ALPHABODIES SPECIFICALLY BINDING TO VIRAL PROTEINS AND METHODS FOR PRODUCING THE SAME

The invention provides methods for the production of single-chain Alphabody polypeptides having detectable binding affinity for, or detectable in vitro activity on, a viral protein of interest, which comprising the step of producing a single-chain Alphabody library comprising at least 100 different-sequence single-chain Alphabody polypeptides, wherein said Alphabody polypeptides differ from each other in at least one of a defined set of 5 to 20 variegated amino acid residue positions, and wherein said variegated amino acid residue positions are located at specific positions in one or more of the alpha-helices of the Alphabody or the linker fragment connecting two consecutive alpha-helices of the Alphabody polypeptides. The invention further provides Alphabodies obtainable by the methods of the invention and uses thereof.

EXAMPLES

Generation of Single-Chain Alphabody Library

The present example demonstrates that a single-chain Alphabody library can be obtained which is well-displayed on phage and which is potentially useful for obtaining single-chain Alphabody sequences that bind to a viral protein of interest.

A single-chain Alphabody random library was designed starting from the annotated amino acid sequence and a 3-D model of a reference Alphabody denoted ‘scAB013_L16’. A simplified 3-D model of this reference Alphabody is illustrated inFIG. 1. The amino acid sequence of scAB013_L16 is also provided herein as SEQ ID No: 1. The sequence is further shown inFIG. 2, wherein the conventional heptad core positions are indicated as well.

An Alphabody groove is formed by two spatially adjacent alpha-helices of a folded Alphabody protein (FIG. 1). Since there are three alpha-helices per Alphabody, there are in principle three candidate grooves which can be randomized. The said 3-D model was inspected first to select the most suitable groove for randomization. It was decided to select the groove between the first alpha-helix (‘A-helix’) and third alpha-helix (‘C-helix’), which run parallel in the 3-D model. Next, the model was further inspected to identify the most suitable amino acid residue positions to be randomized (variegated, varied) in each alpha-helix. It was observed that the groove is actually formed by residues located at heptad c- and g-positions in the A-helix and at heptad b- and e-positions in the C-helix. The g- and e-positions were found to contribute the most (i.e., most directly) to the groove, and are therefore denoted ‘primary groove positions’. The c- and b-positions are located somewhat remotely from the middle of the groove, and are therefore denoted ‘secondary groove positions’. In addition to these primary and secondary groove positions, the bottom of a groove is formed by some core (a- and d-) positions; in particular, the model showed that core d-positions of the A-helix and a-positions of the C-helix might contribute to the shape of the groove as well, especially if the primary groove positions are occupied by tiny amino acid residues such as glycine, alanine or serine. Such core a- and d-positions which may conditionally contribute to the shape of a groove are herein denoted ‘core groove positions’.

FIG. 1shows that there are 3 primary e- and also 3 primary g-positions within the coiled coil part of the scAB013_L16 Alphabody model. It is further seen that there are 4 b- and 4 c-positions at secondary groove positions. Further, there are 4 core d- and 4 core e-positions which may potentially contribute to the groove. Thus, there are in total 22 positions which can influence the shape of a groove when being variegated. When all 22 would be fully randomized into the 20 natural amino acid residues, this would correspond to a sequence space (i.e., the total number of possible combinations) of 2022or about 4×1028distinct sequences. Clearly such huge libraries cannot be made in a form wherein all different sequences are physically present (i.e., such library cannot be ‘complete’). Consequently, and if the envisaged library is aimed to be complete (or nearly complete), then the number of variable positions should be drastically reduced.

It was therefore decided not to vary any of the core groove positions. This decision was further motivated by the (avoidance of) risks associated with mutating core positions in a coiled coil: many such substitutions would be detrimental for the stability and/or correct folding of the respective Alphabody constructs. Further, it was also decided not to vary two secondary groove positions. In particular, the first c-position in the A-helix and the first b-position in the C-helix were kept constant. Finally, the first primary groove e-position in the C-helix was also kept fixed as well. This resulted in the selection of 11 variegated positions within the context of the reference Alphabody scAB013_L16. The theoretic sequence space of such library, when fully randomized, is thus 2011or about 2×1014distinct sequences.

In addition to the variegated positions, two other types of modifications to the reference Alphabody scAB013_L16 were made. First, two lysine to glutamic acid mutations were introduced, i.e., one such mutation at the f-position of the second heptad in each of helices A and C. Second, two arginine to alanine mutations were introduced, i.e., one such mutation at the c-position of the fourth heptad in each of helices B and C. The sequence of this modified single-chain Alphabody, wherein positions to be variegated are indicated by ‘x’-symbols, is shown inFIG. 3. This sequence is also provided as SEQ ID No: 2. The single-chain Alphabody library that was constructed on the basis of this design is hereinafter referred to as ‘scLib_AC11’. Since all variegated positions are located in an Alphabody groove, this library is also referred to as a ‘groove library’.

A second single-chain Alphabody groove library was designed starting from a 3-D model of a smaller Alphabody reference construct denoted ‘scAB140_L14’. The latter essentially corresponds to the scAB013_L16 construct wherein the third heptad in each of the alpha-helices is deleted, the glycine/serine linker sequences are reduced from 16 to 14 residues, and the N-terminal alpha-helix capping residues are substituted by an alternative, less negatively charged, motif. Apart from these differences, exactly the same choices with respect to primary, secondary and core groove positions to be variegated were made when designing the library. In view of the deletion of one heptad unit in each of the helices, this library comprises only 7 variable residue positions. The theoretic sequence space for full randomization is therefore 207or about 109, which should guarantee near-completeness of the actual produced library. The sequence of this single-chain Alphabody groove library, denoted ‘scLib_AC7’, is shown inFIG. 3. This sequence is also provided as SEQ ID No: 3.

A third single-chain Alphabody library was designed to explore the potential of generating Alphabodies that bind to their target via a surface-exposed area on a single alpha-helix. In other words, the purpose of this design was to generate an Alphabody ‘helix library’ (as opposed to the scLib_AC11 and scLib_AC7 libraries which are groove libraries). The 3-D model of scAB013_L16 was again used as the template structure for guiding the selection of positions to be variegated. It was decided to select the C-helix in this structure as the one to be variegated. Further inspection of the model shows that the b-, c- and f-positions together form a contiguous rim with a convex shape. There are 11 such surface positions discernible in this alpha-helix. It was observed that, in principle, some flanking e- and g-positions might potentially aid in the formation of a contiguous binding surface, but this option was discarded in view of the risk to destabilize the Alphabodies and because the number of variable positions would run up too much. Thus, all 11 b-, c- and f-positions in the C-helix were initially considered for variegation, but the two N-terminal glutamates were finally left unaltered in order not to cancel out their capping function and to maintain the library completeness within reasonable bounds. This finally resulted in 9 b-, c- and f-positions to be variegated in the library. This library was accordingly termed ‘scLib_C9’. The sequence is shown inFIG. 3. This sequence is also provided as SEQ ID No: 4.

The actual single-chain Alphabody libraries were ordered at Geneart AG (Regensburg, Germany). A ‘3+3’ monovalent display format (Smith, Gene 128:1-2 (1993)) was adopted using the pCx1 vector, a pHEN-derived phagemid. The libraries were delivered as transformedE. coliTG1 cells with a guaranteed minimum of 108unique clones. The Alphabody sequences were fused to the pill coat protein of M13 phage. They were attached via their C-terminus to the pill coat protein through a linker sequence that contains an amber codon (at the genetic level) and a His6-tag. Exportation of the fused Alphabodies to the periplasm was ensured by the presence of a PelB leader sequence at the N-terminus. The level of display on phage was checked using Western-Blotting and was found to be suitably high. Analysis showed that in general one third of the phage displayed an Alphabody (FIG. 4).

Alphabodies Binding to HIV-1 Env

The present example demonstrates that single-chain Alphabodies of the invention can be obtained by a method of the invention, for example by using a mixture of Alphabody groove and helix libraries as provided in EXAMPLE 1.

The viral fusion protein of interest was chosen to be HIV-1 Env. HIV-1 Env complexes, also known as ‘envelope glycoprotein complexes’ or ‘gp120/gp41 complexes’ or ‘spikes’, are a primary target for treatment of HIV infection. They are displayed at the surface of HIV virions and cells that are engineered so as to express Env spikes. HIV entry into a target cell and cell-cell fusion are primarily mediated by the action of these glycoprotein complexes subsequent to their engagement with specific receptors at the target cell. The ability to block viral entry or cellular fusion by impeding the function of Env complexes is generally thought to be of high value for the treatment of HIV infection. The reference sequence for HIV-1 Env is chosen to be that of the HXB2 strain; this sequence is also provided herein as SEQ ID No: 5 (MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDA KAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLK PCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKL DIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVS TVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRI RIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDP EIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKV GKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNRRSELYKYKVVKI EPLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQ QNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNAS WSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF NITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEE GGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWW NLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL).

The target region of interest within the said HIV-1 Env sequence was chosen to be the gp41 HR2 region, in particular, residues 628 to 661 of the HIV-1 HXB2 Env. The amino acid sequence is ‘WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL’ (SEQ ID NO: 6) in single-letter notation, hereinafter also referred to as ‘C36’. The target sequence (target peptide) used for the biopanning work described in the present example was a C36 derivative that was N-terminally biotinylated and C-terminally amidated and wherein the N-terminal biotin group was attached to the C36 sequence through a 4-residue Gly/Ser linker, the full target sequence having the amino acid sequence of SEQ ID No: 7, written in single-letter notation as ‘biotin-GSGSWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL-NH2’, and herein also referred to as ‘bL4_C36’.

A soluble biopanning protocol was applied to obtain (phage-displayed) Alphabodies which recognize the said bL4_C36 target, as follows. An equal mixture of phage displaying the libraries scLib_AC11, scLib_AC7 and scLib_C9 was prepared and used as input for the selection against the bL4_C36 target. The phage were incubated with the target for 1.5 to 2 hours and then captured on streptavidin magnetic beads for 15 to 30 minutes. Mock experiments where the target was omitted were always performed in parallel. Bound phage were eluted with an acidic pH shock after washing of the beads. Five selection rounds (biopanning rounds) were performed. The selection stringency, which was kept constant during the different rounds, was as follows: (i) amount of input phage: ˜1.3×1012particles; (ii) 50 μl streptavidin-coated magnetic beads, target concentration: 500 nM; (iii) 10 washes (each with 1 ml 0.05% Tween 20-containing buffer).

In total, 162 phage colonies (39 from round 3, 32 from round 4 and 91 from round 5) were randomly picked and tested in phage ELISA, both on immobilized bL4_C36 and on an irrelevant control peptide. Phage were rescued in 96-well plates and tested without purification or quantification. None of the clones showed an absorbance (A450nm) larger than 0.1 on the control peptide. Typically, phage clones showing an A450nmlarger than 10 times the average background signal were sequenced. 63 out of 66 clones were found to originate from the scLib_AC11 library, 3 originated from the scLib_AC7 library and none were found to come from the scLib_C9 library. The high abundance of binders from the groove libraries strongly suggests that Alphabodies preferably bind to the HR2 target sequence via a groove. Also, the higher frequency of binders from the scLib_AC11 library compared to the scLib_AC7 library suggests that the target sequence is ideally captured by an extended binding groove. The scLib_AC11 clones further separated into 12 distinct Alphabody sequences and the scLib_AC7 clones all showed the same sequence (data not shown).

Several of the positive Alphabody clones were selected for soluble expression. To this end, their coding sequences were subcloned into the pET16b vector (Novagen), in such way that they were appended with a N-terminal 10-histidine tag. The resulting constructs were transformed into a hostE. colistrain harboring a chromosomal copy of the T7 polymerase gene under control of the lacUV5 promoter (DE3 lysogens), usually BL21(DE3). Transformed cells were grown in medium supplemented with ampicillin and protein expression was induced by the addition of IPTG to exponentially growing cultures. Cells containing the expressed Alphabodies were collected by centrifugation and the pellets were resuspended in 50 mM Tris, 500 mM NaCl, pH 7.8. Cells were then disrupted by sonication and spun down for cell debris removal. The cleared supernatants were applied onto a HITrap IMAC HP column (GE Healthcare) loaded with Ni2+ions. Bound proteins were eluted by applying an imidazole gradient from 5 to 1000 mM. Alpha-body-containing fractions were pooled, concentrated and loaded on a Superdex 75 size exclusion chromatography (SEC) column (GE Healthcare). During this final purification step, the buffer was changed to 50 mM Tris, 150 mM NaCl, pH 7.8. Typically, the recovered protein fractions contained 0.3 to 2 mg/ml of pure Alphabody. Alphabody preparations can be stored for weeks at 4° C.; for longer term storage, samples are kept at −80° C.

FIG. 5shows the results of an ELISA experiment on one of the purified anti-HR2 Alphabodies. Both the target peptide bL4_C36 and a biotinylated control peptide were immobilized on a neutravidin-coated and skim milk-blocked ELISA plate. The negative control peptide, referred to as ‘bL4_N51’, consisted of the HIV-1 Env HXB2 sequence of SEQ ID No: 1 residues 540 to 590 (‘N51’), preceded by a 4-residue Gly-Ser-Gly-Ser linker (‘L4’) and an N-terminal biotin group (‘b’). The Alphabody of the present invention, herein referred to as ‘scAB_Env03’ (SEQ ID No: 8 andFIG. 6), was subsequently applied to the wells at concentrations ranging from 0.4 μM to 0.4 nM and incubated for 2 h. Detection was performed with the Penta-His-HRP antibody (Qiagen). Significant binding was observed for the scAB_Env03 starting at concentrations of 1.5 to 3 nM; half saturation was obtained at about 6 nM and apparent maximal binding was reached at about 25 nM (FIG. 5, black bars). No binding (A450nm<0.05) was observed to the negative control peptide bL4_N51 (white bars), or to peptide-free neutravidin (data not shown). This result shows that scAB_Env03 binds specifically to the target peptide bL4_C36, where the C36 part is an exact copy of HR2 in HIV-1 Env HXB2.

Analysis of Further HIV-1 Env-Binding Alphabodies

In addition to the scAB_Env03 Alphabody of EXAMPLE 2, three other single-chain Alphabodies, obtained from the same biopanning procedure, were further characterized. The present example demonstrates that multiple Alphabodies can be obtained, that their amino acid sequences can be determined, that they are highly thermostable, that they have a high affinity for HIV-1 Env, that their kinetics can be determined, and that some of them may be antivirally active.

The three additional Alphabodies that were tested are referred to as ‘scAB_Env02’, ‘scAB_Env04’ and ‘scAB_Env05’. Their amino acid sequences are shown inFIG. 7. Some apparently preferred amino acid residues were observed at different variegated positions, although not any position was occupied by a single, unique residue type. For example, three distinct Alphabodies had a proline at the first randomized position in the A-helix (i.e., at position g in the first heptad). The next randomized position seemed to prefer a small side chain with a hydroxyl group (i.e., serine or threonine). The fourth randomized position seemed to prefer a tryptophan. The C-helix showed a less conserved pattern, but some preference for hydrophobic residues was observed at the second, third and fourth randomized position.

FIG. 8shows a summary of thermal unfolding experiments as measured by circular dichroism (CD) at 222 nm in different concentrations of GuHCl denaturant. scAB_Env05 turned out to be the most stable, with a melting temperature (Tm) of about 100° C. even in 4 M GuHCl and a Tm=61° C. at 6 M denaturant. scAB_Env03 was less stable by about 5-8° C. in Tm at 4-5 M GuHCl; this may be related to the presence of a proline at the first randomized position in the A-helix (seeFIG. 7), but yet this proline caused only a mild destabilization of the Alphabody structure. scAB_Env04 behaved somewhat aberrant, in that, a pre-transition was observed in the presence of GuHCl concentrations up to 4 M: upon unfolding, the CD signal decreased only a little, but with a Tm markedly lower than those of the other constructs. Then, at higher GuHCl concentrations, the full transition was observed with a Tm comparable to, though slightly less than, those of scAB_Env03 and scAB_Env05. This might be due to the presence of a glycine at the sixth variable position in helix A, as it is possible that the very last alpha-helical turn unfolds in advance of the rest of the scaffold. In conclusion, all constructs were found to be extremely stable and should be fully folded at room temperature, despite the occasional presence of proline and/or glycine in the alpha-helices.

ELISA experiments for scAB_Env02 to -05 showed very specific binding and a fairly high affinity for C36 (FIG. 9). Especially the scAB_Env03 construct behaved most satisfactory: an onset of binding was observed in the low-nanomolar range, thereby confirming the results from EXAMPLE 2. The next strongest binders were scAB_Env02 and scAB_Env04, followed by scAB_Env05. The irrelevant control Alphabody scAB_PR02 (which was selected from an earlier biopanning procedure on an unrelated target) showed no background binding up to about 1 μM.

Three variants of the best-behaving scAB_Env03 Alphabody were synthesized according to the same protocol as the parental sequence. A first variant, denoted ‘scAB_Env03_PA’, was constructed wherein the proline at the first randomized position in the A-helix was replaced by an alanine (to test whether a helix-destabilizing proline could be substituted by a helix-stabilizing residue without loss of affinity). A second variant, denoted ‘scAB_Env03_noM’, was constructed wherein all methionines were replaced by either glycine or isoleucine (the methionines at the N-terminus of each helix were replaced by glycine, and those at the C-terminus of each helix were replaced by isoleucine). A third variant, denoted ‘scAB_Env03_KC’ was constructed wherein the lysine at the f-position in the second heptad of the B-helix was replaced by a cysteine to be able to test whether disulfide-linked or PEGylated Alphabodies retain their physical and biochemical properties. The sequences of these variants are shown inFIG. 10.

Surface plasmon resonance (SPR) was used to determine the binding kinetics to immobilized bL4_C36 (FIG. 11). All unmodified constructs (scAB_Env02 was not tested) showed Langmuir binding kinetics. The off-rate constants were in agreement with the ELISA results (scAB_Env03<scAB_Env04<scAB_Env05), confirming that scAB_Env03 was the most persistent binder. However the on-rate constants showed a different trend (scAB_Env04>scAB_Env03>scAB_Env05), resulting in about equal affinity for scAB_Env03 and scAB_Env04 (KD 200 nM) and a significant lower affinity for scAB_Env05 (KD 4000 nM). The Env03 Cys-mutant (scAB_Env03_KC) showed very strong binding: although the on-rate was lower than for the parental scAB_Env03 (kOn=0.38×105M−1s−1), the off-rate was extremely good (kOff=2.4×10−5s−1), resulting in a subnanomolar affinity constant (KD=0.62 nM). The main reason for this very high affinity is that Env03_KC dimerizes through the formation of a disulfide bond and binds in a bidentate mode onto the chip surface. This was confirmed in a later experiment (not shown) wherein the sample was pretreated with DTT and wherein the kinetics dropped back to about the same rates as the parental construct. The scAB_Env03_PA and scAB_Env03_noM constructs unexpectedly showed biphasic binding and dissociation kinetics. A first, fast association phase with on-rates comparable to the parental scAB_Env03 (i.e., kOn in the order of 105M−1s−1) was followed by a second, slow phase (with kOn below 10−2s−1). However, this second phase resulted in nearly persistent binding, with off-rate constants in the order of 104s−1. The global dissociation constants (KD=KD1*KD2) were therefore close to about 10 nM for both constructs.

The HIV inhibitory properties of different Alphabodies were analyzed in a 5-day ‘MTT infection assay’ using a cell line (MT4-X4) displaying CD4 and CXCR4 receptors. The virus used in this assay was the laboratory adapted reference strain HXB2 virus using CXCR4 as co-receptor. Cells were infected with 100 TCID50/ml of virus in presence of three-fold dilutions of Alphabody starting at 2.5 μM. Inhibition of HIV infection by Alphabodies was evaluated by monitoring the cell survival using MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide). MTT is reduced to formazan by living cells. Solubilization of the formazan crystals results in a colored product that can be measured by spectrophotometry at 540 nm. The cellular toxicity of the Alphabodies was monitored in the same assay using the same read-out, i.e., cell survival. As positive controls, the clinically approved T-20 peptide (Fuzeon®, Roche) was used as well as the CXCR4 antagonist AMD3100 (Mozobile™, Genzyme). Alphabodies scAB_Env03 and scAB_Env05 were tested using this assay, but showed no antiviral activity up to the highest concentration tested (2.5 μM). In contrast, the dimeric construct scAB_Env03_KC showed a clear antiviral activity with a 50% inhibitory concentration (IC50) of 709 nM (FIG. 12). No toxicity effects were observed.

In conclusion, the present example demonstrates that Alphabodies can be obtained by a method of the present invention which display stable folding, high affinity to a subregion of a viral fusion protein, and significant antiviral activity on the virus displaying this viral fusion protein.