NEW POLYPEPTIDE
20240101605 ยท 2024-03-28
Inventors
- John L?FBLOM (Tullinge, SE)
- Stefan ST?HL (Stockholm, SE)
- Jonas PERSSON (Stockholm, SE)
- Olof Eriksson (Uppsala, SE)
- Olov KORSGREN (Uppsala, SE)
Cpc classification
C07K2319/40
CHEMISTRY; METALLURGY
C07K2319/20
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to a class of engineered polypeptides having a binding affinity for CD69 and provides a CD69-binding polypeptide comprising the sequence EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10 X.sub.11LPNLX.sub.16X.sub.17X.sub.18QK X.sub.21AFKX.sub.25X.sub.26LKD. The present disclosure also relates to the use of such a CD69-binding polypeptide as a therapeutic, prognostic and/or diagnostic agent.
Claims
1. A CD69-binding polypeptide, comprising a CD69-binding motif BM, which motif consists of an amino acid sequence selected from: TABLE-US-00030 i) (SEQIDNO:168) EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16 X.sub.17X.sub.18QKX.sub.21AFKX.sub.25X.sub.26LKD wherein, independently of each other, X.sub.2 is selected from F, H, V and W; X.sub.3 is selected from A, D, E, H, N, Q, S, T, Y; X.sub.4 is selected from A, D, E, H, K, M, N, S, V, W and Y; X.sub.5 is selected from M, W and Y; X.sub.7 is selected from A, H, K, N, Q, R, W and Y; X.sub.10 is selected from L and R; X.sub.11 is selected from A, H, K, R, S and V; X.sub.16 is selected from N and T; X.sub.17 is selected from A, D, K, Q, S and V; X.sub.18 is selected from W and Y; X.sub.21 is selected from E and S; X.sub.25 is selected from H and T; and X.sub.26 is selected from K and S; and ii) an amino acid sequence which has at least 93% identity to the sequence defined in i).
2. The CD69-binding polypeptide according to claim 1, wherein, in sequence i), X.sub.2 is F; X.sub.3 is selected from Q, S and Y; X.sub.4 is selected from H, M, N and W; X.sub.5 is selected from M and W; X.sub.7 is selected from K, Q and W; X.sub.10 is selected from L and R; X.sub.11 is selected from A, H, K and V; X.sub.16 is selected from N and T; X.sub.17 is selected from A, K, Q and S; X.sub.18 is selected from W and Y; X.sub.21 is E; X.sub.25 is T; and X.sub.26 is selected from K and S.
3. The CD69-binding polypeptide according to claim 1, wherein sequence i) fulfills at least five of the ten conditions I-X: I. X.sub.2 is F; II. X.sub.3 is Y; III. X.sub.4 is H, N or W; IV. X.sub.6 is M or W; V. X.sub.10 is L; VI. X.sub.11 is K; VII X.sub.17 is K or 0; VIII. X.sub.18 is Y; IX. X.sub.21 is E; and X. X.sub.25 is T.
4. The CD69-binding polypeptide according to claim 1, wherein sequence i) corresponds to the amino acid sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-72, SEQ ID NO:1-29, SEQ ID NO:1-28, SEQ ID NO:1-26, SEQ ID NO:1-6, SEQ ID NO:1-2 and 6, SEQ ID NO:1-2, and SEQ ID NO:1.
5. The CD69-binding polypeptide according to claim 1, wherein said CD69-binding motif (BM) forms part of a three-helix bundle protein domain, said three-helix bundle protein domain for example being a variant of protein Z, which is derived from domain B of staphylococcal Protein A.
6. The CD69-binding polypeptide according to claim 1, which comprises a binding module (BMod), the amino acid sequence of which is selected from: TABLE-US-00031 iii) K-[BM]-DPSQSX.sub.aX.sub.bLLX.sub.cEAKX.sub.dLX.sub.eX.sub.fX.sub.gQ; wherein [BM] is a CD69-binding motif as defined in claim 1; X.sub.a is selected from A and S; X.sub.b is selected from E and N; X.sub.c is selected from A, S and C; X.sub.d is selected from K and Q; X.sub.e is selected from E, N and S; X.sub.f is selected from D, E and S; and X.sub.g is selected from A and S; and iv) an amino acid sequence which has at least 93% identity to a sequence defined in iii).
7. The CD69-binding polypeptide according to claim 6, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-72, SEQ ID NO:1-29, SEQ ID NO:1-28, SEQ ID NO:1-26, SEQ ID NO:1-6, SEQ ID NO:1-2 and 6, SEQ ID NO:1-2, SEQ ID NO:1 or the group consisting of SEQ ID NO:73-144, SEQ ID NO:73-101 SEQ ID NO:73-100, SEQ ID NO:73-98, SEQ ID NO:73-78, SEQ ID NO:73-74 and 78, SEQ ID NO:73-74, SEQ ID NO:73.
8. The CD69-binding polypeptide according to claim 1, which is capable of binding to CD69 such that the K.sub.D value of the interaction with CD69 is at most 1?10.sup.?6 M, 5?10.sup.?7 M, 1?10.sup.?7 M, 5?10.sup.?8 M, 1?10.sup.?8 M, or 5?10.sup.?8 M.
9. A Fusion protein or conjugate, comprising a first moiety consisting of a CD69-binding polypeptide according to claim 1; and a second moiety consisting of a polypeptide having a desired biological activity.
10. The CD69-binding polypeptide according to claim 1, further comprising a label selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, radionuclides, radioactive particles and pretargeting recognition tags.
11. The CD69-binding polypeptide according to claim 10, for use in labeling or targeting cells and tissues which have a high expression of CD69.
12. The CD69-binding polypeptide according to claim 10 which is labeled, directly or indirectly, with an imaging agent, or a radioactive agent.
13. A Polynucleotide encoding a CD69-binding polypeptide or fusion protein according to claim 1.
14. The Composition comprising a CD69-binding polypeptide according to claim 1 and at least one pharmaceutically acceptable excipient or carrier.
15. The CD69-binding polypeptide according to claim 1 for use as a medicament, as a diagnostic agent in vivo or as a prognostic agent in vivo.
16. The CD69-binding polypeptide according to claim 15 for use as a diagnostic agent in the in vivo diagnosis of a CD69-related disorder.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Summary
[0237] The following Examples disclose the development of novel Z variant molecules targeted to CD69 based on autotransporter-mediated E. coli display and affinity maturation. The Examples further describe the characterization of CD69-binding polypeptides and demonstrate in vitro functionality of said polypeptides.
Example 1
Generation of a Z Variant Library for Display on E. coli Cells
[0238] A 121 nucleotide long, randomized oligonucleotide encoding helix 1 and 2 of the Z variant library was designed and synthesized (Ella Biotech GmbH, Martinsried, Germany). Randomized positions were designed to have the codon distribution shown in Table 2. Note in particular that only five amino acids were allowed in position 31 of the full length sequence.
TABLE-US-00015 TABLE 2 Design of Z variant library for primary selection Position in the Z variant sequence Permitted amino acid residues Proportions 9 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 10 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 11 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 13 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 14 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 17 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 18 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 24 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 25 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 27 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 28 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 31 D, H, I, K, Y I: 60%; D: 10%; H: 10%; K: 10%; Y: 10% 32 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6% 35 A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 5.6%
[0239] The oligo was amplified in eight rounds of PCR. Ligation of the insert into the vector was done by adding a threefold molar excess of insert over vector and T4 DNA ligase.
[0240] The ligated vector was electroporated using 40 separate reactions into E. coli BL-21 cells. Samples of the total library were diluted and spread on agar plates, and the library size was estimated to be around 2.4?10.sup.9 variants. To validate the library, 192 separate clones picked at random were sequenced, finding only unique clones and no observed errors. The validated E. coli library was stored at ?80? C.
Example 2
Selection of a CD69-Binding Z Variant Using E. coli Display
Aim
[0241] From the na?ve Z variant library prepared as described in Example 1, Z variants with affinity for human CD69 were selected using a combination of magnetic-assisted cell sorting (MACS) and fluorescence-activated cell sorting (FACS) as previously described (Andersson et al (2018), supra). The MACS step is used to reduce the library size to make FACS selection feasible.
Methods
[0242] Biotinylation of hCD69: Biotinylation of recombinant extracellular domain of human CD69 (SEQ ID NO:165; #8468-CD-025, R&D Systems), here denoted hCD69, was performed using EZ-Link NBS-Biotin (N-hydroxysuccinimidobiotin) (#20217, Thermo Scientific) according to the supplier's recommendations. The protein buffer was changed to PBS (10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) by dialysis twice, first against 2 l PBS and then against 3 l PBS.
[0243] MACS selection: 500 ?l of streptavidin-coated Dynabeads (DYNABEADS? M-280 Streptavidin, Thermo Scientific) were washed twice in 1?PBSP (phosphate-buffered saline with 0.1% Pluronic F108 NF Prill Poloxamer 338 surfactant). The beads were resuspended in 100 nM of biotinylated hCD69 and incubated for 1 h at room temperature (RT) in a rotamixer. 4?10.sup.10 E. coli cells were pelleted and washed in 1?PBSP and negative selection was carried out by adding 250 ?l of Dynabeads without biotinylated hCD69, followed by incubation for 20 min at RT in a rotamixer and subsequent removal of magnetic beads. The target-coated beads were added to the cell suspension and incubated for 60 min at RT in a rotamixer. The magnetic beads were washed three times using 30 ml of ice-cold PBSP and thereafter inoculated to 50 ml of LB medium containing chloramphenicol. The culture was incubated overnight at 37? C. and an aliquot was spread on agar plates containing chloramphenicol for estimation of enrichment.
[0244] FACS selection: Induced recombinant E. coli cells were washed with 1?PBSP. Cells were resuspended in PBSP containing biotinylated hCD69. The mix was incubated on a rotamixer at RT for 1 h, washed with ice-cold PBSP, and resuspended in 150 nM human serum albumin (HSA)-Alexa 647 conjugate and 0.5 ?g/ml streptavidin conjugated with R-phycoerythrin (SAPE; Invitrogen) or neutravidin conjugated with Oregon Green 488 (NAOG; Life Technologies), followed by incubation on ice for 30 min. The cells were subsequently washed with ice-cold PBSP and resuspended in ice-cold PBSP for sorting in a MoFlo Astrios EQ flow cytometer (Beckman Coulter) or analysis in a Gallios flow cytometer (Beckman Coulter). The E. coli library was sorted in a MoFlo Astrios EQ cell sorter (Beckman Coulter). Bacteria were sorted into a 1.5 ml tube containing LB medium and chloramphenicol. The sorted cells were incubated for 1 h on rotamixer at 37? C. and thereafter inoculated to 50 ml LB medium with chloramphenicol for overnight cultivation.
Results
[0245] The MACS step reduced the library size approximately thousand-fold, from 2.4?10.sup.9 members to approximately 2.4?10.sup.6 members. The reduced library was then enriched in three consecutive rounds of FACS (
Example 3
Biochemical Characterization of CD69-Binding Z Variant
[0246] When the CD69-binding Z variant identified as described in Example 2 was expressed recombinantly, the yield was lower than that which has been generally observed for other Z variants. Using homology alignment against previously reported Z variant domains, five amino acid replacements in the scaffold regions of helix three were identified and mutated. These substitution mutations in ZC69078 were S42A, E43N, S46A, Q50K and S54A, and are all outside of the posited binding surface of the three-helix domain polypeptide. The resulting mutated Z variant, having the amino acid sequence SEQ ID NO:6, is denoted ZC69006 herein. The expression yield for ZC69006 was at least 50-fold higher than that of ZC69078. Note that no mutations have been made to the CD69 binding motif of ZC69078, so ZC69078 and ZC69006 share the same binding motif sequence.
Materials and Methods
[0247] Protein production: The gene encoding ZC69006 was subcloned into three different E. coli expression vectors based on pET22b (GenScript Biotech Corp) under control of a T7 promoter. All constructs had an N-terminal hexahistidine tag incorporated in the sequence MGSS-H.sub.6-YYLE, and contained the gene encoding ZC69006 followed by the dipeptide -VD, introduced for cloning purposes. One construct had an additional C-terminal cysteine, and in another, ZC69006 was followed by a (G.sub.4S).sub.3 linker and then an albumin binding domain (ABD) derived from streptococcal Protein G and having the amino acid sequence SEQ ID NO:164. The three respective expression products were denoted H.sub.6-ZC69006 (SEQ ID NO:160), H.sub.6-ZC69006-Cys (SEQ ID NO:161) and H.sub.6-ZC69006-ABD (SEQ ID NO:162). The ligated vector was transformed into E. coli BL21(DE3) cells (Merck) for expression using standard protocols. The recombinant proteins were purified using HisPur Cobalt Resin (#89966, Thermo Scientific) according to the manufacturer's instructions.
[0248] SPR analysis: Human serum albumin (SEQ ID NO:167, HSA; #A3782, Sigma), extracellular domain from human CD69 (SEQ ID NO:165, hCD69; #8468-CD-025, R&D Systems) and extracellular domain from murine CD69 (SEQ ID NO:166, mCD69; #8469-CD-025, R&D Systems), were each diluted in 10 mM NaAc, pH 4.5 and immobilized on CM5 chip surfaces using EDC/NHS coupling chemistry for use as immobilized targets in a Biacore T200 instrument (GE Healthcare). The surfaces were inactivated using ethanolamine prior to binding studies. A first screening was performed by injecting 100 nM of H.sub.6-ZC69006-ABD over the respective immobilized targets for 120 s, followed by running buffer for 300 s before regeneration of the surfaces. ZC69006 in fusion with ABD was first injected for non-covalent and directed capture on immobilized HSA, followed by injection of respective target molecule. Either 10 mM HCl or 10 mM glycine-HCl pH 2.5 were used for regeneration in the experiments.
[0249] Circular dichroism spectroscopy: Thermal stability and refolding after heat-induced denaturation was measured for H.sub.6-ZC69006 using circular dichroism spectroscopy. All measurements were performed on a Chirascan? instrument (Applied Photophysics Ltd, Surrey, UK). The thermal stability was determined by following the ellipticity at 221 nm during variable temperature measurements (5? C./min from 20? C. to 100? C.). After the heat-induced denaturation, the samples were cooled to RT and left for 15 min before measuring ellipticity at 20? C. from 195 nm to 260 nm in five replicates.
[0250] DOTA conjugation of H.sub.6-ZC69006-Cys: After freeze-drying, H.sub.6-ZC69006-Cys was resuspended in PBS supplemented with an equimolar amount of tris-(2-carboxyethyl)phosphine (TCEP) to 1 mg/ml (118 mM) and incubated for 20 min at 37? C. Following the incubation with TCEP, a 10-fold molar excess of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) maleimide was added and the sample was incubated at 37? C. for 3 h. The progression of the conjugation was monitored by MALDI.
[0251] HPLC purification: The DOTA-conjugated H.sub.6-ZC69006-Cys was diluted using acetonitrile (ACN) with 0.1% trifluoroacetic acid (TFA) to a final concentration of 20% ACN, and then sterile filtered through a 0.2 ?m filter prior to injecting into HPLC for purification on a Semi-Prep C18 column. A gradient from 20% to 56% ACN at 2.5 ml/min over 30 min was used. 500 ?l fractions were collected for the peaks, and subsequently analyzed by MALDI.
[0252] Mass spectrometry (MS): MALDI was used to verify DOTA conjugation during the conjugation as well as after HPLC purification. All samples were analyzed on a 4800 MALDI (Applied Biosystems). 1 ?l sample was mixed with 1 ?l ?-cyano-4-hydroxy-cinnamic acid matrix (Bruker Daltonics), added to the MALDI plate and substituted with another ?l of matrix before loading the plate in the MS.
Results
[0253] Protein production: The Z variant molecules H.sub.6-ZC69006, H.sub.6-ZC69006-Cys and H.sub.6-ZC69006-ABD were purified after expression to high purity using IMAC, as evidenced by SDS-PAGE (
[0254] SPR analysis: H.sub.6-ZC69006-ABD was shown to bind to HSA, hCD69 and mCD69 (
[0255] Circular dichroism spectroscopy: The melting temperature for H.sub.6-ZC69006 was determined by circular dichroism spectroscopy and variable temperature measurements to be 70? C. (
[0256] DOTA conjugation: H.sub.6-ZC69006-Cys was successfully conjugated with DOTA to a high degree as determined by MALDI, and subsequently purified using HPLC.
Example 4
Characterization of ZC69006 in Cell Assays and In Vivo
Aims
[0257] This Example describes the radiolabeling and evaluation of the DOTA conjugated CD69-binding Z variant ZC69006 prepared as described in Example 3. The radionuclide indium-111 was selected for labeling, because it chelates stably with the DOTA chelator and has a radioactive half-life that is suitable for preclinical evaluation, i.e. one labeled batch can be used for several experiments. Furthermore, indium-111 is a photon emitter suitable for SPECT imaging.
Methods
[0258] Z variant radiolabeling: H.sub.6-ZC69006-Cys was expressed and conjugated to DOTA via the cysteine at the C terminus as described in Example 3. The Z variant ZTAQ, raised against bacterial Taq polymerase and not binding to CD69, was similarly conjugated with DOTA for use as negative control. The polypeptides are denoted DOTA-ZC69006 and DOTA-ZTAQ in the following for brevity. DOTA-ZC69006 and DOTA-ZTAQ were labelled with indium-111 in a buffered solution (pH=5.0-5.5) at elevated temperature. The crude products were purified using solid phase extraction cartridges. Product was eluted with 50% ethanol and formulated in phosphate buffered saline. The radiochemical purity was controlled by HPLC with UV and radio detectors coupled in series. .sup.111In-DOTA-ZC69006 and .sup.111In-DOTA-ZTAQ were obtained with a radiochemical purity of >95%.
[0259] In vitro cell binding: .sup.111In-DOTA-ZC69006 was incubated with 0.3-2 million human peripheral blood mononuclear cells (PBMC) or mouse splenic cells, either non-activated or activated by anti-CD3 antibody (1 ?g/ml; ab8090, Abcam) in fetal calf serum (FCS). The fraction of CD69+ cells in each preparation was assessed by flow cytometry. Briefly, radioactive .sup.111In-DOTA-ZC69006 (target amount 500 kBq, corresponding to 10 nM peptide, incubation volume 1 ml) was added to each cell suspension and incubated for 1 h at 37? C. The cells were then washed and the supernatant collected after centrifugation. The samples and relevant controls (background, references) were measured in a gamma counter (Wizard). All sample measurements were performed in triplicates. Afterwards, the background activity and the remaining activity in the empty Eppendorf vials were measured separately. Cell binding was expressed as % of total incubated amount of .sup.111In-DOTA-ZC69006 bound per million cells.
[0260] Animal handling: All procedures involving animals were approved by the Animal Ethics Committee of the Swedish Animal Welfare Agency and carried out in accordance with the relevant national and institutional guidelines (Uppsala university guidelines on animal experimentation, UFV 2007/724). During each imaging session, the animals (rats and mice) were sedated by gas anesthesia (sevoflurane) through a facemask. Temperature was maintained by warm air supply integrated in the scanner bed. All SPECT/CT examinations were performed in a fully quantified nanoSPECT/CT scanner (Mediso, Hungary). SPECT were acquired using the energy windows suitable for indium-111 emission (energy map: primary peak 245.35 kEv, secondary peak 171.30 kEv), and reconstructed using an iterative algorithm (iterations/subsets 48/3). CT acquisition was performed before all SPECT examinations for anatomical co-registration (semicircular multi-field-of-view; duration 7:46 min; 3 rotations; scan length 231.66 mm; 480 projections; binning 1:4; 50 kV; 600 ?A; voxel size 0.25?0.25?0.25 mm).
[0261] In vivo imaging in rats: .sup.111In-DOTA-ZC69006 distribution in healthy rats was assessed by SPECT/CT imaging and compared to the distribution of .sup.111In-DOTA-ZTAQ. Sprague-Dawley rats (n=3, male) were injected in the lateral tail-vein with 2.1-5.3 MBq of .sup.111In-DOTA-ZC69006 and examined by SPECT/CT (nanoSPECT, Mediso, Hungary), just after injection. The animal was positioned by a whole-body CT acquisition. Next, a 20 min whole body static SPECT examination was performed. The SPECT/CT examinations were repeated 3 h, 20 h and 48 h post injection. A second group of rats (n=3) was examined similarly, following administration of the negative control molecule .sup.111In-DOTA-ZTAQ.
[0262] In vivo imaging in mice with islet allograft: NMRI mice (n=5) were transplanted subcutaneously on the left flank with an islet allograft isolated from Balb/c mice. Five or 6 days later, when rejection of the graft was underway, the animals were administered 0.6-0.7 MBq .sup.111In-DOTA-ZC69006 in the tail-vein. One mouse was imaged repeatedly over the first 2 h post injection (4 static whole-body scans, 30 min duration each), to determine the optimal imaging time-point. The remaining mice were imaged with a single 30 min static whole-body examination 1 h post injection.
[0263] SPECT data analysis and predicted dosimetry in human: SPECT image analysis for all four time-points (0, 3, 20 and 48 h post injection) was performed using the PMOD 3.8 software (PMOD Technologies, Zurich, Switzerland). The aorta, kidney, muscle, liver, bladder and hind leg lymph nodes were segmented using co-registered CT projections as support. The uptake values were converted to standardized uptake values (SUV) by correcting for animal weight and administered amount of .sup.111In-DOTA-ZC69006. The biodistribution data over 48 h in healthy rats was used to extrapolate the predicted human dosimetry. Residence times were estimated as described previously and the absorbed doses calculated by the OLINDA/EXM 1.1 software (Vanderbilt, Nashville, USA).
[0264] Statistical analysis: Values are given as averages?standard deviations. Statistical analysis and data processing were performed using GraphPad Prism 8.02 for Macintosh/Windows (GraphPad Software Inc) and Microsoft Office Excel 2016 for Macintosh/Windows (Microsoft).
Results
[0265] In vitro cell binding: .sup.111In-DOTA-ZC69006 binding was higher in CD3 activated human PBMCs compared to resting PBMCs (
[0266] In vivo imaging in rats: .sup.111In-DOTA-ZC69006 distributed rapidly throughout the body, with fast clearance from the blood pool combined with renal excretion in all three rats (
[0267] The uptake of .sup.111In-DOTA-ZC69006 in the blood pool was low, as seen in the major arteries and veins, as well as in the left ventricle. This can also be inferred from the very low background binding in the blood rich liver throughout the 48 h imaging time period (
[0268] Except kidney and bladder, the only tissues that displayed a strong, sustained uptake of .sup.111In-DOTA-ZC69006 were small focal uptake sites consistent with the localization of lymph nodes. In a representative rat, shown in
[0269] Importantly, the negative control molecule .sup.111In-DOTA-ZTAQ did not exhibit any uptake in lymph-nodes in any of the animals (
[0270] The predicted absorbed dose of .sup.111In-DOTA-ZC69006 in human was the highest in the kidneys (4.65 mGy/MBq) and the whole-body effective dose was 0.16 mSv/MBq.
[0271] In vivo imaging in mice with islet allograft: .sup.111In-DOTA-ZC69006 displayed rapid renal excretion and low background in all mice, similarly to rats. In a representative mouse examined at day 5 post-transplantation, there was elevated uptake at the site of the allogenic islet graft (ig) (
[0272] Two of the mice examined with .sup.111In-DOTA-ZC69006 in the morning on day 6 post transplantation also displayed binding at the site of implantation, but of a lower magnitude. This indicated a lower amount of activated immune cells as the rejection process was subsiding. Two additional mice, also transplanted 6 days earlier and scanned at the end of day 6, displayed the lowest binding at the site of the islet allograft, potentially indicating the absence of active immune cells at the site following graft rejection.
Conclusions
[0273] In this Example, DOTA-ZC69006 was radiolabeled with indium-111 to enable optimal preclinical evaluation with regard to long time follow-up of biodistribution. The long half-life allowed repeated in vivo imaging sessions in several animals over the course of a week, in order to follow tracer kinetics. However, indium-111 is a SPECT radionuclide with an undesirable dosimetry profile due to its 3-day radioactive half-life. This was illustrated here by the relatively high radiation dose seen especially in the kidney. The positron-emitting radionuclide gallium-68 in combination with PET would improve sensitivity, resolution, quantification accuracy and reduce radiation dose. Consequently, the further development towards clinical use is contemplated to be focused on .sup.68Ga-labelled analogues.
[0274] Strong selective and sustained binding was demonstrated in a subcutaneous islet allograft undergoing rejection (
[0275] All three rats examined by SPECT/CT exhibited variable but sustained and clearly detectable uptake of .sup.111In-DOTA-ZC69006 in different lymph nodes across the body. This is consistent with the immune response against local, subclinical infection. The specificity of .sup.111In-DOTA-ZC69006 for lymph nodes was indirectly demonstrated by performing the same set of experiments using the negative control .sup.111In-DOTA-ZTAQ, which has an amino acid sequence that does not confer binding to CD69. No detectable uptake was observed in lymph nodes when using .sup.111In-DOTA-ZTAQ. Furthermore, several other Z variants, targeting e.g. HER2 and HER3, have previously been radiolabeled and thoroughly examined in vivo in animals and humans, and focal uptake in lymph nodes has not been observed in such previous studies either. Thus, the lymph node binding of .sup.111In-DOTA-ZC69006 is likely to be an active and specific process. These encouraging results warrant the further development of gallium-68 and fluorine-18 labelled analogues of DOTA-ZC69006 for imaging of activated immune cells by PET.
[0276] In conclusion, .sup.111In-DOTA-ZC69006 is a novel CD69-binding Z variant useful for non-invasive imaging of activated immune cells.
Example 5
Affinity Maturation of the First Generation CD69-Binding Z Variant
[0277] ZC69006 has an affinity of just over 50 nM to human and murine CD69. For PET tracers, the affinity (K.sub.D) is generally inversely correlated to the ability to detect lower number of receptors (B.sub.max). Thus, high affinity is crucial for detecting small changes in CD69-presenting immune cells. In order to achieve a higher affinity, affinity maturation was carried out on ZC69078 identified in Examples 1-2, i.e. the original version of Z variant ZC69006 studied extensively in Examples 3-4.
Methods
[0278] Design of an affinity maturation library: An affinity maturation library was designed with the distribution of varied amino acid positions shown in Table 3 below.
TABLE-US-00016 TABLE 3 Design of Z variant library for affinity maturation selection Position in the Z variant sequence Permitted amino acid residues Proportions 9 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y F: 70%; Rest: 1.9% each 10 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y Y: 70%; Rest: 1.9% each 11 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y H: 70%; Rest: 1.9% each 13 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y M: 70%; Rest: 1.9% each 14 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y K: 70%; Rest: 1.9% each 17 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y L: 70%; Rest: 1.9% each 18 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y K: 70%; Rest: 1.9% each 24 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y K: 70%; Rest: 1.9% each 25 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y Y: 70%; Rest: 1.9% each 27 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y K: 70%; Rest: 1.9% each 28 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y E: 70%; Rest: 1.9% each 31 D, H, I, K, Y K: 60%; D: 10%; H: 10%; I: 10%; Y: 10% 32 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y T: 70%; Rest: 1.9% each 35 A, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, Y K: 70%; Rest: 1.9% each
[0279] DNA encoding the library was obtained from Twist Bioscience. Transformation of the DNA into E. coli BL21(DE3) cells (Merck) gave 6?10.sup.8 transformants. 96 clones were picked at random and sequenced. The sequencing showed that the library was highly functional and no sequence occurred more than once, except for the original variant ZC69078 (SEQ ID NO:78), which occurred 6 times.
[0280] FACS selection: Induced recombinant E. coli cells were washed with 1?PBSP. Cells were resuspended in PBSP containing biotinylated hCD69. The mix was incubated on a rotamixer at RT for 1 h, followed by extended washes with ice-cold PBSP, and resuspended in 150 nM human serum albumin (HSA)-Alexa 647 conjugate and 0.5 ?g/ml streptavidin conjugated with R-phycoerythrin (SAPE) (Invitrogen) or neutravidin conjugated with Oregon Green 488 (NAOG) (Life Technologies), followed by incubation on ice for 30 min. The cells were subsequently washed with ice-cold PBSP and resuspended in ice-cold PBSP for sorting in a MoFlo Astrios EQ flow cytometer (Beckman Coulter) or analysis in a Gallios flow cytometer (Beckman Coulter). The E. coli library cells were sorted in a MoFlo Astrios EQ cell sorter (Beckman Coulter).
[0281] The sort gate was set to sort out the top fraction of cells displaying Z variants (typically 0.1%) showing the highest R-phycoerythrin or Oregon Green 488 to Alexa Fluor? 647 fluorescence intensity ratio.
[0282] Bacteria were sorted into a 1.5 ml tube containing LB medium and chloramphenicol. The sorted cells were incubated for 1 h on rotamixer at 37? C. and thereafter inoculated to 50 ml LB medium with chloramphenicol for overnight cultivation.
Results and Conclusions
[0283] Flow-cytometric sorting for isolation of Z variants with an improved affinity: For isolation of Z variants with an improved affinity for human CD69, the E. coli display library was subjected to four rounds of fluorescence-activated cell sorting (FACS) with alternating rounds of amplification by cell growth. Briefly, cells were incubated with biotinylated hCD69 followed by extensive washes and then incubated with fluorescently labeled streptavidin for subsequent fluorescence-mediated detection of cell-bound hCD69 as well as fluorescently labeled HSA for monitoring of surface expression levels. The incubation of secondary reagents and HSA was performed on ice in order to reduce the dissociation rate of bound hCD69. After an additional washing, the labeled cell library was screened and sorted in a flow cytometer. Selection stringency in terms of target concentration, sorting parameters and sorting gates was increased with each sorting round and typically, the top 0.1% of the library demonstrating the highest ratio of target binding to surface expression, was gated and isolated for amplification and subsequent rounds of sorting. One advantage with cell-based selection systems is the straightforward monitoring of the obtained enrichment throughout the selection process. The visualization of the target-binding properties of the library in the flow cytometer revealed an enrichment of hCD69-positive clones in each sorting round (
[0284] Identification of affinity-matured candidates using FACS: The affinity maturation library was sorted based on surface expression and hCD69-binding. Among the clones isolated from the flow-cytometric sorting, randomly picked variants were sequenced. The amino acid sequences of the 58 amino acid residues long Z variants are listed in
[0285] Clones appearing more than once among the sequences were selected for further characterization. In
Example 6
Biochemical Characterization of Affinity Matured, CD69-Binding Z Variants
Methods
[0286] Protein production: Z variants from Example 5 that were selected for further characterization were subjected to site-directed mutagenesis in analogy to the creation of ZC69006 from ZC69078 in Example 3, i.e. introduction of the scaffold position mutations S42A, E43N, S46A, Q50K and S54A, creating mutated Z variants named ZC69001 (SEQ ID NO:1), ZC69002 (SEQ ID NO:2), ZC69003 (SEQ ID NO:3), ZC69004 (SEQ ID NO:4) and ZC69005 (SEQ ID NO:5). Genes encoding these five Z variants were each subcloned into tree different E. coli expression vectors based on pET22b (GenScript Biotech Corp) under control of a T7 promoter. Again in analogy to Example 3, all constructs had an N-terminal hexahistidine tag incorporated in the sequence MGSS-H.sub.6-YYLE, and the corresponding gene encoding the Z variant sequence in question followed by the dipeptide -VD. For each Z variant, one of the three constructs had a C-terminal cysteine, and one of the three constructs encoded the H.sub.6-tagged Z variant in fusion with a linker and ABD. The three expression products for each Z variant were denoted H.sub.6-ZC6900 #, H.sub.6-ZC6900 #-Cys and H.sub.6-ZC6900 #-ABD, wherein ZCD6900# corresponds to one of the five Z variants listed above (
[0287] SPR evaluation of affinity matured variants: In analogy with Example 3, the target proteins HSA, hCD69 and mCD69 were immobilized on individual CM5 chip surfaces using EDC/NHS coupling chemistry on a Biacore T200 instrument (GE Healthcare), essentially as described in Example 3 for the primary Z variant. The surfaces were inactivated using ethanolamine prior to binding studies. One surface was activated/inactivated for blank subtraction. The five Z variants selected in the affinity maturation procedure described in Example 5 and expressed in the format H.sub.6-ZC6900 #-ABD as described above were injected in the concentration range 1 nM, 5 nM, 25 nM, 50 nM and 100 nM, in triplicates over all four surfaces.
[0288] Circular dichroism spectroscopy: Thermal stability and refolding after heat-induced denaturation was measured for each H.sub.6-Z variant using circular dichroism spectroscopy. All measurements were performed on a Chirascan? instrument (Applied Photophysics Ltd, Surrey, UK). The thermal stability was determined by following the ellipticity at 221 nm during variable temperature measurements (5? C./min from 20? C. to 100? C.). After the heat-induced denaturation, the samples were cooled to RT and left for 15 min before measuring ellipticity at 20? C. from 195 nm to 260 nm in five technical replicates.
Results
[0289] SPR evaluation of affinity matured variants: The affinity towards hCD69 of the five studied Z variants was tested. A representative sensorgram from the SPR analysis is given for Z variant ZC69002 in
TABLE-US-00017 TABLE 4 Affinity constants for tested Z variants from SPR Tested Z variant K.sub.D (nM) in the format H.sub.6-Z-(G.sub.4S).sub.3-ABD vs. hCD69 vs. mCD69 vs. HSA ZC69006 52 67 0.27 ZC69001 34 34 0.17 ZC69002 51 46 0.09 ZC69003 49 ND* 0.49 ZC69004 29 ND 0.78 ZC69005 30 ND 0.52 *ND: Not determined
[0290] Circular dichroism spectroscopy: The melting temperature for four of the five new Z variants was determined by circular dichroism spectroscopy and variable temperature measurements. As shown in Table 5, they all had similar melting temperatures as ZC69006. Measuring the CD spectrum before and after heat-induced denaturation demonstrated complete refolding for all four variants, as shown for ZC69001 in representative
TABLE-US-00018 TABLE 5 Melting temperatures for tested Z variants from CD Tested Z variant in the format H.sub.6-Z Tm (? C.) ZC69006 62 ZC69001 59 ZC69002 59 ZC69003 62 ZC69005 60
Example 7
Characterization of Affinity Matured CD69-Binding Z Variants in an In Vivo Biodistribution Study
Aims
[0291] The primary Z variant ZC69006 (Examples 1-4) and the five affinity matured Z variants ZC69001, ZC69002, ZC69003, ZC69004 and ZC69005 (Examples 5-6) were studied further.
[0292] Outsourced production of large quantities of TCO-conjugated precursor material is expensive. To provide more data for the rational selection of an optimal CD69-binding Z variant, it was decided to produce smaller batches of DOTA-conjugated variants to enable indium-111 radiolabeling and evaluation of critical parameters in vitro and in vivo. This Example describes carrying out this evaluation in order to select an optimal candidate for medical imaging applications.
Methods
[0293] DOTA conjugation: The six tested Z variants were expressed in the format H.sub.6-ZC6900#-Cys and conjugated with DOTA as described for ZC69006 in Example 3. They are referred to as DOTA-ZC6900# for brevity below.
[0294] Indium-111 radiolabeling: All reagents were purchased from Sigma-Aldrich (analytical grade or higher) and used without further purification, unless stated otherwise. Indium-111 chloride (370 MBq/ml) was purchased from Curium. Ammonium acetate buffer (0.2 M, pH 5.5) was prepared in plastic flask (200 ml, HDPE low metal resin from Nalgene) by dissolving 0.771 g ammonium acetate in 200 ml water. pH was adjusted by adding a few drops of glacial acetic acid (>99%, TraceSELECT), while measuring with a pH meter (Mettler Toledo). Chelex 100 sodium was added to the buffer, which was allowed to stand in a refrigerator (4? C.) overnight. A NAP-5 column (illustra; GE Healthcare) was pre-treated with 3 ml 1% bovine serum albumin (BSA) and rinsed with excess 6 ml phosphate buffered saline (PBS). Protein LoBinding Eppendorf tubes (1.5 ml) were used for the reaction and elution collection.
[0295] Analytical HPLC was performed with a VWR Hitachi Chromaster 5110 pump, a Knauer UV detector 40D, a Bioscan Flow count equipped with an Eckert & Ziegler extended range module 106, a Bioscan B-FC-3300 radioactivity probe, a VWR Hitachi Chromaster A/D Interface box and a Vydac 214MS, 5 ?m C4, 50?4.6 mm column. The eluents were: A=0.1% TFA in water, B=0.1% TFA in acetonitrile, with an elution gradient from 5 to 70% B over 15 min ata flow rate of 1.0 ml/min.
[0296] For radiolabeling, a hydrochloric acid solution (0.02 M) of .sup.111InCl.sub.3 (Curium) was buffered with sodium acetate or HEPES, and pH was adjusted to 5.0-5.5. Thereafter, the respective DOTA-conjugated Z variant (3-14 nmol) dissolved in phosphate buffer was added, with the exception that DOTA conjugation of ZC69004 repeatedly exhibited low yields, so this variant was excluded from the radiolabeling experiment. The reaction mixture (in total 400-500 ?l) was incubated at 80? C. for 30-60 min. The crude product was purified on solid phase extraction cartridge (HLB, OASIS; eluted with 1 ml of 50% ethanol) or NAP-5 column (elution with 200 ?l PBS?5). See Table 6 for details of the conditions for each variant. The difference in details are due to progressive optimization of the procedure for this class of Z variants. All radiochemical yields (RCYs) were isolated yields and purity was measured by HPLC.
TABLE-US-00019 TABLE 6 Radiolabeling conditions Z variant ?l .sup.111In MBq Nmol Z Reaction as H.sub.6-Z-DOTA solution .sup.111In variant time Buffer pH Purification ZC69006 280 14 3 30 min sodium 5.5 SPE acetate ZC69001 150 156 13.9 1 h ammonium 5.2 NAP-5 acetate ZC69002 300 103 10 30 min HEPES 5.0 SPE (0.1M) ZC69003 120 120 13.4 1 h ammonium 5.5 NAP-5 acetate ZC69005 100 102 4.7 30 min ammonium 5.5 NAP-5 acetate
[0297] Assessment of biodistribution by SPECT/CT imaging in rats: All procedures involving animals were approved by the Animal Ethics Committee of the Swedish Animal Welfare Agency and carried out in accordance with the relevant national and institutional guidelines (Uppsala university guidelines on animal experimentation, UFV 2007/724).
[0298] .sup.111In-DOTA-Z variant biodistribution in healthy rats was assessed by SPECT/computed tomography (CT) imaging. Sprague-Dawley rats (n=12 in total, n=3 per Z variant, male, weight 310?40 g) were injected in the lateral tail-vein with approximately 8 MBq of indium-111 labeled Z variant (.sup.111In-DOTA-ZC69006: 3.9?1.6 MBq; .sup.111In-DOTA-ZC69001: 12.6?4.6 MBq; .sup.111In-DOTA-ZC69002: 5.5?1.3 MBq; .sup.111In-DOTA-ZC69003: 8.0?2.6 MBq; .sup.111In-DOTA-ZC69005: 9.9?2.6 MBq).
[0299] Each animal was examined by SPECT/CT (nanoSPECT, Mediso, Hungary) immediately post injection as well as 3 h, 20 h, 48 h and 72 h post injection. For each scan, the animal was anesthetized and positioned by a whole-body CT acquisition. Next, a 20-minute whole body static SPECT examination was performed. During each imaging session, the rat was sedated by gas anesthesia (sevoflurane) through a facemask. Temperature was maintained by warm air supply integrated in the scanner bed.
[0300] All SPECT/CT examinations were performed in a fully quantified nanoSPECT/CT scanner (Mediso, Hungary). SPECT were acquired using the energy windows suitable for indium-111 emission (energy map: primary peak 245.35 kEv, secondary peak 171.30 kEv), and reconstructed using an iterative algorithm (iterations/subsets 48/3). CT acquisition was performed before all SPECT examinations for anatomical co-registration (semicircular multi-field-of-view; duration per bed 7:46 min; 3 rotations; scan length 231.66 mm; 480 projections; binning 1:4; 50 kV; 600 ?A; voxel size 0.25?0.25?0.25 mm).
[0301] SPECT image analysis for all time-points was performed using the Nucline software (Mediso, Hungary). The kidneys, liver, heart, lung and muscle were segmented directly on SPECT images using co-registered CT projections as support. Also, distinct uptake in lymph nodes along the main vessels as well as in the hind leg was identified and segmented. The uptake values were decay corrected to the time of administration and converted to standardized uptake values (SUV) by correcting for animal weight and administered amount of .sup.111In-DOTA-Z variant.
[0302] In animals where distinct uptake in one or more lymph nodes was identified, a post mortem examination was performed. After euthanasia, the lymph node (as well as surrounding tissues, e.g. adipose tissue) was localized and excised. The biopsies were measured for radioactivity in an automated gamma counter (2480 Wizard2?, PerkinElmer). Then, the biopsies were snap frozen, embedded in OCT media, processed into 10 ?m sections, placed on SuperFrost object glasses and exposed to a phosphorimager plate with a known reference for quantification (i.e. ex vivo autoradiography).
[0303] Statistical analysis: Values are given as averages?standard deviations. Statistical analysis and data processing were performed using GraphPad Prism 8.02 for Macintosh/Windows (GraphPad Software Inc) and Microsoft Office Excel 2016 for Macintosh/Windows (Microsoft).
Results and Conclusions
[0304] Indium-111 radiolabeling: All Z variants were radiolabeled, except for ZC69004 for which DOTA conjugation repeatedly exhibited low yields. Indium-111 chelation was successful for all other tested variants, i.e. for DOTA-ZC69006, DOTA-ZC69001, DOTA-ZC69002, DOTA-ZC69003 and DOTA-ZC69005, demonstrating acceptable yields and radiochemical purity (Table 7,
TABLE-US-00020 TABLE 7 Results of radiolabeling tested Z variants with indium-111 Z variant as H.sub.6-Z- A.sub.m (MBq/ DOTA A (MBq) Purity nmol) RCY Stability ZC69006 14 99% 5 5% Not performed ZC69001 85.0 99% 6 71% 91% (4 days) ZC69002 92 98% 9 90% 98% (1 day).sup. ZC69003 76.5 100% 6 84% 88% (4 days) ZC69005 83.2 99% 18 93% 67% (4 days)
[0305] Biodistribution: All indium-111 labeled Z variants exhibited rapid excretion though the kidneys (
[0306] For the background binding in liver and muscle tissue, .sup.111In-DOTA-ZC69001 demonstrated the lowest binding, followed by .sup.111In-DOTA-ZC69003. .sup.111In-DOTA-ZC69006, .sup.111In-DOTA-ZC69002 and .sup.111In-DOTA-ZC69005 all had higher background binding in liver and muscle (
[0307] Lymph node targeting: Like the primary Z variant, the affinity matured variants also exhibited strong and sustained uptake in different lymph nodes in the trunk of the body and in the hind body, indicative of an immune response against local, subclinical infection, in some of the animals (
[0308] The binding as measured by SPECT was confirmed by ex vivo autoradiography, demonstrating a strong signal for the lymph nodes that were positive on the SPECT images, but a negligible signal from the surrounding adipose tissue (
Conclusions and Selection of Candidate
[0309] For use in medical imaging in a human, the renal uptake and retention dose should be minimized to reduce the predicted extrapolated absorbed radiation dose to the kidney. Furthermore, the background binding of the radioligand should be minimized for optimal image contrast in lesions or tissues with activated CD69 expressing immune cells. All tested Z variants demonstrated an acceptable kidney dose and background binding. However, based on biodistribution, Z variant ZC69001 seems optimal for further development.
Example 8
PET Radiolabeling and Characterization of ZC69001 in an In Vitro Cell Binding Assay
Aims
[0310] This Example describes the radiolabeling and in vitro evaluation of the CD69-binding Z variant ZC69001 in a cell binding assay. The radionuclides gallium-68 and fluorine-18 was selected for labeling in this setting, because both are suitable radionuclides for quantitative PET imaging, further evaluated in Example 9.
Methods
[0311] Gallium-68 radiolabeling of ZC69001: ZC69001-Cys (SEQ ID NO:1 directly followed by a cysteine residue) was produced by chemical peptide synthesis and conjugated with DOTA on the C-terminal cysteine (Almac). DOTA-ZC69001 was labelled with gallium-68 (produced by a generator) in a buffered solution (pH=4.0-5.5) at elevated temperature. The crude products were purified using solid phase extraction cartridges. Product was eluted with 50% ethanol and formulated in phosphate buffered saline. The radiochemical purity was controlled by HPLC with UV and radio detectors coupled in series.
[0312] Fluorine-18 radiolabeling of ZC69001: ZC69001-Cys (SEQ ID NO:1 directly followed by a cysteine residue) was obtained functionalized with a trans-cyclooctene (TCO) on the C-terminal cysteine via a PEG3 linker (Almac). This Z variant was produced by chemical synthesis rather than by recombinant expression. ZC69001-TCO was labelled with fluorine-18 by the following three-step procedure: labelling of an activated ester on solid support, conversion to a tetrazine-amide derivative, and conjugation of this tetrazine-amide derivative to ZC69001-TCO. The reaction between the labelled tetrazine and the TCO-modified biomolecule was efficient and rapid, and performed in an aqueous phosphate buffer (pH 7.4) at RT. Purity was assessed by HPLC.
[0313] Generation of a CD69 overexpressing cell line: A CHO-K1 cell line was purchased from ATCC and cultured in Ham's F-12 (Biowest), 10% FBS (Sigma) and 1% penicillin/streptomycin (Merck Millipore). The human CD69 cDNA sequence was constructed from the NM_001781.2 NCBI Reference Sequence Database (RefSeq) and purchased from Genscript Biotech Corporation as a pcDNA3.1+/C-(K)DYK vector. The CHO-K1 cells were cultured to 80% confluence before transfection. The CD69 cDNA clone sequence was mixed with Lipofectamine 3000 reagents (Invitrogen) and prepared according to the manufacturer's guideline. Transfected CHO-K1 clones were selected using 1 mg/ml of Geneticin (ThermoFisher) before moving to a pressure concentration of 0.4 mg/ml. Surface expression of CD69 was analyzed by fluorescence activated cell sorting (FACS) using APC conjugated anti-human CD69 antibody (FN50, Biolegend).
[0314] Binding studies: Functional binding of .sup.18F-TZ-ZC69001 was evaluated using the CD69 overexpressing CHO-K1 cell line. Background binding was assessed by blocking the CD69 receptor by pre-incubation with excess of unlabeled ZC69001-Cys. .sup.18F-TZ-ZC69001 binding was evaluated using viable detached cells incubated with different concentrations of tracers using a gamma counter (Wallac).
Results and Conclusions
[0315] Radiolabeling: Both .sup.68Ga-DOTA-ZC69001 and .sup.18F-TZ-ZC69001 were reproducibly obtained with a radiochemical purity of >95%.
[0316] Cell binding studies: .sup.18F-TZ-ZC69001 bound to CD69 transfected CHO-K1 cells in a manner that could be inhibited by addition of excess ZC69001-Cys (
Example 9
In Vivo Imaging of ZC69001 in Disease Models
Aims
[0317] This Example describes the use of the radiolablelled CD69-binding Z variant ZC69001 for in vivo PET imaging of inflammation and of immunotherapy treated animals. ZC69001 radiolabled with either .sup.68Ga or .sup.18F as described in Example 8 is used.
Methods
[0318] In vivo imaging in induced model of rheumatoid arthritis (RA) in mice: A mouse strain (T-cells knockout, on mixed C57/BL6/NOD background) were administered splenic cells from a transgenic mouse stain (KRN T-cells, C57/BL6 background). After administration, the recipient mice usually develop a model of RA after 2-7 days. The RA is progressive until around 2-3 weeks and associated with increased swelling of the paws as well as infiltration of immune cells. Here, recipient mice (n=8) were given splenic cells from donor mice (n=3). Five of the mice were used for longitudinal PET imaging, while three were used for histological morphometry at different stages of RA progression. The group of five mice were examined by .sup.68Ga-DOTA-ZC69001 PET/CT four timesbefore induction of RA (baseline), and 3, 7 and 12 days after induction of RA. All animals were evaluated for clinical symptoms by weight loss and the RA score (sum of grade 0-3 swelling for each of the four paws, total grade from 0 (no symptoms) to 12 (severe RA)).
[0319] For PET/CT, a target dose of 2 MBq .sup.68Ga-DOTA-ZC69001 was administered in the tail vein. After 1 h, each mouse was anaesthetized and examined by a 30 min whole body PET scan (nanoPET/MRI system, Mediso) and a CT scan for anatomical correlation (nanoSPECT/CT system, Mediso) using a detachable bed.
[0320] Binding of .sup.68Ga-DOTA-ZC69001 in hind leg joints was evaluated in image analysis software PMOD (PMOD technologies) and expressed as Standardized Uptake Values (SUV) normalized for injected amount of radioactivity and mouse weight to enable inter- and intra-individual comparison.
[0321] In vivo imaging of lung inflammation in pigs: Pigs (n=3) were anaesthetized, placed on ventilator and prepared for PET imaging of the lung. Two of the pigs were induced with lung inflammation while one was not. For this study, an established ventilator induced model of lung inflammation (VILI) was used, consisting of repeated lung lavages followed by injurious ventilation. Briefly, repeated lavages were performed with 35 mL/kg warm NaCl 0.9%. The lung function was assessed after each set of three lavages by monitoring ventilator readout and arterial blood gases (p/f ratio, Vd/Vt ratio, oxygen saturation). Lavages were performed until the p/f ratio was recorded as below 100 (severe ARDS) or the maximum of 8 lavages was reached. Then, VILI was started (PEEP=0; Target Ppeak=35; Respiratory Rate (RR)=30; Tidal volume 300 mL, FiO2=100) and continued for appr 1 h.
[0322] Each pig was examined by PET over the lungs using a Discovery MI PET/CT (GE healthcare). Two of the pigs, one with induced lung inflammation and one untreated control, were administered 1 MBq/kg .sup.68Ga-DOTA-ZC69001 intravenously and examined by dynamic PET/CT for 90 min and a whole body static scan. The second pig with induced lung inflammation was administered 1 MBq/kg .sup.68Ga-DOTA-ZAM106, a non-CD69-binding control peptide of the same molecular size and scaffold.
[0323] Chimeric antigen receptor T cells (CAR-T) immunotherapy mouse model: .sup.18F-TZ-ZC69001, prepared and tested as described in Example 8, is evaluated in a mouse model of CAR-T cell treatment of lymphoma. Murine B-cell lymphoma cells are injected subcutaneously. When tumors form, the mice are treated with murine CD20-directed CAR-T cells secreting neutrophil-activating protein (NAP) upon target cell recognition. Five to seven days later, activated T cells in the tumor area are imaged with .sup.18F-TZ-ZC69001. Mice are treated with conventional CD20-directed CAR-T cells or mock-transduced T cells as control. 10 MBq .sup.18F-TZ-ZC69001 is administered in the tail vein under sevoflurane anesthesia. Animals are examined by dynamic PET for up to 2 h using a nanoPET/MRI scanner (Mediso). After the PET examination, the animals are euthanized, organs excised, weighed and measured in a gamma counter. Part of the tumor and reference organs are snap frozen, embedded in OCT media, sectioned and exposed to a phosphorimager plate to generate ex vivo autoradiography images of .sup.18F-TZ-ZC69001 distribution in the tumor microenvironment. Part of the tumor is fixed in PFA and stained with e.g. H&E, and immunostained for CD69 and other relevant immune and tumor markers.
Results
[0324] In vivo imaging in induced model of rheumatoid arthritis (RA) in mice: .sup.68Ga-DOTA-ZC69001 binding was low or negligible in all mice at baseline, before induction of RA. PET binding was elevated already on day 3, increasing further on day 7 and finally on day 12 (
[0325] In vivo imaging of lung inflammation in pigs: .sup.68Ga-DOTA-ZC69001 demonstrated elevated binding of .sup.68Ga-DOTA-ZC69001 in the lung of pig with induced lung injury and inflammation, compared to lung binding in a control pig. In contrast, the non-CD69-binding control peptide 88 Ga-DOTA-ZAM106 exhibited negligible binding in a pig with lung inflammation, indicating that the scaffold peptide itself does not accumulate in inflamed tissue in a non-specific manner (
[0326] Chimeric antigen receptor T cells (CAR-T) immunotherapy mouse model: .sup.18F-TZ-ZC69001 is expected to accumulate in the tumors of immunotherapy treated animals, but not in the controls. The PET results are expected to be confirmed by ex vivo autoradiography and correlative staining.
Itemized List of Embodiments
[0327] 1. CD69-binding polypeptide, comprising a CD69-binding motif BM, which motif consists of an amino acid sequence selected from:
TABLE-US-00021 i) (SEQIDNO:168) EX.sub.2X.sub.3X.sub.4AX.sub.6X.sub.7EIX.sub.10X.sub.11LPNLX.sub.16 X.sub.17X.sub.18QKX.sub.21AFKX.sub.25X.sub.26LKD
wherein, independently of each other, [0328] X.sub.2 is selected from F, H, V and W; [0329] X.sub.3 is selected from A, D, E, H, N, Q, S, T, Y; [0330] X.sub.4 is selected from A, D, E, H, K, M, N, S, V, W and Y; [0331] X.sub.6 is selected from M, W and Y; [0332] X.sub.7 is selected from A, H, K, N, Q, R, W and Y; [0333] X.sub.10 is selected from L and R; [0334] X.sub.11 is selected from A, H, K, R, S and V; [0335] X.sub.16 is selected from N and T; [0336] X.sub.17 is selected from A, D, K, Q, S and V; [0337] X.sub.18 is selected from W and Y; [0338] X.sub.21 is selected from E and S; [0339] X.sub.25 is selected from H and T; and [0340] X.sub.26 is selected from K and S;
and
ii) an amino acid sequence which has at least 93% identity to the sequence defined in i).
[0341] 2. CD69-binding polypeptide according to item 1, wherein, in sequence i), [0342] X.sub.2 is F; [0343] X.sub.3 is selected from Q, S and Y; [0344] X.sub.4 is selected from H, M, N and W; [0345] X.sub.6 is selected from M and W; [0346] X.sub.7 is selected from K, Q and W; [0347] X.sub.10 is selected from L and R; [0348] X.sub.11 is selected from A, H, K and V; [0349] X.sub.16 is selected from N and T; [0350] X.sub.17 is selected from A, K, Q and S; [0351] X.sub.18 is selected from W and Y; [0352] X.sub.21 is E; [0353] X.sub.25 is T; and [0354] X.sub.26 is selected from K and S.
[0355] 3. CD69-binding polypeptide according to item 1 or 2, wherein sequence i) fulfills at least five of the ten conditions I-X: [0356] I. X.sub.2 is F; [0357] II. X.sub.3 is Y; [0358] III. X.sub.4 is H, Nor W; [0359] IV. X.sub.6 is M or W; [0360] V. X.sub.10 is L; [0361] VI. X.sub.11 is K; [0362] VII X.sub.17 is K or Q; [0363] VIII. X.sub.18 is Y; [0364] IX. X.sub.21 is E; and [0365] X. X.sub.25 is T.
[0366] 4. CD69-binding polypeptide according to item 3, wherein sequence i) fulfills at least six of the ten conditions I-X.
[0367] 5. CD69-binding polypeptide according to item 4, wherein sequence i) fulfills at least seven of the ten conditions I-X.
[0368] 6. CD69-binding polypeptide according to item 5, wherein sequence i) fulfills at least eight of the ten conditions I-X.
[0369] 7. CD69-binding polypeptide according to item 6, wherein sequence i) fulfills at least nine of the ten conditions I-X.
[0370] 8. CD69-binding polypeptide according to item 7, wherein sequence i) fulfills all of the ten conditions I-X.
[0371] 9. CD69-binding polypeptide according to any one of items 1-8, wherein X.sub.3 is Y, X.sub.4 is H and X.sub.11 is K.
[0372] 10. CD69-binding polypeptide according to any one of items 1-9, wherein X.sub.3 is Y, X.sub.4 is H and X.sub.21 is E.
[0373] 11. CD69-binding polypeptide according to any one of items 1-10, wherein X.sub.3 is Y, X.sub.11 is K and X.sub.18 is Y.
[0374] 12. CD69-binding polypeptide according to any one of items 1-11, wherein X.sub.11 is K, X.sub.18 is Y and X.sub.21 is E.
[0375] 13. CD69-binding polypeptide according to any preceding item, wherein sequence i) corresponds to the amino acid sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-72.
[0376] 14. CD69-binding polypeptide according to item 13, wherein sequence i) corresponds to the amino acid sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-29.
[0377] 15. CD69-binding polypeptide according to item 14, wherein sequence i) corresponds to the amino acid sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-28.
[0378] 16. CD69-binding polypeptide according to item 15, wherein sequence i) corresponds to the amino acid sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-26.
[0379] 17. CD69-binding polypeptide according to item 16, wherein sequence i) corresponds to the amino acid sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-6.
[0380] 18. CD69-binding polypeptide according to item 17, wherein sequence i) corresponds to the amino acid sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-2 and 6.
[0381] 19. CD69-binding polypeptide according to item 18, wherein sequence i) corresponds to the amino acid sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-2.
[0382] 20. CD69-binding polypeptide according to item 19, wherein sequence i) corresponds to the amino acid sequence from position 8 to position 36 in SEQ ID NO:1.
[0383] 21. CD69-binding polypeptide according to any preceding item, wherein said CD69-binding motif (BM) forms part of a three-helix bundle protein domain.
[0384] 22. CD69-binding polypeptide according to item 21, wherein said CD69-binding motif (BM) essentially constitutes two alpha helices with an interconnecting loop, within said three-helix bundle protein domain.
[0385] 23. CD69-binding polypeptide according to item 22, wherein said three-helix bundle protein domain is selected from domains of bacterial receptor proteins.
[0386] 24. CD69-binding polypeptide according to item 23, wherein said three-helix bundle protein domain is selected from the five different three-helical domains of Protein A from Staphylococcus aureus, such as domain B, and derivatives thereof.
[0387] 25. CD69-binding polypeptide according to item 24, wherein said three-helix bundle protein domain is a variant of protein Z, which is derived from domain B of staphylococcal Protein A.
[0388] 26. CD69-binding polypeptide according to any preceding item, which comprises a binding module (BMod), the amino acid sequence of which is selected from:
TABLE-US-00022 iii) K-[BM]-DPSQSX.sub.aX.sub.bLLX.sub.cEAKX.sub.dLX.sub.eX.sub.fX.sub.gQ;
wherein [0389] [BM] is a CD69-binding motif as defined in any one of items 1-12; [0390] X.sub.a is selected from A and S; [0391] X.sub.b is selected from E and N; [0392] X.sub.c is selected from A, S and C; [0393] X.sub.d is selected from K and Q; [0394] X.sub.e is selected from E, N and S; [0395] X.sub.f is selected from D, E and S; and [0396] X.sub.g is selected from A and S; and
iv) an amino acid sequence which has at least 93% identity to a sequence defined in iii).
[0397] 27. CD69-binding polypeptide according to item 26, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-72.
[0398] 28. CD69-binding polypeptide according to item 27, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-29.
[0399] 29. CD69-binding polypeptide according to item 28, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-28.
[0400] 30. CD69-binding polypeptide according to item 29, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-26.
[0401] 31. CD69-binding polypeptide according to item 30, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-6.
[0402] 32. CD69-binding polypeptide according to item 31, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-2 and 6.
[0403] 33. CD69-binding polypeptide according to item 32, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-2.
[0404] 34. CD69-binding polypeptide according to item 33, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in SEQ ID NO:1.
[0405] 35. CD69-binding polypeptide according to item 26, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:73-144.
[0406] 36. CD69-binding polypeptide according to item 35, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:73-101.
[0407] 37. CD69-binding polypeptide according to item 36, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:73-100.
[0408] 38. CD69-binding polypeptide according to item 37, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:73-98.
[0409] 39. CD69-binding polypeptide according to item 38, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:73-78.
[0410] 40. CD69-binding polypeptide according to item 39, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:73-74 and 78.
[0411] 41. CD69-binding polypeptide according to item 40, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:73-74.
[0412] 42. CD69-binding polypeptide according to item 41, wherein sequence iii) corresponds to the amino acid sequence from position 7 to position 55 in SEQ ID NO:73.
[0413] 43. CD69-binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
TABLE-US-00023 v) FN-[BMod]-AP;
wherein [BMod] is a CD69-binding module as defined in any one of items 26-42; and [0414] vi) an amino acid sequence which has at least 90% identity to a sequence defined in v).
[0415] 44. CD69-binding polypeptide according to any one of items 1-42, which comprises an amino acid sequence selected from:
TABLE-US-00024 vii) YA-[BMod]-AP;
wherein [BMod] is a CD69-binding module as defined in any one of items 26-42; and [0416] viii) an amino acid sequence which has at least 90% identity to a sequence defined in vii).
[0417] 45. CD69-binding polypeptide according to any preceding item, wherein the CD69-binding motif forms part of a polypeptide comprising an amino acid sequence selected from
TABLE-US-00025 ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK; ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK; ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK; AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK; VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; VDNKFNK-[BM]-DPSQSSELLSEAKQLNDSQAPK; VDNKFNK-[BM]-DPSQSSELLSEAKKLNDSQAPK AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK; AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK; AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK; AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK; AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK; VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a CD69-binding motif as defined in any one of items 1-20.
[0418] 46. CD69-binding polypeptide according to any one of items 1-44, which comprises an amino acid sequence selected from:
TABLE-US-00026 xvii) VDNKFNK-[BM]-DPSQSSELLSEAKQLNDSQAPK; [0419] wherein [BM] is a CD69-binding motif as defined in any one of items 1-20; and [0420] xviii) an amino acid sequence which has at least 89% identity to the sequence defined in xvii).
[0421] 47. CD69-binding polypeptide according to item 46, wherein sequence xvii) is selected from the group consisting of SEQ ID NO:73-144.
[0422] 48. CD69-binding polypeptide according to item 47, wherein sequence xvii) is selected from the group consisting of SEQ ID NO:73-101.
[0423] 49. CD69-binding polypeptide according to item 48, wherein sequence xvii) is selected from the group consisting of SEQ ID NO:73-100.
[0424] 50. CD69-binding polypeptide according to item 49, wherein sequence xvii) is selected from the group consisting of SEQ ID NO:73-98.
[0425] 51. CD69-binding polypeptide according to item 50, wherein sequence xvii) is selected from the group consisting of SEQ ID NO:73-78.
[0426] 52. CD69-binding polypeptide according to item 51, wherein sequence xvii) is selected from the group consisting of SEQ ID NO:73-74 and 78.
[0427] 53. CD69-binding polypeptide according to item 52, wherein sequence xvii) is selected from the group consisting of SEQ ID NO:73-74.
[0428] 54. CD69-binding polypeptide according to item 53, wherein sequence xvii) is SEQ ID NO:73.
[0429] 55. CD69-binding polypeptide according to any one of items 1-44, which comprises an amino acid sequence selected from:
TABLE-US-00027 xix) VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; [0430] wherein [BM] is a CD69-binding motif as defined in any one of items 1-20; and [0431] xx) an amino acid sequence which has at least 89% identity to the sequence defined in xix).
[0432] 56. CD69-binding polypeptide according to item 55, wherein sequence xix) is selected from the group consisting of SEQ ID NO:1-72.
[0433] 57. CD69-binding polypeptide according to item 56, wherein sequence xix) is selected from the group consisting of SEQ ID NO:1-29.
[0434] 58. CD69-binding polypeptide according to item 57, wherein sequence xix) is selected from the group consisting of SEQ ID NO:1-28.
[0435] 59. CD69-binding polypeptide according to item 58, wherein sequence xix) is selected from the group consisting of SEQ ID NO:1-26.
[0436] 60. CD69-binding polypeptide according to item 59, wherein sequence xix) is selected from the group consisting of SEQ ID NO:1-6.
[0437] 61. CD69-binding polypeptide according to item 60, wherein sequence xix) is selected from the group consisting of SEQ ID NO:1-2 and 6.
[0438] 62. CD69-binding polypeptide according to item 61, wherein sequence xix) is selected from the group consisting of SEQ ID NO:1-2.
[0439] 63. CD69-binding polypeptide according to item 62, wherein sequence xix) is SEQ ID NO:1.
[0440] 64. CD69-binding polypeptide according to any one of items 1-44, which comprises an amino acid sequence selected from:
TABLE-US-00028 xxi) VDNKFNK-[BM]-DPSQSSELLSEAKKLNDSQAPK; [0441] wherein [BM] is a CD69-binding motif as defined in any one of items 1-20; and [0442] xxii) an amino acid sequence which has at least 89% identity to the sequence defined in xxi).
[0443] 65. CD69-binding polypeptide according to any one of items 1-44, which comprises an amino acid sequence selected from:
TABLE-US-00029 xxiii) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; [0444] wherein [BM] is a CD69-binding motif as defined in any one of items 1-20; and [0445] xxiv) an amino acid sequence which has at least 89% identity to the sequence defined in xxiii).
[0446] 66. CD69-binding polypeptide according to any preceding item, which is capable of binding to CD69 such that the K.sub.D value of the interaction with CD69 is at most 1?10.sup.?6 M, such as at most 5?10.sup.?7 M, such as at most 1?10.sup.?7 M, such as at most 5?10.sup.?8 M, such as at most 1?10.sup.?8 M, such as at most 5?10.sup.?8 M.
[0447] 67. CD69-binding polypeptide according to any preceding item, which comprises additional amino acids at the C terminus and/or N terminus.
[0448] 68. CD69-binding polypeptide according to item 67, wherein said additional amino acid(s) improve(s) production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.
[0449] 69. CD69-binding polypeptide according to any preceding item in a multimeric form comprising at least two CD69-binding polypeptide monomer units, the amino acid sequences of which may be the same or different.
[0450] 70. CD69-binding polypeptide according to item 69, wherein said monomer units are covalently coupled together.
[0451] 71. CD69-binding polypeptide according to item 69, wherein said CD69-binding polypeptide monomer units are expressed as a fusion protein.
[0452] 72. CD69-binding polypeptide according to any one of items 69-71 in dimeric form.
[0453] 73. Fusion protein or conjugate, comprising [0454] a first moiety consisting of a CD69-binding polypeptide according to any preceding item; and [0455] a second moiety consisting of a polypeptide having a desired biological activity.
[0456] 74. Fusion protein or conjugate according to item 73, wherein said desired biological activity is a therapeutic activity.
[0457] 75. Fusion protein or conjugate according to item 73, wherein said desired biological activity is a binding activity.
[0458] 76. Fusion protein or conjugate according to item 73, wherein said desired biological activity is an enzymatic activity.
[0459] 77. Fusion protein or conjugate according to item 75, wherein said binding activity is albumin binding activity which increases the in vivo half-life of the fusion protein or conjugate.
[0460] 78. Fusion protein or conjugate according to any one of items 73-77 further comprising at least one linker.
[0461] 79. CD69-binding polypeptide, fusion protein or conjugate according to any preceding item, further comprising a label.
[0462] 80. CD69-binding polypeptide, fusion protein or conjugate according to item 79, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, radionuclides, radioactive particles and pretargeting recognition tags.
[0463] 81. CD69-binding polypeptide, fusion protein or conjugate according to any one of items 79-80, for use in labeling or targeting cells and tissues which have a high expression of CD69.
[0464] 82. CD69-binding polypeptide, fusion protein or conjugate according to any one of items 79-80 which is labeled, directly or indirectly, with an imaging agent, such as a radioactive agent.
[0465] 83. Polynucleotide encoding a CD69-binding polypeptide or fusion protein according to any one of items 1-78.
[0466] 84. Expression vector comprising the polynucleotide according to item 83.
[0467] 85. Host cell comprising the expression vector according to item 84.
[0468] 86. Method of producing the CD69-binding polypeptide, fusion protein or complex according to any one of items 1-78, comprising [0469] culturing the host cell according to item 85 under conditions permissive of expression of said polypeptide from its expression vector; and [0470] isolating the polypeptide.
[0471] 87. Composition comprising a CD69-binding polypeptide, fusion protein or conjugate according to any one of items 1-82 and at least one pharmaceutically acceptable excipient or carrier.
[0472] 88. Composition according to item 87, further comprising at least one additional active agent, such as an immune response modifying agent.
[0473] 89. CD69-binding polypeptide, fusion protein or conjugate according to any one of items 1-82 or a composition according to item 87 or 88 for use as a medicament, as a diagnostic agent in vivo and/or as a prognostic agent in vivo.
[0474] 90. CD69-binding polypeptide, fusion protein, conjugate or composition for use according to item 89 as a medicament in the treatment of a CD69-related disorder.
[0475] 91. CD69-binding polypeptide, fusion protein, conjugate or composition for use according to item 89 as a diagnostic agent in the in vivo diagnosis of a CD69-related disorder.
[0476] 92. CD69-binding polypeptide, fusion protein, conjugate or composition for use according to item 89 as a prognostic agent in the in vivo prognosis of a CD69-related disorder.
[0477] 93. CD69-binding polypeptide, fusion protein, conjugate or composition for use according to any one of items 90-92, wherein said CD69-related disorder is an autoinflammatory disease.
[0478] 94. CD69-binding polypeptide, fusion protein, conjugate or composition for use according to item 93, wherein said autoinflammatory disease is type 1 diabetes (T1D).
[0479] 95. Method of treatment of a CD69-related disorder, comprising administering to a subject in need thereof an effective amount of a CD69-binding polypeptide, fusion protein or conjugate according to any one of items 1-82 or a composition according to item 87 or 88.
[0480] 96. Method of detecting CD69, comprising providing a sample suspected to contain CD69, contacting said sample with a CD69-binding polypeptide, fusion protein or conjugate according to any one of items 1-82 or a composition according to item 87 or 88, and detecting the binding of the CD69-binding polypeptide, fusion protein, conjugate or composition to indicate the presence of CD69 in the sample.
[0481] 97. Diagnostic or prognostic method for determining the presence of CD69 in a subject comprising the steps of: [0482] a) contacting the subject, or a sample isolated from the subject, with a CD69-binding polypeptide, fusion protein or conjugate according to any one of items 1-82 or a composition according to item 87 or 88, and [0483] b) obtaining a value corresponding to the amount of the CD69-binding polypeptide, fusion protein, conjugate or composition that has bound in said subject or to said sample.
[0484] 98. The diagnostic or prognostic method according to item 97, which is a method for diagnosis in vivo or prognosis in vivo, for example via medical imaging, in which said contacting in step a) consists of contacting said subject with said polypeptide, fusion protein, conjugate or composition.
[0485] 99. The diagnostic or prognostic method according to item 97, which is an in vitro method and in which said contacting in step a) consists of contacting a sample previously isolated from said subject with said polypeptide, fusion protein, conjugate or composition.