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
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Abstract |
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Keywords: human CD3/human CD19/linker/single-chain Fv/tandem diabody
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Introduction |
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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., 1995; Baeuerle et al., 2003
), 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., 1999
; Kontermann and Müller, 1999
). 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., 2003
). Moreover, the scBsDb format facilitates the production of relatively stable bispecific constructs from weakly associated Fv fragments (Kipriyanov et al., 2003
).
In contrast to native antibodies, most of the developed BsAb formats, either quadroma-derived or recombinant (such as scFvscFv 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., 1999; Völkel et al., 2001
). Unlike many other BsAb formats, this so-called tandem diabody (tandab) is a homodimer with Mr
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., 1999
; Cochlovius et al., 2000a
).
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Materials and methods |
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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., 1997) and pHOG21-HD37 (Kipriyanov et al., 1996
) 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-scFv6-319 encoding the hybrid VHCD3L6VLCD19 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., 1998
). The plasmid pHOG21-scFv6-193 encoding the hybrid VHCD19L6VLCD3 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., 2004
). The gene coding for the hybrid VHCD3L6VLCD19 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/L6K6, pDISC19 x 3/L6K9 and pDISC19 x 3/L6SL were constructed by ligation of the NcoI/PvuII restriction fragment from pHOG21-scFv6-193 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., 1999
). 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., 2004
). 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 (lac
74 galISII::OP308strA) (Maurer et al., 1980
) (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., 2000b
). 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., 1999
). Protein concentrations were determined by the Bradford dye-binding assay (Bradford, 1976
) 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., 1999
).
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., 1998). 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 13/45/312 (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., 1998), except that the detection of retained antibody was performed using anti-(His)6 mouse mAb 13/45/312 (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., 1979), Edu-2 (anti-CD4) (Chemicon, Hofheim, Germany), UCH-T4 (anti-CD8) (Chemicon) and anti-CD19 mAb HD37 (Pezzutto et al., 1987
) 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.
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Results |
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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., 1996) and anti-CD3 mAb OKT3 (Kipriyanov et al., 1997
) were used. Since the tandab comprises two diabody-like modules (Kipriyanov et al., 1999
) (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., 1998
; Le Gall et al., 1999
) by a natural six amino acid peptide, Ser113Pro118, connecting VH and CH1 domains of the heavy chain in the murine IgG1 and IgG2b (Lefranc, 2001
) led to a 10-fold higher bacterial production of monospecific anti-CD3 diabody assembled in a VH-to-VL orientation (Le Gall et al., 2004
). 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 VHCD3VLCD19 and VHCD19VLCD3 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
(L6, K6 and K9) or comprise additional amino acids (L10, SL and LL), thus potentially contributing to extra flexibility.
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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., 1999). 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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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., 1999, 2003
). 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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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 5798% 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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Discussion |
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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., 1988), 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., 1996
; Turner et al., 1997
). 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., 1999, 2003
; Kontermann and Müller, 1999
). 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., 1999
; Völkel et al., 2001
). 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., 1999
) and since the peptide unit length is
3.8 Å (Huston et al., 1988
), 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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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., 1999
). For example, Völkel et al. (2001)
speculated that the four-domain VHAlinker1VLBlinker2VHBlinker3VLA 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.
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Acknowledgments |
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Received February 13, 2004; revised April 6, 2004; accepted April 16, 2004.
Edited by Phillipp Holliger
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