Parental Monoclonal Antibody Research Articles

Single-chain antibody against human lipocalin-type prostaglandin D synthase: Construction, expression, purification, and activity assay

An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.

Generation and application of a fluorescein-specific single chain antibody

A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K D of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface.

Engineered anti-CD70 antibody with multiple effector functions exhibits in vitro and in vivo antitumor activities

AbstractAntigens expressed on malignant cells in the absence of significant expression on normal tissues are highly desirable targets for therapeutic antibodies. CD70 is a TNF superfamily member whose normal expression is highly restricted but is aberrantly expressed in hematologic malignancies including non-Hodgkin lymphoma (NHL), Hodgkin disease, and multiple myeloma. In addition, solid tumors such as renal cell carcinoma, nasopharyngeal carcinoma, thymic carcinoma, meduloblastoma, and glioblastoma express high levels of this antigen. To functionally target CD70-expressing cancers, a murine anti-CD70 monoclonal antibody was engineered to contain human IgG1 constant domains. The engineered antibody retained the binding specificity of the murine parent monoclonal antibody and was shown to induce Fc-mediated effector functions including antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis in vitro. Further, administration of this antibody significantly prolonged survival of severe combined immunodeficient (SCID) mice bearing CD70+ disseminated human NHL xenografts. Survival of these mice was dependent upon the activity of resident effector cells including neutrophils, macrophages, and natural killer (NK) cells. These data suggest that an anti-CD70 antibody, when engineered to contain human IgG1 constant domains, possesses effector cell–mediated antitumor activity and has potential utility for anticancer therapy.

Production and Characterization of Single Chain Fv Directed against β2-Agonist Clenbuterol

The single chain Fv (scFv) directed against beta2-agonist clenbuterol (CBL) was produced by using phage display technology. The heavy chain and light chain variable region genes (VH) VL) were amplified by the polymerase chain reaction (PCR) from CBL specific hybridoma cell lines 5D1 and assembled as a single chain Fv (scFv) fragment with linker peptide (Gly4Ser)3. Then the scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBL antibody libraries were constructed. Phages displaying scFv were enriched by panning with CBL-ovalbumin (CBL-OVA) conjugate. After only one round of panning, antigen-positive recombinant phage clones were successfully selected by ELISA. The positive phage was used to infect Escherichia coli HB2151, and the expression of soluble scFv was then induced by IPTG. The scFv showed an improved sensitivity (with IC50 of 0.78 +/- 0.005 ng/mL (n = 4)) when compared with the parent monoclonal antibody (MAb) (with IC50 of 1.34 +/- 0.006 ng/mL (n = 4)) in competitive indirect ELISA (CI-ELISA). Cross-reactivity studies showed that the specificity of scFv was similar to that of MAb. The recombinant scFv prepared in this study could be potentially used instead of conventional antisera or MAb for development of a rapid and affordable immunoassay for the detection of residual CBL in biological matrices.

Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of potato virus Y

Three single-chain variable fragment (scFv) antibodies recognizing the nuclear inclusion a (NIa) and capsid proteins of potato virus Y were obtained from two mouse derived hybridoma clones secreting, respectively, an anti-NIa (22-1) and an anti-coat protein (136-13) monoclonal antibodies. The first monoclonal antibody was able to inhibit in vitro the PVY polyprotein cleavage by blocking the NIa protease activity. The amplified scFv cDNAs were first inserted into the TOPO vector and then sequenced. Several recombinant E. coli clones carrying the accurate scFv sequences were selected and the corresponding cDNAs were subcloned in pHEN phagemid and transferred in E. coli strain. The expressed scFv fragments showed an antibody activity that recognized the viral target proteins in infected tissues. Their activity was comparable to the parental monoclonal antibodies.

Humanization of the Bispecific Epidermal Growth Factor Receptor × CD3 Diabody and Its Efficacy as a Potential Clinical Reagent

Bispecific antibodies (BsAb) have been exploited as both cancer immunodiagnostics and cancer therapeutics and show promise in clinical trials of cancer imaging and therapy. For development of BsAbs as clinical reagents, we have focused on construction of small recombinant BsAbs, called bispecific diabodies. Here, we constructed and characterized a humanized bispecific diabody. We have reported significant antitumor activity of an anti-epidermal growth factor receptor (EGFR) x anti-CD3 bispecific diabody (Ex3) in in vitro cytotoxicity assays and in vivo. We humanized the Ex3 diabody (hEx3) by grafting the complementarity-determining region and compared its biological properties with those of Ex3. We also tested its physiologic stability and ability to alter survival in xenografted mice. The final yield of hEx3 was 10 times that of Ex3, and refolded hEx3 and Ex3 showed identical binding profiles in EGFR-positive cell lines and EGFR-transfected Chinese hamster ovary cells. hEx3 showed dose-dependent cytotoxicity to EGFR-positive cell lines, which could be specifically inhibited by parental monoclonal antibody IgGs against EGFR or CD3 antigens. The heterodimeric structure was retained in PBS for 6 months, and growth inhibition was maintained after incubation under physiologic conditions. Coadministration of hEx3 with T-LAK cells and interleukin-2 prolonged the survival of nude mice with human colon carcinoma. The humanized diabody hEx3 is an attractive molecule for cancer therapy and may provide important insights into the development of EGFR-based cancer-targeting reagents.

A Humanized Anti-CD70 Monoclonal Antibody Targets CD70-Expressing Multiple Myeloma.

The tumor necrosis factor family member CD70 (CD27L) is an integral membrane protein transiently expressed on antigen-activated T and B lymphocytes. CD70 regulates lymphocyte survival and differentiation of CD27 positive resting T cells and antigen-primed B cells. Aberrant CD70 expression has been detected on hematologic tumors including diffuse large cell, follicular, and mantle cell lymphomas, chronic lymphocytic leukemia and Reed-Sternberg cells in Hodgkin's disease. Here we report for the first time the expression of CD70 on multiple myeloma (MM). Nine of thirteen MM cell lines examined expressed detectable surface CD70 with receptor copy numbers ranging from 9,000 to 145,000. Furthermore, seven of twelve primary MM isolates and six of six Waldenstrom's macroglobulinemia (WM) isolates tested were positive for CD70. The presence of CD70 on MM and WM, combined with the antigen's limited expression on normal non-hematopoietic tissues makes CD70 an attractive target for antibody-directed therapy of plasma cell disorders. We have engineered chimeric and humanized variants (human IgG1 isotype) of the murine anti-CD70 monoclonal antibody, 1F6. Both chimeric and humanized 1F6 variants retain the binding specificity of the murine parent monoclonal antibody (mAb) and acquired Fc-dependent effector functions including antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytoxicity (CDC) and macrophage-mediated antibody-dependent cellular phagocytic (ADCP) activities against CD70+ tumor cells. Specific lysis (ADCC, CDC) or tumor cell uptake (ADCP) was observed when engineered anti-CD70 mAb were used at ≥10 ng/mL against a panel of CD70-expressing hematologic tumors. The significance of these in vitro observations was further established by the ability of anti-CD70 mAb to significantly improve the survival of mice bearing disseminated CD70-expressing tumors. The in vitro function and in vivo efficacy of anti-CD70 were dependent upon Fc-FcγR interactions, as activity and survival benefit was lost with anti-FcγR blockade or when the targeting antibody was engineered to eliminate FcγR binding. Our data suggest that humanized anti-CD70 mAb possesses potent antitumor activity and has potential utility for therapy of hematologic cancers including MM and WM.

Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody

A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the β′ subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (V H) and light chain (V L) were used as the template for cDNA synthesis. The sequences were joined by the addition of a “linker” sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73.

Thermodynamic basis for antibody binding to Z-DNA: Comparison of a monoclonal antibody and its recombinant derivatives

Antibody engineering represents a promising area in biotechnology. Recombinant antibodies can be easily manipulated generating new ligand and effector activities that can be used as prototype magic bullets. On the other hand, an extensive knowledge of recombinant antibody binding and stability features are essential for an efficient substitution. In this study, we compared the stability and protein binding properties of two recombinant antibody fragments with their parental monoclonal antibody. The recombinant fragments were a monomeric scFv and a dimeric one, harboring human IgG1 CH2–CH3 domains. We have used fluorescence titration quenching to determine the thermodynamics of the interaction between an anti-Z-DNA monoclonal antibody and its recombinant antibody fragments with Z-DNA. All the antibody fragments seemed to bind DNA similarly, in peculiar two-affinity states. Enthalpy–entropy compensation was observed for both affinity states, but a marked entropy difference was observed for the monomeric scFv antibody fragment, mainly for the high affinity binding. In addition, we compared the stability of the dimeric antibody fragment and found differences favoring the monoclonal antibody. These differences seem to derive from the heterologous expression system used.

Cloning, expression and characterisation of a single-chain Fv antibody fragment against domoic acid in Escherichia coli

Domoic acid is a potent neuroexcitatory toxin that causes amnesic shellfish poisoning in humans through ingestion of contaminated shellfish. The variable regions of the heavy chain (V H) and light chain (V L) of an antibody specific for domoic acid were cloned from a mouse hybridoma cell line and used to construct single-chain antibody fragments (scFvs) in a variety of formats. V H–linker–V L scFvs were expressed better in Escherichia coli than the V L–linker–V H format, while use of the commonly used (Gly 4Ser) 3 inter-domain linker resulted in higher yields than a longer (Gly 4Ser) 6 linker variant. Higher soluble protein yields were achieved in E. coli TOP 10 than in E. coli XL1-Blue cells and co-production of the E. coli disulfide bond isomerase enzyme DsbC allowed higher cell densities to be attained during scFv production, leading to increased yields of recombinant protein. The purified scFv exhibited binding similar to the parent monoclonal antibody and is being used to develop an immunosensor to detect domoic acid in contaminated shellfish samples.

Engineering and functional evaluation of a single-chain antibody against HIV-1 external glycoprotein gp120

The HIV-1 envelope glycoprotein surface subunit gp120 is an attractive target for molecular intervention. This is because anti-HIV-1 gp120 neutralizing antibodies display the potential ability to inhibit HIV-1 infection. The present investigation describes the construction of a genetically engineered single chain antibody (scFv102) against HIV-1 gp120, its expression and functional evaluation. The parental hybridoma cell line (102) produces an immunoglobulin directed against the conserved CD4-binding region of gp120. cDNAs encoding the variable regions of the heavy (V(H)) and light (V(L)) chains were prepared by reverse transcription PCR and linked together with an oligonucleotide encoding a linker peptide (Gly(4)Ser)(3) to produce a single chain antibody gene. The resulting DNA construct was cloned into a prokaryotic expression vector (pET28) and recombinant scFv102 was expressed in Eserichia coli as an insoluble protein. The denatured scFv102 was refolded and purified by immobilized metal ion affinity chromatography. Purified scFv102 had the same specificity as the intact IgG in immuno-blotting assays and immuno-fluorescence (IF) detection, but ELISA analyses demonstrated the affinity of scFv102 to be 5-fold lower than that of the parental monoclonal antibody. In neutralization assays, scFv102 at concentrations lower than 40 microg/ml exhibited efficient interference with viral replication and inhibition of viral infection (90%) across a range of primary isolates of subtype B HIV-1. These results suggest that the constructed anti-HIV-1 gp120 scFv102 has good biological activity and can potentially be used for in vitro diagnostic and in vivo therapeutic applications.

A Bispecific Recombinant Immunotoxin, DT2219, Targeting Human CD19 and CD22 Receptors in a Mouse Xenograft Model of B-Cell Leukemia/Lymphoma

A novel bispecific single-chain fusion protein, DT2219, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT(390)) fused to two repeating sFv subunits recognizing CD19 and CD22 and expressed in Escherichia coli. Problems with yield, purity, and aggregation in the refolding step were solved by incorporating a segment of human muscle aldolase and by using a sodium N-lauroyl-sarcosine detergent-based refolding procedure. Problems with reduced efficacy were addressed by combining the anti-CD19 and anti-CD22 on the same single-chain molecule. DT2219 had greater anticancer activity than monomeric or bivalent immunotoxins made with anti-CD19 and anti-CD22 sFv alone and it showed a higher level of binding to patient leukemia cells and to CD19(+)CD22(+) Daudi or Raji cells than did anti-CD19 and anti-CD22 parental monoclonal antibodies. The resulting DT2219, mutated to enhance its avidity, was cytotoxic to Daudi cells in vitro (IC(50) = 0.3 nmol/L). In vivo, DT2219 was effective in a flank tumor therapy model in which it significantly inhibited tumor growth (P < 0.05) and in a systemic model in which it significantly prolonged survival of severe combined immunodeficient mice with established Daudi (P < 0.008) compared with controls. DT2219 has broader reactivity in recognizing B-cell malignancies, has more killing power, and requires less toxin than using individual immunotoxin, which warrants further investigation as a new drug for treating B leukemia/lymphoma.

Combined effects of tamoxifen and a chimeric humanized single chain antibody against the type I IGF receptor on breast tumor growth in vivo.

Proliferative and anti-apoptotic actions of IGFs are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and -II bind with high affinity. We previously reported that alphaIGF-IR scFv-Fc (scFv-Fc) consisting of the alphaIGF-IR scFv and human IgG (1) Fc domain retained general characteristics of the parental 1H7 monoclonal antibody, and significantly suppressed MCF-7 tumor growth. We proposed IGF-IR down-regulation as a possible mechanism for inhibition of MCF-7 tumor growth. To further determine the therapeutic potentials of this approach, in vivo effects of this antibody on breast tumor growth were evaluated in the absence or presence of tamoxifen (Tam) using a T61 human breast tumor model. T61 xenograft growth in athymic mice was compared under five conditions, PBS, scFv-Fc, Tam, scFv-Fc+Tam, and control antibody. While treatment with PBS and control antibody did not affect T61 tumor growth, scFv-Fc, Tam, and scFv-Fc+Tam treatments significantly suppressed the tumor growth during the first two weeks of treatment. Although the growth inhibitory effect of scFv-Fc during the first two weeks was significant, the tumor grew as rapidly as PBS-treated tumors thereafter. This rapid tumor growth was suppressed when scFv-Fc was combined with Tam. Throughout four weeks, the combined Tam+scFv-Fc treatment was more effective in inhibiting the T61 tumor growth than scFv-Fc or Tam treatment alone. scFv-Fc treatment down-regulated IGF-IR which appears to contribute to tumor growth inhibition. This study provides evidence that simultaneous targeting of IGF-IR and the estrogen receptor may enhance the therapeutic effect.

Stability of murine, chimeric and humanized antibodies against pre-S2 surface antigen of hepatitis B virus

We have constructed a humanized antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) by grafting the complementarity determining regions (CDRs) of parental murine monoclonal antibody (mAb) into human anti-Sm antibody framework regions. The humanized antibody has a substitution at position 94 in a framework region of the heavy chain variable region, and exhibits the same antigen binding affinity as the parental murine monoclonal and chimeric antibodies. In order to assess the stability of these antibodies, thermal inactivation of the parental, chimeric and humanized antibodies was analyzed. Fifty percent inactivation of the chimeric and humanized antibodies was observed at 63.7 °C and 68.7 °C, respectively, compared to 55.0 °C for murine antibody. The humanized antibody also exhibited increased stability against denaturant. Guanidine-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that midpoints of the transition of the chimeric and humanized antibodies were 2.47 M and 2.56 M, respectively, whereas that of the murine antibody was 1.36 M.

Monoclonal antibody 667 recognizes the variable region A motif of the ecotropic retrovirus CasBrE envelope glycoprotein and inhibits Env binding to the viral receptor.

Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D(57), is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.

Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants.

We constructed a recombinant antibody fragment--single chain fragment-variable (scFv) antibody--derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene. The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb). Up to 220 ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities. These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.

Functional enhancement of a partially active single-chain variable fragment antibody to Venezuelan equine encephalitis virus.

Previously cloned recombinant A116 single chain fragment variable (scFv) antibody gene has been re-engineered for enhanced reactivity to Venezuelan equine encephalitis virus (VEE) successfully. A PCR-based site-directed mutagenesis approach was adopted to re-introduce the three single-base deletions in the 5' region of the V(L) gene of A116, corresponding to the framework-1 region. The mutagenized A116 was designated as MA116. The introduction of these three bases corrected a localized frame-shift to a consensus framework-1 amino acid sequence. Four MA116 clones (MA116-4, MA116-14, MA116-15, and MA116-16) have been analysed in detail for their reactivity to VEE antigen, and all showed varying degrees of reactivity to VEE antigen. ScFv antibody expressed by MA116-14, MA116-15, and MA116-16 clones showed three to five-fold enhanced enzyme-linked immunosorbant assay reactivity to VEE antigen over the parental A116 clone, while scFv antibody from MA116-4 was less reactive than A116 clone. MA116-15 purified scFv protein showed comparable reactivity to the parental 1A4A-1 monoclonal antibody in recognizing VEE antigen. Sequence analysis revealed that only MA116-15 had incorporated the three intended base insertions. The varying degrees of reactivity of MA116 clones are discussed in light of their molecular changes.

Production of a functional catalytic antibody ScFv-NusA fusion protein in bacterial cytoplasm.

Functional expression of catalytic antibodies in the cytoplasm of E. coli is potentially of great interest in searching for new catalysts by genetic selection. Herein, a catalytic antibody single chain Fv (ScFv) 14D9, which catalyzes a highly enantioselective protonation, was expressed as a NusA fusion protein under the T7 promoter. A functional disulfide-containing ScFv fusion protein was obtained in the oxidizing environment of bacterial cytoplasm. The 14D9 ScFv could not be overexpressed alone without NusA fusion. The highly soluble NusA protein most likely retards aggregate formation of ScFv and indirectly supports correct folding and disulfide bridge formation in the fusion construct ScFv-NusA. The ScFv-NusA fusion product shows highly enantioselective, specific, hapten inhibited catalytic activity comparable to its parent monoclonal antibody, 14D9. The NusA fusion method might be generally helpful for functional antibody expression in vivo and for the new development of biocatalysts by genetic selection.

Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibα

A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Ibα was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIbα. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH–VL orientation, being joined together with a 15-amino-acid (Gly 4Ser) 3 linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-β- d-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP-induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent.

Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody.

A recent study reported on an anti-CD19/anti-CD3 single-chain bispecific antibody (bscCD19xCD3) exhibiting high activity against human B lymphoma cell lines (Löffler et al., Blood 2000;95:2098-103). In the present study, we have explored in detail the in vitro efficacy, T-cell donor variability, binding characteristics, specificity, kinetics and interleukin-2 (IL-2) dependence of bscCD19xCD3. We found that a majority of human donor T cells tested (n = 86) gave half-maximal B-lymphoma cell lysis (ED(50)) within a range of 10-50 pg/ml bscCD19xCD3, corresponding to sub-picomolar concentrations of the bispecific antibody. Under identical experimental conditions, the anti-CD20 monoclonal antibody rituximab had an at least 100,000-fold lower in vitro efficacy. The extreme potency of bscCD19xCD3 was in sharp contrast to the relatively low affinity of the anti-CD3 and anti-CD19 single-chain Fv portions in K(D) ranges of 10(-7) and 10(-9) M, respectively. Cell lysis by bscCD19xCD3 was predominantly mediated by the population of CD8/CD45RO-positive T cells. Both immortalized CD4- and CD8-positive human T-cell clones were highly active effector cells as well. Cell lysis by bscCD19xCD3 was rapid and specific. The respective parental monoclonal antibodies inhibited cell lysis and CD19-negative cells were not harmed by T cells in the presence of high amounts of bscCD19xCD3. The potent T-cell stimulus IL-2 could not markedly augment the activity of bscCD19xCD3-stimulated T cells. In conclusion, bscCD19xCD3 could redirect unstimulated cytotoxic T cells against CD19-positive cells in an unexpectedly potent, rapid and specific fashion.