Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments

We have produced single-chain antibody fragments (scFv) in Saccharomyces cerevisiae at levels up to 20 mg/L in shake flask culture by a combination of expression level tuning and overexpression of folding assistants. Overexpression of the chaperone BiP or protein disulfide isomerase (PDI) increases secretion titers 2-8 fold for five scFvs. The increases occur for scFv expression levels ranging from low copy to ER-saturating overexpression. The disulfide isomerase activity of PDI, rather than its chaperone activity, is responsible for the secretion increases. A synergistic increase in scFv production occurs upon cooverexpression of BiP and PDI.     

Pharmacokinetics and biodistribution of genetically-engineered antibodies

Monoclonal antibodies (MAbs), because of their inherent specificity, are ideal targeting agents. They can be used to deliver radionuclides, toxins or cytotoxic drugs to a specific tissue or malignant cell populations. Intact immunoglobulin (IgG) molecules have several practical limitations of their pharmacology; their relatively large size of approximately 150,000 daltons leads to a slow clearance from the blood pool and the body resulting in significant exposure to normal organs with limited quantities delivered to tumors. The IgG molecule shows a relatively poor diffusion from the vasculature into and through the tumor. Attempts to modify the pharmacology of the Ig molecule have classically involved the use of proteases to generate F(ab')2 and Fab' fragments with molecular weights of approximately 100,000 and 50,000 daltons, respectively. Fv fragments of IgG are one of the smallest size functional modules of antibodies that retain high affinity binding of an antigen. Their smaller size, approximately 25,000 daltons, enables better tumor penetration and makes them potentially more useful than a whole antibody molecule for clinical applications. Molecular cloning and expression of the variable region genes of IgG has greatly facilitated the generation of engineered antibodies. A single-chain Fv (scFv) recombinant protein, prepared by connecting genes encoding for heavy-chain and light-chain variable regions at the DNA level by an appropriate oligonucleotide linker, clears from the blood at much faster rate than intact IgG. The scFv fragment can retain an antigen-binding affinity similar to that of a monovalent Fab' fragment; this however, represents a relative decrease in binding affinity when compared to intact antibodies. The scFv with its faster clearance and lower affinity results in a lower percent-injected dose localizing in tumors when compared to the divalent IgG molecule. This may be adequate for imaging but probably not for therapy. The valency of the MAb fragment is critical for the functional affinity of an antibody to a cell surface or a polymeric antigen. In attempts to generate multivalent forms of scFv molecules, non-covalently linked scFv dimeric and trimeric molecules, disulfide linked dimeric scFvs, as well as covalently linked chimeric scFvs have been studied. These multivalent scFvs generally have a higher functional affinity than the monovalent form resulting in better in vivo targeting. Another way to alter the pharmacology of the scFvs is to modify its net charge. Charge-modified scFvs with desired isoelectric points (pI), have been prepared by inserting negatively charged amino acids on the template of the variable region genes. This can help to overcome undesirable elevations in renal uptake seen with most antibody fragments. In conclusion, genetic manipulations of the immunoglobulin molecules are effective means of altering stability, functional affinity, pharmacokinetics, and biodistribution of the antibodies required for the generation of the "magic bullet".     

Identification of functional domains in murine granulocyte-macrophage colony-stimulating factor using monoclonal antibodies to synthetic peptides

Granulocyte-macrophage (GM)-CSF is an important hematopoietic cytokine that regulates proliferation and differentiation of macrophages, neutrophils, and eosinophils. In this study, we generated mAb to five synthetic peptides that correspond to regions along the murine GM-CSF molecule. The ability of anti-peptide mAb to bind to and inhibit biologic activity of murine (m) GM-CSF was determined. mAb with the highest neutralization titers were derived from mice immunized with peptide II, which correspond to amino acids 27 to 38 of mGM-CSF. Immunochemical studies showed that peptide II specifically blocked binding of anti-peptide II mAb to GM-CSF. mAb to two other peptides in the N-terminal half corresponding to residues 7 to 17 and 47 to 58, respectively, of mGM-CSF also inhibited GM-CSF-dependent proliferation and differentiation of murine bone marrow precursors for macrophages and granulocytes. Anti-peptide mAb also inhibited growth of a murine hematopoietic cell line FDCP1 and a murine T cell line HT-2, which was shown to be dependent on GM-CSF for growth in vitro. Biologic activity of both natural and recombinant mGM-CSF was neutralized by anti-peptide mAb. These findings indicate that epitopes in the N-terminal region of mGM-CSF are important for biologic activity, and the epitope defined by peptide II (residues 27 to 38) lies within a particularly important functional domain of the mGM-CSF molecule.     

Targeting of interleukin 2 to human ovarian carcinoma by fusion with a single-chain Fv of antifolate receptor antibody

To provide a new tool for the immunotherapy of human ovarian carcinoma, we constructed a fusion protein between interleukin-2 (IL-2) and the single-chain Fv (scFv) of MOV19, a monoclonal antibody directed against alpha-folate receptor (alpha-FR), known to be overexpressed on human nonmucinous ovarian carcinoma. This was accomplished by fusing the coding sequences in a single open reading frame and expressing the IL-2/MOV19 scFv chimera under the control of the murine immunoglobulin K promoter in J558L plasmacytoma cells. The design allowed the construction of a small molecule combining the specificity of MOV19 with the immunostimulatory activity of IL-2. This might improve the tissue penetration and distribution of the fusion protein within the tumor, reduce its immunogenicity, and avoid the toxicity related to the systemic administration of IL-2. The IL-2/MOV19 fusion protein was stable on purification from the cell supernatant and was biologically active. Importantly, this construct was able to target IL-2 onto the surface of alpha-FR-overexpressing tumor cells and stimulated the proliferation of the IL-2-dependent CTLL-2 cell line as well as that of human resting peripheral blood lymphocytes. In a syngeneic mouse model, IL-2/MOV19 scFv specifically targeted a-FR gene-transduced metastatic tumor cells without accumulating in normal tissues, due to its fast clearance from the body. Prolonged release of IL-2/MOV19 scFv by in vivo transplanted J558-EF6.1 producer cells protected 60% of mice from the development of lung metastases caused by an i.v. injection of a-FR gene-transduced tumor cells. Moreover, treatment with IL-2/MOV19 scFv, but not with recombinant IL-2, significantly reduced the volume of s.c. tumors. The pharmacokinetics and biological characteristics of IL-2/NMOV19 scFv might allow us to combine the systemic administration of this molecule with the adoptive transfer of in vitro retargeted T lymphocytes for the treatment of ovarian cancer, thereby providing local delivery of IL-2 without toxicity.     

First case of pneumatosis cystoides intestinalis in adult dermatomyositis

The past year has seen a proliferation in the use of recombinant, replication-defective adenoviruses for experimental models of gene therapy. The fact that adenovirus infects most cell types with no requirement for cell division, combined with the high titers and high efficiency of gene transfer obtainable with recombinant adenovirus, make it a promising system for in vivo human gene therapy.     

Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long-term gene therapy in Gunn rats

Recombinant adenoviruses are highly efficient at transferring foreign genes in vivo. However, duration of gene expression is limited by the host antiviral immune response which precludes expression upon viral readministration. We tested the feasibility of prolonging gene expression by induction of central tolerance to adenoviral antigens in bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats. Tolerance was induced by intraperitoneal injection of antilymphocyte serum, followed by intrathymic inoculation of one of the following: a recombinant adenovirus (Ad), adenovirus human UDP-glucuronosyltransferase (Ad-hBUGT1) carrying the hBUGT1 gene; a protein extract of the same virus; or viral infected hepatocytes. Controls received intrathymic injections of normal saline. After 12 d all groups were injected intravenously with 5 x 10(9) pfu of either Ad-hBUGT1 or adenovirus beta-galactosidase (Ad-LacZ) (expressing the Escherichia coli beta-galactosidase [LacZ] gene). In all three groups of tolerized rats, hBUGT1 was expressed in the liver after administration of Ad-hBUGT1, with glucuronidation of biliary bilirubin of above 95%. Serum bilirubin levels decreased from 7.2 to 1.8 mg/dl within 1 wk and remained low for 7 wk. Similar findings were observed following repeat injections given on days 45 and 112. In control rats serum bilirubin levels were reduced for only 4 wk, and viral readministration was ineffective. In all tolerized groups, but not in controls, there was a marked inhibition of appearance of neutralizing antibodies and cytotoxic lymphocytes against the recombinant adenovirus. Injection of wild type adenovirus-5 (Ad5) into the tolerized rats elicited a wild type-specific cytotoxic lymphocyte response. This is the first demonstration of Ad-directed long-term correction of an inherited metabolic disease following central tolerization with thymic antigen.     

Development of recombinant adenoviruses that drive high level expression of the human metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 and -2 genes: characterization of their infection into rabbit smooth muscle cells and human MCF-7 adenocarcinoma cells

Remodelling of the extracellular matrix resulting from increased secretion of metalloproteinase enzymes (MMPs) is implicated in many pathological conditions, including rheumatoid arthritis, restenosis following balloon angioplasty, atherosclerosis and cancer cell invasion and metastasis. Clear definition of the normal and pathological function of individual MMPs will benefit from approaches that use gene transfer to produce increases in MMP levels that mimic those observed in pathological conditions. Similarly, gene transfer methods leading to controlled increases in levels of the tissue inhibitor of metalloproteinases (TIMPs) will help to define the function of MMPs both in vitro and in vivo. Gene transfer of TIMPs may also have therapeutic potential in pathological conditions where inhibition of MMP activity may be beneficial. We have used the adenovirus serotype 5 vector system to generate replication-deficient recombinant adenoviruses capable of expressing the MMP-9, TIMP-1 or -2 genes. High level expression is driven by the cytomegalovirus major immediate early promoter (CMV IEP). Efficient and selective over-production of each recombinant protein was shown by immunofluorescence in either rabbit smooth muscle cells (SMC) or human MCF-7 adenocarcinoma cells. High level secretion directly dependent on the multiplicity of infection (MOI) was observed for each functional transgene by gelatin zymography. Using a quantitative ELISA assay, levels of recombinant TIMP-1 were detected when SMC were infected with as low as three plaque forming units (pfu) of virus per cell in vitro. A linear increase in TIMP-1 secretion was observed up to 1000 pfu/cell of virus (0.75 ng/10(4) cells/24 h at 3 pfu/cell to 1243 ng/10(4) cells/24h at 1000 pfu/cell). Similar levels of secretion of MMP-9 and TIMP-2 were observed by Western blot analysis using the same MOI of adenovirus. Thus, recombinant adenoviruses are an efficient and flexible system for high level expression of MMPs and TIMPs and will be useful tools in the study of matrix remodelling in vivo and in vitro.     

Characterization of human anti-high molecular weight-melanoma-associated antigen single-chain Fv fragments isolated from a phage display antibody library

The human high molecular weight-melanoma-associated antigen (HMW-MAA) meets the criteria to be used as an immunogen for immunotherapy of malignant melanoma, because it is expressed by a large percentage of melanoma lesions with limited heterogeneity and has a restricted distribution in normal tissues. The high immunogenicity of the HMW-MAA in BALB/c mice has resulted in the development of a large number of anti-HMW-MAA monoclonal antibodies (mAbs). In contrast, no human anti-HMW-MAA mAbs have been described. Because the latter may serve as useful probes to characterize the antigenic profile of the HMW-MAA, human anti-HMW-MAA single-chain fragments of the variable region (scFvs) were isolated by panning synthetic scFv library 1 on purified HMW-MAA. Colony hybridization studies and nucleotide sequence analysis revealed that scFv 19, 44, 56, and 61 belong to the V(H)3 gene family and use the DP-38 germ-line gene segment but have a diverse third complementarity-determining region. The human scFvs share some characteristics with mouse anti-HMW-MAA mAb but also display some distinct features. Like mouse mAbs, human scFvs recognize determinants of HMW-MAA with a heterogeneous cellular and molecular distribution in human melanoma cells. Furthermore, like some mouse mAbs, human scFvs react with rat neural cells expressing the chondroitin sulfate proteoglycan NG2, which shows 81% homology to the HMW-MAA. However, at variance with mouse mAbs, the human scFvs show poor reactivity with guinea pig melanoma cells. Lastly, human scFv 61 stains both benign and malignant lesions of melanocytic origin, although with a lower frequency than mouse mAbs. Analysis of the clinical significance of the differential expression of the scFv 61-defined determinant in melanoma lesions will be facilitated by its reactivity with formalin-fixed melanoma lesions. In contrast to mouse mAb, scFv 61 immunoprecipitates the >450-kDa chondroitin sulfate proteoglycan component of the HMW-MAA, but not its 250-kDa subunit from melanoma cells. Thus, contrary to the current view about the structure of HMW-MAA, our results demonstrate that the two components are not associated. The described scFv antibodies, which represent the first example of human anti-HMW-MAA antibodies, have provided novel information about the structure of this antigen. Future studies will assess the impact of these in vitro-assembled antibody fragments on the identification of antigenic determinants of the HMW-MAA that can be recognized by the human immune system.     

A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice

The variable region genes of a murine anti-coronavirus monoclonal antibody (mAb) were joined by assembly polymerase chain reaction and expressed in Escherichia coli in a single chain variable fragment (scFv) configuration. After induction of expression, the expected 32-kDa protein was identified by Western immunoblotting with specific rabbit anti-idiotype antibodies. The scFv fragments were purified from soluble cytoplasmic preparations by affinity chromatography on nickel agarose, which was possible with an N-terminal but not with a C-terminal histidine tag. Purified scFv fragments retained the antigen-binding properties of the parental antibody, could inhibit its binding to viral antigens with apparently higher efficiency than monovalent antigen-binding (Fab) fragments, but neutralized viral infectivity with lower efficiency (about sevenfold at a molar level). To evaluate the usefulness of these smaller and less immunogenic molecules in the treatment of viral diseases, mice were treated with purified recombinant scFv fragments and challenged with a lethal viral dose. A small delay in mortality was observed for the scFv-treated animals. Therefore, even though the scFv could neutralize viral infectivity in vitro, the same quantity of fragments that partially protected mice in the form of Fab only slightly delayed virus-induced lethality when injected as scFv fragments, probably because of a much faster in vivo clearance: the biologic half-life was estimated to be about 6 min. Since a scFv derived from a highly neutralizing and protective mAb is only marginally effective in the passive protection of mice from lethal viral infection, the use of such reagents for viral immunotherapy will require strategies to overcome stability limitations.     

Immunizing patients with metastatic melanoma using recombinant adenoviruses encoding MART-1 or gp100 melanoma antigens

BACKGROUND: The characterization of the genes encoding melanoma-associated antigens MART-1 or gp100, recognized by T cells, has opened new possibilities for the development of immunization strategies for patients with metastatic melanoma. With the use of recombinant adenoviruses expressing either MART-1 or gp100 to immunize patients with metastatic melanoma, we evaluated the safety, immunologic, and potential therapeutic aspects of these immunizations. METHODS: In phase I studies, 54 patients received escalating doses (between 10(7) and 10(11) plaque-forming units) of recombinant adenovirus encoding either MART-1 or gp100 melanoma antigen administered either alone or followed by the administration of interleukin 2 (IL-2). The immunologic impact of these immunizations on the development of cellular and antibody reactivity was assayed. RESULTS: Recombinant adenoviruses expressing MART-1 or gp100 were safely administered. One of 16 patients with metastatic melanoma receiving the recombinant adenovirus MART-1 alone experienced a complete response. Other patients achieved objective responses, but they had received IL-2 along with an adenovirus, and their responses could be attributed to the cytokine. Immunologic assays showed no consistent immunization to the MART-1 or gp100 transgenes expressed by the recombinant adenoviruses. High levels of neutralizing antibody were found in the pretreatment sera of the patients. CONCLUSIONS: High doses of recombinant adenoviruses could be safely administered to cancer patients. High levels of neutralizing antibody present in patients' sera prior to treatment may have impaired the ability of these viruses to immunize patients against melanoma antigens.     

Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen

Antibodies that recognize antigens restricted to leukemia, lymphoma, and normal hematopoietic cells represent a unique opportunity to develop therapeutics, because they have the potential for relatively selective treatment of these diseases. Antibodies that recognize the CD19 antigen found on normal and malignant B cells, but not on stem cells, have been used to develop immunoconjugates. However, these conjugates are large and might be suboptimal in tumor penetration when compared to molecules using smaller single chain Fv (scFv) antibody fragments. scFv has the advantage of being a molecularly engineered homogeneous molecule. In this report, we demonstrate the cloning, expression, and binding of three anti-CD19 antibodies as scFvs. All three scFvs were successfully cloned and expressed. FVS191, derived from cell line B43, and FVS192, derived from SJ25C1, were properly refolded and bound CD19 antigen in FACS competition assays. These anti-CD19 scFv should be useful in the further development of diagnostic and therapeutic molecules.     

High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.     

Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics

By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vkappa regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.     

Cloning and functional characterization of the 5''-flanking region of human methionine adenosyltransferase 2A gene

Electron microscopy of dimeric and trimeric single chain antibody Fv fragments (scFvs) complexed with anti-idiotype Fab fragments was used to reveal the orientation of antigen binding sites. This is the first structural analysis that discloses the multivalent binding orientation of scFv trimers (triabodies). Three different scFv molecules were used for the imaging analysis; NC10 scFv-5 and scFv-0, with five- and zero-residue linkers respectively between the VH and VL domains, were complexed with 3-2G12 anti-idiotype Fab fragments and 11-1G10 scFv-0 was complexed with NC41 anti-idiotype Fab fragments. The scFv-5 molecules formed bivalent dimers (diabodies) and the zero-linker scFv-0 molecules formed trivalent trimers (triabodies). The images of the NC10 diabody-Fab complex appear as boomerangs, not as a linear molecule, with a variable angle between the two Fab arms and the triabody-Fab complexes appear as tripods.     

Localized primary non-Hodgkin lymphoma of the breast

The efficiency of both phage display in Escherichia coli and periplasmic expression of recombinant proteins may be limited by the same periplasmic folding steps. To search for E. coli factors that improve the efficiency of both procedures, a library of E. coli proteins was coexpressed in a phagemid vector that contained a poorly folding single-chain Fv antibody (scFv) fragment fused to g3p. We enriched, by panning for antigen binding, those phagemids in which the amount of displayed scFv is highest. We thus identified the periplasmic protein Skp/OmpH/HlpA as improving phage display of a wide range of scFv fragments. This occurs as a result of an increase in the amount of hybrid protein displayed on the phage. Coexpression of skp also increases the functional yield of scFv fragments when expressed by secretion to the periplasm.     

Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine

The necessity for prolonged tissue culture manipulations limits the clinical application of many form of gene therapy in patients with malignancies. We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a plasmid expression vector could be effectively introduced into resting tumor cells, without the need for tissue culture propagation prior to or following transfection, and that efficient expression of transgenic GM-CSF by the transfected tumor cells would confer an effective immune response against tumors. GM-CSF cDNA in expression vectors was coated onto gold particles and accelerated with a gene gun device into mouse and human tumor cells. Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice. Transfected and irradiated murine B16 melanoma cells produced > or = 100 ng/ml murine GM-CSF/10(6) cells per 24 hr in vitro for at least 10 days. The antitumor efficacy of this nonviral approach was tested using irradiated B16 tumor cells that were transfected with mGM-CSF cDNA and injected into mice as tumor "vaccine". Subsequent challenge of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals wer protected from the tumor by the prior vaccine treatment. In contrast, only 2% of control animals were protected by prior treatment with irradiated B16 cells transfected with the vector containing the luciferase gene. These results suggest that particle-mediated transfection of fresh tumor explants with cytokine cDNA is an effective and clinically attractive approach for cancer therapy.     

Characterization of an anti-RNA recombinant autoantibody fragment (scFv) isolated from a phage display library and detailed analysis of its binding site on U1 snRNA

This is the first study in which the complex of a monoclonal autoantibody fragment and its target, stem loop II of U1 snRNA, was investigated with enzymatic and chemical probing. A phage display antibody library derived from bone marrow cells of an SLE patient was used for selection of scFvs specific for stem loop II. The scFv specificity was tested by RNA immunoprecipitation and nitrocellulose filter binding competition experiments. Immunofluorescence data and immunoprecipitation of U1 snRNPs containing U1A protein, pointed to an scFv binding site different from the U1A binding site. The scFv binding site on stem loop II was determined by footprinting experiments using RNase A, RNase V1, and hydroxyl radicals. The results show that the binding site covers three sequence elements on the RNA, one on the 5' strand of the stem and two on the 3' strand. Hypersensitivity of three loop nucleotides suggests a conformational change of the RNA upon antibody binding. A three-dimensional representation of stem loop II reveals a juxtapositioning of the three protected regions on one side of the helix, spanning approximately one helical turn. The location of the scFv binding site on stem loop II is in full agreement with the finding that both the U1A protein and the scFv are able to bind stem loop II simultaneously. As a consequence, this recombinant monoclonal anti-U1 snRNA scFv might be very useful in studies on U1 snRNPs and its involvement in cellular processes like splicing.