-erbB-2 monoclonal antibody was used as the basis for construction of 741F8 sFv, from which the sFv' and (sFv')2 derivatives were prepared. Recombinant c-erbB-2 extracellular domain (ECD) was prepared in CHO cells and the bivalency of 741F8 (sFv')2 demonstrated by its complex formation with ECD. The tumor binding properties of 125I-labeled anti-c-erbB-2 741F8 sFv, sFv', and (sFv)2 were compared with radiolabeled antidigoxin 26-10 sFv' and (sFv')2 controls. Following intravenous administration of radiolabeled species to severe combined immune-deficient (SCID) mice bearing SK-OV-3 tumors (which over-express c-erbB-2), blood and organ samples were obtained as a function of time over 24 h. Comparative analysis of biodistribution and tumor-to-organ ratios demonstrated the 741F8 sFv, sFv', and (sFv')2 had excellent specificity for tumors, which improved with time after injection. This contrasted with nonspecific interstitial pooling in tumors observed with the 26-10 sFv, sFv', and (sFv')2, which decreased with time after administration. Tumor localization was significantly better for disulfide or peptide crosslinked 741F8 (sFv')2 having Gly4Cys tails than for monovalent 741F8 sFv' or Fab. The superior properties of the 741F8 (sFv')2 in targeting SK-OV-3 tumors in SCID mice suggests the importance of further investigations of divalent sFv analogs for immunotargeting.

Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single-chain Fv.

The in vivo properties of monovalent and divalent single-chain Fv (sFv)-based molecules with the specificity of the anti-c-erbB-2 monoclonal antibody 741F8 were examined in scid mice bearing SK-OV-3 tumor xenografts. 741F8 sFv monomers exhibited rapid, biphasic clearance from blood, while a slightly slower clearance was observed with the divalent 741F8 (sFv')2 comprising a pair of 741F8 sFv' with a C-terminal Gly4Cys joined by a disulfide bond. Following i.v. injection, the 741F8 sFv monomer was selectively retained in c-erbB-2-overexpressing SK-OV-3 tumor, with excellent tumor:normal organ ratios uniformly exceeding 10:1 by 24 h. The specificity of this effect was demonstrated by the lack of retention of the anti-digoxin 26-10 sFv monomer, as evaluated by biodistribution studies, gamma camera imaging, and cryomacroautoradiography studies. The specificity index (741F8 sFv retention/26-10 sFv retention) of 741F8 monomer binding, measured by the percentage of injected dose per g of tissue, was 13.2:1 for tumor, and 0.8 to 2.1 for all tested normal organs, with specificity indices for tumor:organ ratios ranging from 7.0 (kidneys) to 16.7 (intestines). Comparing divalent 741F8 (sFv')2 with the 26-10 (sFv')2, similar patterns emerged, with specificity indices for retention in tumor of 16.9 for the Gly4Cys-linked (sFv')2. These data demonstrate that, following their i.v. administration, both monovalent and divalent forms of 741F8 sFv are specifically retained by SK-OV-3 tumors. This antigen-specific binding, in conjunction with the 26-10 sFv controls, precludes the possibility that passive diffusion and pooling in the tumor interstitium contributes significantly to long-term tumor localization. 741F8 (sFv')2 species with peptide spacers exhibited divalent binding and increased retention in tumors as compared with 741F8 sFv monomers. Since the blood retention of the (sFv')2 is slightly more prolonged than that of the monomer, it was necessary to demonstrate that the increased tumor localization of the peptide-linked (sFv')2 was due to its divalent nature. The significantly greater localization of the divalent bismalimidohexane-linked 741F8 (sFv')2 as compared with a monovalent 741F8 Fab fragment of approximately the same size suggests that the increased avidity of the (sFv')2 is a factor in its improved tumor retention. This is the first report of successful specific in vivo targeting of tumors by divalent forms of sFv molecules. The improved retention of specific divalent (sFv')2 by tumors may have important consequences for targeted diagnostic or therapeutic strategies.

The effect of high- and low-intensity warm-up on the physiological responses to a standardized swim and tethered swimming performance.

This investigation was conducted to determine the effect of high- and low-intensity warm-ups on physiological responses, lactate accumulation, and high-intensity freestyle and tethered swimming performance. Ten male collegiate swimmers were tested for maximal oxygen uptake (VO2 max) followed by two series of three warm-up protocols performed in a randomized order at least 2 days apart. The warm-up protocols were: (1) no warm-up (NWU), (2) a 366-m swim at 70% VO2 max (LWU) and (3) four 46-m swims at 1-min intervals at a speed corresponding to 110% VO2 max (HWU). Five minutes after each warm-up in the first series, the swimmers swam a 183-m standardized freestyle swim at a velocity corresponding to 110% VO2 max, and 5 min after each warm-up in the second series the swimmers completed a tethered swim to exhaustion with a weight attached to the tether to elicit fatigue at about 2 min. Three minutes after each warm-up and 3 min after each standardized and tethered swim, a finger-prick blood sample for lactate measurement was obtained. Heart rate and VO2 were also measured during the warm-up and the standardized and tethered swims. The performance times in the tethered swim were not significantly different between the three conditions (116.8 +/- 46.8, 137 +/- 53.3 and 122.94 +/- 37.2 s for the NWU, LWU and HWU, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen recognition and targeted delivery by the single-chain Fv.

The single-chain Fv (sFv) has proven attractive for immuno-targeting, both alone and as a targeting element within sFv fusion proteins. This chapter summarizes the features of sFv proteins that have sparked this interest, starting with the conservation of Fv architecture that makes general sFv design practical. The length and composition of linkers used to bridge V domains are discussed based on the sFv literature; special emphasis is given to the (Gly4Ser)3 15-residue linker that has proven of broad utility for constructing Fv regions of antibodies and other members of the immunoglobulin superfamily. The refolding properties of sFv proteins are summarized and examples given from our laboratory. Spontaneous refolding from the fully reduced and denatured state, typified by 26-10 sFv, is contrasted with disulfide-restricted refolding, exemplified by MOPC 315 and R11D10 sFv proteins, which recover antigen binding only if their disulfides have been oxidized prior to removal of denaturant. The medical value of sFv proteins hinges on their reliability in antigen recognition and rapidity in targeted delivery. Detailed analysis of specificity and affinity of antigen binding by the 26-10 antidigoxin sFv has demonstrated very high fidelity to the binding properties of the parent 26-10 sFv. These results gave confidence to the pursuit of more complex biomedical applications of these proteins, which is indicated by our work with the R11D10 sFv for the imaging of myocardial infarctions. Diagnostic imaging and therapeutic immunotargeting by sFv present significant opportunities, particularly as a result of their pharmacokinetic properties. Intravenously administered sFv offers much faster clearance than conventional Fab fragments or intact immunoglobulin with minimal background binding.

Medical applications of single-chain antibodies.

A single-chain antibody or single-chain Fv (sFv) incorporates the complete antibody binding site in a single polypeptide chain of minimal size, with an approximate molecular weight of 26,000. In antibodies, the antigen combining site is part of the Fv region, which is composed of the VH and VL variable domains on separate heavy and light chains. Efforts over nearly two decades have indicated that Fv fragments can only rarely be prepared from IgG and IgA antibodies by proteolytic dissection. Beginning in 1988, single-chain analogues of Fv fragments and their fusion proteins have been reliably generated by antibody engineering methods. The first step involves obtaining the genes encoding VH and VL domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis. The single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly4-Ser)3. The linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either VH-linker-VL or VL-linker-VH. In principle, the sFv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site, as demonstrated in our model studies with the 26-10 anti-digoxin sFv. Furthermore, the sFv remains stable at low concentrations that promote VH and VL dissociation from the Fv heterodimer, resulting in loss of Fv binding. Intravenously administered sFv proteins exhibit accelerated biodistribution and exceptionally fast clearance compared to IgG or Fab. These pharmacokinetic properties allow rapid imaging by sFv, which therefore may be labeled with a short-lived isotope such as Tc-99m. Expression of a single gene product from fused sFv and effector genes facilitates immunotargeting of the effector protein, as shown for single-chain Fv toxin fusion proteins.