Sergey Kipriyanov

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.

Bispecific CD3×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 CD3ϵ 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 VH3‐VL19 and VH19‐VL3 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°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. Int. J. Cancer 77:763–772, 1998.

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 (t 1/2 cell surface retention at 37°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.

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.

Treatment of Human B Cell Lymphoma Xenografts with a CD3 × CD19 Diabody and T Cells

The use of anti-CD3 × antitumor bispecific Abs is an attractive and highly specific approach in cancer therapy. Recombinant Ab technology now provides powerful tools to enhance the potency of such immunotherapeutic constructs. We designed a heterodimeric diabody specific for human CD19 on B cells and CD3ε chain of the TCR complex. After production in Escherichia coli and purification, we analyzed its affinity, stability, and pharmacokinetics, and tested its capacity to stimulate T cell proliferation and mediate in vitro lysis of CD19+ tumor cells. The effect of the diabody on tumor growth was investigated in an in vivo model using immunodeficient mice bearing a human B cell lymphoma. The CD3 × CD19 diabody specifically interacted with both CD3- and CD19-positive cells, was able to stimulate T cell proliferation in the presence of tumor cells, and induced the lysis of CD19+ cells in the presence of activated human PBL. The lytic potential of the diabody was enhanced in the presence of an anti-CD28 mAb. In vivo experiments indicated a higher stability and longer blood retention of diabodies compared with single chain Fv fragments. Treatment of immunodeficient mice bearing B lymphoma xenografts with the diabody and preactivated human PBL efficiently inhibited tumor growth. The survival time was further prolonged by including the anti-CD28 mAb. The CD3 × CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.

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.

Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin's lymphoma.

To target NK cells against non-Hodgkin’s lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcγRIII (CD16). Bacterially produced CD19 × CD16 BsDb specifically interacted with both CD19+ and CD16+ cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs. The combination of the CD19 × CD16 BsDb with a previously described CD19 × CD3 BsDb and CD28 costimulation significantly increased the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt’s lymphoma (5 mm in diameter) with human PBLs, CD19 × CD16 BsDb, CD19 × CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target.

Generation and production of engineered antibodies

Various forms of recombinant monoclonal antibodies are being used increasingly, mainly for therapeutic purposes. This review specifically focuses on what is now called antibody engineering, and discusses the generation of chimeric, humanized, and fully human recombinant antibodies, immunoglobulin fragments, and artificial antigen-binding molecules. Since the production of recombinant antibodies is a limiting factor in their availability, and a shortage is expected in the future, different expression systems for recombinant antibodies and transgenic organisms as bioreactors are also discussed, along with their advantages and drawbacks.

Effect of Domain Order on the Activity of Bacterially Produced Bispecific Single-chain Fv Antibodies

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.

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.