Humanised anti kallikrein-2 antibody
11230609 · 2022-01-25
Assignee
Inventors
- Par Oskar Vilhelmsson Timmermand (Lund, SE)
- Amanda Thuy Tran (Malmo, SE)
- Sven-Erik Strand (Lund, SE)
- Urpo Juhani Lamminmaki (Vanhalinna, FI)
- Kjell Sjostrom (Lund, SE)
Cpc classification
C07K2317/24
CHEMISTRY; METALLURGY
A61K51/1045
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61K51/1096
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61K51/10
HUMAN NECESSITIES
Abstract
The present invention provides antibody polypeptides with binding specificity for human kallikrein-2 (hK2), wherein the antibody polypeptide comprises (a) a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 and SEQ ID NO:3 and/or (b) a light chain variable region comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6, and wherein the heavy chain variable region and light chain variable region comprise framework amino acid sequences from one or more human antibodies. The invention further provides use of said antibody polypeptides in the diagnosis and treatment of prostate cancer.
Claims
1. An antibody polypeptide with binding specificity for human kallikrein-2 (hK2), wherein the antibody polypeptide is an intact antibody and comprises (a) a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 and SEQ ID NO:3; and (b) a light chain variable region comprising the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:5 and SEQ ID NO:6, and wherein the heavy chain variable region and light chain variable region comprise framework amino acid sequences from one or more human antibodies.
2. The antibody polypeptide according to claim 1, wherein the intact antibody comprises a heavy chain constant region, and a light chain constant region.
3. The antibody polypeptide according to claim 2, wherein the heavy chain constant region is of an immunoglobulin with a subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.
4. The antibody polypeptide according to claim 2, wherein the light chain constant region is of a kappa light chain or lambda light chain.
5. The antibody polypeptide according to claim 1, wherein the antibody polypeptide is linked, directly or indirectly, to a therapeutic moiety.
6. The antibody polypeptide according to claim 5, wherein the therapeutic moiety is a cytotoxic moiety that comprises or consists of one or more radioisotopes.
7. The antibody polypeptide according to claim 6, wherein the one or more radioisotopes is or are each independently selected from the group consisting of beta-emitters, auger-emitters, conversion electron-emitters, alpha-emitters, and low photon energy-emitters.
8. The antibody polypeptide according to claim 7, wherein the one or more radioisotopes each independently have an emission pattern of locally absorbed energy that creates a high dose absorbance in the vicinity of the antibody polypeptide.
9. The antibody polypeptide according to claim 7, wherein the one or more radioisotopes are each independently selected from the group consisting of long-range beta-emitters; medium range beta-emitters; low-energy beta-emitters; conversion electron-emitters or auger-emitters; and alpha-emitters.
10. The antibody polypeptide according to claim 9, wherein the long-range beta-emitters comprise .sup.90Y, .sup.32P, .sup.186Re/.sup.186Re; .sup.166Ho, .sup.76As/.sup.77As or .sup.153Sm.
11. The antibody polypeptide according to claim 9, wherein the medium range beta-emitters comprise .sup.131I, .sup.177Lu, .sup.67Cu or .sup.161Tb.
12. The antibody polypeptide according to claim 11, wherein the medium range beta-emitters comprise .sup.177Lu.
13. The antibody polypeptide according to claim 9, wherein the low-energy beta-emitters comprise .sup.45Ca, .sup.35S or .sup.14C.
14. The antibody polypeptide according to claim 9, wherein the conversion electron-emitters or auger-emitters comprise .sup.51Cr, .sup.67Ga, .sup.99Tcm, .sup.111In, .sup.123I, .sup.125I or .sup.201Tl.
15. The antibody polypeptide according to claim 9, wherein the alpha-emitters comprise .sup.212Bi, .sup.213Bi, .sup.223Ac, .sup.225Ac or .sup.221At.
16. The antibody polypeptide according to claim 15, wherein the alpha-emitters comprise .sup.225Ac.
17. The antibody polypeptide according to claim 5, wherein the therapeutic moiety is a cytotoxic moiety that comprises or consists of one or more cytotoxic drugs.
18. The antibody polypeptide according to claim 5, wherein the therapeutic moiety and/or detectable moiety is joined to the antibody polypeptide indirectly, via a linking moiety.
19. The antibody polypeptide according to claim 1, wherein the antibody polypeptide further comprises a detectable moiety.
20. The antibody polypeptide according to claim 19, wherein the detectable moiety comprises or consists of a radioisotope or a paramagnetic isotope.
21. The antibody polypeptide according to claim 1, wherein the antibody polypeptide comprises a pair of detectable and cytotoxic radionuclides.
22. The antibody polypeptide according to claim 21, wherein the pair of detectable and cytotoxic radionuclides is capable of simultaneously acting in a multi-modal manner as a detectable moiety and also as a cytotoxic moiety.
23. The antibody polypeptide according to claim 1, wherein the antibody polypeptide further comprises a moiety for increasing the in vivo half-life of the antibody polypeptide.
24. An isolated nucleic acid molecule encoding the antibody polypeptide according to claim 1.
25. A method for producing an antibody, the method comprising culturing a host cell comprising a nucleic acid as defined in claim 24 under conditions which permit expression of the encoded antibody or antigen-binding fragment thereof.
Description
(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
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(17) The following examples are included to demonstrate particular embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute specific modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLES
Example 1—Cloning of 11B6 from Hybridoma Cell Line
(18) Reagents
(19) Monoclonal antibody 11B6 producing hybridoma cell line was used for mRNA extraction and production of antibodies which were further affinity purified for protein sequencing (Väisänen et al., 2004).
(20) Restriction enzymes, FastAP and T4 DNA ligase were from Fermentas, primers from the University of Turku, Department of Biotechnology (WO252) and from Thermo Scientific. DNA purifications were done with Qiagen's Gel extraction and PCR purification kits.
(21) mRNA Extraction and cDNA Synthesis
(22) mRNA was extracted from 11B6 MAb producing hybridoma cells (5E6 cells) with QuickPrep Micro mRNA purification kit (Amersham Biosciences) and cDNA synthesis from the mRNA was done with Applied Biosystems' High-capacity cDNA archive kit according to instructions.
(23) Amplification of Antibody Genes from cDNA
(24) N-terminal sequences of the purified 11B6 MAb heavy (H) and light (L) chains were determined by Edman degradation at the University of Helsinki protein sequencing service. Light chain sequence was DIVLTQSPAS [SEQ ID NO: 16] and the heavy chain sequence DVQLQESGPG [SEQ ID NO: 17]. IMGT database comparison of amino acids identified the genes: IGKV3 and IGHV3, respectively. The complementary regions for forward PCR primers (degenerate) were designed based on the DNA sequences (found by NCBI BLAST) coding the N-terminal amino acids. Reverse primer used to clone the heavy chain was designed to bind C.sub.H1. In the case of the light chain, two reverse primers were used; the one used in the first PCR binds to C.sub.L and the other one used in second PCR to the border of V.sub.L and C.sub.L. All primers also contain the restriction enzyme recognition sites needed for cloning (later underlined).
(25) TABLE-US-00012 Light chain forward primer was Sfil_DIVLTQSPAS [SEQ ID NO: 16]: (5′-TTACTCGCGGCCCAGCCGGCCATGGCGGAYATHGTRYTVACNCART CTCC-3′; [SEQ ID NO: 18]) and reverse primers WO252 (5′-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3′, XbaI; [SEQ ID NO: 19]) and Cpol_JK2 (5′-GATACAGTTGGTGCAGCATCGGTCCGTTTTATTTCCAGCTTGGTCC CCCCT-3′; [SEQ ID NO: 20]) Heavy chain forward primer was NotI_DVQLQESGPG [SEQ ID NO: 17] (5′-TGCTGCTGGCGGCCGCTCCAGCCATGGCTGAYGTVCARCTKCAGGA GTCDGG-3′; [SEQ ID NO: 21]) and reverse primer asCH1_SacI (5′-CGCCACCAGAGCTCTCACAATCCCTGGGCACAATTTTC-3′; [SEQ ID NO: 22])
(26) V.sub.L+C.sub.L fragment was amplified in PCR reaction containing 100 ng cDNA as template, 0.2 mM dNTP's, 0.5 μM primers Sfil DIVLTQSPAS [SEQ ID NO: 16] and WO252, 1× Phusion HF buffer and 0.6 U Phusion DNA polymerase (Finnzymes). Amplification was done by protocol of 98° C. 30 sec, 30 cycles of 98° C. 7 sec, 50° C. 20 sec, 72° C. 20 sec, and final extension of 72° C. 10 min. After sequencing the PCR product and finding out the sequence of V.sub.L-C.sub.L border the PCR for actual cloning was done again from cDNA in a reaction like above except with primers Sfil_DIVLTQSPAS [SEQ ID NO: 16] and Cpol_JK2 to clone only the V.sub.L part. Amplification was done by protocol of 98° C. 30 sec, 10 cycles of 98° C. 7 sec, 60° C. 20 sec, 72° C. 20 sec, 25 cycles of 98° C. 7 sec, 56° C. 20 sec, 72° C. 20 sec, and final extension of 72° C. 10 min.
(27) VH.sub.H+CH.sub.H1 fragment was amplified in a reaction like with V.sub.L except with primers Notl_DVQLQESGPG [SEQ ID NO: 17] and as CH1_Sacl. Amplification protocol was 98° C. 30 sec, 30 cycles of 98° C. 7 sec, 64° C. 20 sec, 72° C. 20 sec, and final extension of 72° C. 10 min.
(28) Cloning
(29) The correct sized products were purified from the preparative agarose gel. V.sub.L was digested with Sfil and Cpol, V.sub.H+CH.sub.H1 with Sacl and Notl. Recipient vector pAK400 5404 FAb Ich (modified from pAK400, Krebber et al., 1997) was digested separately with both enzyme combinations, fragments dephosphorylated with FastAP and purified from the preparative gel.
(30) Digested 11B6 V.sub.L and the corresponding vector fragment were ligated with T4 DNA ligase and transformed by electroporation into Escherichia coli XL1-Blue cells (Stratagene) to produce vector pAK400-11B6-VL. Ligation product of Sacl+Notl digested V.sub.H+C.sub.H1 and vector fragment was called pAK400-11B6-VH+CH1. Correct clones were confirmed by DNA sequencing and comparing sequences to the original protein sequences and to the antibodies found on the database (BLAST search).
(31) To construct the complete 11B6 Fab, both previously made constructs were digested with Notl and Sacl. Vector pAK400-11B6-VL was used as recipient vector to which V.sub.H+CH.sub.H1 from vector pAK400-11B6-VH+CH1 was inserted. Ligation and transformation were done as above. The constructed pAK400 11B6 FAb Ich vector was confirmed with restriction enzyme analysis.
REFERENCES
(32) Barbas C F 3rd, Kang A S, Lerner R A, Benkovic S J. (1991) Assembly of combinatorial antibody libraries on phage surfaces: The gene III site. Proc. Nat. Acad. Sci., Vol. 88, pp. 7978-7982 Biomagnetic Techniques in Molecular Biology: Technical handbook. Dynal A. S, 2.sup.nd edition, 1995 Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard H R, Plückthun A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods. 201(1):35-55 Lilja H, Christensson A, Dahlén U, Matikainen M T, Nilsson O, Pettersson K, Lövgren T. (1991) Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin. Clin Chem. 37(9):1618-25 Pajunen M, Saviranta P, Jauria P, Karp M, Pettersson K, Mäntsälä P, Lovgren T. (1997) Cloning, sequencing, expression and characterization of three anti-estradiol-17beta Fab fragments. Biochim Biophys Acta. 1351(1-2):192-202 Väisänen V, Eriksson S, Ivaska K K, Lilja H, Nurmi M, Pettersson K. (2004) Development of sensitive immunoassays for free and total human glandular kallikrein 2. Clin Chem. 50(9):1607-17
Example 2—Humanisation of the 11B6 Antibody
(33) The variable domain of the murine anti-hK2 antibody 11B6 was humanised using CDR-grafting method. In this approach, the complementarity determining regions (CDR) of the murine antibody were grafted to the variable heavy and light domain frameworks. In addition residues at CDR regions, the residues in the certain critical positions at the framework regions were retained as murine-like rather than turned to human-like in order to maintain the conformation of grafted CDR loops as similar as possible to their conformation in the parental murine antibodies.
(34) Kabat numbering scheme (Kabat et al., 1991) is used throughout this description.
(35) Homology Modelling
(36) An homology model of the murine 11B6 antibody was generated by using automatic
(37) Web antibody modelling—Server (VAM; http://antibody.bath.ac.uk/index.html). The model was used for visual inspection based evaluation of the importance of the residues differing in between the parental murine antibodies and the human immunoglobulin sequences used as frameworks for the variable domain humanization, respectively.
(38) Design of the 11B6 Humanised V-Domain Sequences
(39) V.sub.L Domain Design
(40) The amino acid sequence of the murine 11B6 light chain variable domain was compared to the database of human immunoglobulin germline sequences in NCBI using ClustalW sequence alignment program. 11B6 V.sub.L was found share the highest similarity with the human germline gene B3 (IGKV4-1*01), the only member of the human V.sub.K4 family. Concerning the J-segment encoding the C-terminal part of the variable domain sequence, the human J.sub.K2 was found to be the most similar with the corresponding region of the murine 11B6.
(41) The human B3 gene together with sequence of IGKJ2 were used as a framework for the grafting of the CDR-loops (
(42) V.sub.H Domain Design
(43) The amino acid sequence of the murine 11B6 heavy chain variable domain was compared to the database of human immunoglobulin germline sequences in NCBI using clustalW sequence alignment program. 11B6 V.sub.H was found have the highest similarity with the human V.sub.H4 family member VH4-28. Concerning J-segment encoding the C-terminal part of the variable domain sequence, the human J.sub.H1 was found to be the most similar with the corresponding region of the murine 11B6.
(44) The human VH4-28 gene together with sequence of J.sub.H1 were used as a framework for the grafting of the CDR-loops (
(45) Murine-like residues asparagine and threonine were introduced at the positions 27 and 30 of V.sub.H, respectively. Although not belonging to CDR-H1 according to the Kabat definition (Kabat et al., 1991;
(46) The genes encoding the humanized 11B6 as Fab fragment, where the designed V.sub.H and V.sub.L domains were joined to the human C.sub.H1 and human C.sub.K constant domains, respectively, were purchased as a synthetic construct (Genscript, US). The genes were cloned into the expression vector pAK400Fab modified from pAK400 (Krebber et al., 1997) using Sfil restriction enzyme having recognition sites on the either side of the Fab cassette. The vector was transformed into E. coli XL-1 blue cells for the expression of the humanised Fab fragment.
(47) The sequences of the heavy and light chain variable regions of the exemplary humanised 11B6 Fab fragment of the invention are shown in
REFERENCES
(48) Chothia, C., Lesk, A. M., Tramontano, A., Levitt, M., Smith-Gill, S. J., Air, G., Sheriff, S., Padlan, E. A., Davies, D., Tulip, W. R., Colman, P. M., Spinelli, S., Alzari, P. M., and Poljak, R. J. (1989) Conformations of immunoglobulin hypervariable regions Nature, 342, 877-883 Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S and Foeller, C. (1991) Sequences of Immunoglogical Interest, 5th edit., NIH, Bethesda, Md. Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard H R, Plückthun A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods. 201(1):35-55 Tramontano, A., Chothia, C. and Lesk, A. M. (1990) Framework Residue 71 is a Major Determinant of the Position and Conformation of the Second Hypervariable Region in the V.sub.H Domains of Immunoglobulins. J. Mol. Biol. 215, 175-182
Example 3—Expression and Purification of h11B6
(49) HEK293 cells were expanded in to a 2 L suspension culture in FreeStyle 293 Expression Medium (Life Technologies). The cell density was on the day for transfection 1×10.sup.6 cells/ml.
(50) The nucleotide sequences encoding the component heavy or light chains (i.e. SEQ ID NOs: 14 and 15, respectively) were codon-optimized for expression in mammalian cells, synthesized and cloned to IgG expression vectors. The plasmid DNA (expression vector) containing the nucleotide sequences for the heavy and light chains was then mixed with the transfection agent and incubated for 10 min in RT. The DNA-transfection agent-mix was slowly added to cell culture while slowly swirling the flask. The transfected cell culture was then incubated at 37° C., 8% CO.sub.2 on an orbital shaker platform rotating at approx. 135 rpm, for seven days.
(51) Culture medium was harvest by centrifugation and filtered through 5 μm, 0.6 μm and 0.22 μm filter systems.
(52) Antibodies were purified by Protein G chromatography and the buffer was changed to PBS pH 7.4 by dialysis; subsequently, the antibodies were concentrated by ultrafiltration.
(53) Concentration was measured by absorbance.
(54) DNA: Light chain: p11B6VLhV1hk (4300 bp) amount: 0.35 mg Heavy chain: p11B6VHhV1hIgG1 (4900 bp) amount: 0.6 mg
(55) The DNA amounts were not optimized.
(56) Transfection agent: proprietary (however, suitable commercially-available transfection agents are readily available, such as Xfect™ Transfection Reagent (Clontech), Lipofectamine (Life Technologies), FuGENE® HD Transfection Reagent (Promega), FreeStyle™ Max Reagent (Invitrogen), DEAE-dextran, polyethylenimine and calcium phosphate).
(57) Overall yield: 13.1 mg (˜6.5 mg/L)
Example 4—Characterisation of h11B6: Affinity
(58) Aims of Study
(59) The aim of the study was to investigate the binding kinetics between four variants of the antibody 11B6 and the antigen hK2 by using the technique of Surface Plasmon Resonance (SPR) on a Biacore instrument.
(60) In order to investigate the quality of the protein samples (antibodies and antigen), a SDS-PAGE gel was run prior to the SPR experiments.
(61) In a Pre-Study, different parameters were investigated in order to find the appropriate conditions for the experiments in the Study.
(62) In the Study, multiple binding measurements were performed for the four antibodies and the antigen. From the collected data, the association and dissociation rate constants (k.sub.on and k.sub.off) and the dissociation constants (KD) were calculated and reported here.
(63) Reagents and Instrument Information
(64) Following solutions of the four antibodies and one antigen were provided by Diaprost AB: m11B6 stock: a-ehk211B6 14.12013 PP, 3.41 mg/ml: 0.9% NaCl, 100 μl h11B6 stock: Innovagen Lot 90476.30 2013-04-12, 1 mg/ml: PBS pH 7.4, 320 μl h11B6-DTPA stock: 0.2M Na-acetate pH 5.5, 0.9 mg/ml, 340 μl
(65) h11B6-DFO stock: 5 mg/ml gentisin acid in 0.2M ammonium acetate pH 5.5, 1.6 mg/ml, 400 μl
(66) hK2 stock: 26.6 μg/ml frakt 2 fr 7 SL+protein inh 5/2-02 1% BSA
(67) All the samples were aliquoted and kept in −20° C. freezer prior to analysis.
(68) All binding experiments were performed on CM4 chip on a Biacore 3000 instrument. The chip and all the reagents needed for activation, immobilization, deactivation, binding and regeneration were purchased from GE Healthcare and used according to the guidelines from the manufacturer.
(69) SDS-PAGE
(70) (a) Description of the Experiment
(71) The reagents provided by Diaprost AB were run on a TRIS-Tricine 10-20% acrylamide gel from Novex according to the guidelines from the manufacturer.
(72) Two series of the protein samples, native and reduced, were run simultaneously on the same gel together with a standard sample.
(73) Each sample in the native series contained: 1-1.3 μg of the protein, TRIS-buffer pH 8.8, SDS and loading buffer.
(74) Each sample in the reduced series contained: 1-1.3 μg of the protein, TRIS-buffer pH 8.8, SDS, loading buffer and 0.04% v/v beta2-merkaptoethanol (the reducing agent).
(75) The staining of the gel was performed in commasie brilliant blue solution of acetic acid, ethanol and water with the corresponding proportions of 0.7, 3.0, 6.3.
(76) The destaining of the gel was performed in the solution of acetic acid, ethanol and water with the corresponding proportions of 0.7, 3.0, 6.3.
(77) (b) Results & Conclusions
(78) The results are shown in
(79) It is evident from these results that the antibody and antigen samples are of high quality and purity.
(80) Affinity Study
(81) (a) Immobilisation of Antigen on a CM4 Chip
(82) Activation of the chip CM4-2 was performed according to manufacturer's guidelines for amine coupling using EDC and NHS mixture.
(83) A solution containing 2.96 μg/ml of the antigen hK2 (stock solution of hK2 diluted in 10 mM NaAc-buffert pH 3.8) was flown over channels fc2-4 on the chip CM4-2 in order to immobilize the antigen to the chip. Flow rate: 5 μl/min, volume: 200 μl.
Target RU≤M.sub.w/10 M.sub.w(hk2)=25 900 Da Target RU(hk2)≤2590
(84) Channel fc1 was used as a blank.
(85) The following immobilization was achieved:
fc2=1104 RU fc3=731 RU fc4=688 RU
(86) All channels (fc1-4) were blocked by ethanolamine after activation and immobilization.
(87) These data demonstrate that appropriate immobilization was achieved using 2.96 μg/ml of the antigen.
(88) (b) Investigation of the Association Phase
(89) The association phase of the four antibodies to the chip CM4-2 was followed for 4-5 minutes when solutions of 5 different concentrations of each antibody (stock solutions diluted in HSP-buffer) were flown over the channels fc2-4 on the chip CM4-2 with a rate of 30 μl/min.
(90) The investigated concentrations for each antibody were: 100, 50, 25, 12.5 and 6.25 nM.
(91) Additionally association data was obtained from the experiments where the dissociation process was followed for 480 minutes.
(92) In total, 18 individual association experiments for each antibody were performed.
(93) The signal from the blank, fc1, was subtracted for all the data.
(94) In
(95) We found that after 4-5 minutes, we were able to fit the data for the association processes.
(96) (c) Investigation of the Dissociation Phase
(97) The dissociation phase was followed for 480 minutes for each of the antibodies after flowing a solution of 50 nM of the antibody for 5 minutes over the channels fc2-4 on the chip CM4-2 with a rate of 30 μl/min (
(98) The signal from the blank, fc1, is subtracted in all the data used in the calculations of the dissociation rate constant.
(99) The data indicate that the dissociation processes are very slow. For all four antibodies, the signal in channel fc4 was drifting and the dissociation process could not be followed in that channel.
(100) (d) Estimation of the Dissociation Rate Constant (k.sub.off)
(101) The dissociation phase data was fitted and the dissociation rate constants (koff) were estimated (see Table 2).
(102) TABLE-US-00013 TABLE 2 K.sub.off(10.sup.−5 K.sub.off(10.sup.−5 K.sub.off(10.sup.−5 Std Antibody s.sup.−1)fc2 s.sup.−1)fc3 s.sup.−1)fc4 Mean dev m11B6 1.9 4.9 — 3.4 ±2.1 h11B6 6.4 6.9 — 6.7 ±0.4 h11B6- 6.3 19.1 — 12.7 ±9.1 DTPA h11B6- 5.8 5.5 — 5.7 ±0.2 DFO
(103) Based on the two measurements taken for each antibody, there appears to be no significant difference between the dissociation rate constants (k.sub.off) of the tested antibodies.
(104) (e) Estimation of the Association Rate Constant (k.sub.on)
(105) In order to estimate the association rate constants, the dissociation rate constants (Table 2) were used in the fitted equations.
(106) All fitted data was used in order to calculate an average value of the association rate constant and the standard deviation for each antibody (see Table 3).
(107) TABLE-US-00014 TABLE 3 Antibody No. of expts fitted Mean k.sub.on (10.sup.5M.sup.−1s.sup.−1) Std dev m11B6 18/18 2.48 ±0.85 h11B6 15/18 1.17 ±0.38 h11B6-DTPA 17/18 1.82 ±0.54 h11B6-DFO 18/18 1.11 ±0.22
(108) Based on the 15-18 measurements taken for each antibody, there appears to be no significant difference between the association rate constants (k.sub.on) of the tested antibodies.
(109) (f) Estimation of the Dissociation Constant (k.sub.D)
(110) Dissociation constant (K.sub.D) for each of the tested antibodies are shown in Table 4.
(111) TABLE-US-00015 TABLE 4 Antibody Mean K.sub.D 10.sup.−11 M Std dev m11B6 19 ±15 h11B6 65 ±25 h11B6-DTPA 93 ±78 h11B6-DFO 54 ±13
(112) The dissociation constants (K.sub.D) are in the 10.sup.−12 M range for all four antibodies.
(113) Although not statistically significant, the dissociation constant for the humanised antibody appears to be higher than that of the parent murine antibody.
(114) Conjugation of the humanised antibody does not appear to affect the affinity noticeably since the K.sub.D is not significantly changed for h11B6-DTPA or h11B6-DFO.
(115) Summary The association processes are very fast for all four antibodies and the association rate constants (k.sub.on) are all in the 10.sup.5 M.sub.−1 s.sup.−1 range based on 15-18 experiments for each antibody. The dissociation processes are very slow and almost in the range of technical limitations of Biacore. The dissociation rate constants (k.sub.off) are all in the 10.sup.−5 s.sup.−1 range based on two experiments for each antibody. The dissociation constants (K.sub.D) are in the 10.sup.−12 M range for all four antibodies.
Example 5—Characterisation of h11B6: Aggregation
(116) Executive Summary
(117) Dynamic light scattering (DLS) studies have been carried out on 4 variants of the IgG in order to study their propensity to aggregate. The DLS results show that all constructs have a reasonable size (200 kDa or slightly above 200 kDa assuming a spherical protein) and little or no aggregation.
(118) Objective
(119) To characterise four IgG constructs with respect to oligomeric state using dynamic light scattering. Insulin was used as a control.
(120) Results
(121) Dynamic Light Scattering
(122) Phosphate buffered saline (PBS pH 7.4) was filtrated through 0.22 micron filter. The delivered protein was diluted to 0.1 mg/ml in PBS pH 7.4. Dynamic light scattering was measured at 20° C. in duplicate samples using the Malvern APS equipment. Each sample was measured three times. The dilution buffer was used as control to make sure that the buffer was reasonably free from dust and aggregates,
(123) TABLE-US-00016 TABLE 5 Dynamic light scattering data derived from size distribution Average Polydispersity Mass distribution Construct radius (nm) (%) (%) Insulin 2.8 28 100 h11B6 5.7 15 99.2 M11B6 5.7 17 100 DFO-h11B6 6.0 22 99.9 H11B6-DTPA 6.1 22 100 Polydispersity = Standard deviation of radius/Average radius × 100%
(124) The insulin control (4 mg/ml 20 mM Na2HPO4, 10 mM Na3EDTA) have an average radius of 2.8 nm which is about 37 kDa. In solution insulin is known to form hexamers of about 35 kDa. A radius of 5.7 nm corresponds to a molecular weight of about 200 kDa for a protein having a perfect spherical shape. A radius of 6.1 nm corresponds to a molecular weight of about 230 kDa for a protein having a perfect spherical shape. This is reasonably close to the molecular weight of 150 kDa for IgG molecules, which means that most of the samples primarily consist of monomeric and/or dimeric IgG molecules. The reason for not excluding dimers is that light scattering give a rough size estimate based on molecular shape and this makes it difficult to separate monomers and dimers but easy to separate large aggregates from monomers or monomers from hexamers (as in the insulin case).
(125) Conclusions
(126) Dynamic light scattering shows that all constructs have a reasonable size and little or no aggregation. The size distributions for all four constructs are overlapping (data not shown).
Example 6—Characterisation of h11B6: In Vivo Biodistribution
(127) This study compares biodistribution in vivo of murine 11B6 and human 11B6 when labeled to .sup.177Lu.
(128) Material and Methods Materials
(129) .sup.177Lu was purchased from Mallinkrodt Medical B V, Petten, Holland.
(130) All chemicals were obtained from Sigma Aldrich and buffers were prepared in-house using analytical grade water (unless otherwise noted).
(131) The parent murine antibody m11B6, with specific for the human kallikrein 2, was obtained from the University of Turku, Finland.
(132) TABLE-US-00017 m11B6 heavy chain [SEQ ID NO: 23]: DVQLQESGPGLVKPSQSLSLTCTVTGNSITSDYAWNWIRQFPGNRLEWMG YISYSGSTTYSPSLKSRFSITRDTSKNQFFLQLNSVTPEDTATYFCATGY YYGSGFWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF PEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCN VAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLT PKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSEL PIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKE QMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFV YSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK m11B6 light chain [SEQ ID NO: 24]: DIVLTQSPASLAVSLGQRATISCRASESVEYFGTSLMHWYRQKPGQPPKL LIYAASNVESGVPARFSGSGSGTDFSLNIQPVEEDDFSMYFCQQTRKVPY TFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV KWKIDGSERQNGVLNSVVTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC
(133) A humanised counterpart antibody, h11B6, was produced as described in Examples 2 and 3 above (see
(134) For in vivo studies, the prostate carcinoma cell lines LNCaP expressing hK2 (ATCC, Manassas, Va., USA) and DU145 (ATCC, Manassas, Va., USA) were used. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and PEST (penicillin 100 IU/ml and 100 μg/ml streptomycin). The cells were maintained at 37° C. in a humidified incubator with 5% CO.sub.2 and were detached with trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer, Thermo Scientific). Matrigel matrix from BD-biosciences (San-Jose, Calif., USA) was used when xenografting LNCaP cells. NMRI-Nu, (Charles River) and Balb/c-Nu (in house bread) mice were inoculated with the two cell lines.
(135) Conjugation and Radiolabelling
(136) Conjugation of CHX-A″-DTPA with 1186: Solutions of the murine and humanised 11B6 mAbs in PBS was adjusted to pH 9.2 using 0.07 M sodium borate buffer, prior to being concentrated on an Amicon Ultra-2 centrifugal filter (2 ml, 100 K). The resultant protein solution was then conjugated with chelator CHX-A″-DTPA (Macrocyclics, USA) in a molar ratio of 3:1 chelator to antibody at 40° C. The reaction was terminated after 4 h and CHX-A″-DTPA-11B6 (DTPA-11B6) was separated from free chelate by size-exclusion chromatography on a NAP-5 column (GE Healthcare), equilibrated with 20 ml 0.2 M ammonium acetate buffer, pH 5.5. The conjugated 11B6 antibodies were eluted with 1 ml ammonium acetate buffer.
(137) Radiolabeling of DTPA-11B6: Murine and humanised DTPA-11B6, in ammonium acetate buffer pH 5.5 was mixed with a predetermined amount of .sup.177LuCl.sub.3. A final activity of 0.5-0.6 MBq per subject was used for biodistribution or a final activity of 18-20 MBq per subject was used for SPECT studies. After incubation at room temperature for 2 h, the labeling was terminated and purified on a NAP-5 column, equilibrated with PBS
(138) Animal Studies
(139) All animal experiments were performed in accordance with national legislation on laboratory animals' protection.
(140) Male immunodeficient nude mice, NMRI-Nu, (6-8 wk old) and Balb/c-Nu, were used for this study. All mice were xenografted with LNCaP cells or DU145 on their left or right flank, 8-10 million cells, in 100 μl growth medium and 100 μl Matrigel.
(141) Biodistribution Studies
(142) Biodistribution studies were performed on both h11B6 and m11B6.
(143) Six groups (n=4) of mice were injected intravenously with either 20 μg of h11B6 or 20 μg m11B6 labeled with .sup.177Lu. The animals were sacrificed at 24 h p.i., 48 h p.i and 72 h p.i. and organs of interest were analysed with an automated NaI(Tl) well-counter with a 3-inch NaI (Tl) detector (1480 WIZARD, Wallac Oy, Turku, Finland).
(144) The tissue uptake value, expressed as percent injected dose per gram tissue (% IA/g), was calculated as:
% IA/g=(tissue radioactivity/injected radioactivity)/organ weight×100 wherein for iv injections:
Injected radioactivity=Average radioactivity in control syringes−radioactivity in used syringe−radioactivity in tail
(145) The organs were also weighed following dissection.
(146) Kinetic Data
(147) D The time-% ID curve is represented as a straight line ID(t)=k*t+m up to 48. Based on the data from the second and third time-points (48 respectively 72 h), a mono-exponential curve (ID(t)=ID(0)e.sup.−λt) is applied for the time interval [48, ∞[. If, however, lambda becomes a negative value, i.e. that the ID is increasing between 48 and 72 h, the time ID curve in this time-interval is instead modeled as a straight line, and the pharmaceutical is assumed to be retained in the organ from 72 hours and onwards. To obtain the time-activity curves, the physical half-life is applied.
(148) Note—in some figures, ID is termed “IA”; these expressions are used interchageably herein.
(149) Results
(150) Biodistribution of Humanised .sup.177Lu-11B6
(151) The biodistribution of .sup.177Lu-h11B6 is shown in
(152) The antibody rapidly accumulates in the LNCaP tumour within by 24 hours, and the radioactivity remains high at 72 hours.
(153) Initially, high levels of h11B6 are also evident in the blood and kidneys, which reduce over the following 48 hour period as the antibody is cleared from the body. Such biokinetics are an inevitable and expected consequence of intravenous injection of any radiolabelled antibody.
(154) All other organs, such as bone and muscle, show low levels of radioactivity.
(155) These data demonstrate that .sup.177Lu-h11B6 can effectively target prostate cancer cells in vivo.
(156)
(157) .sup.177Lu-h11B6 exhibits an unexpectedly better therapeutic ratio
(158) Comparison of the biodistribution data for humanised .sup.177Lu-11B6 with that for the parent murine antibody (.sup.177Lu-m11B6) revealed an unexpected and advantageous difference.
(159) As shown in
(160)
(161) Dosimetry Calculations
(162) Calculation of absorbed doses provides a more sophisticated measure of differences in the kinetics of the humanised antibodies of the invention relative to those of the parent murine 11B6 antibody.
(163) The absorbed dose was calculated according to the MIRD-schema D=(r.sub.T←r.sub.s)=Ã(r.sub.s).Math.S(r.sub.T←r.sub.s), where à is the total number of disintegrations in an source organ, and S is the absorbed dose per unit of disintegrations (see Bolch et al., 2009, J. Nucl. Med. 50:477-484, the disclosures of which are incorporated herein by reference). The à was calculated as the time-integral over the time-activity curve. The S-factor was based on mice-specific Monte Carlo simulations using the Moby-phantom (see Larsson et al., 2011, Acta Oncol. 50:973-980 and Keenan et al., 2010, J. Nucl. Med. 50:471-476). To get the total absorbed dose, all organs were considered as being source—as well as target sources.
(164) Calculated absorbed dose values for different tissues are shown in Table 6.
(165) TABLE-US-00018 TABLE 6 Absorbed dose (Gy/MBq) Organ m11B6 h11B6 Blood 0.915101 1.16768 Heart 1.11217 0.756549 Lung 0.445617 0.677907 Liver 0.2652 0.447948 Spleen 0.396808 0.612251 Intestines 0.189362 0.426528 Kidney 0.291966 0.913515 Bone 0.193169 0.179905 Brain 0.0332977 0.0556325 Testes 0.178707 0.516442 Tumour 1.21858 2.20389 Bone 0.309455 0.385407 (marrow)
(166) As can been seen from Table 6, tumour absorbed dose increases from 1.21 Gy/MBq for m11B6 to 2.2 Gy/MBq for h11B6, i.e. an increase with 80%. The ratios of tumour to bone marrow absorbed doses increases from 3.9 for m11B6 to 5.6 for h11B6 about 40%. Herein, the enhanced therapeutic efficacy for h11B6 compared to m11B6 is shown and indicates that higher absorbed doses to the tumor can be given with less normal organ toxicity.
(167) Antibody Clearance from the Blood
(168) Analysis of blood levels for the humanised and murine 11B6 antibodies is shown in
(169) The results suggest that h11B6 may be cleared from the blood slightly quicker than the murine 11B6 antibody. If so, such an enhanced clearance rate for the humanised antibody may also of therapeutic benefit from a safety perspective, potentially allowing higher activities to be administered.
(170) An enhanced clearance rate may also be beneficial for external imaging
(171) Conclusions
(172) The results of this study demonstrate the following: the humanised 11B6 antibody, .sup.177Lu-h11B6, effectively targets prostate tumours in vivo; the humanised 11B6 antibody exhibits an unexpectedly better therapeutic ratio than its parent murine antibody (as determined by the ratio of uptake in tumours to uptake in healthy bone); and the humanised 11B6 antibody may be cleared from the blood slightly quicker than the murine 11B6 antibody.
(173) Taken together, these findings provide compelling evidence of the enhanced therapeutic efficacy of humanised 11B6 antibodies in the treatment (and diagnosis) of prostate cancer.
(174) Since the humanised and murine antibodies are targeted to the same antigen (namely, human kallikrein 2), the calculated difference in the update ratio in tumour to healthy bone marrow cannot readily be predicted or explained (particularly given that the humanised antibody appears to exhibit a lower affinity for the target hK2 antigen compared to the parent murine antibody; see Example 4).
(175) The difference in the relative uptake in tumour compared to healthy bone between the humanised and murine 11B6 antibodies is of considerable importance since this comparison provides a measure of the therapeutic ratio. A higher value for this ratio (as evident for the humanised 11B6 antibody) is indicative of a better therapeutic antibody. In particular, a higher therapeutic ratio means that higher absorbed doses of therapeutically-radiolabelled h11B6 can be administered to achieve a better therapeutic effect (since binding of the humanised antibody to healthy tissue and organs is much lower than for the murine antibody). The higher ratio also indicates that h11B6 will be better than the murine antibody for diagnostic purposes (since it equates to a lower signal to noise ratio, allowing imaging of smaller tumours including metastases).
(176) In conclusion, the data demonstrate that humanisation of the 11B6 antibody gives an enhanced possibility for early diagnosis and an unexpectedly higher therapeutic efficacy in the treatment prostate cancer.
Example 7—Demonstration of Diagnostic and Therapeutic Efficacy
(177) The aim of this study was to confirm the utility of 11B6, a mAb that specifically targets an epitope inside the catalytic cleft of hK2, as a vehicle to deliver highly toxic radionuclides specifically to the sites of prostate cancer growth. In this proof of concept study, we labelled the parent murine 11B6 antibody with 177Lu, a low energy beta particle that also employs gamma emission, enabling SPECT-imaging to be performed.
(178) Materials & Methods
(179) Materials
(180) .sup.177Lu was purchased from Mallinkrodt Medical BV, Petten, Holland. The Cyclone™ Storage Phosphor System and the OptiQuant™ image analysis software (Perkin Elmer, Wellesley, Mass., USA) was used to measure the radioactivity on the ITLC (instant thin layer chromatography) strips (Biodex, US) for determining labeling kinetics and radiochemical purity. All chemicals were obtained from Sigma Aldrich and the buffers were in-house prepared using analytical grade water if not otherwise noted. The mAb 11B6 is an antibody specific for the human kallikrein 2 with an affinity for this antigen of about 1.2 nM; see
(181) Conjugation and Radiolabeling
(182) Conjugation of CHX-A″-DTPA with 11B6: A solution of the mAb 11B6 in PBS was adjusted to pH 9.2 using 0.07 M sodium borate buffer. The sample was concentrated on an Amicon Ultra-2 centrifugal filter (2 ml, 100 K). The protein solution was conjugated with the chelator CHX-A″-DTPA (Macrocyclics, USA) in a molar ratio of 3:1 chelator to antibody at 40° C. The reaction was terminated after 4 h and CHX-A″-DTPA-11B6, from now on called DTPA-11B6, was separated from free chelate by size-exclusion chromatography on a NAP-5 column (GE Healthcare) equilibrated with 20 ml 0.2 M ammonium acetate buffer, pH 5.5. Conjugated 11B6 and 5A10 was eluted with 1 ml ammonium acetate buffer.
(183) Radiolabeling of DTPA-1186: DTPA-11B6 in ammonium acetate buffer pH 5.5 was mixed with a predetermined amount of .sup.177LuCl.sub.3. After incubation at room temperature for 2 h, the labeling was terminated and purified on a NAP-5 column, equilibrated with PBS. Labeling efficiency and labeling kinetics were monitored with ITLC strips, eluted with 0.2 M citric acid. In this system, the radiolabelled conjugate remains at the origin line, while free Lu-177 migrates with the front of the solvent. The radioactivity distribution was determined with a Phosphorlmager system (Perkin Elmer, Wellesley, Mass., USA) using the Optiquant as quantification software (Perkin Elmer, Wellesley, Mass., USA).
(184) Animal Studies
(185) All animal experiments were performed in accordance with national legislation on laboratory animals' protection. The animal study has been approved by the local Ethics Committee for Animal Research. Male immunodeficient nude mice, NMRI, (6-8 wk old) purchased from Taconic Europe (Bomholt, Denmark) were used for this study.
(186) Xenografts of hK2-expressing LNCaP prostate carcinoma cells were subcutaneously implanted in the right flank and/or left flank at about 10*10.sup.6 cells per injection.
(187) Animals that developed LNCaP tumors were divided into groups and injected with either the therapeutic agent 177Lu-DTP-11B6 or with a control, see Table 7 below:
(188) TABLE-US-00019 TABLE 7 Group Animals nr Treatment 5 animals/ 1 NaCl (control) group 2 Unspecific Ab labeled with 177Lu-low 11 groups absorbed dose Total = 55 3 Unspecific ab labeled with 177Lu-high animals absorbed dose 4 Only 177Lu-low absorbed dose 5 Only 177Lu-high absorbed dose 6 177Lu-DTPA-m11B6: A/4 7 177Lu-DTPA-m11B6: A/2 8 177Lu-DTPA-m11B6: A 9 177Lu-DTPA-m11B6: 2*A 10 177Lu-DTPA-m11B6: 3*A 11 Only m11B6 A = 26.7 MBq
(189) All animals included were continuously measured and weighed within an interval of 3-4 days.
(190) Initially some animals got a lower activity (8 MBq) of .sup.177Lu-DTPA-11B6 for investigation of the localization of the therapeutic agent using SPECT. One mouse from group 8 was also studied with SPECT. These animals had their organs removed and an automated NaI(Tl) well-counter with a 3-inch NaI (Tl) detector (1480 WIZARD, Wallac Oy, Turku, Finland) was used to quantify radioactivity in these tissue samples.
(191) To study the effect on the bone marrow blood samples (10 μL) were taken regularly. Blood samples were collected twice a week for 8 weeks post-injection and WBC counts, RBC counts, and platelet counts were analyzed in a Medonic Cell Analyzer-Vet CA530 Vet (Boule Medical, Stockholm, Sweden). At the time of blood sampling, the weight and physical condition of the animals were monitored. Toxicity was evaluated by monitoring animals for loss of body weight, decline in general condition, and hematologic toxicity.
(192) Tumor volume was measured with a caliper. The length l, with w and thickness t were measured and the volume was calculated.
(193) Therapy Planning
(194) Based on the relationship between absorbed dose and biological effect on the bone marrow in rats undergoing Radioimmunotherapy with 90Y and 177Lu (see Larsson et al., 2012, Med. Phys. 39(7):4434-43), it could be estimated that the LD50 for bone marrow would be in the order of 12 Gy. In the literature LD50 for acute irradiation of rats and mice are the same, about 9 Gy (for example, see Radiobiology for the radiologist, Hall & Giacca (Eds), 2006, 6.sup.th edition).
(195) The therapies were then designed from the assumption of a tolerable absorbed dose of 12 Gy to bone marrow. Then, from the dosimetry calculations the activity corresponding to this absorbed dose was calculated.
(196) Corresponding doses/activities were used for the controls.
(197) Results
(198) Animal Tumor Shrinkage
(199)
(200) Radioimmunotherapy Results
(201)
(202) There is a clear trend of decrease of tumor volume in both treatment groups. The onset of tumor shrinkage is seen already a few days after injection of 177Lu-m11B6. In the control group there is an increase of tumor volume after the injection of NaI solution.
(203)
(204) In the SPECT study (8 d pi) the tumor volume is shown with still activity present; see
(205) Conclusion
(206) The present study with exemplary antibody 177Lu-m11B6 clearly demonstrates the therapeutic efficacy of hK2-targeted antibodies against prostate cancer tumours in vivo.
Example 8—Therapeutic Efficacy of an Exemplary .SUP.177.Lu-Labeled Humanised 11B6 Antibody of the Invention in Prostate Cancer Xenografts
(207) Materials & Methods
(208) Antibodies, Conjugation and Radiolabeling
(209) Antibodies: The exemplary humanised monoclonal antibody 11B6 (IgG1/kappa, transient expressed in HEK 293 cells), comprising a heavy chain according to SEQ ID NO:12 and a light chain according to SEQ ID NO:13, was provided by Innovagen AB, Lund (1 mg/ml in PBS pH 7.4, Lot No. 90476.30). A non-specific IgG antibody was utilised as an isotype control (IgG antibody from mouse serum, Sigma I-8765).
(210) Conjugation: The exemplary h11B6 non-specific IgG control antibody were conjugated with the chelator CHX-A″-DTPA (Macrocyclics, USA) as followed: A solution of the antibody was concentrated on an Amicon Ultra-2 centrifugal filter (2 mL, 100 K) and was later adjusted to pH 9.2 using 0.07 M sodium borate buffer (Sigma Aldrich).
(211) Coupling of the chelator compound CHX-A″-DTPA to the protein solution in a molar ratio of approximately 3:1 (chelator to antibody) was performed similarly to a previously described method (see Almqvist et al). The coupling efficiency, i.e. number of obtained chelators per antibody can be determined by a spectrophotometric method (Pippin et al) but was not analysed in this study. However, the coupling preferably should not exceed 3 chelators/antibody in order to avoid damage to the protein. The chelator was added to the protein and the solution was incubated with gentle shaking at 40° C.
(212) The reaction was terminated after 4 h and CHX-A″-DTPA-h11B6, referred to as DTPA-h11B6, was separated from free chelate by size-exclusion chromatography on a NAP-5 column (GE Healthcare) equilibrated with 20 ml 0.2 M ammonium acetate buffer (Sigma Aldrich), pH 5.5. Conjugated h11B6 was eluted with 1 ml ammonium acetate buffer and aliquoted samples were stored at −20° C.
(213) Conjugation of the IgG control antibody was controlled in the similar way as above.
(214) Radiolabeling: Conjugated h11B6 or IgG control antibody (typically 200-300 μL of ˜1 μg/μL in 0.2 M sodium acetate buffer pH 5.5) was mixed with a predetermined amount (˜200-300 MBq) of .sup.177LuCl.sub.3 (IDB Holland) and incubated at room temperature for 1.5-2 h. After incubation, the labeling was terminated and purified on a NAP-5 column (GE Healthcare), equilibrated with PBS (Thermo Scientific). Labeling efficiency was monitored with instant thin layer chromatography (Biodex, USA), eluted with 0.2 M citric acid (Sigma Aldrich). In this system, the radiolabeled conjugate remains at the origin line, while free .sup.177Lu migrates with the front of the solvent. The radioactive distribution was determined with a Cyclone Storage Phosphor System using the Optiquant as quantification software (both from Perkin Elmer).
(215) The radiolabeling of the IgG control antibody was performed in the similar way as above.
(216) Therapy Study
(217) Cell Lines: LNCaP (hK2+) were purchased from American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 medium (Thermo Scientific) supplemented with 10% fetal bovine serum (Thermo Scientific) with penicillin 100 IU/mL and 100 μg/mL streptomycin (Thermo Scientific). The cells were maintained at 37° C. in a humidified incubator at 5% CO.sub.2 and were detached with trypsin-EDTA solution (Thermo Scientific).
(218) All animal experiments were conducted in compliance with the national legislation on laboratory animals' protection, and with the approval of the Ethics Committee for Animal Research (Lund University, Sweden). In-house bred male immunodeficient Balb/c nude mice (6-8 weeks of age) were used. Mice were xenografted with LNCaP cells on their right flank by s.c. injection (8-10 million cells) in 100 μL growth medium and 100 μL Matrigel (BD Matrigel™ Basement Membrane Matrix Growth Factor Reduced, Phenol Red Free, Cat No 356231). Mice with established tumors having a diameter of at least ˜3 mm were included in the study and divided into the three groups described below in Table 8. The animals were i.v. injected in the tail vein. The 20 MBq activity-level was chosen because doses at this amount have been used in a study with m11B6, showing good therapeutic effect (see Example 7).
(219) TABLE-US-00020 TABLE 8 Three groups of animals were included: One group injected with .sup.177Lu-h11B6, one with .sup.177Lu-unsepcific mAb (to show the specificity of h11B6), and one with NaCl (as a control group). Group n Treatment Activity (MBq) 1 12 .sup.177Lu-h11B6 20 2 10 .sup.177Lu-unspecific mAb 20 3 10 NaCl —
(220) The therapeutic efficacy was assessed by repeated measurement of the tumour size using a caliper. The tumour volume was calculated by measuring the length (L) and the width (W) of the tumor and then calculating the volume V as 0.5×L×W×W.
(221) Also, hematological (white blood cell counts, red blood cell counts, platelet number and haemoglobin counts) and weight measurements were taken repeatedly for all animals in order to identify any potential hematological toxicity and to monitor the animals' general condition. The hematological toxicity is especially important to monitor when evaluating radioimmunotherapy since the radioactivity will be distributed in the blood and finally reach the bone marrow, where the blood stem cells are situated.
(222) Mice that developed a tumour length/width exceeding 14 mm, or a weight loss exceeding 15% compared to the initial weight, or otherwise had a negatively affected general condition, or had a combination of all these three parameters, were terminated according to the ethical guidelines.
(223) Results
(224) Assessment of Therapeutic Efficacy
(225) As shown in
(226) The data from the individual animals shown in
(227) Assessment of Hematological Toxicity
(228) Assessment of white blood cell counts, red blood cell counts, platelet number, haemoglobin counts and weight did not reveal any toxicity effect of administration of .sup.Lu-hl11B6 (data not shown).
(229) Discussion
(230) The results of this study reveal a significant therapeutic effect of .sup.177Lu-h11B6 treatment in the prostate cancer xenograft model.
(231) The activity administered to the mico (20 MBq) corresponds to an absorbed dose to the bone marrow of approximately 10 Gy, which was well-tolerated by these animals. Even at this low activity of 20 MBq, a large therapeutic effect was observed. As estimated earlier, a tumour absorbed dose of at least 60 Gy can be expected.
(232) No indications of hematological toxicity were observed.
REFERENCES
(233) Almqvist Y., et al. In vitro and in vivo characterization of .sup.177Lu-huA33: a radio-immunoconjugate against colorectal cancer. Nucl Med Biol. 2006; 33:991-998. Pippin C G et al. Spectrophotometric method for the determination of a bifunctional DTPA ligand in DTPA-monoclonal antibody conjugates. Bioconjug Chem. 1992; 3:342-5.
Example 9—Radionuclide Therapy Dosimetry Planning and Treatment of Prostate Cancer in a Patient
(234) For radionuclide therapy (RNT), the radiation source is distributed in the whole and the radioactivity is normally administered systemically as a radiopharmaceutical. The radioactivity distribution depends on the amount of radiopharmaceutical that accumulates over time in different tissues, something which varies between patients (1).
(235) RNT treatment should be based on a prescribed absorbed dose (2). Then first one should perform a pre-therapy study using a tracer amount of the radiopharmaceutical, and determine the tumor and organ absorbed doses. Usually, this information is expressed as a factor describing the organ absorbed dose per unit administered activity, in units of mGy/MBq; D.sup.P.sub.T(organ).
(236) If the therapeutic administration is then given under similar conditions, this factor can be used to determine the activity that needs to be administered in order to deliver a prescribed absorbed dose to a given organ, tissue or tumor (4,6).
(237) In the case of prostate cancer treatment with radiolabelled h11B6 antibodies, a pre-therapy study should be based on .sup.111In imaging with .sup.111In-h11B6. .sup.111In is best suitable for quantitative (planar/SPECT) imaging when then .sup.177Lu is to be the therapeutic radionuclide. When then the D.sup.P.sub.T(organ) is determined the therapy can be given with a therapy activity A.sub.T giving a prescribed therapy effect. During therapy, the activity distribution and corresponding dose rate should be calculated based on imaging to get the actual therapy absorbed dose given to tumor and normal organs, necessary for evaluation of treatment.
(238) In case of therapy where the bone marrow toxicity level is reached as a result of the treatment planning then bone-marrow support is necessary and based on dosimetry calculations for the bone marrow cavity the time for reinfusion of stem cells has to be determined.
(239) In summary, the following treatment scheme should be planned accordingly:
(240) Pre-Therapy Dosimetry Study 1. 111In-labeled h11B6 (200-300 MBq) injection 2. Blood sampling—activity concentration in blood and plasma determined first week. 3. Imaging (SPECT/Planar) over 1 week (7 times) 4. Organ Dosimetry based on LundaDose scheme (3) 5. Therapy activity determined limited by specified absorbed dose to radiosensitive organs as bone marrow (2-3 Gy), kidneys (20-30 Gy) and liver (12-36 Gy).
(241) Therapy Including Intra-Therapy Dosimetry 1. 177Lu-labeled h11B6 administered (based on pretherapy dosimetry) 2. Blood sampling—activity concentration in blood and plasma 3. Imaging over 1 week (6 times) 4. Organ Dosimetry=>Verification of prescribed therapy absorbed dose.
(242) Specific Comments on Dosimetry
(243) The cumulated activity is the number of decays that occur in a given region over a period of time. The unit is Bq s, or Bq h. When ionizing radiation travels through matter, it interacts and deposits energy. The energy imparted is the sum of all energy deposits in a given volume. The absorbed dose is the ratio of the mean energy imparted and the mass of the volume. The unit of absorbed dose is Gray (Gy), 1 Gy equals 1 J/kg.
(244) From the values of the activity in a tissue at different times, the cumulated activity is determined by integration, and the mean absorbed dose can be determined. Activity measurements are made using planar imaging for whole-organ dosimetry. Quantitative SPECT/CT allows for dosimetry in smaller volumes using voxel-based methods.
(245) From the 3D distribution of activity concentration values, the absorbed dose rate distribution can be calculated using so-called point dose kernels or voxel S values, describing the energy deposition pattern around a point source located in water (or bone). This method assumes that the anatomical region is homogeneous in terms of density, such as soft tissues within the trunk. For body regions where the density is heterogeneous, as in the lungs, a direct Monte Carlo calculation is preferable. Here, the activity distribution from SPECT or PET is used as input to a Monte Carlo dose calculation code.
REFERENCES
(246) 1. Strand S-E, Zanzonico P, Johnson T K. Pharmacokinetic modeling. Med Phys 1993; 20(2):515-27 2. ICRU report nr 67—Dose Specifications in Nuclear Medicine. Adelstein S J, DeLuca P, Feinendegen L E, Green L, Howell R W, Humm J L, Strand S E ICRU; 2002 3. The LundADose Method for Planar Image Activity Quantification and Absorbed-Dose Assessment in Radionuclide Therapy. Sjogreen, K., Ljungberg, M., Wingardh, K., Minarik, D., and Strand, S. E. (2005): Cancer Biother. Radiopharm., 20:92-97 4. Quantitative imaging for clinical dosimetry. Bardies M, Flux G, Lassman M, Monsieurs N, Savolainen S, Strand S-E Nucl Instr and Methods 2006:569:467-471. 5. 177Lu-[DOTA0, Tyr3] octreotate therapy in patients with disseminated neuroendocrine tumors: Analysis of dosimetry with impact on future therapeutic strategy. Garkavij Michael, Nickel Mattias, Sjögreen-Gleisner Katarina, Ljungberg Michael, Ohlsson Tomas, Wingårdh Karin, Strand Sven-Erik, Tennvall Jan. Cancer 2010:116(4 Suppl):1084-92. 6. Dosimetry in patients with B-cell lymphoma treated with [(90)Y]ibritumomab tiuxetan or [(131)I]tositumomab Sjögreen-Gleisner K., Dewaraja Y K., Chisea C., Tennvall J., Lindén O., Strand S E, Ljungberg M. Q J Nucl Med Mol Imaging, 2011 April; 55(2):126-54.