Cure of Burkitt's lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 x CD19 tandem diabody, and CD28 costimulation.

To increase the valency, stability, and therapeutic potential of bispecific antibodies, we have constructed a tetravalent tandem diabody (Tandab) that is specific to both human CD3 (T-cell antigen) and CD19 (B-cell marker; S. M. Kipriyanov et al., J. Mol. Biol., 293: 41-56, 1999). It was generated by the functional dimerization of a single chain molecule that contained four antibody variable domains (V(H) and V(L)) in an orientation that prevented intramolecular pairing. Compared with a previously constructed heterodimeric CD3 x CD19 diabody, the Tandab exhibited a higher apparent affinity to both CD3+ and CD19+ cells and longer blood retention when injected into mice. Biodistribution studies in mice bearing Burkitt's lymphoma xenografts demonstrated specific accumulation of the radioiodinated Tandab in a tumor site with tumor-to-blood ratios of 1.5, 8.1, and 13.3 at 3, 18, and 24 h, respectively. Treatment of severe combined immunodeficiency mice bearing established Burkitt's lymphoma (5 mm in diameter) with human peripheral blood lymphocytes, Tandab, and anti-CD28 MAbs resulted in the complete elimination of tumors in all of the animals within 10 days. In contrast, mice receiving human peripheral blood lymphocytes in combination with either the diabody alone or the diabody plus anti-CD28 MAbs showed only partial tumor regression. These data demonstrate that the CD3 x CD19 Tandab may be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas.

Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa.

BACKGROUND: Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot. METHODS AND RESULTS: The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly(4)Ser)(3) was introduced by overlap PCR. After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the Bbeta15-22 peptide of fibrin (beta-peptide). Hirudin was genetically fused to the C-terminus of the variable region of the light chain. To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv(59D8) and hirudin. The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to beta-peptide, and by thrombin inhibition after Xa cleavage. Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin. CONCLUSIONS: A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa. This recombinant anticoagulant effectively inhibits appositional clot growth in vitro. Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.

Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics.

To increase the valency, stability and therapeutic potential of bispecific antibodies, we designed a novel recombinant molecule that is bispecific and tetravalent. It was constructed by linking four antibody variable domains (VHand VL) with specificities for human CD3 (T cell antigen) or CD19 (B cell marker) into a single chain construct. After expression in Escherichia coli, intramolecularly folded bivalent bispecific antibodies with a mass of 57 kDa (single chain diabodies) and tetravalent bispecific dimers with a molecular mass of 114 kDa (tandem diabodies) could be isolated from the soluble periplasmic extracts. The relative amount of tandem diabodies proved to be dependent on the length of the linker in the middle of the chain and bacterial growth conditions. Compared to a previously constructed heterodimeric CD3xCD19 diabody, the tandem diabodies exhibited a higher apparent affinity and slower dissociation from both CD3(+)and CD19(+)cells. They were also more effective than diabodies in inducing T cell proliferation in the presence of tumor cells and in inducing the lysis of CD19(+)cells in the presence of activated human PBL. Incubated in human serum at 37 degrees C, the tandem diabody retained 90 % of its antigen binding activity after 24 hours and 40 % after one week. In vivo experiments indicated a higher stability and longer blood retention of tandem diabodies compared to single chain Fv fragments and diabodies, properties that are particularly important for potential clinical applications.

Recombinant single-chain Fv fragments carrying C-terminal cysteine residues: production of bivalent and biotinylated miniantibodies.

A murine antibody single-chain Fv (scFv) fragment carrying five C-terminal histidine residues preceded by a cysteine residue and a marker peptide was expressed in Escherichia coli. Its variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody mAb215, which is specific for the largest subunit of RNA polymerase II of Drosophila melanogaster. ScFv' monomers, covalently linked (scFv')2 and non-covalent dimers, as well as aggregated antibody fragments, were isolated from an E. coli cell paste by immobilized metal affinity chromatography in 6 M urea followed by a renaturation procedure that does not use any sulfhydryl agents. In a final step, the components were separated by size exclusion chromatography. All the recombinant antibody fractions demonstrated high antigen-binding activity and specificity as shown by ELISA and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental monoclonal antibodies and four-fold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.

A single expression system for the display, purification and conjugation of single-chain antibodies.

To facilitate the purification and conjugation of single-chain antibodies (scFv) selected from a phage display library, we have incorporated His6, an amber stop codon and a C-terminal Cys into a surface expression vector. The vector also contains a lacIq gene for improving the efficiency of regulation and a sequence coding for a marker peptide.

Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding.

Single chain variable fragments (scFv) of the murine monoclonal antibody HD37 specific to human B-cell antigen CD19 were constructed by joining the VH and VL domains with linkers of 18, 10, 1 and 0 residues. ScFv-18 formed monomers, dimers and small amounts of tetramers; scFv-10 formed dimers and small amounts of tetramers; scFv-1 formed exclusively tetramers; scFv-0 formed exclusively trimers. The affinities of the scFv-10 (diabody) and scFv-1 (tetrabody) were approximately 1.5- and 2.5-fold higher, respectively, than that of the scFv-0 (triabody). The tetrabody displayed a significantly prolonged association with cell-bound antigen (t1/2 cell surface retention at 37 degrees C of 26.6 min) compared to both the diabody (13.3 min) and triabody (6.7 min). This increase in avidity of the tetrabody combined with its larger size could prove to be particularly advantageous for imaging and the immunotherapy of B-cell malignancies.

Mutagenesis directed by phosphotriester analogues of oligonucleotides: a way to site-specific mutagenesis in vivo.

A new approach to induce directed mutations in genes of study through simple cotransfection of E. coli cells by the mixture of primer and template was developed. This method is based on the use of synthetic phosphotriester analogues of oligonucleotides as site-specific mutagenic primers. The achieved yield of mutant clones was 2-3%.

Antibodies to steroids from a small human naive IgM library.

Human antibodies specific for digoxigenin, estradiol, testosterone and progesterone have been isolated from a small combinatorial IgM repertoire (4 x 10(7)) of single chain antibodies (scFv). The affinities of both the anti-estradiol and antiprogesterone scFv were approximately 10(8) M(-1). Naive IgM genes appeared to be highly represented, since only the heavy chain variable domain of the anti estradiol antibody contained differences to corresponding germline sequences. The light chain variable domain of the progesterone receptor was also identical to a germline sequence, showing that it is possible for completely naive antibodies to bind steroids with affinities comparable to those obtained after a secondary immune response.

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures.

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15-25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80-150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20 degrees C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.

Rapid detection of recombinant antibody fragments directed against cell-surface antigens by flow cytometry.

Cloning the correct genes coding for antibody variable domains (especially VL kappa) from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from the myeloma cell line. Indeed, four different VL genes were obtained after the amplification of immunoglobulin genes by PCR from the hybridoma HD37, which produces an antibody against the human CD19 B cell differentiation antigen. Most of the variants (eight out of 15) were derived from the kappa chain of the myeloma MOPC-21. For the rapid functional evaluation of recombinant antibody fragments against cell surface antigens, we established an efficient expression and detection system. First, deleted and mutated genes were eliminated by a colony screening procedure. Bacteria from picked colonies were then induced and grown in the presence of 0.4 M sucrose to increase the accumulation of soluble scFv in the periplasm (5-10 micrograms per ml of bacterial shake-tube culture). Finally, the cell-specific binding of scFv in crude periplasmic extracts was detected by flow cytometry. This procedure facilitated the efficient cloning of a functional anti-CD19 VH/VL combination from the hybridoma cDNA.

Monitoring of scFv selected by phage display using detection of scFv-pIII fusion proteins in a microtiter scale assay.

We describe here a method for the efficient and rapid analysis of antigen binding characteristics of recombinant antibodies (ab) selected by phage display. This novel approach combines the bacterial production of soluble single chain ab (scFv)-pIII fusion proteins on a microtiter scale with the detection of these fusion proteins via a pIII-specific ab. It facilitates the parallel analysis of large numbers of clones and is more efficient than current analysis protocols. Applying this technique, we analysed phage display selection of tetanus toxoid (TTX) specific scFv with respect to: (i) the productive expression of fusion proteins; (ii) the enrichment of specific scFv in subsequent rounds of phage display selection on a polyclonal level; (iii) the antigen specificity of individual scFv clones; (iv) the antigen binding affinity of a selected scFv. A TTX-specific scFv (clone 4.3) was further examined in a mono- and bivalent form by surface plasmon resonance analysis. ScFv 4.3 possesses a subnanomolar affinity and a low off rate constant.

Idiotypic vaccine for treatment of human B-cell lymphoma. Construction of IgG variable regions from single malignant B cells.

Immunoglobulin idiotypes (Id) of malignant B cells represent highly specific markers which can be used for vaccination. PCR-amplification of immunoglobulin genes enables the rapid production of large amounts of Id vaccines. However, the separate amplification and subsequent recombination of heavy and light chains can lead to a loss of the relevant Id. To preserve the original chain pairs, we used single malignant B cells derived from an immunocytoma patient. Cytoplasm was extracted and the mRNA transcribed into cDNA. The VH and VL genes were then amplified by PCR and cloned into a vector for expression in E. coli. Id production was checked using an anti-Id mouse monoclonal Ab raised against the patient's tumor-specific IgG. One out of 3 constructs expressed the relevant Id. Analysis of the first 31 light chain residues revealed an identical sequence for the malignant B cells' IgG and the recombinant Id construct. Exchange of either the heavy or light chain with an unrelated chain resulted in loss of the Id. An unrelated sequence derived from the c-myc protein is coupled to the Id vaccine. The lymphoma patient was shown to have Abs to the c-myc sequence. This sequence therefore, increases the Id+ Ab's antigenicity. CD spectroscopy showed an alpha-helical structure for the c-myc epitope. In conclusion, a B-cell lymphoma autovaccine was produced containing immunogenic sequences that do not alter the steric conformation of the tumor-specific Id.

The antigen binding domain of non-idiotypic human anti-F(ab')2 autoantibodies: study of their interaction with IgG hinge region epitopes.

In previous studies we described a natural human IgG-anti-F(ab')2 autoantibody family with immunoregulatory properties. Genes coding for the variable regions of the heavy and light chains of the Abs were isolated from a natural Ig gene library and scFv Abs were expressed in E. coli. The scFv Abs bound to F(ab')2 but not to Fab fragments. This points to an epitope located in the hinge region since Fab fragments are lacking most of the hinge. In order to verify our hypothesis, double chain peptides comprising the lower-, middle-, and part of the upper hinge subregion of IgG1-IgG4 were synthesized on cellulose membranes and tested for binding to the Abs. The results show binding of Abs to IgG1 and IgG4 hinge region peptides. In order to identify the key residues of the discontinuous epitopes we carried out complete substitutional analyses in which each amino acid of the wt peptides was substituted by all other amino acids except cysteine. The exchange of proline in the IgG1 or IgG4 middle hinge region abrogated the binding, revealing the importance of this subregion for epitope expression. No binding to the IgG2 or IgG3 hinge was detected. These results indicate that scFv anti-F(ab')2 Abs recognize the hinge region of IgG1 and IgG4 and that the expression of the epitope depends on an intact middle hinge subregion.

Generation of recombinant antibodies.

Recombinant antibody technology is opening new perspectives for the development of novel therapeutic and diagnostic agents. In this review we focus on advances in the generation of both genetically engineered humanized and fully human monoclonal antibodies. Methods for their production in different expression systems are also discussed.

Purification, characterization, and biotinylation of single-chain antibodies.

The variable region (Fv) portion of an antibody is comprised of the antibody V(H) and V(L) domains and is the smallest antibody fragment containing a complete antigen-binding site. To stabilize the association of the recombinant V(H) and V(L) domains, they have been linked in single-chain Fv constructs with a short peptide that bridges the approx 3.5 nm between the carboxy terminus of one domain and the ammo terminus of the other (1-3). An NMR comparison of the unlinked Fv fragment of the antibody McPC603 with the corresponding scFv containing a V(H)-(Gly(4)Ser)(3)-V(L) linker has shown no perturbation of the folding of the variable domains by the linker (4,5). In comparison to the much larger Fab', F(ab')(2), and IgG forms of monoclonal antibody (MAb) from which they are derived, scFvs have lower retention times in nontarget tissues, more rapid blood clearance, and better tumor penetration (6-8). ScFvs, therefore, represent potentially very useful molecules for the targeted delivery of drugs, toxins, or radionuclides to a tumor site.

Recent developments in antibody engineering.

Rapid growth in the field of antibody engineering occurred after it was shown that functional antibody fragments could be secreted into the periplasmic space and even into the medium of Escherichia coli by fusing a bacterial signal peptide to the antibody's N-terminus (1,2). These findings allowed scientists to transfer the principles of the immune system for producing specific antibodies to a given antigen into a bacterial system (3). It was now possible to establish antibody libraries in E. coli that could be directly screened for binding to antigen. This was accomplished at first by transforming E coli with plasmids containing polymerase chain reaction (PCR)-amplified immunoglobulin families from the lymphocytes of immunized mice. Immunogen-reactive recombinant antibodies were then selected by an enzyme-linked immunosorbent assay (ELISA) of the bacterial supernatant from isolated bacterial colonies (4). This procedure was subsequently improved upon by inserting the antibody operon into bacteriophage λ. Antibody libraries were then able to be efficiently transfected into E. coli and plaque lift-offs of lysed bacterial colonies on nitrocellulose could be screened for reactivity to a radioactive labeled immunogen (5-7).

Single-chain antibody streptavidin fusions: tetrameric bifunctional scFv-complexes with biotin binding activity and enhanced affinity to antigen.

To increase the avidity of single-chain antibodies (scFv) for their antigen, we have fused them to core-streptavidin. The chimeric protein, expressed by the vector pSTE (plasmid for streptavidin-tagged expression) from Escherichia coli, can form tetrameric complexes, binds its antigen and contains four biotin binding sites per tetrameric complex. An additional cysteine inserted near the carboxy terminus further stabilised the complex. The scFv fusion protein tetramers could be enriched by affinity chromatography using the biotin analog 2-iminobiotin from periplasmic inclusion bodies after refolding. We have also shown that the scFv fusion protein could be used for direct detection of its antigen in ELISA when stained with biotinylated horseradish peroxidase. The affinity of the scFv-antibody complex was substantially increased by avidity effects due to the tetrameric structure. The biotin binding sites may be used for coupling other antibodies and molecules to form bispecific and bifunctional reagents.

Bispecific CD3 x CD19 diabody for T cell-mediated lysis of malignant human B cells.

For the treatment of minimal residual disease in patients with leukemias and malignant lymphomas, we constructed a heterodimeric diabody specific for human CD19 on B cells and CD3epsilon chain of the T cell receptor complex. The bispecific diabody was expressed in Escherichia coli using a vector containing a dicistronic operon for co-secretion of V(H)3-V(L)19 and V(H)19-V(L)3 single-chain Fv fragments (scFv). It was purified in one step by immobilized metal affinity chromatography (IMAC) from the periplasmic extract and culture medium. Flow cytometry experiments revealed specific interactions of the diabody with both CD3 and CD19 positive cells, to which it bound with affinities close to those of the parental scFvs. It was less stable than anti-CD3 scFv but more stable than anti-CD19 scFv when incubated in human serum at 37 degrees C. In cytotoxicity tests, the diabody proved to be a potent agent for retargeting peripheral blood lymphocytes to lyse tumor cells expressing the CD19 antigen. The efficiency of cell lysis compared favorably with that obtained with a bispecific antibody (BsAb) of the same dual specificity that was prepared by the quadroma technique.

Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines.

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.

Apoptosis of a human melanoma cell line specifically induced by membrane-bound single-chain antibodies.

CD28 is a key regulatory molecule in T cell responses. Ag-TCR/CD3 interactions without costimulatory signals provided by the binding of B7 ligands to the CD28R appear to be inadequate for an effective T cell activation. Indeed, the absence of B7 on the tumor cell surface is probably one of the factors contributing to the escape of tumors from immunological control and destruction. Therefore, to increase the immunogenicity of tumor cell vaccines, we have expressed anti-CD3 and anti-CD28 single-chain Abs (scFv) separately on the surface of a human melanoma SkMel63 cell line (HLA-A*0201). A mixture of cells expressing anti-CD3 with cells expressing anti-CD28 resulted in a marked activation of allogeneic human PBL in vitro. The apparent induction of a Th1 differentiation pathway was accompanied by the proliferation of MHC-independent NK cells and MHC-dependent CD8+ T cells. PBL that had been cultured together with transfected SkMel63 tumor cells were able to specifically induce apoptosis in untransfected SkMel63 cells. In contrast, three other tumor cell lines expressing HLA-A*0201, including two melanoma cell lines, showed no significant apoptosis. These results provide valuable information for both adoptive immunotherapy and the generation of autologous tumor vaccines.