Engineering and characterization of a divalent single-chain Fv angiotensin II fusion construct of the monoclonal antibody CC49

For the therapy of solid tumors, co-administration of angiotensin II (AngII) results in an increased uptake of drugs into the tumor interstitium. We have engineered a dimeric sc(Fv)(2)-AngII fusion construct that combines the superior kinetics of covalent dimeric scFvs [sc(Fv)(2)], recognizing the pancarcinoma tumor-associated antigen 72 (TAG-72), with the advantageous intrinsic activity of AngII. The binding characteristics of the fusion construct were unaltered by the addition of the AngII sequence [affinity constant K(A) 1.18 x 10(7) and 8.42 x 10(6) M(-1) for sc(Fv)(2) and sc(Fv)(2)-AngII, respectively]. The binding of the fusion construct to the angiotensin receptor (AT(1)) was similar to AngII, and the arterial contraction was 16 +/- 1% of the response observed with norepinephrine. In animal studies, the radiolabeled sc(Fv)(2)-AngII construct exhibited similar uptake and a more homogeneous distribution within the tumor as compared to sc(Fv)(2).     

Pharmacokinetics and biodistribution of radioimmunoconjugates of anti-CD19 antibody and single-chain Fv for treatment of human B-cell Malignancy

The comparative advantages and disadvantages of intact antibodies and single-chain Fv as immunotoxins and radioimmunoconjugates have been widely discussed but not directly compared. In this study, the in vivo properties of anti-CD19 B43 monoclonal antibody and its derived single-chain Fv (FVS191) were studied in athymic nude mice bearing CD19-positive human lymphomas. B43 mab and FVS191 were labeled with iodine-125 using iodine-beads, and immunoreactivities were determined to be 57% and 72%, respectively. Scatchard analysis showed a similar high affinity for both. The results of pharmacokinetic studies revealed that FVS191 had a rapid biphasic clearance from the circulation (T1/2α = 2.5 min, T1/2β = 3.7 h); The T1/2α and T1/2β phases of B43 mab were determined to be 0.72 h and 57 h respectively. Biodistribution studies compared the uptake of labeled antibodies by CD19-positive and by CD19-negative tumors. The peak percentages of injected dose were 5.7% at 12 h for B43 and 2.45% at 1 h for FVS191. Radiolocalization indices (RI) demonstrated tumor-specific uptake for both, but higher uptake for B43. The optimal RI was seen at 15 min for FVS191 and 6 h for B43. FVS191 was unstable in vivo, approximately 50% of the injected dose being degraded in blood in 100 min. Radioactivity detected in the urine was present mainly as the deiodinized form of FVS191. The results suggest that B43 mab is favored over FVS191 in biodistribution properties and in vivo stability. Because B43 Mab showed early tumor-specific uptake, high RI values, and favorable tissue-to-blood ratios, it is a potential candidate for radioimmunotherapy and immunotoxin therapy of B-cell leukemia and lymphoma. Received: 17 June 1997 / Accepted: 17 June 1998     

Pharmacokinetics and biodistribution of a new anti-episialin monoclonal antibody 139H2 in ovarian-cancer-bearing nude mice

Summary The new murine anti-episialin monoclonal antibody (mAb) 139H2 has been selected for its strong reactivity with a series of human ovarian cancer xenografts. In the present report we describe the characteristics of mAb 139H2 investigated in vitro as well as in vivo. Scatchard plot analysis using the human ovarian cancer cell line NIH:OVCAR-3 showed an affinity constant of 1 × 10⁸ M^–1 and the expression of 7 × 10⁶ antigenic sites/cell. Reactivity with OVCAR-3 xenograft tissue was intense, localized at the cell membrane, heterogeneously distributed, and mainly detectable at the apical site of the cell. Administration of radiolabelled mAb 139H2 to nude mice bearing s.c. OVCAR-3 xenografts showed specific uptake in the tumour up to 9% of the injected dose/g. The maximum uptake in the tumour was retained for 3.5 days and mAb 139H2 cleared from the tumour with a half-life of 5.5 days. The half-life in blood was 50 h and no antibody-antigen complex formation could be detected. Poor uptake and no retention in episialin-negative WiDr colon cancer xenografts demonstrated specificity. Administration of an excess of an unlabelled irrelevant mAb did not influence the uptake in the OVCAR-3 xenografts or in other tissues. In contrast, tumour uptake decreased after addition of 300 µg or more unlabelled mAb 139H2 to a tracer dose of radiolabelled mAb 139H2. The uptake of mAb 139H2 in OVCAR-3 xenografts appeared inversely related to the tumour size.Supported by the Dutch Cancer Society     

Development of single-chain Fv of antibody to DNA as intracellular delivery vehicle

Application of antibodies in most therapeutic area is limited to extracellular or membranous targets because of their impermeability of membrane. For the purpose of biotechnological and therapeutic application, developing intracellular localizing antibody is the invaluable research field. A new recombinant single-chain variable fragment of an anti-dsDNA monoclonal antibody G2-6, IgG of which has been previously shown to have a cell-penetrating activity, was engineered and produced for the use as a delivery vehicle of biomolecule(s). The penetrating capacity of single-chain variable fragment in three mammalian cell lines, L929, NIH/3T3, and COS-7 was analyzed using flow cytometry and confocal microscopy. The results demonstrated that the single-chain variable fragment can effectively internalize all three cell lines, although the internalization level varied. It was also shown that the internalization was time- and concentration-dependent. Moreover, the single-chain variable fragment was located in nuclei as well as cytoplasm of L929 cells. Overall, the G2-6 single-chain variable fragment might be a candidate vehicle which could be used to deliver specific genes or biomolecules for therapy or diagnosis into the cytoplasm or cell nucleus.     

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.     

Functional expression of single-chain antibody to leukotriene C4

Leukotriene C₄ (LTC₄) is synthesized by binding of glutathione to LTA₄, an epoxide derived from arachidonic acid, and further metabolized to LTD₄ and LTE₄. We previously prepared a monoclonal antibody with a high affinity and specificity to LTC₄. To explore the structure of the antigen-binding site of a monoclonal antibody against LTC₄ (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC. The heavy and light chains consisted of 461 and 238 amino acids including a signal peptide with molecular weights of 51,089 and 26,340, respectively. An expression plasmid encoding a single-chain antibody comprising variable regions of mAbLTC heavy and light chains (scFvLTC) was constructed and expressed in COS-7 cells. The recombinant scFvLTC showed a high affinity with LTC₄ comparable to mAbLTC. The scFvLTC also bound to LTD₄ and LTE₄ with 48% and 17% reactivities, respectively, as compared with LTC₄ binding, whereas the antibody showed almost no affinity for LTB₄.     

Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152)

A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.     

Biosynthetic antibody binding sites: Development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315

The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein inE. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values forK _a,app of 1.5 to 2.2×10⁶ M^–1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv³¹⁵ fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.     

Production of engineered IgM-binding single-chain antibodies inEscherichia coli

Summary Two single-chain antibodies were engineered and tested as novel binding proteins with specificity for immunoglobulin M. Genes for the two single-chain Fv proteins were assembled from the variable light chain cDNA and variable heavy chain cDNA of monoclonal antibodies DA4.4 and Bet 2, which specifically bind human IgM and mouse IgM, respectively. Both single-chain Fv proteins were designed with a 14-amino acid linker which bridged the variable light chain and variable heavy chain domains. The two proteins were expressed inEscherichia coli, purified and assayed for IgM-binding activity. Both proteins demonstrate a binding specificity for their corresponding IgM which is similar to the monoclonal antibodies from which they were derived. These small IgM-binding proteins may have applications in the investigation of the immune response and in the detection and purification of monoclonal antibodies, cell-associated antibodies, and IgM from serum.     

Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli

The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined. Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen. However, immediately after induction at either 30°C or 37°C there was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant. The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic \-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis. Bacteria incubated at 30°C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A ₆₆₀ at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded. Osmotic-shock procedures did not release L6 sFv. However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location. French press disruption resulted in the release of correctly but not incorrectly folded material. Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane. These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis. It is also suggested that membrane association in the periplasmic space may impede proper folding.     

Production and validation of the pharmacokinetics of a single-chain Fv fragment of the MGR6 antibody for targeting of tumors expressing HER-2

The HER-2 antigen, which is overexpressed in many breast carcinomas, is an ideal target for monoclonal antibodies due to its low expression in normal tissue and its homogeneous distribution in the tumor mass. We have developed and characterized the murine MAb MGR6 against HER-2, which is able to inhibit proliferation of tumor cells overexpressing HER-2. On the basis of these preclinical results, phase I studies in breast carcinoma patients were conducted and radiolocalization data indicated an antibody half life which directly paralleled that of other whole antibodies and thus resulting in a limited in vivo diagnostic capacity. To obtain a smaller reagent with possibly improved in vivo properties, a single chain variable fragment (scFv) of the original MGR6-producing hybridoma was generated by phage display technology. Biologically active MGR6 scFv was purified rapidly and at high yield by metal affinity chromatography. Competition FACS and ELISA analyses identified an epitope on the HER-2 extracellular domain that was shared by the scFv and the parental MAb. BIAcore analysis indicated a K_off of 9.3 × 10⁻⁴ s⁻¹, similar to that of the intact MGR6 MAb. Distribution and elimination half-lives of MGR6 scFv, calculated from in vivo preclinical evaluations, were much faster (13 min and 6.2 h, respectively) than previously published results for the intact MAb (mean t1/2β of 46 h). This represents a theoretical improvement in pharmacokinetics with respect to the parental murine MAb and points to the potential for utilizing this fragment in redirecting therapeutic agents, such as radioisotopes, to different human carcinomas overexpressing HER-2. Received: 10 August 2000 / Accepted: 19 October 2000     

Applications of single-chain variable fragment antibodies in therapeutics and diagnostics

Antibodies (Abs) are some of the most powerful tools in therapy and diagnostics and are currently one of the fastest growing classes of therapeutic molecules. Recombinant antibody (rAb) fragments are becoming popular therapeutic alternatives to full length monoclonal Abs since they are smaller, possess different properties that are advantageous in certain medical applications, can be produced more economically and are easily amendable to genetic manipulation. Single-chain variable fragment (scFv) Abs are one of the most popular rAb format as they have been engineered into larger, multivalent, bi-specific and conjugated forms for many clinical applications. This review will show the tremendous versatility and importance of scFv fragments as they provide the basic antigen binding unit for a multitude of engineered Abs for use as human therapeutics and diagnostics.     

Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab)₂ (n=28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18;n=24, 3 mg). Serum samples were taken before injection and 2–3 and 6–14 weeks after administration. A double-antigen or bridging assay was developed to detect responses against both murine as well as chimeric antibodies. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) as well as three commercially available assays were used to study antibody response against the murine antibody OV-TL 3. With both the double-antigen (bridging) assay and the indirect ELISA 1 of the 28 patients (4%) injected with murine OV-TL 3 F(ab)₂ showed a HAMA reaction 6 weeks after injection, which was demonstrated to be a mixed anti-isotypic and anti-idiotypic response. None of the 24 patients injected with the chimeric MOv18 showed an anti-chimeric antibody response. The various commercially available assays demonstrated conflicting results. The double-antigen-or bridging assay is a reliable method to detect anti-murine and antichimeric antibodies. The assay can be easily adapted for use with human antibodies. The immunogenicity of OV-TL 3 F(ab)₂ and cMOv18 in patients is low, making both antibodies candidates for immunotherapy.This work was supported by a clinical research grant of the Netherlands Organization for Scientific Research (NWO 900-716-020) and by the Biocare Foundation (grant 92-05).     

Bivalent Fv antibody fragments obtained by substituting the constant domains of a fab fragment with heterotetrameric molybdopterin synthase

The antibody Fv fragment is the smallest functional unit of an antibody but for practical use, the VH/VL interface requires stabilization, which is usually accomplished by a peptide linker that joins the two variable domains to form a single chain Fv fragment (scFv). An alternative format to scFv is proposed that (i) allows stabilization of the Fv fragment, and (ii) restores the bivalency of the antibody as a pseudo-F(ab')2 format. This new antibody fragment was constructed by replacing the CHI and CL domains of the Fab fragment with heterotetrameric molybdopterin synthase (MPTS). We found that this format, named MoaFv, improved significantly the cytoplasmic expression of the Fv as a soluble protein in BL21 or Origami Escherichia coli strains. This MoaFv format is expressed as a homogeneous heterotetrameric protein with a Mr value of 110 kDa containing two functional binding sites as revealed by active site titration. In its native condition at 37 degrees C or in the presence of urea, this format was nearly as stable as the corresponding scFv, indicating that non-covalent interactions between the MPTS subunits can replace the covalent peptide linker in scFv. Finally, this MoaFv construct could be a useful format when bivalency is desirable to improve the functional avidity.     

Expression and long-term stability of a recombinant single-chain Fv antibody fragment in transgenic Nicotiana tabacum seeds

A functionally active anti-hepatitis B surface antigen single-chain Fv antibody fragment (scFv) was expressed in seeds of transgenic tobacco plants using genetic constructs for expression in the vacuole or the apoplastic fluid. Antibody levels close to 0.2% of the total soluble protein were found. After storage of the transgenic tobacco seeds for one year and a half a year at room temperature, the scFv maintained its antigen-binding activity in full.     

Production and characterization of a bacterial single-chain antibody fragment specific to B-cell-activating factor of the TNF family

An active form of a single-chain antibody fragment (scFv) from the murine monoclonal antibody ABL-1, which is specific for B-cell-activating factor of the TNF family, was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker, using an assembly polymerase chain reaction. The construct VH-linker-VL was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli BL21(DE3) as inclusion bodies. After extraction from the E. coli cells, the inclusion bodies were solubilized and denatured in the presence of 8M urea. The expressed scFv fusion proteins were purified by Ni(2+)-IDA His-bind resin and finally renatured by dialysis. The purity and activity of the purified scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and enzyme-linked immunosorbent assay. The result revealed that the ABL-1 scFv retains the specific binding activity to BAFF with an affinity constant of 0.9x10(-8)molL(-1).     

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational,structure-guided Fv engineering

Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies.     

Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance

Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins’ respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field.     

pH-dependent antigen-binding antibodies as a novel therapeutic modality

Monoclonal antibodies have become a general modality in therapeutic development. However, even with infinite binding affinity to an antigen, a conventional antibody is limited in that it can bind to the antigen only once, and this results in antigen-mediated antibody clearance when the a membrane-bound antigen is targeted, or in antibody-mediated antigen accumulation when a soluble antigen is targeted. Recently, a pH-dependent antigen-binding antibody that binds to an antigen in plasma at neutral pH and dissociates from the antigen in endosome at acidic pH has been reported to overcome this limitation and to reduce antigen-mediated antibody clearance and antibody-mediated antigen accumulation. A pH-dependent binding antibody against a soluble antigen can be further improved by Fc engineering to enhance the Fc receptor binding. Various approaches, including histidine-based engineering, direct cloning from immunized animals, and synthetic and combinatorial libraries, have been successfully applied to generate pH-dependent binding antibodies against various antigens. This review discusses the features, approaches, advantages, and challenges of developing a pH-dependent binding antibody as a novel therapeutic modality. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Recent advances in the generation of human monoclonal antibody

The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs.