Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody

Fabrice Le Gall, Uwe Reusch, Melvyn Little and Sergey M. Kipriyanov1

Affimed Therapeutics AG, Im Neuenheimer Feld 582, D-69120 Heidelberg, Germany

1 To whom correspondence should be addressed. E-mail: s.kipriyanov{at}affimed.com


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities connected by three linkers. When assembled in the order VHA–linker1–VLB–linker2–VHB–linker3–VLA, the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary VH and VL domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains. We generated a number of single-chain constructs using four VH and VL domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6–27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites.

Keywords: human CD3/human CD19/linker/single-chain Fv/tandem diabody


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the most promising immunotherapeutic strategies is based on the activation of host immune mechanisms using bispecific antibodies (BsAb) (Peipp and Valerius, 2002Go). The BsAb makes a bridge between the tumor cell and the immune effector cell, which then triggers the cytotoxic responses that include perforin and granzyme release, Fas-mediated apoptosis and cytokine production. The BsAbs can limit complement activation, which is responsible for side-effects in many therapeutic settings, and profoundly enhance target selectivity. BsAb have mainly been produced using murine hybrid hybridomas (quadromas) or by chemical cross-linking (Segal et al., 1999Go). A major limitation of the hybrid hybridoma technology procedure is the production of inactive antibodies due to the random heavy and light chain associations. Only ~15% of the antibodies produced by the quadroma are of the desired specificity (Milstein and Cuello, 1983Go). The correct BsAb must then be purified in a costly procedure from a large quantity of other, very similar molecules. A further limitation of the quadroma BsAbs obtained from rodent cell lines is their immunogenicity. Repeated doses of rodent antibodies elicit an anti-immunoglobulin response, referred to as HAMA (human anti-murine antibody). The HAMA response and release of inflammatory cytokines are the major drawbacks of BsAb derived from rodent monoclonal antibodies (mAb) in clinical use (Manzke et al., 2001Go). Recent advances in recombinant antibody technology have provided several alternative methods for constructing and producing BsAb molecules (Kipriyanov and Le Gall, 2004Go). For example, bispecific F(ab')2 have been created either by chemical coupling from Fab' fragments expressed in Escherichia coli (Shalaby et al., 1992Go) or by heterodimerization through leucine zippers (Kostelny et al., 1992Go). Analogously, scFv fragments have been genetically fused with the adhesive polypeptides (de Kruif and Logtenberg, 1996Go) or protein domains (Müller et al., 1998Go) to facilitate the formation of heterodimers. An alternative BsAb fragment is the scFv heterodimer diabody (Holliger et al., 1993Go). The bispecific diabody (BsDb) was obtained by the non-covalent association of two single-chain fusion products consisting of the VH domain from one antibody connected by a short linker to the VL domain of another antibody (Holliger et al., 1996Go; Kipriyanov et al., 1998Go). It has been shown by crystallographic analysis that two antigen-binding sites are located on opposite sides of the diabody, assembled both in VH-to-VL (Perisic et al., 1994Go) and in VL-to-VH (Carmichael et al., 2003Go) orientation, such that they are able to cross-link two cells. The bispecific diabodies are potentially less immunogenic than quadroma-derived BsAbs and can be easily produced in bacteria in relatively high yield (Zhu et al., 1996Go; Cochlovius et al., 2000bGo). We have previously shown that CD19 x CD3 BsDbs are more effective than quadroma-derived BsAb in mediating T-cell cytotoxicity in vitro against tumor cells (Kipriyanov et al., 1998Go; Cochlovius et al., 2000bGo). Although BsDb was relatively rapidly cleared from the blood through the kidneys, its antitumor activity in animal tumor models was closely similar to that of the quadroma-derived BsAb (Arndt et al., 1999Go; Cochlovius et al., 2000bGo). The rapid clearance of the BsDb was probably compensated for by better tumor penetration and a more efficient induction of cell lysis.

However, the BsDb is formed by heterodimerization of two different gene products, which must be expressed in the same cell in similar amounts. Moreover, the co-secretion of two hybrid scFv fragments can give rise to two types of dimer: active heterodimers and inactive homodimers. The problem of quantitative heterodimer formation can be overcome by linking four variable domains (VH and VL) derived from antibodies of two specificities into a single-chain construct. Depending on the variable domain order and on the length of peptides separating them, the single-chain molecule either forms an (scFv)2, two scFv modules composed of two adjacent VH and VL of the same specificity (Mack et al., 1995Go; Baeuerle et al., 2003Go), or folds head-to-tail with the formation of a of a diabody-like structure, a so-called single-chain bispecific diabody (scBsDb) (Kipriyanov et al., 1999Go; Kontermann and Müller, 1999Go). Recently, we demonstrated that, at least in E.coli, the head-to-tail folding and formation of functional diabody-like molecules are favored (Kipriyanov et al., 2003Go). Moreover, the scBsDb format facilitates the production of relatively stable bispecific constructs from weakly associated Fv fragments (Kipriyanov et al., 2003Go).

In contrast to native antibodies, most of the developed BsAb formats, either quadroma-derived or recombinant (such as scFv–scFv tandems, BsDb or scBsDb), have only one binding domain for each specificity. However, bivalent binding is an important means of increasing the functional affinity and possibly the selectivity of antibodies and antibody fragments for particular cell types carrying densely clustered antigens. A tetravalent bispecific antibody devoid of any immunoglobulin constant domains, which are responsible for side-effects in many therapeutic settings, can be formed by dimerization of a single-chain molecule comprising four antibody variable domains (VH and VL) in an orientation preventing intramolecular pairing (Kipriyanov et al., 1999Go; Völkel et al., 2001Go). Unlike many other BsAb formats, this so-called tandem diabody (tandab) is a homodimer with Mr {approx} 115 kDa, which is composed only of antibody variable domains, and its formation is determined by the association of complementary VH and VL domains (Figure 1a). Compared with bispecific diabody, the tandab exhibited a higher functional affinity for both antigens, improved pharmacokinetics and elevated biological activity both in vitro and in vivo (Kipriyanov et al., 1999Go; Cochlovius et al., 2000aGo).



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Fig. 1. Structural model of the tandem diabody (a) and a ribbon diagram of a murine Fab fragment showing the location of the peptides for L6, K6 and K9 linkers (b). In (a), the atoms of middle linkers connecting two diabody-like structures are represented as filled spheres. ß-Sheets are shown in yellow, ß-turns in blue and irregular peptide sequences in green. The locations of VH and VL domains of two different specificities (A and B) are indicated. In (b), the antibody light chain is shown in blue and VH and CH1 domains in red. The heavy chain Ser113–Pro118 peptide (L6) is shown in yellow. The light chain Arg108–Pro113 peptide (K6) is shown in green. The further three amino acid Thr114–Ser116 extension of K6 into K9 is shown in cyan. The numeration is given according to Kabat et al. (1991)Go. (a) and (b) were generated on the basis of the structural model of a T10/SL tandab (Kipriyanov et al., 1999Go) and of the structure of intact IgG1 (PDB entry 1IGY) (Harris et al., 1998Go), respectively, using the molecular visualization program RasMol v2.7.2 (R.Sayle, Biomolecular Structures Group, Glaxo Welcome, Stevenage, Hertfordshire, UK).

 
In the present study, we analyzed the influence of length and amino acid composition of the peptide linkers connecting individual VH and VL domains on the folding efficacy, stability and biological activity of the tandab molecule. We generated a number of single-chain constructs using four VH and VL domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6–27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell (PBMC) cultures from patients suffering from B-cell chronic lymphocytic leukemia (B-CLL), we demonstrated that tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between CD19 and CD3 binding sites.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasmid constructions

For all cloning steps and isolation of DNA, the E.coli K12 strain XL1-Blue (Stratagene, La Jolla, CA) was used. The plasmids pHOG21-dmOKT3 (Kipriyanov et al., 1997Go) and pHOG21-HD37 (Kipriyanov et al., 1996Go) encoding the scFv fragments derived from hybridoma OKT3 specific for human CD3 and hybridoma HD37 specific for human CD19, respectively, were used as a source of genetic information for assembly of genes encoding the bispecific antibodies. The plasmid pHOG21-scFv6-3–19 encoding the hybrid VHCD3–L6–VLCD19 scFv was generated by PCR amplification of the anti-CD3 VH gene with the primers Bi3sk, 5'-CAG CCG GCC ATG GCG CAG GTG CAA CTG CAG CAG and OKT-5, 5'-TAT TAA GAT ATC GGG TGT TGT TTT GGC TGA GGA G, followed by digestion of the PCR fragment with NcoI and EcoRV, and ligation with the NcoI/EcoRV-linearized plasmid pHOG3-19 (Kipriyanov et al., 1998Go). The plasmid pHOG21-scFv6-19–3 encoding the hybrid VHCD19–L6–VLCD3 scFv was generated by PCR amplification of the anti-CD19 VH gene with the primers DP1p (5'-GAA TTC ATT AAA GAG GAG AAA TTA ACC) and OKT-5, followed by digestion of the PCR fragment with NcoI and EcoRV and ligation with the NcoI/EcoRV-linearized plasmid pHOG21-dmOKT3 + Not (Le Gall et al., 2004Go). The gene coding for the hybrid VHCD3–L6–VLCD19 scFv was amplified by PCR with the primers Bi3sk and either K6-Pvu (5'-CTG CTG CAG CTG CAC CTG TGG TGC AGC ATC AGC CCG TTT GAT TTC C) to generate a six amino acid intramolecular linker K6 (PCR fragment 1) or K9-Pvu (5'-CTG CTG CAG CTG CAC CTG GGA TAC AGT TGG TGC AGC ATC AGC CCG) to generate a nine amino acid linker K9 (PCR fragment 2) or Li-2 (5'-TAT ATA CTG CAG CTG CAC CTG CGA CCC TGG GCC ACC AGC GGC CGC AGC ATC AGC CCG) to generate a 12 amino acid linker SL (PCR fragment 3). The expression plasmids pDISC19 x 3/L6–K6, pDISC19 x 3/L6–K9 and pDISC19 x 3/L6–SL were constructed by ligation of the NcoI/PvuII restriction fragment from pHOG21-scFv6-19–3 comprising the vector backbone and the NcoI/PvuII-digested PCR fragments 1, 2 and 3, respectively. Construction of genes encoding the bispecific CD19 x CD3 T10/SL and T10/LL tandabs has been described previously (Kipriyanov et al., 1999Go). For high-level bacterial expression, the genes coding for bispecific molecules followed by His6 tail were transferred as the NcoI/XbaI fragments into a plasmid pSKK3 containing the hok/sok plasmid-free cell suicide system and an skp gene encoding the Skp/OmpH periplasmic factor (Le Gall et al., 2004Go). The sequences of all newly constructed genes and plasmids were verified by restriction digests and sequencing.

Expression and purification of recombinant proteins

The E.coli K12 strain RV308 ({Delta}lac{chi}74 galISII::OP308strA) (Maurer et al., 1980Go) (ATCC 31608) was used for functional expression of single-chain antibodies. The bacteria transformed with the expression plasmids were grown in shaking flasks and induced essentially as described previously (Cochlovius et al., 2000bGo). The recombinant proteins were isolated from soluble periplasmic fractions by immobilized metal affinity chromatography (IMAC) followed by ion-exchange chromatography as described (Kipriyanov et al., 1999Go). Protein concentrations were determined by the Bradford dye-binding assay (Bradford, 1976Go) using a Bio-Rad (Munich, Germany) protein assay kit. The purified material was subjected to analytical size-exclusion chromatography on a calibrated Superdex 200 HR10/30 column (Amersham Pharmacia, Freiburg, Germany) in PBSI buffer (15 mM sodium phosphate, 0.15 M NaCl, 50 mM imidazole, pH 7.0). For T10/LL variant, the dimeric (tandab) and monomeric (scBsDb) species were separated by preparative size-exclusion chromatography on the Superdex 200 HR10/30 column (Amersham) essentially as described (Kipriyanov et al., 1999Go).

Flow cytometry

The human CD3+/CD19 acute T-cell leukemia line Jurkat and the CD19+/CD3 B-cell line JOK-1 were used for flow cytometry as described previously (Kipriyanov et al., 1998Go). In brief, the cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin G sodium and 100 µg/ml streptomycin sulfate (all from Invitrogen, Groningen, The Netherlands) at 37°C in a humidified atmosphere with 5% CO2. A total of 1 x 106 cells were incubated with 100 µl of phosphate-buffered saline (PBS; Invitrogen) supplemented with 2% FCS and 0.1% sodium azide (Roth, Karlsruhe, Germany) (referred to as FACS buffer) and containing diluted recombinant antibodies for 45 min on ice. After washing with FACS buffer, the cells were incubated with 100 µl of 10 µg/ml anti-(His)6 mouse mAb 13/45/31–2 (Dianova, Hamburg, Germany) in the same buffer for 45 min on ice. After a second washing cycle, the cells were incubated with 100 µl of 15 µg/ml FITC-conjugated goat anti-mouse IgG (Dianova) under the same conditions as before. The cells were then washed again and resuspended in 0.5 ml of FACS buffer containing 2 µg/ml propidium iodide (Sigma-Aldrich, Taufkirchen, Germany) to exclude dead cells. The fluorescence of 1 x 104 stained cells was measured using a Beckman-Coulter Epics XL flow cytometer (Beckman-Coulter, Krefeld, Germany). The mean fluorescence (F) was calculated using System-II and Expo32 software (Beckman-Coulter) and the background fluorescence was subtracted.

In vitro cell surface retention

Cell surface retention assays were performed at 37°C under conditions preventing internalization of cell surface antigens as described previously (Adams et al., 1998Go), except that the detection of retained antibody was performed using anti-(His)6 mouse mAb 13/45/31–2 (10 µg/ml; Dianova) followed by FITC-conjugated goat anti-mouse IgG (15 µg/ml) (Dianova). The kinetic dissociation constant (koff) and t1/2 values for dissociation of scFv and mAb were deduced from a two-phase exponential decay fit (R2 > 0.99) of experimental data using the software program PRISM (GraphPad Software, San Diego, CA).

Analysis of stability in vitro

The recombinant antibodies were diluted in RPMI 1640 medium supplemented with 10% FCS to a concentration of 10 µg/ml and sterilized by filtration. Aliquots (250 µl) were prepared immediately under sterile conditions and stored at 37°C. At given time points, the aliquots were frozen and kept at –80°C. The antigen-binding activities of the samples after storage were determined by flow cytometry using CD19+ JOK-1 cells and CD3+ Jurkat cells.

Isolation of PBMCs from patients' blood

The patients fulfilling the clinical, morphological and immunophenotypic criteria of B-CLL were selected at the Department of Internal Medicine of the University of Heidelberg and blood samples were obtained after informed consent. PBMCs were isolated by density gradient centrifugation. The blood samples were diluted 2-fold with PBS (Invitrogen, Breda, The Netherlands), layered on a cushion of Histopaque-1077 (Sigma-Aldrich, Deisenhofen, Germany) and centrifuged at 800 g for 25 min. The PBMCs located in the interface were collected and washed three times with PBS before use.

Autologous T-cell activation and depletion of leukemia cells

PBMCs from patients with B-CLL were seeded in individual wells of a 12-well plate in 2 ml of RPMI-medium containing either 10% FCS (Invitrogen) or 10% autologous human serum at a density of 2 x 106 cells/ml. The recombinant antibodies were added at concentrations of 0.1, 1 and 5 µg/ml. After 6 days of incubation, the cells were harvested, counted in the presence of trypan blue and stained with anti-CD3 mAb OKT3 (Kung et al., 1979Go), Edu-2 (anti-CD4) (Chemicon, Hofheim, Germany), UCH-T4 (anti-CD8) (Chemicon) and anti-CD19 mAb HD37 (Pezzutto et al., 1987Go) for flow cytometry. Then, 104 living cells were analyzed using a flow cytometer, the absolute amounts of CD3+, CD4+, CD8+ and CD19+ cells were determined, and the ratios of T cells to B cells were calculated.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Design of the linkers connecting individual variable domains and construction of single-chain molecules

For the generation of bispecific CD19 x CD3 single-chain constructs, the VH and VL domains derived from anti-CD19 mAb HD37 (Kipriyanov et al., 1996Go) and anti-CD3 mAb OKT3 (Kipriyanov et al., 1997Go) were used. Since the tandab comprises two diabody-like modules (Kipriyanov et al., 1999Go) (Figure 1a), the design of the peptide linkers connecting variable domains was based on a rough structural similarity of the diabody with a Fab fragment, where VH and VL domains are separated from the adjacent CH1 and CL domains, respectively, with the six amino acid long flexible elbow-like peptides (Figure 1b). We assumed that the peptide used by nature for connecting individual antibody domains will not interfere with the folding of the VH and VL domains in the bacterial periplasm, thus favoring formation of functional diabody. Previously, we demonstrated that substitution of a 10 amino acid long L10 linker (Kipriyanov et al., 1998Go; Le Gall et al., 1999Go) by a natural six amino acid peptide, Ser113–Pro118, connecting VH and CH1 domains of the heavy chain in the murine IgG1 and IgG2b (Lefranc, 2001Go) led to a 10-fold higher bacterial production of monospecific anti-CD3 diabody assembled in a VH-to-VL orientation (Le Gall et al., 2004Go). We therefore constructed a number of symmetric four-domain single-chain molecules, each comprising two hybrid scFv where the VH and VL have different specificities and are separated either by six (L6) or 10 (L10) amino acid residue linkers, which prevent formation of scFv modules by the adjacent domains. In turn, these hybrid VHCD3–VLCD19 and VHCD19–VLCD3 scFv were connected by a middle linker of six (K6), nine (K9), 12 (SL) and 27 (LL) amino acids (Figure 2). The interdomain linkers used either solely represent the natural continuations of the corresponding VH and V{kappa} (L6, K6 and K9) or comprise additional amino acids (L10, SL and LL), thus potentially contributing to extra flexibility.



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Fig. 2. Schematic representation of constructed genes encoding the CD19 x CD3 bispecific molecules. (a) Designation of putative dimeric protein products. (b) Genetic constructs. (c) Amino acid sequences of peptide linkers connecting VH and VL domains. The locations of promoter/operator (P/O), pelB leader sequence (PelB), antibody variable domains (VH and VL), peptide linkers (L6, L10, K6, K9, SL and LL) and His6 are indicated. The amino acid stretches corresponding to the beginning of murine CH1 or C{kappa} domains are underlined.

 
Bacterial expression, purification and analysis of molecular forms

The constructed four-domain single-chain molecules were expressed as soluble secreted proteins in E.coli under conditions favoring dimerization of diabody-like molecules (Kipriyanov et al., 1999Go). The recombinant proteins were isolated from the soluble periplasmic extracts in two chromatographic steps with a purity >95% (data not shown). Analysis of expression levels demonstrated that using natural linkers led to 2- to 2.5-fold higher production of antibody fragments (Table I). Size-exclusion chromatography on a calibrated Superdex 200 column demonstrated that all molecules, except for T10/LL, were in a dimeric (tandab) form (Figure 3; Table I). As expected, the single-chain construct containing the 27 amino acid long middle linker (T10/LL) effectively forms both monomeric (single-chain diabody) and dimeric (tandab species), which could be easily separated either by ion-exchange chromatography or by gel filtration (data not shown). For further analyses, homogeneous preparations of dimeric tandab molecules were used.


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Table I. Expression yield and molecular forms of CD19 x CD3 four-domain constructs

 


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Fig. 3. Elution profiles of purified single-chain recombinant proteins from a calibrated Superdex 200 gel filtration column. For T10/LL, the profiles of separated dimeric (D) and monomeric (M) species are shown.

 
Functional affinity of CD19 x CD3 tandabs

Flow cytometry demonstrated that all the constructed tandabs specifically interacted with both human CD3- and CD19-expressing cells (Figure 4a). All of the examined tandab variants exhibited higher fluorescence intensities when interacting with CD3+ Jurkat cells than with CD19+ JOK-1 cells, a phenomenon distinguishing tetravalent CD19 x CD3 molecules from the bivalent diabody and the single-chain diabody (Kipriyanov et al., 1999Go, 2003Go). The variants with the longest linkers between the VH and VL domains, T10/SL and T10/LL, demonstrated the strongest binding among the molecules examined (Figure 4a).



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Fig. 4. Flow cytometric analysis of bispecific tandabs binding to CD19+/CD3 JOK-1 and CD3+/CD19 Jurkat cells. (a) Fluorescence dependence on antibody concentration. (b) In vitro cell surface retention assay. Values are expressed as a percentage of the initial mean fluorescence intensity.

 
In order to investigate the biological relevance of the differences in direct binding experiments, the in vitro retention of the tandabs on the surface of both CD19+ and CD3+ cells at 37°C was determined by flow cytometry (Figure 4b). In contrast to the direct binding experiments, the cell surface retention and deduced affinity constants are not dependent on the accuracy of determining the concentration of biologically active protein. All examined tandab variants exhibited a relatively quick dissociation from the surface of CD3+ Jurkat cells with a half-life (t1/2) of 2–3 min and a significantly longer retention on the surface of CD19+ JOK-1 cells (Figure 4b; Table II), thus reflecting higher affinities to CD19 than to CD3 for all molecules. Comparison with bispecific CD19 x CD3 bivalent diabody (BsDb) and single-chain diabody (scBsDb) having only one binding site for each specificity demonstrated that all examined tandabs demonstrated monovalent binding to CD3+ cells. In contrast, all tandabs except T6/K6 demonstrated significantly longer retention on the surface of CD19+ cells than BsDb and scBsDb. The best binding characteristics to CD19 were shown by T10/LL tandab with the combination of longest linkers between individual VH and VL domains (Figure 4b; Table II).


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Table II. Cell surface retention and stability of bispecific antibody molecules

 


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Fig. 5. Stability of the tandab molecules in a serum-containing medium at 37°C as determined by flow cytometry using (a) CD19+ JOK-1 cells and (b) CD3+ Jurkat cells. Activities of the samples at point zero were taken as 100%.

 
Stability of CD19 x CD3 tandabs in vitro

ScFv antibody fragments and their multivalent derivatives often exhibit poor stability properties that may preclude their clinical applications. We therefore analyzed the CD19+ and CD3+ cell-binding activities of different tandab variants after prolonged storage at a fairly low concentration in a serum-containing medium at 37°C. A concentration of 10 µg/ml was chosen in order to avoid the fluorescence plateau in cell-binding experiments (Figure 4a). The highest stability (half-life >10 days), as determined by binding to both CD19- and CD3-positive cells, was demonstrated by the T10/LL tandab with the combination of longest linkers between individual VH and VL domains (Figure 5; Table II). The poorest stability (half-life ~12 h) was shown by T6/SL tandab. For almost all tandab variants except T6/K6, there was a good correlation between the stability of CD19 and CD3 binding portions of the molecules (Figure 5; Table II).

Biological activity of CD19 x CD3 tandabs

The ability of different tandab variants to mediate killing of tumor cells by autologous T lymphocytes was tested using 6-day PBMC cultures from six B-CLL patients, which contained 57–98% CD19+/CD5+ leukemia cells. All examined tandab variants, except T6/SL, induced vigorous proliferation of both CD4+ and CD8+ T cells followed by depletion of CD19+ tumor cells in a dose-dependent manner, although the effect showed considerable variation between the B-CLL patients (Table III). Figure 6 presents a typical experiment with PBMCs from a B-CLL patient, No. 01. Separate comparisons of the tandab variants for each donor demonstrated that of all the variants examined, the best T-cell proliferative and anti-tumor activity was possessed by the T10/SL tandab. Even at a concentration of 0.1 µg/ml it caused significant depletion of malignant B-CLL cells without any additional T-cell stimulation (Figure 6; Table III). Within the T-cell fraction, significant tandab-mediated proliferation of both CD4+ and CD8+ cells was observed (Figure 6).


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Table III. CD19 x CD3 tandab-mediated depletion of CD19+ leukemia cells and T-cell proliferation in PBMC cultures from the B-CLL patients as reflected by the ratio of CD3+ T cells to CD19+ leukemia cells

 


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Fig. 6. Depletion of primary malignant CD19+ B-CLL cells and proliferation of autologous T cells induced by CD19 x CD3 tandabs. Freshly isolated PBMCs from patient No. 01 with B-CLL were incubated for 6 days without antibodies (w/o Ab) or in the presence of the indicated concentrations of different tandab variants. The relative amounts of CD3+, CD4+, CD8+ and CD19+ cells were determined by flow cytometry and plotted.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Unlike many other bispecific antibody formats, the tandem diabody is a homodimer comprising only antibody variable domains and its formation is determined by the association of complementary VH and VL domains located on different polypeptide chains. The tandab is twice as large as a diabody and (scFv)2 is able to bind bivalently both effector and target cells, and possesses improved pharmacokinetic characteristics, higher stability and enhanced biological activity both in vitro and in vivo (Kipriyanov et al., 1999Go; Cochlovius et al., 2000aGo). However, the formation of monomeric (single-chain diabody) and dimeric (tandab) four-domain VHA–linker1–VLB–linker2–VHB–linker3–VLA single-chain antibodies depends on the length and probably the amino acid composition of the three linkers joining the variable domains (Kipriyanov et al., 1999Go; Völkel et al., 2001Go). To prevent pairing of the adjacent VHA/VLB and VHB/VLA domains with the formation of non-functional scFv modules, the linker1 and linker3 should generally be shorter than 12 amino acid residues (Todorovska et al., 2001Go). The previously designed 10 amino acid long linker L10 (Figure 2c) completely satisfied this criterion and led to the quantitative formation of a heterodimeric CD19 x CD3 diabody from the hybrid VHCD3–L10–VLCD19 and VHCD19–L10–VLCD3 scFv fragments co-expressed in the same bacterial cell (Kipriyanov et al., 1998Go; Cochlovius et al., 2000bGo). On the other hand, the capacity of a single-chain molecule to form non-covalent dimers (diabody or tandem diabody) is strongly influenced by the tightness of the VH/VL interface formed by the cognate variable domains. For example, the OKT3-derived (anti-CD3) scFv10 fragment containing L10 linker between VH and VL was solely monomeric (Le Gall et al., 2004Go), while the HD37-derived (anti-CD19) scFv10 formed a mixture of dimers and tetramers (Le Gall et al., 1999Go). Furthermore, it was recently demonstrated that the four-domain single-chain molecule VL–linker–VH–linker–VL–linker–VH, where the VL and VH domains were derived from anti-TAG-72 mAb CC49, formed 20–30% tetrameric non-covalent dimers when expressed in Pichia pastoris, although the variable domains were separated by a relatively long (25 amino acids) 205C linker (Goel et al., 2000Go).

To optimize the structure of the CD19 x CD3 tandab, we analyzed the influence of the length and amino acid composition of peptide linkers connecting individual VH and VL domains on the folding efficacy and biological activity of the tandab molecule. The most common linkers contain a combination of glycine and serine residues to provide flexibility and protease resistance (Huston et al., 1988Go), although the linker sequence can also be optimized by phage display to improve the antigen binding and production yield of single-chain antibody molecules (Tang et al., 1996Go; Turner et al., 1997Go). In the present study, we substituted the L10 linker by a six amino acid long flexible elbow-like peptide corresponding to the N-terminal part of the IgG CH1 domain (linker L6; Figure 2c). We assumed that the peptide used by nature for connecting individual antibody domains will not interfere with the folding of VH and VL in the bacterial periplasm, thus favoring formation of functional diabody-like structures. Indeed, this modification led to a 2-fold higher production of the constructed four-domain single-chain molecules in bacteria. Moreover, all variants containing L6 linkers were completely in a dimeric (tandab) form.

The middle linker (linker2) is critical for the structure of the whole four-domain single-chain molecule. If the linker is long (in general, 15 or more amino acid residues) and flexible, the molecule can fold head-to-tail into a diabody-like structure (single-chain diabody), which has two antigen-binding sites on different sides of the molecule (Kipriyanov et al., 1999Go, 2003Go; Kontermann and Müller, 1999Go). If the middle linker is short and rigid, the molecule cannot fold head-to-tail and has to dimerize with the formation of a tetravalent homodimer (tandab) (Kipriyanov et al., 1999Go; Völkel et al., 2001Go). On the other hand, the middle linker mainly determines the distance between the two antigen-binding sites of the same specificity. Too short distances might preclude bivalent binding of the tandab to the cell surface, although the probability of the antibody binding to the cell surface bivalently also depends on the antigen density and accessibility. The middle linker also influences the flexibility of the molecule, the possibility of acquiring the most suitable conformation for binding to the antigen. Assuming that the tandab has a bird wing-shaped symmetric structure (Kipriyanov et al., 1999Go) and since the peptide unit length is ~3.8 Å (Huston et al., 1988Go), one may roughly estimate the distances between CD19 and CD3 binding sites in different tandab variants (Figure 7). Thus, the CD19 binding parts of the tandab molecules can theoretically span distances of 28 Å for T6/K6, 39 Å for T6/K9, 50 Å for T6/SL and T10/SL and 107 Å for T10/LL variants. Similarly, the two CD3 binding sites of the examined variants are separated by distances of 103 Å for the T6/K6, 114 Å for T6/K9, 125 Å for T6/SL, 155 Å for T10/SL and up to 212 Å for T10/LL variants (Figure 7). The results of cell surface retention experiments indicated that at least a fraction of the tandab molecules having >35 Å between the two CD19 binding sites is able to bind CD19+ cells bivalently. In contrast, the span between two CD3 binding moieties of the tandab had no influence on the retention of the molecules on CD3+ cells, although the variants with longer linkers (T10/SL and T10/LL) demonstrated significantly better binding to Jurkat cells in direct cell-binding experiments.



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Fig. 7. Structural model of the tandem diabody T10/SL (top view). Anti-CD3 VH and VL domains are shown in red and green, anti-CD19 VH and VL in magenta and cyan, respectively. The linkers between variable domains and the middle linkers are shown in yellow and white, respectively. To indicate the antigen-binding sites, the residues of the most hypervariable CDR-3 loops of either variable domain are shown in a space-fill mode. The calculated distances between the CD19 and CD3 binding sites of different variants are shown above and below the model, respectively. The figure was generated using the molecular visualization program RasMol.

 
The single-chain antibody molecules and particularly their multivalent non-covalently associated derivatives often demonstrate poor stability properties that may preclude their clinical applications. For example, it has been demonstrated previously that scFvs can easily aggregate when stored at 37°C (Reiter et al., 1994Go; Willuda et al., 1999Go). Similarly, the humanized anti-Lewis Y diabody completely lost its immunoreactivity after 16 h of incubation in human plasma at 37°C (Tahtis et al., 2001Go). The overall stability of the single-chain antibodies depends on the intrinsic structural stability of the comprised VH and VL domains and also on the extrinsic stabilization provided by their interaction (Wörn and Plückthun, 1999Go). All single-chain constructs described in the present study comprise the same four variable domains arranged in the same order. All molecules form functional bispecific dimers with antigen-binding sites composed of cognate VH and VL pairs. The VH/VL interface can be disturbed by the linkers connecting variable domains. Measurements of stability of two- and four-domain single-chain molecules demonstrated that neither of the used linkers alone caused a deleterious effect on the stability of the whole molecule. For example, we have recently shown no loss of antigen-binding activity of OKT3-derived scFv monomer and non-covalent dimer comprising L10 and L6 linkers, respectively, after incubation for 10 days in a serum-containing medium (Le Gall et al., 2004Go). The present study demonstrates that combinations of certain linkers, such as L6 and SL, can destabilize the tandab molecule, probably by causing dissociation of some VH/VL interfaces and leading to aggregation.

Analysis of the biological activity of tandabs in the 6-day PBMC cultures demonstrated a correlation to some extent of their anti-tumor effect with their stability and with the calculated distances between CD3 and especially CD19 binding sites. For example, the least stable T6/SL tandab with a half-life of ~12 h demonstrated only marginal cytotoxic activity. The T6/K9 variant was more active than T6/K6 but less active than T10/SL. Surprisingly, the most stable T10/LL variant, which in addition demonstrated the best antigen-binding characteristics, appeared to be less active than the T10/SL tandab in activating and recruiting T cells against tumor cells. It is probable that the T10/LL tandab where two halves of the molecule are separated by a very long 27 amino acid linker can assume a set of conformations, some of them being different from the previously postulated tandem diabody structure (Kipriyanov et al., 1999Go). For example, Völkel et al. (2001)Go speculated that the four-domain VHA–linker1–VLB–linker2–VHB–linker3–VLA single-chain molecule having a long and flexible middle linker may adopt a circular tetrabody-like structure. In this case, the geometry of the whole molecule and orientation of antigen-binding sites of the same specificity would be different from the tandab, thus obviously having an effect on the biological activity. However, this assumption is speculative and further studies are needed to demonstrate the correct structure of these bispecific tetravalent molecules.

In conclusion, our data indicate the presence of certain threshold requirements for the linkers connecting individual antibody variable domains in a four-domain single-chain molecule necessary not only for effective dimerization of the molecule but also for biological activity and suitability for clinical applications.


    Acknowledgments
 
This work was supported by the German BioRegio Program (Grant BEO32/AZ12389). We thank Dr Manfred Hensel (Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany) for providing the blood samples from B-CLL patients and also Brita Geweniger and Stefanie Wolff for their excellent technical assistance.


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Received February 13, 2004; revised April 6, 2004; accepted April 16, 2004.

Edited by Phillipp Holliger





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