Radio-pharmaceutical complexes
09827336 · 2017-11-28
Assignee
Inventors
Cpc classification
A61K51/0455
HUMAN NECESSITIES
A61K51/0478
HUMAN NECESSITIES
A61K51/1027
HUMAN NECESSITIES
A61K51/0474
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61P35/00
HUMAN NECESSITIES
Abstract
A tissue-targeting complex comprising a tissue targeting moiety, an octadentate hydroxypyridinone-containing ligand comprising four HOPO moieties and the ion of an alpha-emitting thorium radionuclide, where at least one of the four HOPO moieties is substituted at the N-position with a hydroxyalkyl solubilising group.
Claims
1. A tissue-targeting complex comprising a tissue targeting moiety selected from the group consisting of a monoclonal or polyclonal antibody, an antibody fragment, a construct of said antibody or fragment, and an antibody mimetic, an octadentate hydroxypyridinone-containing ligand comprising four chelating 3,2-HOPO moieties of formula I ##STR00028## wherein R.sub.1 is a hydroxyalkyl moiety; groups R.sub.4 to R.sub.6 are each H, R.sub.3 is OH, and R.sub.2 is ═O, and the 4+ion of the alpha-emitting thorium radionuclide .sup.227Th.
2. The complex as claimed in claim 1 wherein the N-substituents on each of the four HOPO groups are each independently chosen from HOCH.sub.2—, HOCH.sub.2CH.sub.2—, HO—CH.sub.2CH.sub.2CH.sub.2—, HO—CH(CH.sub.3)CH.sub.2—, HO—CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, HO—CH(CH.sub.3)CH.sub.2CH.sub.2—, HO—CH(CH.sub.2CH.sub.3)CH.sub.2—, HO—C(CH.sub.3).sub.2CH.sub.2—, HO—CH(CH.sub.3)CH(CH.sub.3)—and HOCH.sub.2CH(CH.sub.2CH.sub.3)—.
3. The complex as claimed in claim 1 comprising a ligand moiety of formula VI: ##STR00029## wherein R.sub.L is a linker moiety for attachment of the ligand moiety to the tissue targeting moiety.
4. A complex as claimed in claim 1 wherein the antibody fragment (is a Fab, a F(ab′).sub.2, a Fab′, or a scFv.
5. A method of treatment of a human or non-human animal in need thereof comprising administration of a tissue targeting moiety selected from the group consisting of a monoclonal or polyclonal antibody, an antibody fragment, a construct of said antibody or fragment, and an antibody mimetic, an octadentate hydroxypyridinone-containing ligand comprising four chelating 3,2-HOPO moieties of formula I ##STR00030## wherein R.sub.1 is a hydroxyalkyl moiety; groups R.sub.4 to R.sub.6 are each H, R.sub.3is OH, and R.sub.2 is ═O, and the 4+ion of the alpha-emitting thorium radionuclide .sup.227Th.
6. The method as claimed in claim 5 for the treatment of hyperplastic or neoplastic disease, a carcinoma, a sarcoma, a myeloma, leukemia, a lymphoma or a mixed type cancer.
7. A pharmaceutical composition comprising a tissue-targeting complex as defined in claim 1 together with at least one pharmaceutical carrier or excipient.
8. A kit for use in a method according to claim 5, said kit comprising a tissue targeting moiety, conjugated or conjugatable to an octadentate hydroxypyridinone-containing ligand comprising four 3,2-HOPO moieties, where all four HOPO moieties are substituted at the N-position with a hydroxyalkyl solubilising group, said kit including the alpha-emitting thorium radionuclide .sup.227Th.
9. A method of formation of a tissue-targeting complex, said method comprising coupling a tissue targeting moiety to an octadentate hydroxypyridinone-containing ligand in aqueous solution, the complex comprising four 3,2-HOPO moieties and the ion of an alpha-emitting thorium radionuclide, where all four HOPO moieties are substituted at the N-position with a hydroxyalkyl solubilising group.
10. The method of claim 9 comprising preparing a first aqueous solution of octadentate hydroxypyridinone-containing ligand and a second aqueous solution of said tissue targeting moiety and contacting said first and said second aqueous solutions.
11. The method of claim 10 wherein said contacting is conducted at below 40° C.
12. The method of claim 10 wherein said contacting is conducted in the substantial absence of any organic solvent.
13. The method of claim 9 wherein said coupling yields an amide, ester, ether or amine bond between the ligand moiety and the targeting moiety.
14. The method of claim 13 wherein said amide or ester linkage is formed by means of at least one activated ester group.
15. The method as claimed in claim 5, wherein said antibody fragment is a Fab, a F(ab′).sub.2, a Fab′, or a scFv.
16. The method of claim 13, wherein said amide or ester linkage is formed by means a N-hydroxy maleimide, carbodiimide, or azodicarboxylate coupling reagent.
Description
BRIEF SUMMARY OF THE FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13) The invention will now be illustrated by the following non-limiting Examples. All compounds exemplified in the examples form preferred embodiments of the invention (including preferred intermediates and precursors) and may be used individually or in any combination in any aspect where context allows. Thus, for example, each and all of compounds 2 to 4 of Example 2, compound 10 of Example 3 and compound 7 of Example 4 form preferred embodiments of their various types.
(14) In the Examples, the following ligands, antibodies and antibody conjugates are referred to: AG0003—Comparative ligand (structure 13 below) AG0015—High solubility ligand of the invention. AG0700—Anti-CD33 antibody as generated in Example 5 AG0715—AG0700 conjugated to a high-solubility ligand (12) AGC1100—Anti-DC22 antibody as generated in Example (13) AGC1115—AGC1100 conjugated to a high-solubility ligand (12)
EXAMPLE 1
Isolation of Pure Thorium-227
(15) Thorium-227 is isolated from an actinium-227 cow. Actinium-227 was produced through thermal neutron irradiation of Radium-226 followed by the decay of Radium-227 (t½=42.2 m) to Actinium-227. Thorium-227 was selectively retained from an Actinium-227 decay mixture in 8 M HNO.sub.3 solution by anion exchange chromatography. A column of 2 mm internal diameter,
(16) length 30 mm, containing 70 mg of AG®1-X8 resin (200-400 mesh, nitrate form) was used. After Actinium-227, Radium-223 and daughters had eluted from the column, Thorium-227 was extracted from the column with 12 M HCl. The eluate containing
(17) Thorium-227 was evaporated to dryness and the residue resuspended in 0.01 M HCl.
EXAMPLE 2
Synthesis of Compound IX
(18) Step 1
(19) ##STR00013##
(20) 2-benzyloxyethylamine (31 g, 207 mmol) and glycolonitrile (16 mL, 70% solution in water, 207 mmol) was dissolved in 300 mL EtOH (abs) and refluxed for 4 h. The volatiles were removed under reduced pressure. The crude product (24.7 g, 130 mmol) was carried on to the next step without further purification.
(21) .sup.1H-NMR (CDCl.sub.3, 400 MHz): 2.92 (m, 2H), 3.58-3.62 (m, 4H), 4.51 (s, 2H), 7.25-7.37 (m, 5H)
(22) Step 2
(23) ##STR00014##
(24) 1 (24.7 g, 130 mmol) was dissolved in dry ether. HCl (g) was bubbled through the solution for 30 minutes. The precipitate was filtered off and dried under reduced pressure, giving the desired product (27.8 g, 122.6 mmol. The product was carried on to the next step without further purification or analysis.
(25) Step 3
(26) ##STR00015##
(27) 2 (27.8 g, 122.6 mmol) was dissolved in 230 mL chlorobenzene at room temperature. Oxallyl chloride (45 mL, 530 mmol) dissolved in 100 mL chlorobenzene was added drop wise over 30 minutes at room temperature. The reaction mixture was stirred at room temperature for 45 hours. The reaction was carefully quenched by drop wise addition of 100 mL water. The phases were separated, and the aqueous phase was extracted with 3*100 mL DCM. The organic phases were combined and washed with 100 mL brine. The organic phase was dried over Na.sub.2SO.sub.4, filtered and the volatiles were removed under reduced pressure. The crude product was purified by dry flash chromatography on SiO.sub.2 using a gradient of MeOH (0-2%) in DCM, yielding the desired product (21.2 g, 70.8 mmol).
(28) .sup.1H-NMR (CDCl.sub.3, 400 MHz): 3.71-3.76 (m, 2H), 4.06-4.12 (m, 2H), 4.47 (s, 2H), 7.217-7.22 (m, 2H), 7.26-7.36 (m, 4H)
(29) MS(ESI-pos, m/z,): 321.0
(30) Step 4
(31) ##STR00016##
(32) Sodium hydride (60% dispersion, 3.60 g, 90 mmol) was stirred in 50 mL THF at 0° C. and benzyl alcohol (8.3 mL, 80 mmol) was added drop wise over 10 minutes. The reaction mixture was stirred for 30 minutes at 0° C. before 3 (21.2 g, 70.8 mmol) dissolved in 100 mL THF was added drop wise at 0° C. The reaction mixture was stirred in the dark over night at room temperature. 50 mL HCl in dioxane (4M) was added drop wise before the reaction mixture was reduced in vacuo. 500 mL DCM was added, followed by 200 mL water. The phases were separated and the aqueous phase was extracted with 200 mL DCM. The organic phases were combined and washed with 100 mL brine. The organic phase was dried over Na.sub.2SO.sub.4, filtered and the volatiles were removed under reduced pressure. Dry flash chromatography on SiO.sub.2 using a gradient of MeOH (0-6%) in DCM gave the desired product (25.6 g, 69 mmol).
(33) .sup.1H-NMR (CDCl.sub.3, 300 MHz): 3.69-3.75 (m, 2H), 4.01-4.07 (m, 2H), 4.46 (s, 2H), 5.37 (s, 2H), 6.97 (s, 1H), 7.19-7.39 (m, 8H), 7.44-7.51 (m, 2H)
(34) MS(ESI-pos, m/z): 371.1, 763.2
(35) Step 5
(36) ##STR00017##
(37) 4 (25.6 g, 69 mmol) and ethyl propiolate (41 mL, 0.4 mol) was heated at 140° C. for 5 hours. The reaction mixture was cooled down to room temperature and the reaction mixture was purified by dry flash chromatography on SiO.sub.2. A gradient of MeOH (0-10%) in DCM gave the desired product as an inseparable mixture of the desired 4-isomer together with the 5-isomer. This mixture (28.6 g, .sup.˜65 mmol) was used directly in the next step without further purification.
(38) Step 6
(39) ##STR00018##
(40) 5 (28.6 g, .sup.˜65 mmol), as obtained in the previous step, was dissolved in 300 mL THF at 0° C. 100 mL KOH (1M, aq) was added, and the reaction mixture was stirred for 40 hours at room temperature. HCL (1M, aq) was added until pH.sup.˜2 (125 mL) and the aqueous phase was extracted with 3*250 mL CHCl.sub.3. The organic phases were combined and washed with 100 mL brine, filtered and the volatiles were removed in vacuo. The obtained material (25.9 g, .sup.˜65 mmol) was used without in the next step without further purification or analysis.
(41) Step 7
(42) ##STR00019##
(43) 6 (25.9 g, .sup.˜64 mmol), as obtained in the previous step, was partially dissolved in 400 mL DCM. 2-Thiazoline-2-thiol (8.94 g, 75 mmol) and DMAP (0.86 g, 7 mmol) was added, followed by DCC (15.48 g, 75 mmol). The reaction mixture was stirred at room temperature over night. The reaction mixture was filtered through a Celite-pad and the Celite-pad was washed with 100 mL DCM. The volatiles were removed in vacuo. The product mixture was purified by dry flash chromatography on SiO.sub.2 using first a gradient of DCM (50-100%) in heptane, followed by a gradient of THF (0-15%) in DCM. The appropriate fractions were reduced in vacuo, giving a mixture of products. This inpure mixture was purified by flash chromatography on SiO.sub.2 using a gradient of EtOAc (25-75%) in heptane. The appropriate fractions were reduced in vacuo, giving a mixture of products. Finally, to get the desired product, the product mixture was purified by dry flash chromatography on RP18-silica using a gradient of MeCN (25-75%) in water. This gave the desired product (8.65 g, 18 mmol).
(44) .sup.1H-NMR (CDCl.sub.3, 300 MHz): 2.90 (t, J=7.3 Hz, 2H), 3.77-3.84 (m, 2H), 4.18-4.23 (m, 2H), 4.35 (t, J=7.3 Hz, 2H), 4.51 (s, 2H), 5.33 (s, 2H), 6.11 (d, 7.0 Hz, 1H), 7.21-7.48 (m, 11H)
(45) MS(ESI-pos, m/z): 503.1
(46) Step 8
(47) ##STR00020##
(48) 7 (5.77 g, 12 mmol) and 8 (1.44 g, 2.4 mmol) were partially dissolved in 40 mL DMPU. DBU (2.7 mL, 18 mmol) was added drop wise. The reaction was stirred for 4 days at room temperature. Purification by dry flash chromatography on SiO.sub.2 using a gradient of DCM and MeOH in EtOAc gave the desired product (3.93 g, 2.15 mmol).
(49) .sup.1H-NMR (CDCl.sub.3, 400 MHz): 2.20-2.32 (m, 10H), 2.44-2.50 (m, 2H), 3.05-3.20 (m, 10H), 3.23-3.27 (m, 1H), 3.69-3.77 (m, 8H), 4.06-4.15 (m, 8H), 4.43 (s, 8H), 5.24 (s, 8H), 6.62 (d, J=7.2 Hz, 4H), 7.13 (d, J=7.2 Hz, 4H), 7.16-7.38 (m, 42H), 7.82-7.93 (m, 6H)
(50) Step 9
(51) ##STR00021##
(52) 9 (3.93 g, 2.15 mmol) was dissolved in 300 mL EtOH at room temperature. 60 mL water was added, followed by NH.sub.4Cl (5.94 g, 32.3 mmol). The reaction mixture was to 60° C. before iron powder (1.80 g, 32.3 mmol) was added. The reaction mixture was stirred at 60° C. for 1 hour. The reaction mixture was cooled down to room temperature and 400 mL DCM and 100 mL water was added. The reaction mixture was filtered, and the organic phase was washed with 100 mL water and 100 mL brine. The aqueous phases were combined and back extracted with 3*100 mL DCM. The organic phases were combined, dried over Na.sub.2SO.sub.4, filtered and the volatiles were removed under reduced pressure. The product mixture was purified by dry flash chromatography on SiO2 using a gradient of MeOH (0-7%) in DCM gave the desired product (3.52 g, 1.96 mmol).
(53) MS(ESI-pos, m/z): 899.2
(54) Step 10
(55) ##STR00022##
(56) 10 (1.00 g, 0.56 mmol), Pd(OH).sub.2/C (Pearlman's catalyst, 1.00 g) and 10 mL AcOH was placed in a pressure reactor. The reactor was evactuated by water aspirator and H.sub.2 was introduced (7 bar). The reaction mixture was stirred for 1 hour before the pressure was released and 5 mL HCl (6M, aq) was added to the reaction mixture. The reactor was evacuated as before and H.sub.2 was once again introduced (7 bar). After stirring for 7 days, HPLC indicated full conversion. The reaction mixture was filtered and the volatiles were removed under reduced pressure. The residue was dissolved in MeOH/MeCN (1:1) and the product was precipitated by addition of Et.sub.2O. The solids were collected by centrifugation and decanting the supernatant before the product was dried in vacuo (484 mg, 0.45 mmol).
(57) .sup.1H-NMR (D.sub.2O, 400 MHz): 2.70-2.95 (m, 2H), 3.00-3.10 (m, 2H), 3.15-3.65 (m, 19H), 3.75-4.23 (m, 16H), 6.25 (bs, 4H), 7.04 (d, J=7.0 Hz, 4H), 7.44 (d, J=8.2 Hz, 2H), 7.57 (d, J=8.2 Hz, 2H)
(58) MS(ESI-pos, m/z): 1076.4
(59) Step 11
(60) ##STR00023##
(61) Compound 11 (20 mg, 18 μmol) was dissolved in 3 mL MeCN and 3 mL water. 20 μL thiophosgene was added. The reaction mixture was stirred rigidly for 1 hour. The volatiles were removed under reduced pressure and the residue was dissolved in 4 mL MeCN. The product was precipitated by adding the acetontrile phase to 40 mL Et.sub.2O. The solids were collected by centrifugation and decating the supernatant before the product was dried in vacuo (10 mg, 9 μmol).
(62) MS(ESI-pos, m/z): 1118.4
EXAMPLE 3
Conjugation
(63) Filtered trastuzumab in 1.0 mL 0.9% NaCl solution (9.5 mg/mL), and novel chelator (formula IX) in 5-35 μL metal free water (10 mg/mL) were added to 1.0 mL sterile filtered borax buffer (70 mM, pH 9). The reaction mixture was stirred gently at 37° C. over night, and the resulting conjugate was purified and concentrated in 0.9% NaCl solution using an Amicon Ultra-4 (30 k MWCO) centrifugal filter unit. Successful conjugation was confirmed by LC/MS analysis.
EXAMPLE 4
Chelation
(64) The conjugate of Example 3 in 50 μL 0.9% NaCl (5 μg/μL) was added 100 μL sodium acetate buffer (0.5 M, pH 5.5), and then .sup.227Th-solution (approx. 0.5-1 MBq in 1-4 μL 0.05 M HCl). Reaction was done under gentle mixing at 37° C. for 1 hour or at room temperature (approx. 20° C.) for 15 minutes, and the resulting product purified on NAP-5 column using sodium acetate buffer as eluting buffer. The spent column containing retained free radiometals and the eluted fractions containing labelled protein were measured on a HPGe-detector GEM (15), to determine reaction yields and specific activity of the product (Table 1).
(65) TABLE-US-00003 TABLE 1 Summary, chelation reactions Reaction Radiochemical conditions yield (%) 37° C., 1h 96 37° C., 1h 93 20° C., 15 min 95 20° C., 15 min 95
EXAMPLE 5
Generation of the Anti-CD33 Monoclonal Antibody (AGC0700)
(66) The sequence of the monoclonal antibody (mAb) HuM195 as described in (1) and published in (2) served as template for the generation of AGC0700. The codon encoding the C-terminal lysine (Lys) was omitted from the IgG1 heavy chain gene. The resulting protein is one of three variants that are present in the antibody when produced from the full length genes, the other two variants having a lysine attached at one or both of the heavy chains, respectively. It is anticipated that removal of this Lys-residue allows a more precise determination of the conjugate to antibody ratio (CAR) as outlined in Example 6. An overview of the complete amino acid sequence of AGC0700 is presented in Table 2.
(67) The genes encoding AGC0700 were generated using standard molecular biology techniques. Briefly, the amino acid sequence of each chain was back-translated into DNA sequence using Vector NTI® Software (Invitrogen/Life-Technologies Ltd., Paisley, United Kingdom). The optimized DNA sequence was codon optimized for mammalian expression and synthesized by GeneArt (GeneArt/Life-Technologies Ltd., Paisley, United Kingdom). The VH- and VL-domains were sub-cloned via endonuclease restriction digest into an expression vector by Cobra Biologics (Sodertalje, Sweden). Chinese hamster ovarian suspension (CHO-S) cells were stably transfected with the plasmid encoding the VH- and VL-domains of AGC0700 and grown in presence of standard CD-CHO medium (Invitrogen/Life-Technologies Ltd., Paisley, United Kingdom), supplemented with puromycin (12.5 mg/I; Sigma Aldrich). Stable clones, expressing AGC0700, were selected via limiting dilution over 25 generations. Clone stability was assessed by measuring protein titers from supernatants. A cell bank of the most stable clone was established and cryo-preserved. Expression of the mAb was carried out at 37° C. for approximately 14 days in a single-use bioreactor. The monoclonal antibody was harvested after filtration of the supernatant. AGC0700 is further purified by protein A affinity chromatography (MabSelect SuRe, Atoll, Weingarten/Germany), followed by an ion exchange step. A third purification step based on electrostatic and hydrophobicity is used to further remove aggregates and impurities from production. The identity of AGC0700 will be confirmed by isoelectric focusing and SDS-PAGE analysis. Sample purity will be further analyzed by size-exclusion chromatography (SEC).
(68) TABLE-US-00004 TABLE 2 Amino acid sequence of AGC0700. Fragment QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNG variable V.sub.H GTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLV domain TVSS (SeqID 1) Fragment DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAAS variable V.sub.L NQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK domain (SeqID 2) Complete V.sub.H QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNG domain GTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLV (SeqID 3) TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Complete V.sub.L DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAAS domain NQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKR (SeqID 4) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
EXAMPLE 6
Conjugation of AGC0700 with Chelator AGC0015
(69) The naked antibody AGC0700 was conjugated (coupled) with the water soluble chelator AGC0015 (12). AGC0015 was prepared in a solution of metal-free. The chelator (12) is presented below:
(70) ##STR00024##
(71) The reaction was performed in a 1:1 (v/v) mixture of PBS, mixed with 70 mM borate buffer at a pH of 8.5. A nominal molar chelator to antibody ratio of 1.3:1 was used and the reaction was incubated for 20 hours at 21° C. At the end of reaction time the antibodies were separated from free chelator by size exclusion chromatography on an ÄKTA Purifier (Amersham), using HiLoad Superdex 200 16/600 PG column (GE Healthcare; part.no. 29-9893-35) and 50 mM histidine buffer of pH 6.0 as a mobile phase. The final chelator-antibody-ratio (CAR) of purified conjugates was determined by size exclusion chromatography-mass spectrometry (SEC-MS) analysis. Samples were enzymatically deglycosylated using recombinant EndoS (IgGZERO, Genovis, Sweden) prior to LC-MS analysis. Briefly, the chromatography was done on an Acquity UPLC system (Waters) and the column was a TSK Gel Super SW 3000, 2.0×300 mm, 4 μm particles (part no. 21485) maintained at room temperature. The mobile phase was: 50% acetonitrile in water, 0.1% (v/v) trifluoroacetic acid (isocratic elution). The injection volume was up to 15 μL and the LC flow rate was 75 μL/min or 50 μl/min, for intact conjugates and reduced conjugates, respectively. The total SEC run time was 16 minutes. The Xevo QTOF mass spectrometer (Waters) was equipped with an electrospray ionization (ESI) source. The ion source was operated in positive ion mode and the scan range was 2000-4000 Da. Multiply charged ions were transformed to singly charged species by using the Maximum Entropy software. The mass spectrometer was previously calibrated with sodium iodide in the given mass range. Intact conjugate gave one peak the SEC-MS chromatogram, containing all conjugate species (chelators 0-n). Representative results of a CAR-determination are presented in
EXAMPLE 7
Chelation of Antibody-Chelator Conjugate AGC0715 with Th-227
(72) Thorium-227 as a 4+ ion was isolated from an actinium-227 generator system. Briefly, Th-227 was selectively retained from a Ac-227 decay mixture in 8 M HNO3 by anion exchange chromatography (negatively charged nitrate complexes are formed with .sup.227Th4+). After Ac-227, Ra-223 and daughters had been washed from the column, Th-227 was eluted using 12 M HCl. The Th-227 eluate was evaporated to dryness and the residue dissolved in 0.5 M HCl.
(73) The antibody-conjugate AGC0715 was incubated for one hour in histidine buffer, pH 6.0 at 37° C. in the presence of 1 MBq of Th-227 per 0.5 mg antibody. The high molecular fraction containing radio labelled antibody-conjugates was separated from free Th-227 and daughter nuclides by size exclusion chromatography using NAP-5 DNA Grade columns (GE Healthcare). The labelling efficiency was typically 96-98%, including potential losses in the NAP-5 desalting step.
EXAMPLE 8
Binding Studies of AGC0700 and AGC0715 to CD33-Positive Cells by Flowcytometry
(74) Binding to CD33-positive HL-60 cells were studied by flow cytometry. The determination of EC.sub.50 based on the plotted curve gives an approximate value for the affinity. The commercially available mouse anti human CD33 (BD Pharmingen; #555450, 1 mg/mL) was used as a reference antibody for assessment of the affinity, including a setup with a 50:50 mixture with the mAb to be analyzed. This assay was also used to confirm that target binding affinity was not negatively affected by conjugation with the chelator.
(75) HL-60 cells were grown in Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen; #12440-046) in the presence of 20% of fetal bovine serum and penicillin/streptomycin. Approximately 20 mL cell culture was harvested by centrifugation at 4° C. for 5 min at 300 g. Cells were re-suspended in 10 mL PBS, supplemented with 1% of fetal bovine serum (FBS), and pelleted by centrifugation at 4° C. for 5 min at 300 g. Subsequently, 20 μL of the preparation of resuspended cells was diluted 1:500 in Coulter Isoton II Diluent, and counted using a Beckman Coulter Z2 instrument (Beckman Coulter; CA, USA). The preparation was adjusted to a cell density of 3-4×10.sup.6 cells/mL, and 100 μL was transferred to each well in a round or V-shaped bottom 96-well plate (Nunc/Fisher Scientific; NH, USA). Cells were spun down and re-suspended after decantation, which resulted in an approximate volume of 50 μL cell suspension per well.
(76) The mAb or conjugate to be analyzed was prepared fresh form frozen stock by shifting the storage buffer to PBS, kept at 4° C., and used within a few days after preparation. F(ab).sub.2′ Alexa488 conjugated goat anti-human IgG Fc (Jackson Immuno Research; #109-546-170) was used as a secondary antibody reagent for detection of human mAb. The secondary antibody reagent was prepared at 0.015 mg/mL in PBS, supplemented with 0.1% BSA. The mAb stock was diluted in 10-fold dilution steps, starting from 5 mg/mL. An isotype control mAb (trastuzumab) was prepared accordingly. 20 uL from each dilution step of the anti-human CD33 mAb was added to wells containing HL-60 cells. After incubation for 1 h at 4° C. the cells were spun down and washed three times with 200 μl cold PBS, supplemented with 0.1% BSA. A solution containing 4% goat serum was added as a blocking agent, and incubated for 15 minutes. 20 μl from the secondary antibody reagent was subsequently added to each well, before incubation for 30 min at 4° C. in the dark.
(77) The cells were washed twice, as described above, and re-suspended in 200 μL PBS, supplemented with 0.1% BSA. All samples were analysed in 96-well round or conical bottom plates. Fluorescent signals were recorded on a Beckham Coulter Cell lab Quant SC flow cytometer (Beckman Coulter; CA, USA). Median values were exported to an Excel graph sheet and plotted against the concentration. Data were fitted using the “one-site specific” binding model in Graph Pad Prism (PrismSoftware; CA, USA) (
(78) The negative control with secondary but no primary antibody showed low background, mean fluorescent intensity (MFI) values of approximately 5 (1-2% of the high positive values).
(79) The binding affinities of the conjugate AGC0715 was comparable to the non-conjugated AGC0700. Thus, conjugation did not result in reduced binding affinity (
EXAMPLE 9
Internalisation of AGC0715
(80) The extent of internalisation of a radioimmunoconjugate after attachment to outer cell membrane is one factor determining the cell killing potency. Internalization into HL-60 cells of the radioimmunoconjugate AGC0715-Th-227 and the negative control trastuzumab-AC0015-Th-227 was studied.
(81) The conjugated mAbs were labelled in parallel according to the procedure described in Example 5, to a specific activity of about 20 Bq/ug. Wells containing 200 000 cells in 500 μL growth medium (Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen; #12440-046) supplemented with 20% of fetal bovine serum and penicillin/streptomycin) were added equal amounts of radioimmunoconjugate, corresponding to 12 kBq.
(82) Samples of cells were harvested after 0, 30, 60, 120, and 240 minutes incubation time at 37° C. and 5% CO.sub.2. After harvest the cells were washed with glycine pH 2.5, 0.9% NaCl to remove membrane bound antibody-conjugates. Analysis by flow cytometry showed that no membrane bound antibody remained after a 2-4 minutes acid wash. Internalisation was measured on cell pellets on a gamma counter (Wizard) for 60 seconds and the measured counts per minute were plotted against time (
EXAMPLE 10
Th-227-Induced Cell Cytotoxicity by AGC0715-Th-227
(83) In vitro cell cytotoxicity was investigated in CD33 positive HL-60 cells. AGC0715 and the control trastuzumab conjugated AGC0015 were used to chelate Th-227 to a specific activity of 44 kBq/μg. HL-60 cells were grown at 37° C. with 5% CO.sub.2, and split 1:5 three times a week. The day before the assay the culture medium (Iscove's Modified Dulbecco's Medium (IMDM) with 20% FBS and 1% Penicillin/Streptomycin) was replaced by new medium and the volume adjusted to give 400 000 cells per mL.
(84) About, 1 600 000 cell (4 mL) were added to each well in a 6 well plate. The plate was incubated until next day for addition of labelled mAb, or culture medium. After adding labelled mAb, or culture medium, the plate was incubated for 4 additional hours. In one experiment AGC0715 or trastuzumab-AGC0015 was added to each well to a final concentration of 3 nM. In another experiment the final concentration was 0.3 nM.
(85) Following incubation, the cells were washed twice in culture medium, and the ATP in the supernatant and in the pellet was measured. The cells were then split 1:2 and incubated in culture medium at 37° C. with 5% CO.sub.2. The same procedure, but with only one wash, was repeated at days 2, 4, and 7. A quantification of ATP was used as a measure of cell viability at different sample times (CellTiter-Glo Luminescent cell viability assay from Promega), resulting in the growth curves shown in
(86) The HL-60 tumour cell binding AGC0715-Th-227 resulted in cellular toxicity, in contrast to the trastuzumab construct, not binding to HL60 cells. The loss of viability from day 4 to day 7 in the culture medium control is believed to be due to a too long interval between media replacement.
EXAMPLE 11
Effective Tumour Targeting by AGC0715 in a Human Xenograft Model
(87) Female NMRI nude mice were xenografted with cells from the human HL-60 tumour cell line. HL-60 cells are derived from a patient with acute promyelocytic leukemia, and express CD33 according to Sutherland et al (3). This cell line has been proven to be tumourigenic when inoculated subcutaneously into nude mice (4). 54 female NMRI nude mice (Taconic, Europe) were used in the study.
(88) The animals were allowed an acclimation period of at least 5 days before entering the study and were at an age of 4 weeks before tumour inoculation. The mice had an approximated body weight of 20 grams at the start of the study. Animals were kept in individually ventilated cages (IVC, Scanbur) with HEPA filtered air and had ad libitum access to “Rat and mouse nr.3 Breeding” diet (Scanbur BK) and water acidified by addition of HCl to a molar concentration of 1 mM (pH 3.0). HL-60 cells (ATCC/United Kingdom; Catalog Number CCL-240) were grown and prepared for subcutaneous inoculation in IMDM (Invitrogen; #12440-046) in presence of 20% FCS and penicillin/streptomycin. Stocks were made at passage number four (P4) and frozen down for storage in liquid nitrogen at 3×107 cells/vial in the culture media containing 5% DMSO.
(89) On the day of inoculation, the cells were thawed quickly in a 37° C. water bath (approx. 2 min), washed and re-suspended in PBS, supplemented with 2% FCS (centrifugation at 1200 rpm for 10 min). Cells were mixed thoroughly every time cells before aspiration into the dosing syringe. A volume of 0.1 mL of cell suspension was injected s.c. at the back using a fine bore needle (25G) while the animals were under light-gas anaesthesia (N.sub.2O). Animals were returned to their cages and the tumours were allowed to grow for 15 days. Dosing of test article (0.1 mL) was performed as an intravenous bolus via the tail vein.
(90) Animals were randomized into 3 groups (n=6) after 21 days of tumour growth, and injected with the test compound .sup.227Th-AGC0715 at a dose of 15 kBq/animal. Animals were euthanized at predetermined time points post injection, and blood, muscle, femur, kidneys, lung, liver, stomach, small intestine, large intestine and tumour were collected. Tissues and blood samples were weighed, and the radioactivity in each sample was measured using gamma spectroscopy (HPGe15p or HPGe50p germanium detectors).
(91) The measured radioactivity (Bq) was related to the radioactivity measured in 10% injection standard samples, and the percentage of Th-227 and Ra-223 were calculated and presented as % ID or % ID per gram of tissue. Two animals were excluded from the analysis because of large tumours compared to the rest of the group, as retrospective analysis showed an inverse relation between tumour uptake and tumour size (data not shown). The data confirm specific tumour uptake of the AGC0715-Th-227. Previous studies have shown that non-specific retention of a mAb in the tumour due to vascular leakage is washing out over time and do not exceed a few % ID/g at day 7 (data not shown).
(92) The results of the biodistribution study are presented in the
EXAMPLE 12
In Vivo Efficacy of AGC0715-Th-227 in Xenografted Mice
(93) Cell preparation and xenografting of female NMRI nude mice are done as described in Example 9. The animals are allowed an acclimation period of at least 5 days before entering the study and are at an age of 4 weeks before tumour inoculation. The mice have an approximated body weight of approximately 20 grams at the start of the study. Animals are kept in individually ventilated cages (IVC, Scanbur) with HEPA filtered air and have ad libitum access to “Rat and mouse nr.3 Breeding” diet (Scanbur BK) and water acidified by addition of HCl to a molar concentration of 1 mM (pH 3.0). Mice will be inoculated with tumour cells about 10 days before dosing in order to have mice with average size of tumours in the range from 75-150 mm3. Animals are randomized into 4 treatment groups and 3 control groups (n=10/group).
(94) Mice in the treatment groups are injected into the tail vein with 100 μL containing 75 kBq/mL, 150 kBq/mL, 225 kBq/mL or 300 kBq/mL AGC0715-Th-227, to achieve 250, 500, 750 or 1000 kBq/kg body weight (b.w.). The mice in the control groups are administered either with vehicle only (buffer), non-radioactive antibody (AGC0715) or trastuzumab-Th-227, labelled to a specific activity of 500 kBq/kg (isotype control).
(95) Appearance of tumours will carefully be monitored, and the tumours will be scored (if not large enough to be measured) or measured thrice a week according to the following scheme:
(96) TABLE-US-00005 Scores: 0 Tumour cannot be detected 1 Tumour is palpable 2 Just before measurements will be possible Marks: R Red W Signs of wound tissue N Necrosis B Blue
(97) Tumour diameters will be measured in two dimensions using a digital caliper and the volume will be estimated by the following formula: L×W×% W (Length×Width×½ Width). Measurements/observations will start at day 0, i.e. the day of inoculation. Measurements of tumour volumes will be three times a week, Monday, Wednesday and Friday. Body weights will be recorded once a week. The data will be presented in figures and descriptive statistics will be conducted.
(98) The mice will be terminated following maximal tumour size of 15 mm diameter. This diameter is equal to a volume of 1688 mm.sup.3 when assuming a spherical form. No adverse clinical sigs is expected in this study. In case of observations of adverse clinical signs, these will be recorded as note to files.
(99) After the study different treatment groups are compared by Caplan Meyer survival curve. Treatment-induced tumour growth is also plotted and growth delays are calculated after nonlinear regression of mean growth versus time and compared using Student T Test.
(100) The data of the efficacy study are expected to be similar to previously published efficacy data obtained after administration of the monoclonal antibodies trastuzumab and rituximab labelled with thorium-227.
EXAMPLE 13
Generation of the Anti-CD22 Monoclonal Antibody (AGC1100)
(101) The sequence of the monoclonal antibody (mAb) hLL2, also called epratuzumab, here denoted AGC1100, was constructed as described in (1). The mAb used in the current examples was produced by Immunomedics Inc, New Jersey, USA. Production of this mAb could for example be done in Chinese hamster ovarian suspension (CHO-S) cells, transfected with a plasmid encoding the genes encoding the light and the heavy chain. First stable clones would be selected for using standard procedures. Following approximately 14 days in a single-use bioreactor, the monoclonal antibody may be harvested after filtration of the supernatant. AGC1100 would be further purified by protein A affinity chromatography (MabSelect SuRe, Atoll, Weingarten/Germany), followed by an ion exchange step. A third purification step based on electrostatic and hydrophobicity could be used to remove aggregates and potentially remaining impurities. The identity of AGC1100 would be confirmed by isoelectric focusing, SDS-PAGE analysis, N-terminal sequencing and LC/MS analysis. Sample purity would be further analyzed by size-exclusion chromatography (SEC).
(102) Known sequences of CD22 binding antibodies (murine and humanised) include the following (where CDRs are bold and predicted contact regions outside of CDRs are underlined):
(103) TABLE-US-00006 Light Chain: (SeqID5) DIQLTQSPSSLAVSAGENVTMSCKSSQSVLYSANHKNYLAWYQQKPGQSP KLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVQVEDLAIYYCHQYLSS WTFGGGTKLEIKR (SeqID6) DIQLTQSPSSLASAAVEDRTMSCKSSQSVLYSANHKNYLAWYQQKPGQKA KLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSS WTFGGGTKLEIKR Heavy Chain: (SeqID7) QVQLQESGAELSKPGASVKMSCKASGYTFTSYWLHWIKQRPGQGLEWIGY INPRNDYTEYNQNFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARRD ITTFYWGQGTTLTVSS (SeqID8) QVQLQQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGY INPRNDYTEYNQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRD ITTFYWGQGTTVTVSS (SeqID9) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGY INPRNDYTEYNQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRD ITTFYWGQGTTVTVSS
REFERENCES (1) Leung, Goldenberg, Dion, Pellegrini, Shevitz, Shih, and Hansen. Molecular Immunology 32: 1413-27, 1995.
EXAMPLE 14
Conjugation of AGC1100 with the Chelator AGC0015
(104) The antibody AGC1100 was conjugated with the water soluble chelator AGC0015. (12) The conjugation reaction was performed in a 1:1 (v/v) mixture of PBS mixed with 70 mM borate buffer pH 8.5. The chelator, AGC0015 is as shown below:
(105) ##STR00025##
(106) The chelator, AGC0015 (12 above), was dissolved in metal-free water before it was added to the conjugation reaction. A nominal molar chelator to antibody ratio of 1.3:1 was used and the reaction was incubated for 22 hours at 21° C. At the end of reaction time the antibody fraction was separated from free chelator by size exclusion chromatography on an ÄKTA Purifier (GE Healthcare), using a HiLoad Superdex 200 16/600 PG column (GE Healthcare; code.no. 28-9893-35) and 0.9% NaCl 100 mM citrate buffer pH 5.5 as mobile phase. The final chelator-antibody-ratio (CAR) of purified conjugate was determined by HPLC size exclusion chromatography-UV (SEC-UV) analysis. The CAR determination was done on an Agilent 1200 series HPLC system (Agilent Technologies), column TSKgel SuperSW 3000, 4.6×300 mm, 4 μm particles (Tosoh Bioscience, part no. 18675) maintained at room temperature and mobile phase 300 mM NaCl 200 mM ammonium acetate pH 6.8 (isocratic elution) with a total run time of 15 minutes. The injection volume was 5 μl and the LC flow rate was 0.35 ml/min. The UV signals were monitored at 280 and 335 nm, corresponding to mAb and chelator absorbance maximum, respectively. Representative results of a CAR-determination are presented in
EXAMPLE 15
Chelation of Antibody/Chelator Conjugate AGC1115 with Th-227
(107) Thorium-227 (.sup.227Th) as a 4+ ion was isolated from an actinium-227 (.sup.227Ac) generator system. .sup.227Th was selectively retained from a .sup.227Ac decay mixture in 8 M HNO.sub.3 by anion exchange chromatography, where negatively charged nitrate complexes are formed with .sup.227Th.sup.4+. .sup.227Ac and daughter nuclides were washed off the column and .sup.227Th was eluted in 12 M HCl. The .sup.227Th-eluate was evaporated to dryness and the residue dissolved in 0.5 M HCl.
(108) In the chelation reaction the antibody-conjugate AGC1115 was incubated for 15 minutes in 0.9% NaCl 100 mM citrate buffer, pH 5.5 at 21° C./room temperature in the presence of 1 MBq .sup.227Th per 0.5 mg antibody conjugate. The high molecular fraction containing radio labelled antibody-conjugate was separated from free .sup.227Th and daughter nuclides by size exclusion chromatography using NAP-5 DNA Grade columns (GE Healthcare). The labelling efficiency was typically 96-98%, including potential loss in the NAP-5 desalting step.
EXAMPLE 16
Binding Analysis of AGC1115 and AGC1100 to CD22-Positive Raji Cells by Flow Cytometry
(109) Binding of AGC1115 and AGC1100 (anti-human CD22, Immunomedics; hLL2, #1003164, 10 mg/ml) to CD22-positive Raji cells (ATCC, #CCL-86) was analysed by flow cytometry. The EC.sub.50 value determined from the fitted curve was used for comparison of the antibody versus the antibody conjugate binding potency. This analysis was used to confirm that antibody conjugate binding potency to CD22 was unaffected by the conjugation procedure.
(110) Raji cells were grown in RPMI 1640 (PAA; #E15-840) in the presence of 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. For the flow cytometry analysis 50 ml cell culture was harvested by centrifugation at 4° C. for 5 min at 340×g. Cells were resuspended and washed twice in 10 ml PBS, supplemented with 1% FBS, and pelleted by centrifugation at 4° C. for 5 min at 340×g. Subsequently, 20 μl of the preparation of resuspended cells was diluted 1:500 in Coulter Isoton II Diluent, and counted using Beckman Coulter Z2 instrumentation (Beckman Coulter; CA, USA). The preparation was adjusted to a cell density of 1×10.sup.6 cells/ml and 100 μL was transferred to each well in a V-shaped bottom 96-well plate (Nunc/Fisher Scientific; NH, USA). Cells were spun down and re-suspended after decantation, which resulted in an approximate volume of 50 μl cell suspension per well.
(111) AGC1115 and AGC1100 was diluted to 50 μg/ml and titrated in twelve points in 3-fold dilution steps. An isotype control antibody (trastuzumab) was prepared accordingly. 100 μl from each dilution of the antibody was added to the wells containing Raji cells. After incubation for 1.5 h at 4° C., the cells were spun down and washed twice with 200 μl cold PBS, supplemented with 1% FBS. PE conjugated mouse anti-human IgG Fc (BioLegend; #409304) was used as a secondary antibody reagent for detection of human mAb. The secondary antibody reagent was prepared at 1 μg/ml in PBS, supplemented with 1% FBS. 100 μl from the secondary antibody reagent was subsequently added to each well, before incubation for 1 h at 4° C. in the dark. The cells were washed twice, as described above, and resuspended in 200 μl PBS, supplemented with 1% FBS. All samples were analysed in a V-shaped bottom 96-well plate. Fluorescent signal was recorded on a Beckman Coulter Cell Lab Quanta SC MPL flow cytometer (Beckman Coulter; CA, USA). Median values (MFI) were exported to an Excel sheet and plotted against the concentration ([nM]).
(112) Data was fitted using the “log(agonist) vs. response—Variable slope (four parameters)” binding model in Graph Pad Prism (PrismSoftware; CA, USA) and the EC.sub.50 values was calculated from the fit (
(113) The calculated EC.sub.50 values of the fitted titration curves of AGC1100 and AGC1115 were 9 nM and 6 nM, respectively, and indicated that the binding potency of the conjugate AGC1115 was comparable to AGC1100.
EXAMPLE 17
Th-227-Induced Cell Cytotoxicity by AGC1115-Th-227
(114) In vitro cell cytotoxicity was investigated in CD22 positive Ramos cells (see Example 6). AGC1115 and the control trastuzumab conjugated with AGC0015 were used to chelate Th-227 to a specific activity of 44 kBq/μg.
(115) Ramos cells were grown at 37° C. with 5% CO.sub.2, and split 1:5 three times a week. The day before the assay the culture medium (Iscove's Modified Dulbecco's Medium (IMDM) with 20% FBS and 1% Penicillin/Streptomycin) was replaced by new medium and the volume adjusted to give 400 000 cells per mL. About, 1 600 000 cell (4 mL) were added to each well in a 6 well plate. The plate was incubated until next day for addition of labelled mAb, or culture medium.
(116) After adding labelled mAb, or culture medium, the plate was incubated for 4 more hours. In the experiment AGC1115 or trastuzumab-AGC0015 was added to each well to a final concentration of 3 nM. Following incubation, the cells were washed twice in culture medium, and the ATP in the supernatant and in the pellet was measured. The cells were then split 1:2 and incubated in culture medium at 37° C. with 5% CO.sub.2. The same procedure, but with only one wash, was repeated at days 3, 5 and 7.
(117) A quantification of ATP was used as a measure of cell viability at different sample times (CellTiter-Glo Luminescent cell viability assay from Promega), resulting in the curves shown in
EXAMPLE 18
Acid Derivative
(118) Making an acid derivative of the water soluble chelator enabling alternative coupling chemistries.
(119) This example shows the successful synthesis of an acid derivative. This derivative of the chelator enables, for example, formation of an amide bond with an epsilon amine of the tumour targeting protein.
(120) The present example shows the synthesis of the soluble chelator and starts out from substance 11 (Example 2). 43 mg (˜0.04 mmol) of substance 11 was dissolved in 4 mL DMSO, 4 mL acetonitrile, and 30 μL NEt.sub.3. 6 mg of succinic anhydride was added (0.06 mmol). LC/MS analysis of the reaction mix after 22 hours reaction at room temperature showed that substance 15 had formed. Some contaminant diacylated side product was formed. Adding the anhydride in portions should minimize the ester formation and improve molar yield of product 14. HPLC analysis of the resulting reaction mixture is shown in
(121) ##STR00026##
EXAMPLE 19
Conjugation of AGC0003 and AGC0015 to a Small Protein
(122) Since lysine coupling was going to be used, the free cysteine of the Affibody fusion protein was chemically blocked before conjugation with the chelators. The Affibody fusion protein PEP9237 (SeqID 10) made in E. coli by essentially as described by Tolmachev et al. (1), was dissolved to 1.32 mg/ml in PBS (Biochrom L1825; 0.9% NaCl, phosphate buffer, pH7.4), containing 2 mM tris(2-chloroethyl)phosphine (TCEP) to hinder disulphide formation. One ml of the affibody fusion protein solution was mixed with 0.985 ml 0.14 M borate buffer, and the pH confirmed to be about 8.4. 15 μL of 1 mg/ml solution of iodoacetamide (Sigma 1149-5 g) was added and the reaction mix incubated for 1 hour at room temperature.
(123) Before conjugation, the 4 ml solution containing sulfhydryl blocked Affibody fusion protein was concentrated to 0.5 mL using Amicon spin filters, and excess reactants removed on a NAP5 desalting column (GE Healthcare, lot#83892624) equilibrated with borate buffer. The eluted 1 mL high molecular weight fraction was spilt in two parts for reacting with either AGC0003 or AGC0015.
(124) The chelator AGC0003 (8.3 μL DMF (Sigma 227056, lot# STBB4668)—13 below-containing 10 mg/ml) was added to a vial containing 0.5 mL sulfhydryl blocked Affibody fusion protein. The chelator AGC0015 (9.3 μL H.sub.2O containing 10 mg/ml) was added to a separate vial containing 0.5 mL sulfhydryl blocked Affibody fusion protein. Both vials containing chelator protein reaction mixtures were incubated over night at 30° C. The resulting clear solution was buffer exchanged to PBS using a NAP5 column.
(125) ##STR00027##
EXAMPLE 20
Solubility Analyses of the Small Protein Conjugates with AGC0003 and AGC0015
(126) The clear PBS solution of the two Affibody fusion protein chelator conjugates produced in Example 19 were frozen at −20° C., before thawing. A distinct milky precipitation was seen with the solution containing AGC0003 conjugate, but not with the in AGC0015 conjugate. The precipitation was more visible after cooling down to on ice.
(127) The clear solution from either reaction was analysed by FPLC-SEC, to investigate possible aggregates (
(128) Next, the protein to chelator ratio was investigated by LC/MS analysis. The unconjugated Affibody fusion protein (AGC200) was included as a control. During LC absorption was monitored at 280 and 335 nm (chelator absorbs at approx. 335 nm), showing that chelator was present in the two conjugate solutions but not in the AGC200 preparation (
REFERENCES
(129) Tolmachev, Orlova, Pehrson, Galli, Baastrup, Andersson, Sandström, Rosik, Carlsson, Lundqvist, Wennborg, Nilsson. Radionuclide therapy of HER2-positive microxenografts using a 177Lu-Labeled HER2-specific Affibody molecule. Cancer Res. 67: 2773-82, 2007.