Radio-labelled antibody fragments for use in the prognosis, diagnosis of cancer as well as for the prediction of cancer therapy response
11660356 · 2023-05-30
Assignee
Inventors
Cpc classification
C07K2317/569
CHEMISTRY; METALLURGY
A61K51/1078
HUMAN NECESSITIES
A61K51/1072
HUMAN NECESSITIES
A61K51/1069
HUMAN NECESSITIES
A61K51/1045
HUMAN NECESSITIES
A61K51/1051
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K51/10
HUMAN NECESSITIES
Abstract
The application provides polypeptides comprising or essentially consisting of at least one heavy chain variable domain of a heavy chain antibody (V.sub.HH) or a functional fragment thereof, wherein said V.sub.HH or a functional fragment thereof specifically binds to a target protein that is present on and/or specific for a solid tumor, e.g. HER2. The application further provides nucleic acids encoding such polypeptides; methods for preparing such polypeptides; host cells expressing or capable of expressing such polypeptides; compositions, and in particular to pharmaceutical compositions, that comprise such polypeptides, nucleic acids and/or host cells. The application further provides such polypeptides, nucleic acids, host cells and/or compositions, for use in methods for detection, imaging, prognosis and diagnosis of cancer as well as for predicting patient response(s) to therapeutics.
Claims
1. A method for identifying a HER-2 positive metastatic brain lesion in a human subject, comprising (a) selecting a human subject previously identified as having cancer, (b) administering to the subject of step (a) a radiolabelled heavy chain antibody (V.sub.HH) or a functional fragment thereof, which specifically binds to HER2, wherein the amino acid sequence of said V.sub.HH or functional fragment thereof comprises the combination of a CDR1 region having SEQ ID NO: 1, a CDR2 region having SEQ ID NO: 2, and a CDR3 region having SEQ ID NO: 3, and (c) measuring using PET/CT imaging the ability of said V.sub.HH or functional fragment thereof to bind HER2 in the brain of the subject, wherein the V.sub.HH or a functional fragment thereof is labelled with a radioisotope selected from the group consisting of 68Ga, 18F, 64Cu, 86Y, 76Br, 82Rb, 209At, and 210At, wherein the subject is identified as having a HER-2 positive metastatic brain lesion when the V.sub.HH or functional fragment thereof binds to HER2 in the brain of the subject.
2. The method of claim 1, wherein said V.sub.HH or functional fragment thereof is labelled with a radioisotope selected from the group consisting of 68Ga, and 64Cu.
3. The method of claim 1, wherein said V.sub.HH or functional fragment thereof has at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof.
4. The method of claim 1, wherein said subject of step (a) was previously identified as having breast cancer.
5. The method of claim 1, wherein said V.sub.HH or functional fragment thereof is administered intravenously or intraperitoneally.
6. The method of claim 1, wherein said V.sub.HH or functional fragment thereof is in a monovalent format.
7. The method of claim 1, wherein said V.sub.HH has an amino acid sequence set forth as SEQ ID NO: 4, or a functional fragment thereof.
8. The method of claim 1, wherein said V.sub.HH or functional fragment thereof is labelled with 68Ga.
9. The method of claim 1, wherein said V.sub.HH or functional fragment thereof is formulated to provide a calculated mean effective dose of between 0.002 and 0.1 mSv/MBq in said human subject.
10. The method of claim 1, wherein the radioisotope has a physical half-life of less than two hours.
11. The method of claim 1, wherein the V.sub.HH or a functional fragment thereof further comprises a chelating agent.
12. The method of claim 11, wherein the chelating agent is selected from the group consisting of 1,4,7-triazacyclononane-N-succinic acid-N′,N″-diacetic acid (NODASA), 1,4,7-triazacyclononane-N-glutamic acid-N′,N″-diacetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives thereof.
13. A method for identifying a HER-2 positive cancer lesion in a human subject, comprising (a) selecting a human subject initially identified as HER-2 negative in a standard in vitro assay for identifying a HER-2 positive cancer lesion, (b) administering to the subject of step (a) a radiolabelled heavy chain antibody (V.sub.HH) or a functional fragment thereof, which specifically binds to HER2, wherein the amino acid sequence of said V.sub.HH or functional fragment thereof comprises the combination of a CDR1 region having SEQ ID NO: 1, a CDR2 region having SEQ ID NO: 2, and a CDR3 region having SEQ ID NO: 3, and (c) measuring using PET/CT imaging the ability of said V.sub.HH or functional fragment thereof to bind HER2 in the subject, wherein the V.sub.HH or a functional fragment thereof is labelled with a radioisotope selected from the group consisting of 68Ga, 18F, 64Cu, 86Y, 76Br, 82Rb, 209At, and 210At, wherein the subject is identified as having a HER-2 positive cancer lesion when the V.sub.HH or functional fragment thereof binds to HER2 in the subject.
14. The method of claim 13, wherein the standard in vitro assay is a FISH assay for Her2 gene amplification.
15. The method of claim 14, wherein the subject of step (a) was initially diagnosed to be HER-2 negative in the FISH assay by yielding a score of less than about 2.0.
16. The method of claim 13, wherein said V.sub.HH or functional fragment thereof is labelled with a radioisotope selected from the group consisting of 68Ga, 18F, and 64Cu.
17. The method of claim 13, wherein said V.sub.HH or functional fragment thereof has at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 4, or a functional fragment thereof.
18. The method of claim 13, wherein said cancer lesion is a breast cancer lesion.
19. The method of claim 13, wherein said V.sub.HH or functional fragment thereof is administered intravenously or intraperitoneally.
20. The method of claim 13, wherein said V.sub.HH or functional fragment thereof is in a monovalent format.
21. The method of claim 13, wherein said V.sub.HH has an amino acid sequence set forth as SEQ ID NO: 4, or a functional fragment thereof.
22. The method of claim 13, wherein said V.sub.HH or functional fragment thereof is labelled with 68Ga.
23. The method of claim 13, wherein said V.sub.HH or functional fragment thereof is formulated to provide a calculated mean effective dose of between 0.002 and 0.1 mSv/MBq in said human subject.
24. The method of claim 13, wherein the radioisotope has a physical half-life of less than two hours.
25. The method of claim 13, wherein the V.sub.HH or a functional fragment thereof further comprises a chelating agent.
26. The method of claim 25, wherein the chelating agent is selected from the group consisting of 1,4,7-triazacyclononane-N-succinic acid-N′,N″-diacetic acid (NODASA), 1,4,7-triazacyclononane-N-glutamic acid-N′,N″-diacetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives thereof.
27. A method for a performing positron emission tomography (PET) imaging study in a human subject, comprising (a) administering to the subject a radiolabelled heavy chain antibody (V.sub.HH) or a functional fragment thereof, which specifically binds to HER2, wherein the amino acid sequence of said V.sub.HH or functional fragment thereof comprises the combination of a CDR1 region having SEQ ID NO: 1, a CDR2 region having SEQ ID NO: 2, and a CDR3 region having SEQ ID NO: 3, wherein the V.sub.HH or a functional fragment thereof is labelled with a radioisotope selected from the group consisting of 68Ga, 18F, or 64Cu; and (b) measuring the ability of said VHH or functional fragment thereof to bind HER-2 positive cancer lesion in the subject.
28. The method of claim 27, wherein the radioisotope has a physical half-life of less than two hours.
29. The method of claim 27, wherein the V.sub.HH or a functional fragment thereof further comprises a chelating agent.
30. The method of claim 29, wherein the chelating agent is selected from the group consisting of 1,4,7-triazacyclononane-N-succinic acid-N′,N″-diacetic acid (NODASA), 1,4,7-triazacyclononane-N-glutamic acid-N′,N″-diacetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2) Images were acquired 60 min after tracer injection.
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(10) The following non-limiting Examples describe methods and means according to the invention. Unless stated otherwise in the Examples, all techniques are carried out according to protocols standard in the art. The following examples are included to illustrate embodiments of the invention. 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 concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
(11) Thus, the Figures, Sequence Listing and the Experimental Part/Examples are only given to further illustrate the invention and should not be interpreted or construed as limiting the scope of the invention and/or of the appended claims in any way, unless explicitly indicated otherwise herein.
EXAMPLES
Example 1: Preliminary Analysis of Phase I Study of .SUP.68.Ga-HER2-Nanobody (.SUP.68.Ga-Anti-HER2 V.SUB.HH.) for PET/CT Assessment of HER2-Expression in Breast Cancer
(12) The primary objective of this study was the assessment of safety, human biodistribution and dosimetry as well as the evaluation of tumor uptake in HER2-expressing lesions in breast carcinoma patients.
(13) Methods
(14) The anti-HER2 V.sub.HH (SEQ ID NO:4) was labeled with .sup.68Ga via a NOTA derivative (pSCNBnNOTA) with >97% radiolabeling yield after 5 min at RT. In total, 15 female patients with breast carcinoma lesions showing intermediate or high HER2 expression on immunohistochemistry were studied (9/15 showed amplification on FISH analysis).
(15) In one patient, the entire tumor lesion had been removed at the primary biopsy. Among the FISH negative patients, 4/5 still showed V.sub.HH uptake. Thus, V.sub.HH imaging can identify patients that may benefit from Her2-targeted diagnosis despite an initial FISH negative score (i.e. to “fish” for false FISH negative patients). In patients in which a Her2 signal can be detected using V.sub.HH imaging, a repeat biopsy could be performed to perform a second FISH analysis.
(16) Three different study groups (n=5 for each) were included in a dose-escalating step-by-step approach, receiving respectively 0.01, 0.1 and 1.0 mg of anti-HER2 VHH. Radioactive dose was kept constant over the groups and was on average 105±39 MBq. PET/CT scans were performed at 10, 60 and 90 min. Blood and urine samples were analyzed for radioactive content and metabolites. Blood analysis (hematology and clinical chemistry) was performed before and 120 min after tracer injection. Patients were asked for changes in physical and emotional state. Physical examination was performed before and at multiple time points after injection. Biodistribution was analyzed by time activity curves of 10 organs using MIM contouring software in patients with normal liver and kidney function (n=11). Dosimetry was assessed using OLIN DA/EXM.
(17) Results
(18) Radiochemical purity of 68GaHER2 was >98% after purification. No related adverse events were observed during the 24 h follow-up period. Blood analysis showed a fast blood clearance, with on average 10.1±2.2% of injected activity remaining in the total blood volume at 60 min post injection. The compound was stable in vivo (plasma, urine). Tracer uptake at 60 min was highest in liver (10.7±3.6% IA) and kidneys (7.6±0.9% IA) (
(19) The V.sub.HH imaging reveals whole-body expression of Her2 in primary tumors and metastases. The Her2 signal that is detected in metastases is hereby often higher than in primary tumor, thus offering the possibility to guide the selection of which (metastatic) lesions are most appropriate for a repeat biopsy (“image-guided biopsy”). In a number of large lesions (>4 cm) a heterogeneous tracer uptake was detected. Hereby, the pattern of heterogeneity in HER2 expression as detected via the Her2 V.sub.HH's did not always match the heterogeneity in metabolic activity as detected via FDG distribution, suggesting superiority in identifying optimal areas for a repeat biopsy, thus further fine-tuning the potential for image-guided biopsy.
(20) Remarkably, in one patient, a focal Her2 signal spot was also detected in the brain, showing the possibility to detect Her2 positive brain lesions.
(21) Conclusion
(22) 68GaHER2 PET/CT is a safe procedure with a radiation dose comparable to other PET/CT imaging procedures. The wide range in uptake intensity of lesions and the heterogeneous distribution in large lesions show potential for the in vivo assessment of HER2 expression levels.
(23) The results more importantly showed that V.sub.HH imaging can accurately identify cancer lesions through specifically binding to a tumor-specific antigen.
(24) In addition, the HER-2 binding VHH's as disclosed herein are able to identify patients that may benefit from Her2-targeted diagnosis despite an initial FISH negative score.
Example 2: Detailed Analysis of Phase I Study of .SUP.68.Ga-HER2-Nanobody (.SUP.68.Ga-Anti-HER2 V.SUB.HH.) for PET/CT Assessment of HER2-Expression in Breast Cancer
(25) In the remaining examples sections the term VHH or VHH domain is used interchangeably with the term Nanobody (Nb).
(26) All terms .sup.68Ga-HER-Nanobody, .sup.68Ga-NOTA-anti-HER2 Nanobody and .sup.68Ga-anti-HER2-VHH on the one hand; HER-Nanobody, anti-HER2-Nanobody, HER2-VHH and anti-HER2-VHH on the other hand, refer to the same compound (either labelled with .sup.68Ga or unlabeled).
(27) Methods
(28) In total, 20 female patients with primary or metastatic breast carcinoma (HER2 IHC 2+ or 3+) were included. Anti-HER2-Nanobody is labeled with .sup.68Ga via a NOTA derivative. Administered activities were 53-174 MBq (average 107 MBq). PET/CT scans (for dosimetry assessment) were obtained at 10, 60 and 90 min post administration. Physical evaluation and blood analysis were performed for safety evaluation. Biodistribution was analyzed for 11 organs using MIM software; dosimetry was assessed using OLINDA/EXM. Tumor targeting potential was assessed in primary and metastatic lesions.
(29) Results
(30) No adverse reactions occurred. A fast blood clearance was observed, with only 10% of injected activity remaining in the blood at 1 h post injection. Uptake was mainly seen in kidneys, liver, and intestines. The effective dose was 0.043 mSv/MBq, resulting in an average of 4.6 mSv per patient. The critical organ was the urinary bladder wall with a dose of 0.406 mGy/MBq. In patients with metastatic disease, tracer accumulation well above background was demonstrated in the majority of identified sites of disease. Primary lesions were more variable in tracer accumulation.
(31) Conclusion
(32) .sup.68Ga-HER2-Nanobody PET/CT is a safe procedure with a radiation dose comparable to other routinely used PET tracers. Its biodistribution is favorable, with the highest uptake in kidneys, liver and intestines, but very low background levels in all other organs that typically house primary breast carcinoma or tumor metastasis. Tracer accumulation in HER2-positive metastases is high, compared to normal surrounding tissues, and warrants further assessment in a phase II trial.
(33) Materials and Methods
(34) Study Design
(35) This was an open-label phase I study in HER2-expressing breast carcinoma patients (n=20). The supplemental data as represented in Example 3 provide details on patient selection and approvals. Three subgroups, receiving respectively 0.01 mg (group 1: pt 1-7), 0.1 mg (group 2: pt 8-15) and 1.0 mg (group 3: pt 16-20) NOTA-Anti-HER2-Nanobody were evaluated. The activity administered was similar for the different patient groups and ranged between 53 and 174 MBq.
(36) Imaging Methods and Safety Assessment
(37) Details on radioligand synthesis, safety assessment and PET/CT protocol are described in the supplemental data represented in Example 3.
(38) A p-SCN-Bn-NOTA chelator was conjugated to the Nanobody. .sup.68Ga (250-400 MBq) was incubated with the NOTA-Anti-HER2-Nanobody in acid conditions for 5-7 min at room temperature. .sup.68Ga-HER2-Nanobody was purified and filtered prior to injection. Quality controls included analysis of appearance, presence of .sup.68Ge, pH, radiochemical purity and radiochemical identity, filter integrity (bubble point test). For the different patient groups the necessary amount of cold NOTA-anti-HER2-Nanobody was added prior to final filtration.
(39) .sup.68Ga-HER2-Nanobody was injected as an intravenous bolus. For safety evaluation, vital sign were recorded and clinical laboratory testing was performed before and 2 h after injection. Subjective adverse experiences were assessed using open questions up to 24 h post injection (p.i.). Whole body PET/CT imaging (low-dose CT) was performed with a Philips Gemini TF at 10, 60 and 90 minutes p.i.
(40) Summary Blood, Urine and Image Analysis
(41) Details on image processing and analysis as well as blood and urine analysis are described in the supplemental data, (see Example 3).
(42) Blood samples were obtained at different time points p.i., assessed for radioactive content and expressed as a percentage of the injected activity (% IA) in total blood volume using Nadler's formula. Urine samples were collected at about 45 min and 2 h p.i. Blood and urine were assessed for metabolites.
(43) Uptake in 11 organs (liver, kidneys, intestines, thyroid, whole body, bladder and urinary activity in ureters, spleen, heart muscle, lungs, hematopoietic bone marrow and breast tissue) was measured on each PET/CT using MIM contouring software (MIM-software Inc.) and expressed as % IA. Dosimetric calculations for the adult female were made using the OLINDA/EXM software 1.0 (16).
(44) Uptake in tumor lesions was measured using the mean Standard Uptake Value (SUV.sub.mean) in a 10 mm spheroid Region of interest (ROI) positioned over the area with the highest uptake. If available, the uptake in the primary lesion and in the metastasis showing the highest SUV.sub.mean is reported.
(45) Results
(46) Patient Characteristics
(47) Between April 2012 and July 2014, 20 patients completed the study protocol. The patients received on average 107±37 MBq (range 53-174 MBq).sup.68Ga-HER2-Nanobody. Patient and study drug characteristics are summarized in table 3.
(48) TABLE-US-00003 TABLE 3 Patient characteristics Injected (Range of) SUV.sub.mean of lesion(s) activity Tumor HER2 HER2 FISH Primary Local Patient no. Age (MBq) type ER/PR IHC (ratio; copies/cell) tumor ADP Distant M+ (type) 0.01 mg 1 43 77 IDC +/+ 2+ + (2.2; 6.2) ISR* A A 2 60 66 IDC +/+ 3+ + (>2; macroclusters) CR A .sup. 3.1 (bone) 3 68 53 IDC +/+ 3+ + (12.2; 20.0) 3.2 A A 4 53 76 IDC +/+ 2+ − (1.3; 3.7) 2.2 A A 5 74 84 IDC +/+ 2+ − (1.3; 3.8) 2.3 A A 6 34 83 IMeC +/− 3+ + (2.8; 8.0) 0.9 A A 7 34 80 IDC +/+ 2+ − (1.0; 1.4) 2.0 A A 0.1 mg 8 67 92 IDC +/+ 2+ − (1.4; 3.4) 5.0 3.2-4.3 1.0-5.6 (bone) 9 57 111 IDC +/+ 3+ + (1.3; 6.1) 2.3 A A 10 61 100 IDC +/− 3+ + (9.4; 15.0) SR SR 4.1-5.7 (bone) 11 65 90 IDC +/+ 3+ + (2.3; 5.1) 2.9 6.3 A 12 46 82 IDC +/+ 3+ + (8.1; 15.6) 1.4 A A 13 32 153 IDC −/− 2+ + (9.4; 17.4) 3.2 1.7 A 14 53 103 IDC −/− 3+ + (4.7; 9.2) 11.8 13.0 A 15 78 148 IDC +/+ 2+ − (1.0; 2.1) 4.9 A A 1.0 mg 16 76 96 ILC +/+ 2+ − (1.0; 1.7) SR SR 2.2-3.9 (bone) 17 74 138 IDC −/− 2+ − (1.2; 4.3) 1.8 A A 18 62 167 IDC +/− 2+ + (2.6; 4.5) SR SR 3.5-6.0 (ADP mediastinum) 19 62 174 IMiC +/+ 2+ + (2.8; 8.0) 4.4 5.1-5.9 3.6-3.9 (bone) 20 48 170 IDC +/+ 3+ + (7.8; 15.6) .sup. 0.7.sup.† A .sup. 4.7-5.4 (bone).sup.† IDC = invasive ductal carcinoma; ILC = Invasive lobular carcinoma; ER = estrogen receptor; PR = progesterone receptor; IMeC = Invasive medullary carcinoma; IMiC = Invasive Mixed carcinoma; ADP = adenopathy; ISR = incomplete surgical removement; SR = surgically removed; A = Absent; *= patient was scanned after incomplete surgical removement but additional surgical resection could not demonstrate remaining tumor cells; .sup.†= after 4 cycles of epirubicine-cyclophosphamide;
(49) Safety Assessment
(50) After the administration of .sup.68Ga-HER2-Nanobody, no symptoms or signs were reported. Clinical laboratory testing of blood, taken before and 120 min after injection, showed no significant changes that could be related to the study drug.
(51) Pharmacokinetics and Biodistribution
(52)
(53) The uptake of .sup.68Ga-HER2-Nanobody in individual organs is presented in
(54) TABLE-US-00004 TABLE 4 Uptake of .sup.68Ga-HER2-Nanobody in single organs. Uptake (% IA) Organ 10 min 60 min 90 min Liver 25.41 ± 8.39 12.47 ± 4.88 8.18 ± 3.07 Kidneys 12.74 ± 2.5000 8.14 ± 1.55 6.36 ± 1.23 Intestines 11.76 ± 1.62 6.72 ± 1.11 4.72 ± 0.80 Central bone 3.65 ± 1.02 1.80 ± 0.53 1.20 ± 0.37 Lungs 2.83 ± 0.56 1.48 ± 0.33 0.98 ± 0.18 Breasts 1.00 ± 0.54 0.58 ± 0.31 0.39 ± 0.19 Spleen 0.48 ± 0.16 0.22 ± 0.09 0.15 ± 0.07 Heart muscle 0.40 ± 0.12 0.19 ± 0.06 0.12 ± 0.04 Thyroid 0.12 ± 0.08 0.05 ± 0.03 0.03 ± 0.02
(55) All images showed uptake in kidneys and excretion of the tracer into the urine. Although liver and intestinal uptake was visible, there were no signs of hepatobiliary excretion, such as accumulation in the gall bladder or duodenum. At 1 h p.i., 50% of the tracer had been eliminated from the body, resulting in an estimated biological half-life of 1 h (
(56) Effect of Injected Mass on Liver Uptake
(57) Based on preclinical results, an effect of the injected mass of the compound on the non-specific binding was expected. Therefore, liver uptake was assessed in the three patient subgroups receiving different amounts of Nanobody mass. Overall, liver uptake was quite variable between patients. There is a trend towards lower liver uptake at 90 min p.i. or the 1.0 mg mass group, with an average uptake of 5.5% IA compared to 9.0 and 9.5% IA for 0.1 and 0.01 mg respectively, but with overlapping 95% confidence intervals (3.3-7.6; 5.7-12.3 and 7.4-11.5 respectively). A one-way ANOVA indicates no significant difference, F(2,15)=3.60, p=0.053.
(58) The findings that the background liver uptake is inherently quite variable between patients and that there is a lack of significant correlation of liver uptake with the amount of injected Nanobody mass indicate that imaging in human patients can be performed using low amounts of injected mass and that, despite the expectations based on preclinical data, there is no benefit or need to increase the injected mass.
(59) Dosimetry
(60) Table 5 summarizes the individual organ doses and individual effective dose (ED) results for all subjects with normal liver and renal function. The urinary bladder wall shows the highest organ dose of 0.406 mGy/MBq, followed by the kidneys (0.216 mGy/MBq), liver (0.0778 mGy/MBq), lower large intestine wall (0.0759 mGy/MBq) and upper large intestine wall (0.0619 mGy/MBq).
(61) TABLE-US-00005 TABLE 5 Organ doses and effective doses. Patient Urinary Effective dose no. Bladder Wall Kidneys Liver LLI Wall ULI Wall Thyroid (mSv/MBq) 1 0.406 0.191 0.0515 0.0843 0.0606 0.0233 0.0425 3 0.406 0.161 0.114 0.0757 0.0679 0.0326 0.0458 4 0.405 0.219 0.0788 0.0295 0.0787 0.0257 0.0371 5 0.407 0.181 0.116 0.0962 0.0566 0.0093 0.0472 6 0.406 0.297 0.0957 0.0798 0.0535 0.0282 0.0453 7 0.405 0.259 0.0788 0.0423 0.0840 0.0020 0.0371 8 0.405 0.141 0.114 0.0788 0.0715 0.0137 0.0433 9 0.406 0.273 0.0740 0.0686 0.0675 0.0200 0.0421 10 0.406 0.229 0.0922 0.0626 0.0991 0.0035 0.0425 12 0.407 0.220 0.0594 0.0816 0.0772 0.0327 0.0435 13 0.406 0.225 0.113 0.0632 0.0812 0.0496 0.0442 14 0.407 0.222 0.0849 0.0719 0.113 0.0513 0.0448 15 0.406 0.278 0.0719 0.0086 0.0071 0.0035 0.0335 16 0.407 0.192 0.0473 0.140 0.0356 0.0124 0.0485 17 0.407 0.216 0.0610 0.141 0.0518 0.0132 0.0504 18 0.406 0.193 0.0583 0.118 0.0554 0.0110 0.0469 19 0.406 0.181 0.0422 0.0087 0.0067 0.0220 0.0317 20 0.406 0.211 0.0469 0.116 0.0464 0.0177 0.0447 Mean ± SD 0.406 ± 0.001 0.216 ± 0.041 0.0778 ± 0.0252 0.0759 ± 0.0384 0.0619 ± 0.0274 0.0207 ± 0.0143 0.0428 ± 0.0050 Patient no. 2 and 11 were not taken into account because of altered liver and/or kidney function. LLI = Lower large intestines; ULI = Upper large intestines. Organ dose (mGy/MBq)
(62) Uptake in Tumor Lesions
(63) Uptake in tumor lesions could be evaluated in 19 patients, 9 of which only had a primary lesion, six both a primary lesion and local or distant metastases, and 4 only local or distant metastases (Table 5).
(64) Uptake in Primary Lesions
(65) Tracer uptake was visible above background in the majority of primary tumors, with SUV.sub.mean values ranging between 0.7 and 11.8. Uptake was absent in the primary tumor of two patients (no. 6 and 20). Representative images showing .sup.68Ga-HER2-Nanobody uptake in primary lesions are presented in
(66) Uptake in Local and Distant Metastases
(67) All patients with metastatic lesions showed clear tracer accumulation in at least one lesion, with SUV.sub.mean ranging from 3.1 to 6.0.
(68) Heterogeneous Uptake Pattern
(69) In patient 8, a heterogeneous uptake pattern was observed in the primary tumor (
(70) Conclusion
(71) .sup.68Ga-HER2-Nanobody PET/CT is a safe procedure with a radiation dose comparable to other routinely used PET tracers. Its biodistribution is favorable, with the highest uptake in kidneys, liver and intestines, but very low background levels in all other organs that typically house primary breast carcinoma or tumor metastasis. Tracer accumulation in metastases of HER2 overexpressing patients is high, compared to normal surrounding tissues, and warrants further assessment in a phase II trial.
Example 3: Supplemental Data for the Phase I Study of .SUP.68.Ga-HER2-Nanobody (.SUP.68.Ga-Anti-HER2 V.SUB.HH.) for PET/CT Assessment of HER2-Expression in Breast Carcinoma
(72) Supplemental methods in addition to the details set out in Example are set out hereunder.
(73) Approvals
(74) The Belgian federal agency for medicines and health products, the regional ethics committee of UZ Brussel and the radiation protection agency of Belgium approved this study. The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Guidelines for Good Clinical Practice. Written informed consent was obtained from all participants. The study was registered as a clinical trial with the identifier EudraCT 012-001135-31.
(75) Patient Characterization and Study Subgroups
(76) Twenty adult female breast carcinoma patients with local, locally advanced or metastatic breast carcinoma that showed a moderate or high expression of HER2 on immunohistochemistry (2+ or 3+) were included in the study. Patients were allowed to enter the study at first diagnosis, at relapse or under treatment (with the exclusion of any HER2-targeted treatment) if there was at least one documented breast carcinoma lesion. No additional work-up for the detection of potential additional metastasis was required for this study, since tumor targeting potential was not the primary aim. Exclusion criteria were male gender, pregnancy, breast feeding, HER2-targeted therapy in the last 30 days before administration, known abnormal liver or kidney function, serious active infection, recent gastro-intestinal disorder with diarrhea, other life-threatening illness, unability to communicate reliably or give informed consent, patients unlikely to cooperate with the requirements of the study or patients who already participated in the study.
(77) Three subgroups of patients, receiving respectively 0.01 mg (group 1: pt 1-7), 0.1 mg (group 2: pt 8-15) and 1.0 mg (group 3: pt 16-20) .sup.68Ga-HER2-Nanobody were evaluated for a potential difference in normal biodistribution, to investigate a potential decrease in non-specific binding in non-target organs with increasing mass of tracer.
(78) The activity administered was similar for the different patient groups and ranged between 66 and 174 MBq (the allowed range was 37-185 MBq).
(79) Patients 2 and 11 were withdrawn from the biodistribution and dosimetry study because of decreased renal function and altered liver enzymes, >1.5× normal values at time of imaging. Their images are however evaluated for tumor targeting potential.
(80) Safety Assessment
(81) For safety evaluation, all patients underwent vital signs measurement (blood pressure, heart rate and temperature), clinical laboratory testing (standard hematologic and comprehensive metabolic panels that included hemoglobin, white blood cells, neutrophils, lymphocytes, platelets, creatinine, blood urea nitrogen, calcium, sodium, potassium, carbon dioxide, lactate dehydrogenase, alanine transaminase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, and albumin), before administration, as well as 2 h after injection of the compound. Subjective adverse experiences were assessed using open questions before injection, throughout the 2 h that patients were present in the nuclear medicine department and using telephone follow-up at 24 h post-injection
(82) Conjugation of p-SCN-Bn-NOTA to Anti-HER2 Nanobody
(83) The anti-HER2 Nanobody was produced according to GMP standards. In order to allow complexation of the .sup.68Ga radiometal, a p-SCN-Bn-NOTA chelator was conjugated to the Nanobody as described earlier in Xavier C, Vaneycken I, D'Huyvetter M, et al. Synthesis, preclinical validation, dosimetry, and toxicity of .sup.68Ga-NOTA-anti-HER2 Nanobodies for iPET imaging of HER2 receptor expression in cancer. J Nuci Med. 2013; 54:776-784. Briefly, Nanobody in 0.05 M sodium carbonate buffer pH 8.7 was added to p-SCN-Bn-NOTA (10-fold molar excess) and incubated for 2 h at room temperature. The coupling reaction was quenched by adjusting the pH to 7-7.4 using HCl 1 N. The conjugate was then purified by size-exclusion chromatography (SEC) on a Superdex 75 10/300 GL (GE Healthcare) using ammonium acetate 0.1 M pH 7 as eluent or 0.01 M PBS.
(84) Synthesis of .sup.68Ga-HER2-Nanobody
(85) .sup.68Ga was obtained from a .sup.68Ge/.sup.68Ga generator (Eckert and Ziegler), eluted with 0.1N HCl (Merck). The 1.5 ml peak fraction (250-400 MBq) was added to 1 M sodium acetate buffer pH5 (1 ml) containing NOTA-Anti-HER2 Nanobody (1.1-3.8 nmol), the final pH was 4-4.5. The reaction mixture was incubated for 5-7 min at room temperature. Next, the product was purified by gel filtration on a disposable PD10 column (GE Healthcare), equilibrated with 0.01 M PBS pH 7.4. The compound was finally filtered through a 0.22 μm membrane filter (13 mm, Millipore, Brussels, Belgium).
(86) The final solution was analyzed by instant thin layer chromatography (iTLC-SG) performed on silica gel (SG) (Agilent) using 0.1 M sodium citrate pH 5.0 as eluent to evaluate radiochemical purity. ITLC-SG: .sup.68Ga-HER2-Nanobody R.sub.f=0, unbound .sup.68Ga R.sub.f=1. The final product was also analyzed by reverse phase high performance liquid chromatography (RP-HPLC) using a polystyrene divinylbenzene copolymer column (PLRP-S 300 Å) to evaluate radiochemical purity and radiochemical identity. RP-HPLC: t.sub.R=12.8 min.
(87) In addition, quality control of .sup.68Ga-HER2-Nanobody involved analysis of appearance of the solution, radionuclide identity (gamma spectrum), presence of .sup.68Ge, pH, filter integrity (bubble point test), endotoxin (LAL test) and sterility (microbiology).
(88) For the different patient groups different masses of .sup.68Ga-HER2-Nanobody were injected (0.01 mg/0.1 mg/1 mg). For the higher masses, the necessary amount of cold NOTA-anti-HER2 Nanobody (in 0.01M PBS pH7.4) was added to the .sup.68Ga-HER2-Nanobody prior to final filtration.
(89) .sup.68Ga-HER2-Nanobody PET/CT Imaging
(90) Images were acquired using a Philips Gemini TF PET/CT (LySO-based PET scanner with Time-of-flight with 18 cm axial and 70 cm transaxial field-of-view (FOV), 64-slice CT). The scanner is accredited by the EANM via the EARL program.
(91) Whole-body images were acquired 10, 60 and 90 minutes after administration of .sup.68Ga-HER2-Nanobody. The time per bed position was 1 min (for a total scan time of about 25 min).
(92) Low-dose CT was performed for attenuation correction and localization of hotspots on PET, consisting of slices of 512 by 512 pixels (FOV: 600 mm) at 5 mm slice thickness, acquired at 120 kV and 50 mAs, resulting in a radiation dose (CTDI) of 2.9 mGy/scan.
(93) PET images were reconstructed to 144×144 matrix with 4 mm slice thickness (4 mm isotropic pixels) using the vendor's standard BLOB-OS-TOF reconstruction with 3 iterations and 33 subsets (at a kernel width of 14.1 cm) with attenuation, scatter and randoms correction.
(94) Blood and Urine Samples
(95) Blood samples were taken from a peripheral vein at 2, 5, 10 and 40 min, and at 1 and 2 h post injection (p.i.) of the compound. Urine sample were collected at about 45 min and 2 h p.i.
(96) Whole blood and plasma samples were counted against appropriate standards of known dilution in an automatic gamma well counter and, after correction for decay and background activity, expressed as a percentage of the injected activity (% IA). The blood volume of each volunteer was estimated according to body weight and height, using Nadler's formula and the patient's hematocrit. Blood activity results of 8 patients were not further used in the analysis because of altered liver or kidney tests (no. 2, 11), because of injection of the tracer and blood sampling on the same arm (no. 10, 16, 18, 19, 20), or because of possible interference with .sup.99mTc blood activity due to a cardiac blood pool scan two days prior to this study (no. 14). Blood half-lives were calculated with a two-phase exponential decay model using GraphPad Prism software (GraphPad Software, La Jolla, Calif., USA). Plasma and urine aliquots were analyzed by SEC or RP-HPLC to identify possible metabolites.
(97) Volume-of-Interest Definition
(98) The .sup.68Ga-HER2-Nanobody uptake in different organs was determined using Region of Interests (ROIs), drawn using MIM contouring software (MIM software Inc., 2014). For biodistribution and dosimetry purposes, organs with tracer uptake were delineated in a semi-automatic (region grow) fashion and manually corrected to assure that the activity on the PET images that was contributing to the organ was included in the ROI (kidneys, intestines, thyroid, whole body, bladder and urinary activity in urethra). For 5 organs not showing substantial tracer uptake, organ delineation was based on CT data (spleen, heart muscle, lungs, central bone (covering bone from top of the skull until proximal femora and representing hematopoietic bone marrow) and unaffected breast). Care was taken that activity originating from kidneys, liver, intestines, bladder and urine was not included into the CT-based ROI's. To reflect normal biodistribution in breast tissue, the activity in the unaffected breast was multiplied by two. For assessment of liver activity, the liver volume was determined using CT-based delineation and multiplied by the average activity in liver parenchyma, as determined by a spheroid ROI of 40 mm diameter, positioned in the right liver lobe. This approach was chosen to overcome the influence of potential motion artifacts in the liver due to breathing or inhomogeneity in liver activity due to underlying liver metastasis.
(99) Dosimetry
(100) For dosimetric calculations, the OLINDA/EXM software 1.0 (Organ Level Internal Dose Assessment/EXponential Modeling, Vanderbilt University) was used (Stabin M G, Sparks R B, Crowe E. OLINDA/EXM: the second-generation personal computer software for internal dose assessment in nuclear medicine. J Nucl Med. 2005; 46:1023-1027.). OLINDA/EXM software entails the EXM code that performs kinetic analysis of biokinetic data for input into the dose calculations algorithms. The organ uptake values per patient were put into the EXM analysis software and a bi-exponential fit was performed per organ for each individual patient. OLINDA subsequently calculates the disintegrations per source organ as well as the radiation dose for all target organs from these data. Other input variables were the excretion parameters, put at 100% renal excretion with a biological half-life of 60 min and a voiding bladder interval of 60 min.
(101) Uptake in Tumor Lesions
(102) A sphere-shaped ROI with a diameter of 10 mm was placed within each discernable tumor lesion that measured at least 10 mm on the low dose CT, obtained for study purposes, or on other available imaging data, using MIM contouring software. For large lesions, the ROI was positioned over the area with the highest uptake. The mean Standard Uptake Value (SUV.sub.mean), corrected for the body weight, within this ROI is reported as the uptake value for a lesion. If available, the uptake in the primary lesion and in the metastasis showing the highest SUV.sub.mean is reported. Osirix software (Pixmeo) was used for image processing.
(103) Statistical Analysis
(104) Values are reported as mean±standard deviation (SD). A one-way ANOVA was conducted to compare the effect of injected Nanobody mass (3 patient subgroups) on the tracer uptake in liver.