scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies.

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the VH and VL domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody). Direct ligation of VH and VL domains further restricts association and forces three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructing a series of scFvs from anti-neuraminidase antibody NC10 in which the linker varied from one to four glycine residues. NC10 scFv molecules containing linkers of three and four residues showed a strong preference for dimer formation (diabodies), whereas a linker length of one or two glycine residues prevented the formation of diabodies and directed scFv association into trimers (triabodies). The data suggest a relatively strict transition from dimer (diabody) to trimer (triabody) upon reduction of the linker length from three to two glycine residues. Modelling studies are consistent with three residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multimers and an anti-idiotype Fab' showed that the dimer was bivalent for antigen binding and the trimer was trivalent.     

Construction of a diabody (small recombinant bispecific antibody) using a refolding system

Diabodies are the recombinant bispecific antibodies (BsAbs), constructed from heterogeneous single-chain antibodies. Usually, diabodies have been prepared from bacterial periplasmic fraction using a co-expression vector (i.e. genes encoding two chains were tandemly located under the same promoter). Some diabodies, however, cannot be expressed as a soluble material owing to inclusion body formation, which limits the utilization of diabodies in various fields. Here we report an improved method for the construction of diabodies using a refolding system. As a model, a bispecific diabody binding to adenocarcinoma-associated antigen MUC1 and to CD3 on T cells was studied. One chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). The second was composed of a VL specific for MUC1 linked to a VH specific for CD3. The two hetero scFvs were independently obtained from intracellular insoluble fractions of Escherichia coli, purified, mixed stoichiometrically (at an equivalent molar ratio of 1:1) and refolded. The refolded two hetero scFv has a hetero-dimeric structure, with complete specificity for both target cells [i.e. MUC1 positive cells and CD3 positive lymphokine-activated killer cells with a T cell phenotype (T-LAK)]. Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach approximately 98% at an effector:target ratio (E:T ratio) of 10, almost identical with that with anti-MUC1xanti-CD3 chemically synthesized BsAbs (c-BsAbs). This is the first report of the construction of a diabody using a refolding system.     

Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD

In an attempt to generate recombinant anti-D reagents for possible diagnostic and therapeutic use we cloned the genes encoding the variable (V) domains of a human anti-D antibody secreted by the lymphoblastoid cell line BTSN4. A single-chain Fv (scFv) fragment was constructed using a 21 amino acid linker to join the genes encoding the variable domains of the BTSN4 heavy (VH) and light chains (VL). A diabody construct was also generated by reducing the length of the scFv linker from 21 to 10 residues. The scFv and diabody constructs were cloned into the pFLAG-CTS vector, expressed in E. coli host cells and the recombinant proteins were affinity-isolated from bacterial culture medium. Analysis of the recombinant proteins indicated that they retained the D antigen binding specificity of the parental BTSN4 IgG. Furthermore, both fragments mediated agglutination of papain-treated D positive erythrocytes in the absence of a cross-linking second antibody. While the agglutinating property of BTSN4 diabody was readily explained by the non-covalent association of this protein as a bivalent dimer, oligomeric forms of BTSN4 scFv were not detected when the protein was analysed by size exclusion chromatography. Thus, the agglutinating property of the scFv is not the result of the formation of non-covalently associated multimeric forms of the antibody fragment.     

The effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody

In recent years a variety of recombinant methods have been developed for efficient production of bispecific antibodies (BsAb) in various formats. Bispecific diabody (bDAb), a 55-60 kDa molecule comprising two non-covalently associated cross-over single chain Fv (scFv) polypeptides, represents one of the most promising as well the most straightforward approaches to BsAb production. Here we constructed a bDAb, using two human scFv, 11F8 and A12, directed against the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR), respectively, as the building blocks. A total of 8 scFv and diabody constructs were prepared comprising the same two variable heavy (V(H)) and variable light (V(L)) chain domains but arranged in different orientations. V(H)/V(L) orientation, i.e., V(H)-linker-V(L) or V(L)-linker-V(H), showed significant effects on the expression and antigen-binding activity of scFv and monospecific diabody of both 11F8 and A12. Further, only 2 out of the 4 possible V(H)/V(L) orientations/arrangements in bDAb construction yielded active products that retain binding activity to both EGFR and IGFR. Both active bDAb preparations retained their original antigen-binding activity after incubation at 37 degrees C in mouse serum for up to 7 days, indicating excellent stability of the constructs. Taken together, our results underscore the importance of identifying/selecting optimal V(H)/V(L) orientation/arrangement for efficient production of active bDAb.     

Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers

By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells.     

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).     

Dimeric and trimeric antibodies: high avidity scFvs for cancer targeting

Recombinant antibody fragments can be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies (dimers), triabodies (trimers) and tetrabodies (tetramers). In particular we discuss the role of linker length between V-domains and the orientation of the V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), and how the size, flexibility and valency of each molecules is suited to different applications for in vivo imaging and therapy. Single chain Fv antibody fragments joined by polypeptide linkers of at least 12 residues irrespective of V-domains orientation predominantly form monomers with varying amounts of dimer and higher molecular mass oligomers in equilibrium. A scFv molecule with a linker of 3-12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, approximately 60 kDa). Reducing the linker length below three residues can force scFv association into trimers (triabodies, approximately 90 kDa) or tetramers ( approximately 120 kDa) depending on linker length, composition and V-domain orientation. A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared with the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv diabodies that have undergone successful pre-clinical trials for in vivo stability and efficacy. We also briefly review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. Bi-specific diabodies formed by association of different scFv molecules have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics). The more challenging trispecific multimers (triabodies) remain to be described.     

Improved biodistribution and radioimmunoimaging with poly(ethylene glycol)-DOTA-conjugated anti-CEA diabody

Diabodies are single chain antibody fragments (scFvs) that spontaneously form bivalent dimers of molecular size 50-55000. Radiolabeled diabodies are almost ideal tumor targeting agents due to their high avidity (bivalent) binding to tumor antigens and small size (50-55000) that leads to improved tumor-to-blood ratio compared to intact antibodies (150000). However, due to their high retention and metabolism in the kidney, radioiodine is the current radiolabel of choice for diabodies since radioiodine is rapidly excreted from the kidney once metabolized. We have previously shown that 111In-DOTA-diabody gives higher tumor uptake in nude mouse xenografts than 125I-diabody, but has extremely high kidney retention since its 111In-labeled metabolites are retained by and only slowly excreted from the kidney. When a diabody is conjugated to a bifunctional PEG-3400 derivative followed by reaction with cysteinyl-DOTA, the resulting product has an apparent molecular size of 75000 and a Stokes radius of 35 angstroms on size exclusion chromatography, compared to a Stokes radius of 25 angstroms for intact diabody. When radiolabeled, the conjugate gives high yields of 111In-labeled product, retains high immunoreactivity, and gives improved biodistributions (30-40%ID/g, 12-48 h) compared to 111In-DOTA-diabody (12-13%ID/g, 6-12 h). We show that the improved biodistribution is due to an increase in Stokes radius caused by the linear PEG-3400 since conjugation of diabody with multiple (PEG)12 linkers followed by reaction with cysteinyl-DOTA does not reduce kidney accumulation. We also show that 111In-cysteinyl-DOTA-PEG3400-diabody gives excellent tumor images in the nude mouse xenograft model and that 125I-PEG3400-diabody gives equivalent images to 125I-minibody (molecular size, 80000), but improved tumor-to-liver ratios, suggesting that this imaging agent can be used to image liver metastases.     

Production and characterization of an anti-(MUC1 mucin) recombinant diabody

A recombinant diabody fragment based on the anti-MUC1 monoclonal antibody, C595 has been produced in a bacterial expression system. Substitution of a 7-amino-acid linker sequence (Gly₆Ser) for the original single-chain (sc)Fv 15-amino-acid linker (Gly₄Ser)_3, using polymerase-chain-reaction-based strategies, forces variable heavy (V_H) and light (V_L) domains to pair with complementary domains on neighbouring scFv molecules, forming a scFv dimer (diabody). This recombinant protein shows similar binding characteristics to the parental C595 monoclonal antibody. The ability to bind to MUC1 mucin on carcinoma cell surfaces will allow its potential as a diagnostic and therapeutic reagent of clinical utility to be investigated. Received: 16 September 1998 / Accepted: 2 December 1998     

Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody: comparison to radioiodinated fragments

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.     

Site-specific conjugation of monodispersed DOTA-PEGn to a thiolated diabody reveals the effect of increasing peg size on kidney clearance and tumor uptake with improved 64-copper PET imaging

Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t(1/2) = 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope (64)Cu (t(1/2) = 12 h). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site-specific attachment of monodispersed PEGn of increasing molecular size (n = 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake, and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were preconjugated to the chelator DOTA, the conjugates were able to bind radiometals such as (111)In and (64)Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80%ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24 h when radiolabeled with (111)In and 37.9% ID/g of tumor uptake (T/B = 8) at 44 h when radiolabeled with (64)Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 h observed with diabodies that were pegylated on surface lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cys-conjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting.     

The importance of framework residues H6, H7 and H10 in antibody heavy chains: experimental evidence for a new structural subclassification of antibody V(H) domains

The N-terminal segment (FR-H1) of the heavy chain (V(H)) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study, we have established a causal relationship between the local sequence and the structure of this framework region and linked this relationship to important biophysical properties such as affinity, folding yield and stability. We have generated six mutants of the scFv fragment aL2, covering some of the most abundant amino acid combinations in positions H6, H7 and H10 (according to a new consensus nomenclature, Kabat H9). For the aL2 wild-type (w.t.) with the sequence 6(Q)7(P)10(A) and for two of the mutants, the X-ray structures have been determined. The structure of the triple mutant aL2-6(E)7(S)10(G) shows the FR-H1 backbone conformations predicted for this amino acid combination, which is distinctly different from the structure of the w.t, thus supporting our hypothesis that these residues determine the conformation of this segment. The mutant aL2-6(E)7(P)10(G) represents a residue combination not occurring in natural antibody sequences. It shows a completely different, unique structure in the first beta-strand of V(H), not observed in natural Fv fragments and forms a novel type of diabody. Two V(H) domains of the mutant associate by swapping the first beta-strand. Concentration-dependent changes in Trp fluorescence indicate that this dimerization also occurs in solution. The mutations in amino acids H6, H7 and H10 (Kabat H9) influence the dimerization behavior of the scFv and its thermodynamic stability. All the observations reported here have practical implications for the cloning of Fv fragments with degenerate primers, as well as for the design of new antibodies by CDR grafting or synthetic libraries.     

A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells

Background Although cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy with bispecific diabodies could be a promising novel treatment option for prostate cancer. Methods A heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv V_HCD3-V_LPSMA and V_HPSMA-V_LCD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model. Results By Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor xenografts with the diabody and PBL efficiently inhibited tumor growth. Conclusions The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal residual disease. P. Bühler and P. Wolf equally contributed to the work.     

Improvement in soluble expression levels of a diabody by exchanging expression vectors

Accumulating evidence suggests that bispecific antibody fragments (BsAbs) seem set to join monoclonal antibodies as powerful therapeutic and diagnostic agents, particularly for targeting cancer. One type of recombinant BsAbs are diabodies, which are constructed from heterogeneous single-chain antibodies and can increase antigen-specific cytotoxicity in T cells by cross-linking tumor antigens with T cell associated antigens. Some diabodies, however, cannot be solubly expressed in sufficient quantities, which limits their use in clinical therapeutics. Previously we constructed an anti-CD20 x CD3 diabody, which effectively directs the lytic potential of cytolytic T cells toward CD20(+) malignant B cells and shows marked antitumor efficacy in vivo. Here, to increase the amount of soluble product for clinical trials, we used an alternative expression vector under the T7 promoter but retained the stII signal sequences to ensure that the expressed protein is secreted to the periplasm of Escherichia coli. We achieved a periplasmic, soluble product by optimizing the conditions for induction with a yield of 8-9mg/L after affinity chromatography purification. This is nearly a five times greater yield than obtained with the previous vector. The diabodies generated from this modified vector retain dual binding specificity for both CD20-positive and CD3-positive cell lines. Taken together, these results suggest that changing expression vectors may be an alternative strategy to accomplish high-level expression of active BsAb proteins from E. coli.     

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.     

Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells

The use of anti-CD3 x 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 CD3epsilon 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 x 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 x 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 Fv multimers of the anti-neuraminidase antibody NC10: the residue at position 15 in the V(L) domain of the scFv-0 (V(L)-V(H)) molecule is primarily responsible for formation of a tetramer-trimer equilibrium

Single-chain variable fragment of the murine monoclonal antibody NC10 specific to influenza virus N9 neuraminidase, joined directly in the V(L) to V(H) orientation (scFv-0), forms an equilibrium mixture of tetramer and trimer with the tetramer as the preferred multimeric species. In contrast, the V(H)-V(L) isomer was previously shown to exist exclusively as a trimer. Computer-generated trimeric and tetrameric scFv models, based on the refined crystal structure for NC10 Fv domain, were constructed and used to evaluate factors influencing the transition between V(L)-V(H) trimer and tetramer. These model structures indicated that steric restrictions between loops spanning amino acid residues L55-L59 and L13-L17 from the two adjacent V(L) domains within the V(L)-V(H) trimer were responsible for four scFv-0 molecules assembling to form a tetramer. In particular, leucine at position L15 and glutamate at position L57 appeared to interfere significantly with each other. To minimize this steric interference, the site-directed mutagenesis technique was used to construct several NC10 scFv-0 clones with mutations at these positions. Size-exclusion chromatographic analyses revealed that several of these mutations resulted in the production of NC10 scFv-0 proteins with significantly altered tetramer-trimer equilibrium ratios. In particular, introduction of a polar residue, such as asparagine or threonine, at position L15 generated a highly stable NC10 scFv-0 trimer.     

ScFv multimers of the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in V(L) to V(H) orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers

Synthetic genes encoding single-chain variable fragments (scFvs) of NC10 anti-neuraminidase antibody were constructed by joining the V(L) and V(H) domains with linkers of fifteen, five, four, three, two, one and zero residues. These V(L)-V(H) constructs were expressed in Escherichia coli and the resulting proteins were characterized and compared with the previously characterized NC10 scFv proteins assembled in V(H)-V(L) orientation. Size-exclusion chromatography and electron microscope images of complexes formed between various NC10 scFvs and anti-idiotype Fab' were used to analyse the oligomeric status of these scFvs. The result showed that as the linker length between V(L) and V(H) was reduced, different patterns of oligomerization were observed compared with those with V(H)-V(L) isomers. As was the case for V(H)-V(L) orientation, the scFv-15 V(L)-V(H) protein existed mainly as a monomer whereas dimer (diabody) was a predominant conformation for the scFv-5, scFv-4 and scFv-3 V(L)-V(H) proteins. In contrast to the V(H)-V(L) isomer, direct ligation of V(L) to V(H) led to the formation of predominantly a tetramer (tetrabody) rather than to an expected trimer (triabody). Furthermore, the transition between dimers and higher order oligomers was not as distinct as for V(H)-V(L). Thus reducing the linker length in V(L)-V(H) from three to two residues did not precisely dictate a transition between dimers and tetramers. Instead, two-residue as well as one-residue linked scFvs formed a mixture of dimers, trimers and tetramers.     

Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria

The equilibrium denaturation and unfolding kinetics of the domains V(L) and V(H) have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimeric assembly even further. Domain dissociation and domain unfolding are coupled processes, giving rise to a highly cooperative unfolding transition. For the Fv fragment, cooperative unfolding is only observed in the presence of antigen. At low protein concentrations and in the absence of antigen, the Fv fragment is significantly destabilized, leading to quantitative domain dissociation before significant domain unfolding takes place. The kinetic unfolding of V(H), V(L) and the scFv fragment is monophasic. Unfolding of the scFv fragment is much slower, when extrapolated to zero denaturant, than either of the isolated domains, suggesting that the higher thermodynamic stability of the scFv fragment is at least partially due to a high-energy transition state for unfolding. These studies emphasize the enormous importance of mutual domain stabilization in engineering stable antibodies.