Oligopeptide and methods for producing conjugates thereof
10787505 · 2020-09-29
Assignee
- F. Hoffmann-La Roche AG (Basel, CH)
- Institut Pasteur (Paris, FR)
- Centre National De La Recherche Scientifique (Cnrs) (Paris, FR)
- Commissariat A L'energie Atomique Et Aux Energies Alternatives (Paris, FR)
Inventors
- Pierre LAFAYE (Malakoff, FR)
- Sylvie Bay (Paris, FR)
- Benoit Delatour (Cachan, FR)
- Marc Dhenain (Limours, FR)
- Charles Duyckaerts (Saint-mande, FR)
- Tengfei Li (Courbevoie, FR)
- Matthias Vandesquille (Fontenay-aux-roses, FR)
- Christian Czech (Grenzach-Wyhlen, DE)
- Fiona Grueninger (Arlesheim, CH)
Cpc classification
C07K2317/569
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
International classification
A61K51/10
HUMAN NECESSITIES
Abstract
The present invention relates to an oligopeptide and conjugates thereof. The present invention also relates to the use of these oligopeptide conjugates for treating or diagnosing disorders mediated by amyloid deposits. Finally, the present invention also relates to a coupling method for obtaining oligopeptide coupled with a substance of interest (functional conjugates).
Claims
1. An oligopeptide of formula P-C-Z or Z-C-P, wherein: P is a 8 to 800 amino acid peptide having no reduced cysteine residue, C is a cysteine residue, Z represents a 1-10 amino acid spacer, wherein the amino acid residues of Z are identical or different and wherein Z does not contain a cysteine residue, characterized in that said cysteine residue C is linked by thio-addition to a substance of Interest through a maleimido compound of formula (I) bearing said substance of interest: ##STR00004## wherein: B, B.sub.2, and B, identical or different, are Independently single bonds or polyols and polyalkyls having 2 to 12 carbon atoms, D, D and D, identical or different, are independently selected from amine, amide, thiourea, carbamate, ester, ether, thioether, and oxime groups, A is a single bond or a chelating agent, SI is the substance of interest selected from the group consisting of a diagnostic compound and a therapeutic compound, X is an acid, amide, or ester function, and n=2 to 100, the chelating agent A being elected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA), diethylene triamine pentaacetic acid (DTPA), 1,4,7-tris(carboxymethylaza)cyclododecane 10-azaacetylamide (DO3A), nitrilotriacetic acid (NTA), D-penicillamine (Pen), 2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), triethylenetetramine (Trien), ethylenediaminetetraacetic acid (EDTA), and 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (IB4M).
2. The oligopeptide according to claim 1, characterized in that Z consists of a 2 amino acid sequence.
3. The oligopeptide according to claim 1, characterized in that P comprises a peptide P selected from the group consisting of a variable domain of a camelid heavy-chain antibody (VHH), a Fab, F(ab).sub.2Fv or scFv fragment of a conventional antibody, an immunoglobulin new antigen receptor (IgNAR), a nanofitin, a DARPin, an anticalin, an affibody, an affilin, an avimer, a monobody and a kunitz domain.
4. The oligopeptide according to claim 1, characterized in that the amino acid peptide P of the oligopeptide of formula P-C-Z has at its C-terminus a 1-10 amino acid spacer Y, or the amino acid peptide P of the oligopeptide of formula Z-C-P has at its N-terminus a 1-10 amino acid spacer Y, wherein the amino acid residues of said amino acid spacer Y are identical or different, and wherein said amino acid spacer Y does not contain a cysteine residue.
5. The oligopeptide according to claim 4, characterized in that Y represents a 4 neutral amino acid spacer.
6. The oligopeptide according to claim 1, characterized in that the amino acid peptide P of the oligopeptide of formula P-C-Z has at its N-terminus a 1-50 amino acid sequence X or the amino acid peptide P of the oligopeptide of formula Z-C-P has at its C-terminus a 1-50 amino acid sequence X, wherein the amino acid residues of said amino acid sequence X are identical or different, and wherein said amino acid sequence X does not contain a cysteine residue.
7. The oligopeptide according to claim 6, characterized in that X comprises a tag and an enzyme cleavage site.
8. The oligopeptide according to claim 3, characterized in that the peptide P is a VHH and the oligopeptide formula is VHH-C-Z, VHH-Y-C-Z, X-VHH-C-Z, X-VHH-Y-C-Z, Z-C-VHH, Z-C-Y-VHH, Z-C-VHH-X, or Z-C-Y-VHH-X, wherein Y is a 1-10 amino acid spacer, the amino acid residues of said amino acid spacer Y being identical or different, providing that Y does not contain a cysteine residue, and wherein X is a 1-50 amino acid sequence X, the amino acid residues of said amino acid sequence X being identical or different, providing that said amino acid sequence X does not contain a cysteine residue.
9. The oligopeptide according to claim 1, characterized in that the diagnostic compound is selected from the group consisting of a NMR contrast agent, a MRI contrast agent, and a radioisotope.
10. The oligopeptide according to claim 9, characterized in that the diagnostic compound is a NMR or MRI paramagnetic contrast agent selected from the group consisting of gadolinium (Gd), dysprosium (Dy) and manganese (Mn).
11. The oligopeptide according to claim 1, characterized in that the therapeutic compound selected from the group consisting of a peptide, an enzyme, a nucleic acid, a virus, an analgesic compound, an anti-inflammatory compound, an antidepressant compound, an anticonvulsant compound, a cytotoxic compound and an anti-neurodegenerative compound.
12. The oligopeptide according to claim 1, characterized in that the substance of interest SI is gadolinium (Gd), and the chelating agent A is DOTA.
13. The oligopeptide according to claim 1, characterized in that the maleimido compound is of formula (I): ##STR00005##
14. A maleimido compound characterized in that it is of formula (I): ##STR00006## wherein B, B.sub.1, B.sub.2 and B, identical or different, are independently single bonds or polyols and polyalkyls having 2 to 12 carbon atoms, A is a single bond or a chelating agent, SI is the substance of interest selected from the group consisting of a diagnostic compound and a therapeutic compound, and n=2 to 100.
15. The oligopeptide according to claim 1, characterized in that n is 2 or 3.
16. The oligopeptide according to claim 1, characterized in that said oligopeptide comprises or consists of the amino acid sequence selected from the group consisting of: SEQ ID NO. 4, corresponding the full-length form of R3VQ, SEQ ID NO. 5, corresponding the full-length form of R3VE, SEQ ID NO. 6, corresponding the short form of R3VQ, and SEQ ID NO. 7, corresponding the short form of R3VE.
17. The oligopeptide according to claim 1, characterized in that: n is 2 or 3, the substance of interest SI is gadolinium (Gd), and the chelating agent A is DOTA, and P comprises a peptide P consisting of a variable domain of a camelid heavy-chain antibody (VHH).
18. The compound according to claim 14, characterized in that the diagnostic compound is selected from the group consisting of a NMR contrast agent, a MRI contrast agent, and a radioisotope.
19. The compound according to claim 18, characterized in that the diagnostic compound is a NMR or MRI paramagnetic contrast agent selected from the group consisting of gadolinium (Gd), dysprosium (Dy) and manganese (Mn).
20. The compound according to claim 14, characterized in that the therapeutic compound selected from the group consisting of a peptide, an enzyme, a nucleic acid, a virus, an analgesic compound, an anti-inflammatory compound, an antidepressant compound, an anticonvulsant compound, a cytotoxic compound and an anti-neurodegenerative compound.
Description
(1) In addition to the preceding features, the invention further comprises other features which will emerge from the following description, which refers to examples illustrating the present invention, as well as to the appended figures.
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EXAMPLE 1: GENERATION OF ANTI-ABETA VHHS COUPLED TO GADOLINIUM CONTRAST AGENT AND THEIR EVALUATION IN VITRO/IN VIVO
(31) Materials and Methods
(32) 1. Production, Selection and Purification of VHH R3VQ
(33) Antigen Preparation and Induction of a Humoral Immune Response in Alpaca
(34) A-beta 42 peptide (A42) (1 mg-Bachem) was dissolved in 900 l H.sub.2O and vortexed. 100 l PBS 10 was added and the mixture was incubated at room temperature for one month before use. 250 l of the mixture was mixed with 250 l of Freund complete adjuvant for the first immunization, and with 250 l of Freund incomplete adjuvant for the following immunizations. One young adult male alpaca (Lama pacos) was immunized at days 0, 21 and 35 with 250 g immunogen. At day 50 a serum sample was taken and the immune response monitored by ELISA using A42 as antigen.
(35) Library Construction and Panning
(36) 250 ml of blood of the immunized animal was collected at day 50 and the peripheral blood lymphocytes isolated by centrifugation on a Ficoll (Pharmacia) discontinuous gradient and stored at 80 C. until further use. Total RNA and cDNA was obtained as previously described in Lafaye P. et al. (1995, Res Immunol., 146:373-382), and DNA fragments encoding VHH domains amplified by PCR using CH2FORTA4 and VHBACKA6 primers, which anneal to the 3 and 5 flanking region of the VH genes, respectively. The amplified product was used as template in a second round of PCR using either the primers VHBACKA4 and VHFOR36 or the primers VHBACKA4 and LHH (5 GGACTAGTTGCGGCCGCTGGTTGTGGTTTTGGTGTCTTGGG-3) (SEQ ID NO. 13) specific for the long hinge homodimeric antibody. The primers were complementary to the 5 and 3 ends of the amplified product and incorporated SfiI and NotI restriction sites at the ends of the VHH genes. The PCR products were digested and ligated into phage expression vector pHEN1. The resulting library was composed of two sub-libraries, one derived from VHH DNA-encoding genes with no hinge and the other from long hinge antibody genes. Phages were produced and isolated using both sub-libraries, and subsequently pooled.
(37) The library was panned for reactivity in parallel with a biotinylated A1-42, A1-40 or A1-16 peptide, as previously described (Lafaye P. et al., 2009, Mol Immunol. 46:695-704). The library (10.sup.13 transducing units) was panned by incubation with each biotinylated peptide for 1h at 37 C. under gentle agitation, then the mixture was incubated with streptavidin beads for 15 at 37 C. A different blocking agent was used at each of the three rounds of panning: 2% skimmed milk, Licor diluted 1:4, and 4% BSA were respectively used. The concentration of biotinylated peptides used decreased at every round of panning with respectively 100 nM, 50 nM and 10 nM. Phage clones were screened by standard ELISA procedures using a HRP/anti-M13 monoclonal antibody conjugate (GE Healthcare) for detection (see below).
(38) Expression of VHHs
(39) The coding sequence of the selected nanobodies in vector pHEN1 was sub-cloned into a modified bacterial expression vector pET23 containing a 6-Histidine tag using NcoI and NotI restriction sites. Transformed E. coli BL21 (DE3) LysS cells express VHH in the cytoplasm after overnight induction with IPTG (0.5 mM) at 16 C. Purified VHHs were isolated by IMAC from cytoplasmic extracts using a HiTrap crude column charged with Ni.sup.2+(GE Healthcare), according to the manufacturer's instructions, followed by size exclusion chromatography with a Superdex 75 column (GE Healthcare). The VHHs (in particular the R3VQ(His)-NH.sub.2; compound 1 in
(40) 2. Characterization of Biochemical Properties of VHH R3VQ
(41) Immunoblots
(42) A-beta 42 peptide was resuspended in NuPAGE LDS sample buffer (Invitrogen) containing 8M urea. Following separation by polyacrylamide gel electrophoresis (PAGE) using NuPAGE Novex 4-12% Bis-tris gel (Invitrogen), semi-dry transfer onto Hybond-C (Amersham) and western blotting were carried out using the Xcell II blot module (Invitrogen). Prior to the immunochemical reaction, membranes were blocked in a 4% skimmed milk solution. Immunoblotting of membranes was accomplished with VHH and revealed by rabbit anti-His tag (eBioscience) polyclonal antibodies followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abcam). Finally, peroxidase activity was visualized using a chemiluminescent kit (GE Healthcare).
(43) ELISA
(44) Streptavidin-coated microtiter plates (Thermo Scientific, Denmark) were coated by incubation overnight at 4 C. with 1 g/ml of biotinylated A-beta 40 or A-beta 42 (preferably A-beta 40) diluted in PBS. Plates were washed with buffer 0.1% Tween 20 in PBS. VHH R3VQ was diluted in buffer 0.5% gelatin 0.1% Tween 20 in PBS. After 2 h incubation at 37 C., plates were washed again before adding respectively a rabbit anti-His tag polyclonal antibody (eBiosciences), followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abcam), and finally revealed by OPD (o-phenylendiamine dihydrochloride, Dako) according to manufacturer's protocol.
(45) Determination of Dissociation Constants by ELISA
(46) The binding affinity of VHHs was determined as previously described (Friguet B. et al., 1985, Immunol Methods, 77:305-19). Briefly, various concentrations of A peptides (A fragments 1-16, 10-20, 15-25, 22-35 and 29-40) were incubated in solution overnight at 4 C. with a known quantity of VHH until equilibrium was reached. The VHH concentration used was determined by preliminary ELISA calibrations. Each mixture (100 l) was transferred to a well of a microtiter plate previously coated with antigen and was incubated for 20 min at 4 C. The plates were washed with buffer 0.1% Tween 20 in PBS and bound VHHs were detected by adding beta-galactosidase-conjugated goat anti-rabbit Igs (Biosys, Compigne, France) and 4-methylumbelliferyl -D galactoside (Sigma). Fluorescence was read (Fluoroskan, Labsystem, Finland) at 460 nm, after excitation at 355 nm. KD was estimated from the slope of the regression curve obtained by plotting the reciprocal of the fraction of bound antibody versus the reciprocal of the molar concentration of antigen.
(47) Sequences Analysis
(48) VHH encoded DNAs were sequenced by GATC Biotech and sequences were treated with DNA strider.
(49) Determination of pI
(50) The pI of VHHs was determined by isoelectric focusing using IEF 2-9 Gel (Invitrogen). NEPGHE (non equilibrium pH gradient gel electrophoresis) with sample application at the anode was used because it allows optimal protein analysis in the basic range of the gel including pH 8.5 to 10.5. The protocol was detailed in SERVAGel IEF 3-10 instruction manual.
(51) 3. VHH R3VQ Coupling to MRI Contrast Agents and Characterization of the MRI Properties of the Synthesized Contrast Agents
(52) VHH R3VQ was conjugated to gadolinium (MRI contrast agent) with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) (chelating agent). Two strategies based on non-site specific and site specific coupling were used: The first strategy comprises the steps of (i) conjugation with the chelating agent DOTA to lysine residues of VHH (R3VQ-NH.sub.2 1), and (ii) subsequent chelation with a MRI contrast agent, i.e. gadolinium (Gd) (see
(53) 3.1. General Synthesis Methods
(54) Unless otherwise specified, the amino-acid derivatives and the reagents are purchased from Novabiochem and Sigma-Aldrich, respectively. The concentration of the peptide and VHH solutions (net protein content) was determined by quantitative amino acid analysis (AAA) using a Beckman 6300 analyser after hydrolysis of the compounds with 6N HCl at 110 C. for 20 h. The RP-HPLC/MS analyses were performed on an Alliance 2695 system coupled to a UV detector 2487 (220 nm) and to a Q-Tofinicro spectrometer (Micromass) with an electrospray ionisation (positive mode) source (Waters). The samples were cooled to 4 C. on the autosampler. The linear gradient was performed with acetonitrile+0.025% formic acid (A)/water+0.04% TFA+0.05% formic acid (B) over 10 or 20 min. The column used was a XBridge BEH300 C18 (3.5 m, 2.1100 mm) (Waters) (gradient 10-100% A). The source temperature was maintained at 120 C. and the desolvation temperature at 400 C. The cone voltage was 40 V. The samples were injected at 0.4-1 mg/ml concentration in their respective buffer added with B. The expected Mr values correspond to the average mass of proteins with N-ter deleted Met and one disulfide bond. The Mr analyses were recorded on the same spectrometer in the positive mode by direct infusion (source temperature and desolvation temperature were maintained at 80 C. and 250 C., respectively). The samples were dissolved at 5 M concentration in water/acetonitrile (1/1) with 0.1% formic acid. The purity of 4 was analyzed by RP-HPLC using an Agilent 1200 pump system with a UV detector at 220 nm. The column used was a Kromasil C18 (100 , 5 m, 4.6250 mm) (AIT) and the gradient was performed with acetonitrile (VWR) (C)/water+0.1% TFA (VWR) (D) over 20 min.
(55) 3.2. Non-Site Specific Approach
(56) The molar equivalents of all reagents are indicated relative to reactive groups (5 NH.sub.2 per R3VQ and an average of 1 DOTA/R3VQ conjugate). The overall yields (see Table 2 below) include all the synthetic steps from the starting protein 1 in the affinity column elution buffer. They were calculated by dividing the actual amount of the final products 2a-f by their expected amount (net protein contents).
(57) The R3VQ(His)-NH.sub.2 VHH 1 eluted from the affinity column was dialyzed in PBS buffer containing 300 mM NaCl (PBS/NaCl). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) (274 g, 4 eq relative to amino groups) dissolved in PBS/NaCl (120 l) was added to 1 (480 l, 0.60 mg/ml) and the solution was stirred at room temperature. Aliquots (10 d) were withdrawn every 15 min, diluted with 100 mM Tris buffer pH 7.3 (90 l) and analyzed by HPLC/MS to monitor the reaction progress. After 3 h, the solution was cooled to 4 C. and the buffer was exchanged to 0.4M Na acetate buffer pH 5 by using Vivaspin 500 centrifugal filter device (3,000 MWCO PES) (Sartorius). The resulting DOTA-VHH conjugate (480 l) was added with GdCl.sub.3 (149 g, 45 eq relative to average DOTA groups) in the same buffer (5 l). The solution was stirred at room temperature for 2.5 h. The buffer was exchanged to PBS/NaCl at 4 C. with the same Vivaspin device as above and the solution was concentrated to afford the R3VQ(His)-N-(DOTA/Gd).sub.0-2 conjugate 2f (105 l, 1.48 mg/ml). The overall yield is 67%.
(58) The conjugate 2e was obtained using the same protocole except that DOTA-NHS was added to 1 portionwise (0.5 eq every 45 min, total of 5.5 eq relative to amino groups). The solution was stirred at room temperature for 8 h 15. The overall yield is 60%.
(59) R3VQ(His)-NH.sub.2 1
(60) AAA: Ala 15.8 (16), Arg 9.1 (9), Asp+Asn 13.4 (13), Glu+Gln 16.0 (15), Gly 13.4 (14), His 5.7 (7), Ile 3.1 (3), Leu 8.4 (8), Lys 4.1 (4), Phe 4 (4), Pro 6.1 (7), Ser 11.3 (13), Thr 10.0 (11), Tyr 4.8 (5), Val 11.1 (11).
(61) MS: 15753.0996 (C.sub.681H.sub.1053N.sub.209O.sub.216S.sub.4 calcd 15752.3949).
(62) R3VQ(His)-N-(DOTA/Gd).sub.0-2 2f
(63) AAA: Ala 16.0 (16), Arg 10.0 (9), Asp+Asn 12.8 (13), Glu+Gln 15.0 (15), Gly 13.8 (14), His 6.7 (7), Ile 3.0 (3), Leu 8.2 (8), Lys 4.5 (4), Phe 4 (4), Pro 8.5 (7), Ser 10.5 (13), Thr 10.1 (11), Tyr 4.8 (5), Val 11.1 (11).
(64) MS: 16293.1328 ((DOTA/Gd)I: C.sub.697H.sub.1076N.sub.213O.sub.223S.sub.4Gd calcd 16293.0263). 16833.5586 ((DOTA/Gd).sub.2: C.sub.713H.sub.1099N.sub.217O.sub.230S.sub.4Gd.sub.2 calcd 16833.6576).
(65) 3.3. Site Specific Approach
(66) Production of R3VQ-SH 3
(67) The coding sequence of a Cys-engineered VHH (R3VQ-SH 3) was cloned into a modified bacterial expression vector pET23 using NcoI and XhoI restriction sites. Transformed E. coli BL21 (DE3) pLysS cells express 3 in the cytoplasm after overnight induction with IPTG (0.5 mM) at 16 C. Purified VHHs were isolated by IMAC from cytoplasmic extracts using a HiTrap crude column charged with Ni.sup.2+(GE Healthcare), according to manufacturer's instructions. 3 was eluted in PBS/NaCl containing 500 mM imidazole.
(68) AAA: Ala 16.1 (16), Arg 10.2 (10), Asp+Asn 13.6 (13), Glu+Gln 11.9 (11), Gly 19.1 (20), His 6.0 (7), Ile 3.1 (3), Leu 8.5 (8), Lys 2.2 (2), Phe 4 (4), Pro 4.3 (4), Ser 15.5 (18), Thr 9.5 (10), Tyr 4.8 (5), Val 12.9 (12).
(69) MS: 15723.4268 (C.sub.671H.sub.1041N.sub.213O.sub.217S.sub.5 calcd 15724.2820).
(70) Synthesis of maleimido-(DOTA/Gd).sub.3 4
(71) The synthesis of 4 was performed stepwise on solid-phase from Fmoc-Gly-Wang resin (143 mg, 0.093 mmol). The building blocks 1,4,7,10-tetraazacyclododecane-1,4,7-tris-tbutyl-acetate-10-(N--Fmoc-N--acetamido-L-lysine) [Fmoc-Lys(DOTA(OtBu).sub.3))OH] (1.1 eq) (Macrocyclics) and 6-maleimidohexanoic acid (3 eq) were incorporated manually using 2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (1.06 and 2.9 eq, respectively)/diisopropylethylamine (DIEA) (2.2 and 6 eq, respectively) as coupling reagents and dimethylformamide (DMF) (Applied Biosystems) as solvent. Fmoc-Gly-OH (3 eq) was incorporated with DIC (3 eq) in DMF. The coupling steps with the Lys, Gly and maleimido derivatives were monitored by the Kaiser test (E. Kaiser et al (1980) Anal. Biochem. 34, 595-598) and were completed in, respectively, 3 h, 2 h and 1 h. Fmoc protection was removed with 20% piperidine in DMF. After the third lysine derivative, the last coupling with 6-maleimidohexanoic acid was carried out on three-quarters of the product (0.07 mmol). The peptide-resin was suspended in 10 ml of TFA (Applied Biosystems)/water/triisopropylsilane (95/2.5/2.5 v/v/v) at 4 C. and stirred for 4 h at RT. After filtration of the resin, the solution was concentrated and the crude product precipitated with diethyl ether. After centrifugation, the pellet was dissolved in water and lyophilized to yield 119 mg of the crude DOTA-peptide which was analyzed by NMR, MS, and RP-HPLC (gradient 10-40% C, retention time 9.2 min).
(72) .sup.1H NMR (D.sub.2O): 6.69 (s, 2H, CH Mal), 4.18 (m, 2H, 2CH), 4.08 (m, 1H, CH), 3.87-3.78 (m, 6H, CH.sub.2 Gly), 3.74-3.48 (b, 24H, CH.sub.2CO DOTA), 3.34 (t, 2H, CH.sub.2 6-Mal, J.sub.5,6=0.017 Hz), 3.30-2.99 (b, 48H, CH.sub.2CH.sub.2N DOTA), 3.06 (b, 6H, CH.sub.2), 2.14 (m, 2H, CH.sub.2 2-Mal), 1.74-1.53 (m, 6H, CH.sub.2), 1.49-1.34 (m, 10H, CH.sub.2, CH.sub.2 3-Mal, CH.sub.2 5-Mal), 1.29-1.18 (m, 6H, CH.sub.2), 1.16-1.06 (m, 2H, CH.sub.2 4-Mal).
(73) .sup.13C NMR (D.sub.2O): 177.11 (1C, CONH Mal), 175.15, 174.63, 174.36 (3C, CO Lys), 173.26 (2C, CO Mal), 172.93 (1C, COOH Gly), 171.48, 171.21 (2C, CO Gly), 163.04-162.68 (4C, CONH DOTA), 134.22 (2C, CH Mal), 120.59, 117.69, 114.79, 111.90 (TFA), 55.20-53.10 (12C, CH.sub.2CO DOTA), 54.04, 53.80, 53.47 (3C, CH), 52.20-46.80 (24C, CH.sub.2CH.sub.2N DOTA), 42.38, 41.00 (3C, CH.sub.2 Gly), 39.10 (3C, CH.sub.2), 37.37 (1C, CH.sub.2 6-Mal), 35.04 (1C, CH.sub.2 2-Mal), 30.48, 30.24, 30.23 (3C, CH.sub.2), 27.67, 27.33, 24.67 (3C, CH.sub.2 3-Mal, CH.sub.2 5-Mal, CH.sub.2), 25.43 (1C, CH.sub.2 4-Mal), 22.43, 22.33, 22.15 (3C, CH.sub.2).
(74) MS: [M+H].sup.+ 1925.9888, [M+K].sup.+ 1963.9391 (C.sub.82H.sub.136N.sub.22O.sub.31 calcd [M+H].sup.+ 1927.1195, [M+K].sup.+ 1965.2098).
(75) The DOTA-peptide intermediate (99 mg) was dissolved in 0.4M Na acetate buffer pH 5 (41 ml) and added with Gd(OAc).sub.3.xH.sub.2O (123 mg, 2 eq relative to DOTA). After stirring at 95 C. for 25 min, the solution was cooled and loaded on a C18 reverse-phase column (2 g, diameter 1.5 cm). The column was washed with four volumes of water and the product was eluted with three volumes of water/acetonitrile 1/1 affording 79 mg of product after lyophilisation. The crude DOTA/Gd peptide was purified by reverse-phase flash chromatography (30200 mm) using a gradient with acetonitrile+0.1% TFA/buffer D over 40 min, from 5/95 to 35/65 (20 ml/min, retention time 18 min). After lyophilization of the main fraction, 61 mg of 4 were obtained with an overall yield of 44% (the overall yield includes all the synthetic steps, it was calculated on the net peptide content of the isolated product 4 based on the first Gly residue loading on the resin). 4 was analyzed by MS and RP-HPLC (gradient 5-35% C, retention time 12.2 min, purity >90%).
(76) MS: 2388.8889 (Cs.sub.2H.sub.127N.sub.22O.sub.31Gd.sub.3 calcd 2388.7901).
(77) Synthesis of R3VQ-S-(DOTA/Gd).sub.3 5
(78) The R3VQ-SH VHH 3 eluted from the affinity column was dialyzed in PBS buffer containing 300 mM NaCl (PBS/NaCl). 4 (1.35 mg, 3 eq relative to 1 thiol group per VHH) in aqueous solution (135 l) was added to 3 (1.5 ml, 2 mg/ml in PBS/NaCl pH 6.8) and the solution was stirred at 4 C. for 3 h. The solution was then diafiltered using Vivaspin 2000 centrifugal filter device (3,000 MWCO PES) (Sartorius). Aliquots (20 l) of 3 and 5 were diluted with buffer B (20 l) for RP-HPLC/MS analyses. Moreover, aliquots (10 l) of 3 and 5 were diluted in 100 mM Tris buffer pH 7.3 (90 l) for ELISA analyses. 1 ml of 5 (2.36 mg/ml) was obtained with a yield of 70%. It was calculated by dividing the actual amount of the final product 5 by its expected amount (net protein contents).
(79) The same reaction was also performed directly in the affinity column elution buffer (PBS/NaCl containing 500 mM imidazole), and gave a 83% overall yield. The process for obtaining the R3VQ/Gd conjugates is thus improved when the conjugation is directly performed in the affinity column elution buffer (PBS/NaCl/Imidazole)): i) the number of steps is decreased to two, and ii) the overall yield is increased until 83%. This process improvement has also been validated with another VHH (data not shown).
(80) AAA: Ala 14.9 (16), Arg 10.2 (10), Asp+Asn 12.2 (13), Glu+Gln 11.1 (11), Gly 24.6 (23), His* (7), Ile 3.1 (3), Leu 8.5 (8), Lys 11.7* (5), Phe 4 (4), Pro 4.8 (4), Ser 14.9 (18), Thr 9.1 (10), Tyr 5.0 (5), Val 12.6 (12). [*His cannot be determined due to co-elution with ammonium. Lys is overestimated due to co-elution with maleimido derivative in the conditions of the analysis.]
(81) MS: 18113.7383 (C.sub.753H.sub.1168N.sub.235O.sub.248S.sub.5Gd.sub.3 calcd 18113.0720).
(82) SDS-PAGE Electrophoresis
(83) Polyacrylamide Gel electrophoresis (PAGE) was performed using NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) according to manufacturer's instructions.
(84) Determination of pI
(85) The pI of VHHs was determined by isoelectric focusing using IEF 2-9 Gel (Invitrogen). NEPGHE (non equilibrium pH gradient gel electrophoresis) with sample application at the anode was used because it allows optimal protein analysis in the basic range of the gel including pH 8.5 to 10.5. The protocol was detailed in SERVAGel IEF 3-10 instruction manual.
(86) ELISA
(87) Streptavidin-coated microtiter plates (Thermo Scientific, Denmark) were coated by incubation overnight at 4 C. with 1 g/ml of biotinylated A-beta 40 or A-beta 42 (preferably A-beta 40) diluted in PBS. Plates were washed with buffer 0.1% Tween 20 in PBS. For the non-site specific strategy, R3VQ-NH.sub.2 1, R3VQ-N-(DOTA/Gd).sub.1-2 2e were diluted in buffer 0.5% gelatin 0.1% Tween 20 in PBS. After 2 h incubation at 37 C., plates were washed again before adding respectively a rabbit anti-His tag polyclonal antibody (eBiosciences), followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abcam), and finally revealed by OPD (o-phenylendiamine dihydrochloride, Dako) according to manufacturer's protocol. For the site specific strategy, R3VQ-SH 3 and R3VQ-S-(DOTA/Gd).sub.3 5 were diluted in the same buffer as described before, after incubation with biotinylated A-beta 40 or A-beta 42 (preferably A-beta 40), a monoclonal anti-His tag antibody (H1029-Sigma) was added, followed by peroxidase labeled goat anti-mouse antibody (ab97265-Abcam), and revealed by the same substrate.
(88) Affinity Determination
(89) The binding properties of 3 and 5 were determined by measuring the amount of soluble A-beta 40 or A-beta 42 (preferably A-beta 40) peptide able to give 50% inhibition of immobilized A-beta 40 or A-beta 42 (preferably A-beta 40) recognition. Briefly, various concentrations of A-beta 40 or A-beta 42 (preferably A-beta 40) were incubated overnight at 4 C. with a defined quantity of 3 or 5 until equilibrium was reached. The VHH concentration used has been deduced from preliminary ELISA calibrations. Each mixture (100 l) was transferred to a well of microtiter plate previously coated with antigen and was incubated for 15 min at 4 C. After washing with PBS containing 0.1% Tween 20, unbound VHH were detected by the addition of an anti-His mAb (H1029-Sigma) followed by p-galactosidase goat anti-mouse Igs and 4-methylumbelliferyl p-D-galactoside. Fluorescence was read (Fluoroskan, Labsystem, Finland) at 460 nm, after excitation at 355 nm.
(90) Immunohistochemistry
(91) Immunohistochemistry was performed on paraffin coronal brain sections (5 m in thickness), obtained from transgenic mouse models of amyloidosis (PS2APP mice). The sections were made with a microtome (Microm HM340E). Sections were de-paraffinized in xylene (5 min, 3 times), rehydrated through ethanol (100%2, 90 and 70%; 5 min/step) and brought to water. They were then pretreated with 98% formic acid for 5 min and finally immersed in water for 5 min. Endogenous peroxidases were neutralized with 3% hydrogen peroxide and 20% methanol, and nonspecific binding sites were blocked with TBS-0.5% tween pH8 BSA 2% for 30 min. In the following steps, the sections were rinsed 3 times, 5 min/time in TBS-tween between steps. The sections were then incubated overnight at 4 C. with the primary antibody R3VQ-SH, diluted to 2 g/ml in TBS-tween. Sections were then treated with mouse monoclonal anti-His-tag antibodies (H1029-Sigma) for 2 h at room temperature, and finally developed with Dako REAL system Peroxidase/DAB Kit (Glostrup, Denmark) according to manufacturer's protocol. After washing with water, sections were counter-stained with Harris hematoxylin and re-rinsed in water. Before being mounted, sections were dehydrated in graded ethanol solution (70, 90 and 100%) and cleared in xylene.
(92) 3.4. MRI Properties of the Synthesized Contrast Agents
(93) MRI properties of the contrast agents were assessed on a 7 T-Spectrometer (Agilent, USA) interfaced with a console running VnmrJ 2.3. The spectrometer was equipped with a rodent gradient insert of 700 mT/m. A quadrature birdcage coil (diameter: 23 mm) was used for emission and reception. The longitudinal and transverse relaxivities r1 and r2 (change in the relaxation rate per unit concentration of an agent, meaning effectiveness as a MRI contrast agent) were determined from linear fits of R1 (i.e. 1/T1) and R2 (i.e. 1/T2) as a function of contrast agent concentration for concentrations of 0.2, 0.15, 0.1, 0.05, 0.025, 0.01, and 0 mmol/l by using the following equations: R1(C)=R1(0)+r1C where R1(C) is the R1 in the tubes containing the contrast agent at a concentration C, R1(0) is the R1 in the tube without the contrast agent, and C is the concentration of the contrast agent. R2(C)=R2(0)+r2C was used to calculate r2. The samples were imaged in hematocrit tubes.
(94) T1 calculation was based on seven successive 2D multi-slice spin echo images with twenty TR values (TR=0.021, 0.04, 0.06, 0.08, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5 sec), TE=14 ms, Nex=4, FOV=1010 mm2, Mtx=6464, 1 slices, slice thickness=3 mm, bandwidth=50 kHz). Parametric maps of relaxation times were calculated from exponential regression curves (S=1exp(TR/T1)) where S is the signal intensity, TR is the repetition time and T1 is the longitudinal relaxation time (ImageJ, MRI Analysis Calculator, Karl Schmidt).
(95) T2 calculation were based on 2D multi-echo multi-slice spin echo images by using 16 Echo times (TE=10 to 160 msec); TR=3300 ms; Nex=2; bandwidth=100 kHz, FOV=1010 mm2, Mtx=6464, 1 slices, slice thickness=3 mm. Parametric maps of relaxation times were calculated from exponential regression curves (S=exp(TE/T2)) where S is the signal intensity, TE is the echo time and T2 is the longitudinal relaxation time (ImageJ, MRI Analysis Calculator, Karl Schmidt).
(96) 4. In Vitro Characterization of VHH R3VQ, and VHH R3VQ Conjugates by Immunohistochemistry, Biochemistry and In-Vitro MRI
(97) Subjects
(98) Human cortical brain tissues from AD patients (Braak stage V and VI) were obtained from the NeuroCEB brain bank. This bank is associated to a brain donation program run by a consortium of patients associations (including France Alzheimer Association) and declared to the Ministry of Research and Universities, as requested by French Law. An explicit written consent was obtained for the brain donation in accordance with the French Bioethical Laws.
(99) Preclinical experiments were performed on B6TgPS2APP (Richards J. G., 2003, J Neurosci., 23:8989-9003) and APP/PS1dE9 (Garcia-Alloza M., 2006, Neurobiol Dis., 24:516-24) transgenic mice. Animal experimental procedures were performed in strict accordance with the recommendations of the EEC (86/609/EEC) and the French national committee (decree 87/848) for the care and use of laboratory animals. The animals were sacrificed using a high dose of sodium pentobarbital (100 mg/kg) and then perfusion-fixed with 10% buffered formalin. Their brains were then removed, immersed in formalin for at least 24 hours and stored at 4 C.
(100) Tissue Extracts
(101) Tissue extraction was performed according to Gong, Y. et al. (2003, Proc Natl Acad Sci USA, 100:10417-10422). Frontal cortex from AD brain (0.2 g) was homogenized in 20 volumes of phenol red-free Ham's F12 medium (Gibco) or buffer A (PBS, pH 7.4, 0.32 M sucrose, 50 mM Hepes, 25 mM MgCl2, 0.5 mM DTT) containing protease inhibitors (200 g/ml PMSF, 2 g/ml pepstatin A, 4 g/ml leupeptin, 30 g/ml benzamidine hydrochloride), and was centrifuged at 100,000g for 1 h. The pellet was re-homogenized in 10 volumes of phenol red-free Ham's F12 medium or buffer A plus protease inhibitors and was re-centrifuged. The protein concentration of the combined supernatants was determined. An aliquot of protein was then concentrated to a volume of 60 l or less, by using a Centricon-10 concentrator.
(102) Immunoblots
(103) Brain extracts or A-beta 42 peptide were resuspended in NuPAGE LDS sample buffer (Invitrogen) containing 8M urea. Following separation by polyacrylamide gel electrophoresis (PAGE) using NuPAGE Novex 4-12% Bis-tris gel (Invitrogen), semi-dry transfer onto Hybond-C (Amersham) and western blotting were carried out using the Xcell II blot module (Invitrogen). Prior to the immunochemical reaction, membranes were blocked in a 4% skimmed milk solution. Immunoblotting of membranes was accomplished with VHH and revealed by rabbit anti-His tag (eBioscience) polyclonal antibodies followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abcam). Finally, peroxidase activity was visualized using a chemiluminescent kit (GE Healthcare).
(104) Immunohistochemistry
(105) Immunohistochemistry was performed on fixed tissues (paraffin-embedded or frozen sections), or alternatively on unfixed fresh tissues (frozen sections). Standard IHC protocols were applied and adapted for each tissue conditions. As most of immunostaining experiments were performed using paraffin sections, a detailed protocol for paraffin-embedded material is presented here. Immunostaining of brain tissue was performed on 4 m thick paraffin sections. Both human and mouse tissues were used (Human AD patient, TauPS2APP mice [Grueninger F. et al., 2010, Neurobiol Dis., 37:294-306] and PS2APP transgenic mice [Richards, J. G., et al 2003, The Journal of neuroscience 23, 8989-9003]). Sections were de-paraffinized in xylene, rehydrated through ethanol (100%, 90%, and 70%), 5 min for each solution and finally brought to running tap water for 10 min. They were then incubated in 98% formic acid for 5 min, washed again under running tap water, quenched for endogenous peroxidase with 3% hydrogen peroxide and 20% methanol, and finally washed in water. Non-specific binding was blocked by incubating the sections for 30 min in 2% bovine serum albumin in TBS+0.5% Tween. Appropriate dilutions of primary antibodies (5-10 g/ml of VHH His or Strep tag) were then applied and slices incubated overnight in a humidified chamber at room temperature. Slides were washed with TBS-Tween and incubated with secondary antibodies rabbit anti-His Tag for 1/1000 or home-made biotinylated anti-strep mAb C23-21 in TBS-Tween at room temperature for 1 h. Slides were then incubated with reagents of Dako REAL Detection System, Peroxidase/DAB+ according to manufacturer's instructions. Chromogenic (DAB) revelation was developed until a good signal-to-noise ratio was obtained (about 5 min). After washing with TBS-Tween, slides were counter-stained with hematoxylin. For labeling of plaques, biotinylated 4G8 (Wisniewski T et al., 1996, B. Biochem J., 313:575-80) mAb (1/10000) or 6F/3D (Akiyama H. et al., 1996, Neurosci let., 206:169-72) mAb (1/200) was used as a positive control in parallel.
(106) In Vitro MRI
(107) MRI were performed on the brains of B6TgPS2APP (Richards J. G., 2003, J Neurosci., 23:8989-9003) and APP/PS1dE9 (Garcia-Alloza M., 2006, Neurobiol Dis., 24:516-24) transgenic mice (n=2, females). MRI were recorded on a 7 T-Spectrometer (Agilent, USA) interfaced with a console running VnmrJ 2.3. The spectrometer was equipped with a rodent gradient insert of 700mT/m. A birdcage coil (RapidBiomed, GmbH, Germany) and a mouse brain surface coil (RapidBiomed GmbH, Germany) were used for emission and reception, respectively.
(108) After 4 h of membranes permeabilization in a solution of Triton 0.2% in PBS, the brain samples were soaked in a solution of phosphate buffered saline (PBS) and tested contrast agent and stored at 4 C. for at least 24 hours prior to imaging. For scanning, the brains were placed in a tight plastic tube filled with Fluorinert (3M, Cergy-Pontoise, France), an aprotonic perfluorocarbon-based fluid that provides a black background in MR images.
(109) MR images were based on a 3D gradient-echo sequence was used (FLASH) to acquire T2*w images (TR=40 ms, TE=15 ms, FA=200, Bw=50 kHz, Nex=16, matrix=512512128, FOV=131313 mm.sup.2, yielding a resolution of 2525100 m.sup.3 for a total Tacq of 11 h 39 min).
(110) Gd-Staining Method: A Gold Standard Method for Amyloid Plaques Detection
(111) This procedure was used to make colocalization between hypointense spots seen on MR images after the in vitro procedure, and a gold standard method revealing amyloid plaques (Petiet A. et al., 2012, Neurobiol Aging, 33:1533-44). Briefly, following in vitro experiments, the samples were soaked in a solution of PBS and 0.5 M gadoterate meglumine at a dilution of 1:200 (2.5 mM) and stored at 4 C. for at least 24 hours prior to imaging. MR images were realized in the same conditions as used for in vitro experiments.
(112) 5. In-Vivo Evaluation of VHH R3VQ, and VHH R3VQ Conjugates
(113) Subjects
(114) In vivo evaluation of VHH R3VQ, and VHH R3VQ conjugates was performed on TauPS2APP (Grueninger F. et al., 2010, Neurobiol Dis., 37:294-306) transgenic mice under the authorization previously described.
(115) In Vivo Stereotaxic Injection of VHH
(116) Stereotaxic injections were performed in TauPS2APP (n=2 female) transgenic anesthetized mice with 2 l of VHH per injection at the rate of 0.5 l/min. The mice were anesthetized with a mixture of isoflurane (1-2%) and air (1 L/min). They were placed on a stereotaxic frame and the skull was bilaterally perforated with a Dremel. Blunt Hamilton syringes were used to inject MR contrast agent. Each mouse received 4 injections, in the frontal cortex and the hippocampus in each hemisphere. The stereotaxic coordinates in the frontal cortex were +0.86 mm anterior from bregma, 1.5 mm lateral from the midline, 0.65 mm ventral from dura. The stereotaxic coordonates in the hippocampus were 2.18 mm posterior from bregma, 1.5 mm lateral from the midline, 1.8 mm ventral from dura. Two or 24 hours after the injection, mice were euthanized and perfused intracardially with 4% paraformaldehyde in PBS (pH 7.6). Brains were removed and postfixed in the same fixative overnight at 4 C. 4 m thick paraffin sections were prepared. The presence of the VHH in cerebral tissue was detected using either of the standard immunohistochemical procedures described above.
(117) Intracarotid Perfusion of VHH in Mouse Models of Amyloidosis
(118) Intracarotid administration of VHH was performed in anesthetized TauPS2APP mice (n=2, females). The anesthesia was performed by injecting once intraperitoneally a mixture of ketamine hydrochloride (Imalgen, 50 l of a solution diluted 1/10) and xylazine (Rompun, 50 l of a solution diluted 1/40). The common carotid artery was exposed and cannulated with fine silicon tubing (PP25_100FT; Portex, Ashford, UK). VHH was infused into the carotid at a constant rate using a peristaltic pump (Model PHD 2000; Harvard Apparatus, Boston, Mass., USA).
(119) Lateral Tail Vein Injection
(120) Mice were placed in an appropriate inverted beaker with hole for tail access. The animals were warmed before the injection and the tail was soaked with lukewarm water to cause vasodilatation of the vein. Injections before the MR images were performed after insertion of a catheter (27 G, Microflex, Vygon, France) into the tail vein of the animals, in order to warrant the proper administration of the contrast agent. Two mg of VHH, VHH R3VQ-DOTA, or VHH R3VQ-S-(DOTA/Gd).sub.3 dissolved in 200 Ail PBS was then injected into the lateral tail vein.
(121) In Vivo MRI
(122) In vivo MRI was performed on a 7 T-Spectrometer (Agilent, USA) interfaced with a console running VnmrJ 2.3 as previously described (see in vitro MRI chapter). During the MRI experiment the animals were anesthetized with a mixture of isoflurane (0.75-1.5%) and carbogen (95% O2-5% CO2) and their breathing rate was monitored. Carbogen was used to reduce the signal coming from circulating blood (Thomas et al., 2003).
(123) MR images were recorded using a high-resolution 3D-Gradient Echo sequence (29*29*117 m.sup.3, FOV: 15*15*15 mm.sup.3, Mtx=512*512*128, TR=30 ms, TE=15 ms, flip angle=20, Nex=1, bandwidth=25 kHz, Acquisition Time: 32 min (Petiet, A. et al., 2012, Neurobiol Aging, 33:1533-44).
(124) The T1 calculation was based on seven successive 2D multi-slice spin echo images with five TR values (TR=0.4, 0.75, 1.5, 2.5, and 5 s, TE=14 ms, Nex=1, FOV=2525 mm2, Mtx=128128, 6 slices, slice thickness=1 mm, bandwidth=50 kHz). Parametric maps of relaxation times were calculated from exponential regression curves (S=1exp(TR/T1)) where S is the signal intensity, TR is the repetition time and T1 is the longitudinal relaxation time (ImageJ, MRI Analysis Calculator, Karl Schmidt). Relaxation times were measured from cortical regions in the frontal part of the brain.
(125) Ex Vivo MRI
(126) 6 h after in vivo intracerebroventricular injections of VHH-S-(DOTA/Gd).sub.3 (1 g/side), mice were perfused (PFA 4%) and their brains were extracted prior to ex vivo MR images. For each procedure, controls were performed by using an equivalent solution of Gd (i.e: 0.1 mM).
(127) Results
(128) 1. Library Construction, and Selection of Specific Anti-A VHH
(129) VHHs were amplified by PCR and cloned in vector pHEN1. Subsequent transformations yielded a library of about 10.sup.8 clones. VHHs displaying the best affinity were selected by phage display through 3 panning cycles with biotinylated A1-42, A1-40 or A1-16 peptides. 46 individual clones were tested by ELISA from A42 panning, 192 clones from A40 and 192 clones from A16. 46/46, 110/192 and 163/192 were found positive from A42, A40 and A16 pannings, respectively. These positive clones were sequenced and 45, 65 and 118 VHH sequences were respectively identified. Finally 3 families of VHH were selected (A7/B10, F12 and R3VQ) (see Table 1 below). A7/B10 VHHs were found 11, 64 and 117 times after pannings against, respectively, biotinylated A1-42, A1-40 or A1-16. VHH R3VE/Q were found respectively 34 times and once after panning against biotinylated A1-42 and A1-40 while VHH F12 were found once after A16 panning.
(130) These VHHs were subcloned in vector pET23 or in vector pASK IBA2 to allow a high level of expression of VHH with, respectively, a His-tag or a Streptavidin-tag. Yields of 1-2 mg/1l of bacterial culture were obtained. The single domain products were shown to be pure to homogeneity by SDS-PAGE (data not shown); their pI values were above 8.5. Subsequent experiments were performed with the two constructs.
(131) R3VQ is kept at 4 C. or for long term storage at 20 C. with glycerol. It is not stable frozen without glycerol.
(132) DLS experiments showed that R3VQ is monomeric and not aggregated after purification.
(133) Amino-acid sequence alignment of anti-A VHHs A7, B10, R3VE, R3VQ and F12 is shown in
(134) 2. Recognition of Amyloid Plagues by VHH R3VQ
(135) Immunoreactivity of VHH R3VQ for A and Amyloid Lesions
(136) It was examined the distribution of VHH-specific immunoreactivity in human AD brains and transgenic TauPS2APP mice. R3VQ showed good ability to immunodetect A plaques and cerebral amyloid angiopathy (CAA) in human paraffin sections after antigen retrieval pretreatment (
(137) To confirm the immunoreactivity of VHH R3VQ on brain tissues, western-blot immunoassays were performed on brain extracts obtained from AD patients. Four principal bands, corresponding to A oligomers between 40 and 55 kDa, were immunodetected with VHH R3VQ. In parallel R3VQ recognized three bands with A42 peptide between 6 and 17 kDa corresponding to monomers, dimers and trimers (
(138) R3V Recognizes the Central Region of A142
(139) VHH R3VQ was shown to be specific for fibrillar synthetic A42 peptide in inhibition assays using A42. VHH R3VQ had a KD of 17 nM. To further determine the epitope recognized by VHH R3VQ, the concentration required for 50% inhibition (IC50) was determined for A40 and for peptides corresponding to different A fragments (1-16, 10-20, 15-25, 22-35 and 29-40). VHH R3VQ did neither recognize fragments 1-16 nor 29-40. The IC50 of VHH R3VQ for A 16-35 was 16 nM, suggesting that VHH R3VQ recognizes an epitope located in the central part of A42. R3VQ did not recognize APP by flow cytometry using the H4 stable clone provided by Roche (data not shown).
(140) VHH R3VQ Labels Amyloid Plaques In Vivo after Stereotaxic Injection
(141) Two g of VHH R3VQ were injected stereotaxically into the hippocampus or the cortex of the left hemisphere of the mouse brain (2 mice). 2 h or 24 h after the injection, the animals were sacrificed and brain sections were collected. Immunostaining of amyloid plaques was observed indicating that VHH R3VQ labeled fibrillar A in vivo. Moreover, it was noticed a brown halo in the cortex indicating the diffusion of VHH R3VQ into brain tissues (
(142) VHH R3VQ Crosses the BBB In Vivo
(143) VHH R3VQ was tested in vivo for its ability to cross the BBB. Four mgs of VHH was injected via the left carotid artery over a period of 60 min. Following the injection, the diffusion of VHH into cerebral tissues was allowed for 1 hour before the mice were euthanized and perfused with fixative. Immunostaining of amyloid plaques was observed in cortex, hippocampus and thalamus. This staining was faint in the right hemisphere contralateral to the injected carotid.
(144) The experiments showed that unlabeled R3VQ 1) was able to cross BBB following slow intra-carotid infusion, 2) specifically recognized amyloid plaques in vivo, and 3) is a good candidate for MRI studies.
(145) Comparison Between VHH R3VQ and Known VHHs Directed Against Amyloid
(146) Table 1 below summarizes the labeling of VHHs directed against amyloid obtained by immunization of an alpaca with the peptide A42, the peptide A1-10 coupled to the ovalbumin or fibrillar form of A42. The selections of the VHHs have been carried out by phage display, except for VHHs R1.3, R1.5 and R3.3 which have been selected by ribosome display.
(147) TABLE-US-00001 TABLE 1 Labeling of VHHs directed against amyloid obtained by immunization of an alpaca with the peptide A42, the peptide A1-10 coupled to the ovalbumin or fibrillar form of A42. VHHs L1-3, L35, 61-3, V31-1 are disclosed in Lafaye P. et al., 2009, Molecular Immunology, 49: 695-704. The other VHHs were obtained according to the method disclosed above. Labeling Amyloid VHHs Immu- angiopathy Amyloid Amyloid clones nogens Selection (CAA) oligomers plaques A42 A42 oligomer coated tubes L1-3 0 0 0 L35 0 0 0 61-3 0 0 0 V31-1 0 ++ 0 A1-10 coated tubes L3 0 0 0 L4 0 0 0 L7 0 0 0 V2 0 0 0 V17 0 0 V11 0 0 0 OVA A1-16 A1-10 coated tubes NN1 ++ 0 0 NN3 ++ 0 + NN4 Fibrillar A42 A42 coated tube 2D5 0 0 0 3F7 0 0 0 2A5 0 0 0 3B4 0 0 0 3B10 0 0 0 3H7 0 0 0 Fibrillar Ribosome A42 display + A42 coated tubes R1.3 ++ 0 0 R1.5 0 0 0 R2.3 ++ 0 0 R3.3 ++ 0 + Fibrillar Biotinylated A42 A40 or biotinylated A16 biotinyl + magnetic beads B10 0 0 0 A7 0 0 0 F12 0 0 0 Fibrillar Biotinylated A42 A42 + magnetic beads R3VE ++ 0 +++ R3VQ Control AcM +++ +++ 6F/3D
(148) The results show that among the 27 VHHs tested only VHH R3VE/Q is able to label amyloid angiopathy and amyloid plaques but not amyloid oligomers.
(149) 3. Antibody Coupling to MRI Contrast Agent
(150) Non-Site Specific Approach:
(151) Conjugation was performed with NHS-activated DOTA to VHH lysine residues. Subsequent chelation with Gd has resulted in conjugates with variable ratios of DOTA/Gd on the protein (Table 2). Monitoring of both steps by HPLC/MS has allowed to optimize the process. Nearly complete chelation could be achieved at room temperature.
(152) Two initial conjugates (2a and b) have showed no A binding activity due to instability during storage at 20 C. without glycerol. By varying experimental conditions, four new conjugates have been prepared in 0.5-1 mg scale with a good recovery (64-74%). The first series (2c and d) has a higher DOTA/Gd density than the second series (2e and f). Compared to the original VHH 1 A recognition in ELISA, the binding of 2c and 2d is low (10%) while the binding of 2e and 2f is hardly affected (50 to 100%). This difference might be due to the overall lower density of DOTA/Gd and/or inherent stability of the VHH.
(153) TABLE-US-00002 TABLE 2 Characteristics of R3VQ(His)-N-(DOTA/Gd) conjugates Average Average Overall ELISA vs Experimental conditions DOTA/ Gd/ yield original Compound Initial storage Conjugation.sup.a Chelation.sup.a protein.sup.b protein.sup.b (%) VHH 2a 20 C. 12 2 eq, 12 h 20 C., (3) 4 5 (6) (3) 4 (5) 67 2 h 30 2b 20 C. 12 2 eq, 12 h 60 C., (3) 4 (5) (3) 4 (5) 65 15 min 2c 20 C. + 12 2 eq, 12 h 20 C., (1) 2 (3) (1) 2 (3) nd + glycerol 2 h 30 2d 20 C. + 12 2 eq, 12 h 60 C., (1) 2 (3) (1) 2 (3) nd + glycerol 20 min 2e +4 C. 11 0.5 eq, 20 C., (0) 1 2 (3) (0) 1 2 (3) 60 ++ 8 h 15 2 h 30 2f +4 C. 4 eq, 3 h 20 C., (0) 1 (2) (0) 1 (2) 67 ++ 2 h 30 .sup.aThe reactions are performed at room temperature. .sup.bDetermined by MS. The minor compounds are in brackets.
(154) Site Specific Approach:
(155) This strategy involves the labeling of the Cys-engineered R3VQ VHH (R3VQ-SH 3) (SEQ ID NO 8) with a maleimido-(DOTA/Gd).sub.3 compound 4 (see
(156) DOTA is represented as the monoacyl moiety. The overall yield is indicated in brackets.
(157) When conjugated to 4 by thioaddition, 3 was totally converted into the well-defined compound R3VQ-S-(DOTA/Gd).sub.3 5, as shown by RP-HPLC/MS, with 79% yield. The pI of 5 was slightly reduced compared to the one of the unlabeled R3VQ-SH. The binding characteristics of R3VQ-SH and R3VQ-S-(DOTA/Gd).sub.3 were determined in competitive inhibition experiments involving A40 bound to the ELISA plate and soluble A40. The concentration of A40 giving 50% binding inhibition was calculated to be 1 g/ml for both R3VQ-SH and R3VQ-S-(DOTA/Gd).sub.3 suggesting that the addition of DOTA/Gd does not affect the VHH binding properties. Further, following the distribution of VHH-specific immunoreactivity in transgenic B6.PS2APP mice, R3VQ-SH showed good ability to immunodetect A plaques in mouse paraffin sections after antigen retrieval pretreatment.
(158) R3VQ-SH was constructed with a C-terminal Cys residue for coupling to Maleimido-DOTA/Gd (
(159) 4. Detection of Amyloid Plaques with VHH R3VQ Conjugated with Gd Contrast Agent
(160) R3VQ-N-(DOTA/Gd).sub.2 Labels Amyloid Plaques In Vivo after Stereotaxic Injection
(161) Two g of VHH R3VQ-N-(DOTA/Gd).sub.1-2 (2e) were injected stereotaxically into the hippocampus or the cortex of the left hemisphere of the mouse brain (2 mice). 4 h after the injection, the animals were sacrificed and brain sections were collected. Immunostaining of amyloid plaques was observed indicating that R3VQ-N-(DOTA/Gd).sub.n-labeled fibrillar A in vivo (
(162) In-Vitro Imaging
(163) Imaging of brains of TauPS2APP mice soaked in a solution of R3VQ-N-(DOTA/Gd).sub.1-2 (2e contrast agent at a final concentration of 0.02 mg/ml, equivalent to a 0.01 mM of Gd) revealed several hypointense spots (n=2;
(164) Ex-Vivo Imaging after Intracerebral, Intracarotid or IV Injection
(165) After intracerebroventricular injection, the anti-A VHH-Gd 2e (R3VQ-N-(DOTA/Gd).sub.1-2) showed hypointense spots on ex vivo images in the hippocampus (
(166) Evaluation of R3 V-S-(DOTA/Gd).sub.3 by In Vitro MRI
(167) R3VQ-S-(DOTA/Gd).sub.3 was synthesized by site specific approach as described above. The HPLC/MS, pI, and IHC assays confirm the biochemical properties (i.e. purity by HPLC/MS, pI, and IHC reactivity against AP) of R3VQ-S-(DOTA/Gd).sub.3 (see
(168) The potential of R3VQ-S-(DOTA/Gd).sub.3 to induce MR contrast modification was evaluated after in vitro incubation of brains from PS2APP mice (Richards, J. G., et al., 2003, The Journal of neuroscience: the official journal of the Society for Neuroscience 23, 8989-9003) (n=2) with R3VQ-S-(DOTA/Gd).sub.3 at 0.1 mg/ml as described above. Images acquired at 7 T revealed hypointense spots in the cortex as compared to brains of PS2APP mice under negative control condition (
(169) Evaluation of R3VQ-S-(DOTA/Gd).sub.3 by Ex-Vivo MRI after Peripheral (Intravenous) Injections
(170) R3VQ-S-(DOTA/Gd).sub.3 ability to reveal amyloid plaques by MRI was then investigated after intravenous injection in the tail vein (20 mg/kg and 50 mg/kg) of 18-month-old PS2APP mice as described above. The MR images acquired at 11.7 T of the PS2APP mice injected intravenously with R3VQ-S-(DOTA/Gd).sub.3 were acquired after brain extraction 5 hours following injection. Contrary to MR images of the control condition (PS2APP mice injected with PBS) (
(171) These hypointense spots were co-registered with contrast anomalies corresponding to amyloid plaques as detected with a gold-standard Gd-staining procedure (
EXAMPLE 2: GENERATION OF ANTI-ABETA VHHS COUPLED TO A FLUOROPHORE AGENT AND ITS EVALUATION IN VITRO/IN VIVO
(172) 1. Materials and Methods
(173) Unless explicitly mentioned hereafter the materials and methods are the same as described for Example 1.
(174) 2. In Vivo Targeting of all-Positive Lesions
(175) Conjugation of R3VQ-SH with AF488 Fluorophore
(176) In order to realize the coupling between R3VQ VHH and a fluorophore, a site specific conjugation was implemented as described above. Briefly, an additional cysteinee residue was inserted in the C terminal part of the sequence of R3VQ (referred to as R3VQ-SH), thus allowing a C terminal thio-addition of a maleimido-Alexa Fluor 488 (AF488). By this way, it was obtained a well defined conjugate, referred to as R3VQ-S-AF488, with a single AF488 on the VHH (
(177) SDS-PAGE and HPLC/MS analyses showed the expected molecular weight (increase of 698 Da) corresponding to the addition of AF488 to the molecule. These data confirm the labeling and the purity of the conjugate (
(178) The isoelectric point (pI) of R3VQ-SH and R3VQ-S-AF488 were analyzed by NEPHGE using IEF 3-10 gel. The pI of R3VQ-SH was between 8.5 and 9.5 (
(179) Diffusion of R3VQ-S-AF488 after Topic Brain Infusion (Two-Photon Imaging)
(180) Craniotomy was performed on a PS2APP mouse to obtain a skull window over the right posterior cortex. 15 g (10 l) of R3VQ-S-AF488 was applied directly onto the exposed brain after peeling off the dura. R3VQ-S-AF488 diffusion was followed up for about 2 hours by two-photon microscopy. Typical specific staining of amyloid plaques and CAA was detected in brain parenchyma, suggesting that R3VQ-S-AF488 was able to diffuse and detect in vivo extracellular and vascular AB-positive lesions after pericortical infusion (
(181) Diffusion of R3VQ-S-AF488 after Intravenous Injection (Two-Photon Imaging)
(182) The integrity of the blood-brain barrier (BBB) of the tested mice was first checked by MRI (see control experiments below) to ensure absence of leakages that may artificially favor brain penetration of the intravenous (iv) injected VHHs.
(183) A 50-mg/kg dose of R3VQ-S-AF488 was injected in the tail vein of one mouse. The conjugate extravasation and staining in the brain was recorded for 3.5 hours post injection using two-photon microscopy on brain window (z=from the surface up to 360 m deep).
(184) Basic pI of VHH is a Key Factor for its Ability to Transmigrate Across the BBB
(185) Maleimido-AF488 conjugated R3VE was also prepared, whose pI was around 7.5 (
(186) Control Experiments
(187) Evaluation in Amyloid-Free Mouse
(188) R3VQ-S-AF488 was intravenously injected in a wild type, amyloid-free, C57BL/6 mouse. No specific in vivo staining in the brain parenchyma was observed using two-photon microscopy assay (data not shown).
(189) Comparison with Conventional IgG Antibody
(190) Injection of mAb 4G8-AF488 iv in a PS2APP mouse only allowed to detect CAA by two-photon imaging but not amyloid plaques indicating no significant extravasation of this standard anti-A immunoglobulin (
(191) Assessment of Blood-Brain Barrier Integrity in Mice Used for Two-Photon Imaging
(192) BBB permeability of the PS2APP mice (2-year-old) used for two-photon experiments was tested using DOTAREM iv injection (0.2 ml Gd 500 mM). This MRI contrast agent is unable to cross the BBB with the exception of pathological conditions leading to local leakages of the barrier. This MRI exam, used also in human, shows an increase of signal in areas where the BBB is disrupted (V. M. Runge et al., American Journal of Roentgenology 1994, 162, 431-435; M. A. Ibrahim et al., Investigative radiology, 1998, 33, 153-162). The absence of signal modification in the tested mice suggested the integrity of their BBB. Two other age-matched PS2APP mice were also MRI-assessed using the same method and no disruption of the BBB was observed (data not shown).