HIGH AFFINITY ANTICALINS DIRECTED AGAINST HUMAN CD98hc
20220153790 · 2022-05-19
Inventors
- Arne SKERRA (Dachau, DE)
- Friedrich-Christian DEUSCHLE (Freising, DE)
- André SCHIEFNER (Nürnberg, DE)
- Volker MORATH (München, DE)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K2318/20
CHEMISTRY; METALLURGY
C07K2319/31
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K16/2896
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a cluster of differentiation 98 heavy chain (CD98hc)-specific binding protein, wherein the CD98hc-specific binding protein is a lipocalin 2 (Lcn2)-derived binding protein and binds to CD98hc with a K.sub.D of 200 nM or lower.
Claims
1. A cluster of differentiation 98 heavy chain (CD98hc)-specific binding protein, wherein the CD98hc-specific binding protein is a lipocalin 2 (Lcn2)-derived binding protein and binds to CD98hc with a K.sub.D of 200 nM or lower.
2. The CD98hc-specific binding protein of claim 1, wherein the CD98hc-specific binding protein specifically binds to an epitope of CD98hc comprising one or more amino acids from amino acid positions 128 to 137 of SEQ ID NO: 1 and/or one or more amino acids from amino acid positions 374 to 404 of SEQ ID NO: 1.
3. The CD98hc-specific binding protein of claim 1, wherein the CD98hc-specific binding protein (a) comprises or consists of an amino acid sequence as represented in formula I: TABLE-US-00011 QDSTSD(X1)(X2)PAPPLSKVPLQQNFQDNQF(Q/H)GKWY(X3)VG (X4)AG(X5)(X6)(X7)(X8)(X9)E(X10)(X11)(X12)(X13) (X14)(X15)M(X16)ATIYELKEDKS(Y/F)NVT(X17)V(X18) (X19)(X20)(X21)(K/T)KC(X22)(Y/N)(X23)(X24)(X25) (T/S)(X26)VPG(C/S)QPGE(F/Y)(T/N)(X27)G(N/K)I(X28) S(Y/R/G/A)P(X29)(X30)(X31)S(X32)L(X33)RVVSTNYNQ (H/Y)A(M/L)VF(X34)K(X35)(V/E)(X36)(X37)N(X38)E (X39)(X40)(X41)I(X42)L(X43)GRTKELTSELKE(N/I/Y)FIR FSKSLGLPE(X44)(X45)IVFPVPIDQCIDG, wherein (X.sub.1) is L; (X.sub.2) is I; (X.sub.3) is V; (X.sub.4) is R; (X.sub.5) is N; (X.sub.6) is L or T, preferably T; (X.sub.7) is G; (X.sub.8) is L; (X.sub.9) is R; (X.sub.10) is D; (X.sub.11) is K; (X.sub.12) is D; (X.sub.13) is P; (X.sub.14) is A or G, preferably G; (X.sub.15) is K; (X.sub.16) is F; (X.sub.17) is Y; (X.sub.18) is W; (X.sub.19) is F or S, preferably S; (X.sub.20) is D or G, preferably G; (X.sub.21) is L or Q, preferably Q; (X.sub.22) is K or M, preferably M; (X.sub.23) is S; (X.sub.24) is I (X.sub.25) is H, G or V, preferably V; (X.sub.26) is F; (X.sub.27) is L; (X.sub.28) is K; (X.sub.29) is G; (X.sub.30) is H, R or Q, preferably R; (X.sub.31) is T; (X.sub.32) is W; (X.sub.33) is V; (X.sub.34) is F; (X.sub.35) is W or S, preferably S; (X.sub.36) is G or T, preferably T; (X.sub.37) is Q; (X.sub.38) is R; (X.sub.39) is N, G or E, preferably G; (X.sub.40) is F; (X.sub.41) is A; (X.sub.42) is T; (X.sub.43) is Y; (X.sub.44) is N; and (X.sub.45) is H; (b) an amino acid sequence which is at least 80% identical to the amino acid sequence of (a), provided that amino acid positions (X.sub.1) to (X.sub.45) are maintained as defined in item (a); (c) an amino acid sequence which is at least 80% identical to the amino acid sequence of (a), provided that amino acid positions (X.sub.1) to (X.sub.45) are maintained as defined in item (a) or are changed by one or more conservative amino acid substitutions; or (d) an amino acid sequence which is at least 80% identical to the amino acid sequence of (a), provided that no more than ten amino acid positions of (X.sub.1) to (X.sub.45) as defined in item (a) are changed by amino acid substitutions, wherein the amino acid substitutions are preferably conservative amino acid substitutions.
4. The CD98hc-specific binding protein of claim 1 or 2, wherein the CD98hc-specific binding protein (a) comprises or consists of the amino acid sequence of TABLE-US-00012 (SEQ ID NO: 14) QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGIAGNSMLREDKDPF KMTATIYELKEDKSYNVTRVRFDDKKCLYRILTFVPGSQPGEFTLGNIK SRPGVVTSWLVRVVSTNYNQHAMVFFKRVHQNRETFWITLYGRTKELTS ELKENFIRFSKSLGLPENHIVFPVPIDQCIDG (b) an amino acid sequence which is at least 80% identical to the amino acid sequence of (a), provided that the underlined amino acids are maintained as defined in item (a); (c) an amino acid sequence which is at least 80% identical to the amino acid sequence of (a), provided that the underlined amino acids are maintained as defined in item (a) or are changed by one or more conservative amino acid substitutions; or (d) an amino acid sequence which is at least 80% identical to the amino acid sequence of (a), provided that no more than ten of the underlined amino acids as defined in item (a) are changed by amino acid substitutions, wherein the amino acid substitutions are preferably conservative amino acid substitutions.
5. The CD98hc-specific binding protein of claim 1 or 2, wherein the CD98hc-specific binding protein comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs 2 to 14; or (b) an amino acid sequence which is at least 80% identical to the amino acid sequence of (a).
6. A nucleic acid molecule encoding the CD98hc-specific binding protein of claim 1.
7. A vector comprising the nucleic acid molecule of claim 6.
8. A host cell transformed with the vector of claim 7.
9. A method for the production of a CD98hc-specific binding protein comprising culturing the host cell of claim 8 under suitable conditions and isolating the CD98hc-specific binding protein produced.
10. A protein conjugate or fusion protein comprising the CD98hc-specific binding protein of claim 1.
11. The protein conjugate or fusion protein of claim 10, wherein the CD98hc-specific binding protein is conjugated to or is part of a fusion protein wherein the fusion partner is (a) a fluorescent dye or a fluorescent protein, (b) a radionuclide, (c) a toxic compound, (d) a photosensitizer, (e) an enzyme or truncated version thereof, (f) a membrane protein or functional fragment thereof retaining the enzyme function, (g) a contrast agent, (h) a cytokine, (i) a chemokine, (j) a pro-coagulant factor, (k) an acetylcholineesterase inhibitor, (l) an inhibitor of Aβ aggregation, (m) a nucleic acid molecule, or (n) a nanoparticle.
12. The protein conjugate or fusion protein of claim 10, wherein the CD98hc-specific binding protein is conjugated to a binding protein, wherein the binding protein is preferably selected from an antibody or immunoglobulin, an antibody fragment and an antibody mimetic, wherein the antibody mimetic is preferably selected from the group consisting of an Anticalin, Affibody, Adnectin, DARPin, Avimer, Nanofitin, Affilin, β-Wrapin, ADAPT, Monobody, Rasln, FingR, Pronectin, Centyrin, Affimer, Adhiron, Affitin, αRep, Repebody, i-body, Fynomer and a Kunitz domain protein.
13. A pharmaceutical composition or a diagnostic composition comprising at least one of (i) the CD98hc-specific binding protein of claim 1; (ii) a nucleic acid molecule encoding the CD98hc-specific binding protein of (i); (iii) a vector comprising the nucleic acid molecule of (ii); (iv) a host cell transformed with the vector of (iii), and/or (v) a protein conjugate or fusion protein comprising the CD98hc-specific binding protein of (i).
14. The CD98hc-specific binding protein of claim 1 for use in therapy and/or diagnosis, preferably for use in treating or diagnosing a disease in the brain and/or the spinal cord.
15. The CD98hc-specific binding protein of claim 1 for use in the therapy and/or diagnosis of a tumor or a neurological disease, wherein the tumor or neurological disease is preferably located in the brain and/or the spinal cord.
16. The nucleic acid molecule of claim 6, a vector comprising the nucleic acid molecule, or a host cell transformed with the vector for use in therapy and/or diagnosis, preferably for use in treating or diagnosing a disease in the brain and/or the spinal cord.
17. The nucleic acid molecule of claim 6, a vector comprising the nucleic acid molecule, or a host cell transformed with the vector for use in the therapy and/or diagnosis of a tumor or a neurological disease, wherein the tumor or neurological disease is preferably located in the brain and/or the spinal cord.
18. The protein conjugate or fusion protein of claim 10 for use in therapy and/or diagnosis, preferably for use in treating or diagnosing a disease in the brain and/or the spinal cord.
19. The protein conjugate or fusion protein of claim 10 for use in the therapy and/or diagnosis of a tumor or a neurological disease, wherein the tumor or neurological disease is preferably located in the brain and/or the spinal cord.
Description
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TABLE-US-00004 TABLE 7 Protein mass determination of PASylated D11vs variants via ESI MS Mass Theoretical Measured difference to Protein mass [Da] mass [Da] uncoupled [Da] D11vs-PAS200 37933.5 37933.6 (Δ0.1) — D11vs-PAS200-Cys 38036.7 n.d. — D11vs-PAS200-DFO 38748.5 39749.4 (Δ0.9) 712.7 DFO: 711.8 D11vs-PAS200-Cy5.5 39060.9 39062.1 (Δ1.2) 1025.4 S-Cy5.5: 1024.3
[0273] The examples illustrate the invention.
EXAMPLE 1—MATERIAL AND METHODS
[0274] Phage display selection, identification and production of hCD98hcED-specific lipocalin variants Selection of CD98hc-specific lipocalin variants was performed via filamentous phagemid display from a combinatorial library based on Lcn2 randomized at 20 positions within the structurally variable loops comprising 1×10.sup.10 variants [50]. Biotinylated recombinant hCD98hcED (cf. Supplementary Information) was immobilized on streptavidin- or NeutrAvidin-coated paramagnetic beads (Sigma-Aldrich, Munich, Germany and Thermo Fisher Scientific, Waltham, Mass., respectively), incubated with the phagemid library (starting titer 1×10.sup.12), and bound phagemids were eluted under denaturing conditions using 4 M urea. After five consecutive cycles, pooled phasmid DNA from the enriched library was prepared and subcloned on pNGAL98 in order to perform high-throughput ELISA screening from microcultures expressing the soluble lipocalin variants, as previously described [50]. Briefly, periplasmic extract was prepared and applied to 96-well MaxiSorp plates (Thermo Fisher Scientific) coated with 250 nM of hCD98hcED. After 1 h incubation bound lipocalin variants were detected by means of their C-terminal Strep-tag II using an ExtrAvidin/alkaline phosphatase (AP) conjugate (Sigma-Aldrich). Signals were developed using 0.5 mg/ml p-nitrophenyl phosphate in AP buffer (0.1 M NaCl, 5 mM MgCl2, 0.1 M Tris/HCl, pH 8.8) and absorbance measured at 405 nm with an Infinite 200 PRO microplate reader (Tecan, Mannerdorf, Switzerland). For clones showing significant binding towards hCD98hcED the expression cassette on the plasmid DNA was sequenced, and the soluble lipocalin variant was produced in a preparative scale as soluble, secretory protein in E. coli JM83 with a C-terminal His.sub.6-tag using the plasmid pNGAL118 [50]. After periplasmic protein extraction the recombinant proteins were purified by immobilized metal ion affinity chromatography (IMAC) using a Ni(II)-charged HisTrap HP column (GE Healthcare, Munich, Germany) and subsequently subjected to size-exclusion chromatography (SEC) in PBS (4 mM KH.sub.2PO.sub.4, 160 mM Na.sub.2HPO.sub.4, 115 mM NaCl pH 7.4) on a 24 ml Superdex 75 10/300 GL column (GE Healthcare). Furthermore, plasmid construct design and production of recombinant D11vs-PAS200, D11vs-PAS200-Cys and Lcn2-PAS200-Cys was performed as previously described [51] and purified as described above.
[0275] Protein Crystallization and Structure Determination
[0276] For protein crystallization a variant of hCD98hcED with N-terminal Strep-tag II was produced in E. coli stain BL21 as described previously (Deuschle et al. submitted) (
TABLE-US-00005 TABLE 4 Data collection and refinement statistics Data collection Space group C2 Unit cell parameters a = 202.81 Å, b = 46.05 Å, c = 137.09 Å, α = γ = 90°, β = 106.64° Wavelength [Å] 0.9184 Resolution Å 30.0 − 1.80 (1.90 − 1.80) Completeness [%] 99.8 (99.9) Unique reflections 113253 (16844) Multiplicity 6.8 (7.0) Mean I/σ (I) 23.9 (2.3) R.sub.meas [%] 4.9 (93.4) Wilson B-factor [Å] 37.4 Refinement Resolution [Å] 30.0 − 1.80 (1.85 − 1.80) Reflections (working) 111018 (8149) Reflections (test).sup.b 2234 (187) R.sub.cryst [%] 18.4 (60.8) R.sub.free [%] 22.6 (67.0) Protein molecules per au 4 Number of atoms: protein/solvent.sup.c 9356/909 B-values of atoms: protein/solvent 33.3/39.2 [Å.sup.2] Ramachandran plot.sup.d: favored/outliers 96.8/0.2 [%] RMSD bonds [Å]/angles [°] 0.02/1.76 .sup.aValues in parentheses refer to the highest resolution shell. .sup.bTest set corresponds to 2% of all reflections .sup.cSolvent refers to waters, ions as well as ordered buffer or cryoprotectant molecules .sup.dRamachandran statistics were calculated with MolProbity
[0277] The crystal structure was solved by molecular replacement with Phaser [54] using coordinates of the hCD98hcED (PDB entry 2DH2) [55] and of the Anticalin N7A (PDB entry 4GH7) [50] as search models. Manual rebuilding and refinement were done with Coot [56] and Refmac5 [57], respectively (Table 4). Translation, libration and screw (TLS) groups were determined with TLSMD [58]. The asymmetric unit of space group C2 contained two P3D11.circle-solid.hCD98hcED complexes, of which the complex comprising chain pairs A and B, with overall lower B-factors, was used for analysis.
[0278] Biomolecular Interaction Analysis Via Surface Plasmon Resonance (SPR) Spectroscopy
[0279] Real time surface plasmon resonance (SPR) spectroscopy was performed on a BIAcore 2000 system (BIAcore, Uppsala, Sweden) at 25° C. using HBS-T (20 mM Hepes/NaOH pH 7.5, 150 mM NaCl, 0.005% v/v Tween20) as running buffer. The purified biotinylated m/hCD98hcED (3 μg/ml protein solution in HBS-T produced in HEK cells or E. coli) was immobilized (ARU-225) via streptavidin on a CAP sensorchip of the Biotin CAPture kit (GE Healthcare). SPR single cycle kinetic experiments were performed using five consecutive injections of a 1:2 dilution series of the purified lipocalin variant at a flow rate of 25 μl/min, with 288 s contact time and 3500 s dissociation time after the fifth injection. For multi cycle kinetic experiments, eight samples from a consecutive 1:2 dilution series of the purified lipocalin variant were measured. Rate constants of association and dissociation were calculated from reference-corrected sensorgrams by fitting to a global 1:1 Langmuir binding model using BIAevaluation software (BIAcore). The equilibrium dissociation constants (K.sub.D value) were calculated as the quotient k.sub.off/k.sub.on. To test competitive binding of the lipocalin variants P3D11, P3A12 and P1E4 to glycosylated hCD98hcED, 100 nM of P3D11 was first injected and followed by a subsequent injection of either 100 nM P3A12 or 100 nM P1E4. HBS-T injections served as negative control.
[0280] Error-Prone Library Construction and Bacterial Cell Surface Display Selection
[0281] Stability and affinity engineering of the lipocalin variant P3D11 was accomplished by means of directed evolution and bacterial surface display (BSD) as previously published [51, 59, 60]. Briefly, error-prone PCR was executed using the GeneMorph II random mutagenesis kit (Agilent, Santa Clara, Calif.) with 10 μg of the central coding region for the lipocalin variant P3D11 used as template. After digest of the PCR product with BstXI, the randomized DNA fragment was ligated with the backbone of pNGAL146 and used for electrotransformation of E. coli JK321, yielding 5×10.sup.9 transformants plated on LB agar medium supplemented with 100 μg/ml ampicillin. The bacterial lawn was scraped from the plate(s), resuspended in 50 ml LB/Amp medium, followed by inoculation to an initial OD.sub.550 of 0.15 at 37° C. Gene expression was induced at OD.sub.550=0.5 with 10 ng/ml anhydrotetracycline (aTc) for 2.5 h. Approximately 2×10.sup.8 cells were sedimented and resuspended in PBS with biotinylated hCD98hcED at different concentrations (cycles 1 and 2: 100 nM; cycles 3 and 4: 10 nM; cycles 5 and 6: 1 nM) for 1 h at 4° C. After one washing step with PBS, the bacteria were incubated with 25 μg/ml streptavidin/phycoerythrin (PE) conjugate (Biolegend, San Diego, Calif.) and 3 μM dye-labeled Fab A3C5-DY634 [61] in PBS for 30 min followed by a last washing step in PBS. Bacteria were sorted on a FACSAria Ilu instrument (BD Bioscience, Heidelberg, Germany) using a 488 nm LASER diode with a 585/42 band pass filter or a 633 nm HeNe LASER in combination with a 660/20 band pass filter for excitation/detection of PE and DY634 fluorescence, respectively. Sorted bacteria were plated on LB/Amp agar, incubated at 37° C. for 12 h and then subjected to a new sorting cycle. After BSD cycle 6, single clone analysis was performed by cytofluorimetric analysis of individual colonies. Data were processed using FlowJo v10 software (FlowJo, Ashland, Oreg.).
[0282] Flow Cytofluorimetry
[0283] The human cancer cell lines Ramos, Raji, SU-DHL-4, PC3 and DU-145 were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium containing 2.0 g/L NaHCO.sub.3 and stable L-glutamine (Biochrom, Berlin, Germany), supplemented with 10% (v/v) fetal bovine serum (FBS) (PAA Laboratories, Pasching, Austria) at 37° C. under humidified 5% CO.sub.2 atmosphere. Adherent cells (PC3, DU-145 and Caco-2) were washed with PBS (w/o Ca.sup.2+ and Mg.sup.2+) (Biochrom) and detached using PBS (w/o Ca.sup.2+ and Mg.sup.2+) supplemented with 0.5 mM EDTA for 10 min at 37° C. Cells growing in suspension (Ramos, Raji and SU-DHL-4) were directly washed with PBS (w/o Ca.sup.2+ and Mg.sup.2+). For each measurement, 250,000 viable cells were resuspended in 250 μl FACS buffer (PBS+10% (v/v) FBS) containing 1 μM D11vs-PAS200-Cy5.5 (cf. Supplementary information) and incubated for 1 h at 4° C. Competition experiments were performed by adding a 10-fold molar concentration of either unlabeled D11vs-PAS200 or the soluble glycosylated hCD98hcED. After incubation, cells were washed three times in PBS and finally resuspended in 250 μl FACS buffer. Flow cytofluorometric experiments were performed on a FACSAria Ilu instrument using an excitation wavelength of 650 nm and an emission band-path filter of 780/60 nm.
[0284] Immunofluorescence Microscopy of CD98hc Expressing Cells
[0285] PC-3 and Caco-2 cells were cultivated at 37° C. on poly-D-lysine (PDL) coated Lab-Tek II chamber slides (Thermo Fisher Scientific, Waltham, Mass.) in RPMI 1640 medium containing 2.0 g/L NaHCO.sub.3 and stable L-glutamine, supplemented with 10 (v/v) % FBS, until 60-70 confluence was reached. After three times washing with PBS, the cells were incubated with 1 μM D11vs-PAS200-Cy5.5 for 1 h at 37° C., followed by another three washing steps. Ramos cells, on the other hand, were grown in suspension and stained in the same manner using a reaction tube, followed by transfer to a PDL coated Lab-Tek II chamber slide. For control experiments, the D11vs-PAS200-Cy5.5 conjugate was premixed with a 10-fold molar concentration of unlabeled D11vs-PAS200 or the equivalent conjugate of wtLcn2 (Lcn2-PAS200-Cy5.5) was applied. Cells were fixed and counterstained by 5 min incubation with ice-cold methanol containing 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Digital fluorescence images were recorded on an Axiovert 40 CFL microscope equipped with an AxioCam MRm camera (Carl Zeiss Microscopy, Jena, Germany) using 365/12 nm (DAPI) and 716/40 nm (Cy5.5) band-pass filters with identical exposure times.
[0286] Circular Dichroism (CD) Spectroscopy and Thermal Denaturation
[0287] CD spectra and thermal unfolding of purified Lcn2 variants were recorded using a Jasco J-810 spectropolarimeter (Jasco, Pfungstadt, Germany) controlled by Spectra manager software (ver. 1.53.05) equipped with a PT-423S Peltier element. Proteins were dialyzed against 20 mM KP.sub.i pH 7.5 and 50 mM K.sub.2SO.sub.4 and applied at a concentration of 1 μM. In order to identify the wavelength with maximum change in CD signal for thermal denaturation studies, spectra were measured in a 1 mm path length quartz cuvette (Hellma, Müllheim, Germany) from 190-250 nM at 20° C. and 90° C. Thermal unfolding of the lipocalin variants was measured at a wavelength of 214 nm by heating from 20° C. to 90° C. at a rate of 60° C./h. Data were fitted to an equation for a one-step unfolding transition, and the melting temperature (T.sub.m), enthalpy of unfolding (ΔH.sub.m) and Gibb's free energy of unfolding (ΔG.sup.0) at ambient temperature was calculated as previously described [62].
[0288] In Vivo Xenograft Studies
[0289] Animal experiments were conducted with permission from the District Government of Upper Bavaria (application no.: 55.2-1-54-2532-216-15). CB17-SCID mice at an age of 6 weeks (♂) and (♀) were purchased from Charles River Laboratories (Wilmington, Mass.) and housed under specific pathogen free (SPF) conditions. Mice were injected subcutaneously with 100 μl RPMI 1640 medium (without supplements) containing 6×10.sup.6 PC-3 cells above the right shoulder. On day 12 p.i. the tumor had reached a diameter of ˜0.5 cm and the mice were used for in vivo studies.
[0290] .sup.89Zr Positron Emission Tomography
[0291] For .sup.89Zr-labeling of Dfo-conjugated [63] D11vs-PAS200 (for protein production and coupling cf. Supplementary information), 250 μg of the protein dialyzed against 250 mM Na-acetate pH 5.5 was incubated with 200 μl 0.5 M Hepes/NaOH pH 7.0, 50 μl 0.5 M gentistic acid and 111 MBq .sup.89Zr in 1 M oxalic acid (neutralized with 3 M Na.sub.2CO.sub.3) (Perkin Elmer, Waltham, Mass.). After 1 h incubation at 37° C., radiolabeling was confirmed by radio thin layer chromatography (radio-TLC) on strips using Na-citrate pH 5.0 as mobile phase. The radio-labeled protein tracer was separated by gel filtration using a PD-10 column (GE Healthcare) equilibrated with 0.9% NaCl solution (B. Braun, Melsungen, Germany). Radiochemical purity of the protein tracer was confirmed by radio-TLC prior to its use for injections.
[0292] For the biodistribution study, mice were injected with the protein tracer via the tail vein at a dose of 3.96±0.12 MBq. For blocking experiments, a 100-fold (
[0293] Biodistribution Analysis and Autoradiography
[0294] Tumor, blood and organs were dissected from the sacrificed animals at the end of the experiment in order to determine weight and quantify radioactivity using a 2480 Wizard.sup.2 automatic gamma counter (PerkinElmer). A 1% fraction of the injected protein tracer dose was used as reference. Uptake values were calculated as % ID/g and corrected for radioactive decay from the time point of injection.
[0295] After radioactivity measurement, the tumor was transversally bisected and used for immunohistochemistry and autoradiography analysis. For autoradiography the tumor was embedded in Tissue-Tek O.C.T. (Sakura Finetek, Alphen aan den Rijn, The Netherlands) in an embedding mold and frozen. Subsequently, 10 μm sections were prepared using a CM1950 cryostat (Leica Biosystems, Nußloch, Germany) and the sections were mounted on a superfrost microscopic slide (Thermo Fisher Scientific). The slide was exposed, together with a standard dilution row in silicon isolators (Grace Bio-Labs, Bend, Oreg.), to a storage phosphor screen BAS-IP super resolution film (GE Healthcare) for one week, followed by quantification using a CR 25 BIO autoradiography scanner (Durr Medical, Bietigheim-Bissingen, Germany) and data analysis with AIDA software ver. 4.24.036 (Raytest, Straubenhardt, Germany). Chosen pictures show the sections with the highest exposure level to allow comparison of blocked and unblocked tumor sections.
[0296] Immunohistochemistry
[0297] Tumor tissue was fixed for 48 h in 4% paraformaldehyde in PBS and stored in PBS at 4° C. until radioactivity decayed. Tissue was dehydrated under standard conditions (ASP300S; Leica Biosystems) and embedded in paraffin. Serial 2-μm-thin sections prepared with a rotary microtome (HM355S; Thermo Fisher Scientific) were collected and subjected to histological and immunohistochemical analysis. Hematoxylin-eosin staining was performed on deparaffinized sections.
[0298] Immunohistochemistry was done under standard conditions using appropriate antibodies (AT-2; Leica Biosystems). Representative images were collected using Aperio Imagescope software (version 12.3; Leica Biosystems).
[0299] Statistics
[0300] For mean comparison between two groups of animals, the Student's t test for unpaired data was used. P values 0.05 were considered statistically significant (* P≤0.05; ** P≤0.005). Experimental data were analyzed using Prism 6 (GraphPad Software, San Diego, Calif.) and standard deviations (S.D.) are indicated.
[0301] Soluble Production and Purification of the Monobiotinylated Human and Murine CD98hc Ectodomains
[0302] The human and murine CD98hcED (UniProt ID P10852-1, residues Glu105-Ala526 and UniProt ID P08195-2, residues Glu111-Ala529, respectively) were produced in E. coli strain BL21 using pASK-IBA5(+)-BAP-m/hCD98hcED and in human embryonic kidney (HEK) 293E cells (MEXi expression system, IBA Lifesciences, Goettingen, Germany) using the expression plasmid pDSG-BM-His.sub.6-BAP-m/hCD98hcEDg-Igk-BirA-StrepDEL (StrepDEL=Fusion of the Strep-tag II amino acid sequence WSHPQFEK and the endoplasmic reticulum retention signal sequence KDEL).
[0303] For expression in E. coli, BL21 was co-transformed with pBirAcm encoding biotin ligase (Avidity LLC, Aurora, Colo.), and heterologous gene expression was induced with 0.5 mg/L anhydrotetracycline (aTc) and 1 mM isopropyl-f3-D-thiogalactopyranoside (IPTG) for 12 h at 26° C. in 2 L 2xYT medium. Cells were harvested by centrifugation, resuspended in anion-exchange chromatography (AEX) buffer (20 mM Tris/HCl pH 8.0, 1 mM EDTA) and disrupted with a PandaPLUS 2000 homogenizer (GEA Niro Soavi, Parma, Italy). The recombinant ectodomain was purified from the whole cell extract by AEX using a MacroCap Q column (GE Healthcare, Munich, Germany) equilibrated with AEX buffer using a linear concentration gradient of 0-500 mM NaCl. Appropriate fractions were pooled, dialyzed against 100 mM Tris/HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, and applied to a 5 ml column carrying a streptavidin mutant which allows binding of biotinylated proteins and competitive elution via an excess of free biotin (unpublished). After elution using 5 mM biotin in the same buffer, preparative size-exclusion chromatography (SEC) was performed on a 24 ml Superdex 200 10/300 GL column (GE Healthcare) using PBS (4 mM KH.sub.2PO.sub.4, 16 mM Na.sub.2HPO.sub.4, 115 mM NaCl, pH 7.4) as a running buffer.
[0304] Expression procedure of the m/hCD98hcEDg in MEXi cells was essentially performed as recommended by the manufacturer. Briefly, cells were cultivated in MEXi culture medium supplemented with 50 mg/I G-418 and 8 mM L-alanyl-L-glutamine at 37° C. under humidified 5% CO.sub.2 atmosphere. 250 ml containing 5×10.sup.6 cells/mi were transfected using 5 μg plasmid DNA/1×10.sup.6 cells, and mixed at a 1:3 mass ratio with polyethylenimine in MEXi transfection medium. After 4 h incubation at 37° C., 500 ml of MEXi cultivation medium was added, and secretory expression was continued for 4 days. After sedimentation of the HEK cells by centrifugation, the supernatant was dialyzed against an immobilized metal ion affinity chromatography (IMAC) buffer (20 mM Tris/HCl pH 8.0, 500 mM NaCl), and IMAC was performed on a Ni(II)-charged HisTrap HP column (GE Healthcare). Elution fractions obtained after applying a linear concentration gradient of 0-300 mM imidazole/HCl in IMAC buffer were pooled and further purified via streptavidin affinity chromatography and preparative SEC as described above.
[0305] Analytical SEC and SDS-PAGE
[0306] Analytical SEC was performed using a 24 ml Superdex 200 10/300 GL column (GE Healthcare) in PBS pH 7.4 at a flow rate of 0.5 ml/min. For apparent molecular weight determination, the column was calibrated with the following standard proteins (Sigma-Aldrich, Munich, Germany): thyroglobulin (669 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa) and aprotinin (6.5 kDa). The void volume of the column was determined using Blue dextran (Sigma-Aldrich). Based on the elution volumes, the partition coefficients K.sub.av were calculated and used to interpolate the apparent molecular sizes of the analyzed proteins.
[0307] SDS-PAGE was performed using a high molarity Tris buffer system with or without the addition of 2-mercaptoethanol in the sample buffer, followed by staining with Coomassie brilliant blue or direct detection of Cy5.5 fluorescence (for corresponding protein conjugates) using an Ettan DIGE fluorescence scanner (GE Healthcare) with an excitation wavelength of 635/30 nm and a 680/30 nm emission band-path filter
[0308] Enzymatic Cleavage of N-Linked Sugars from m/hCD98hcEDg Using PNGase F
[0309] N-linked glycans were enzymatically removed from m/hCD98hcEDg produced in HEK cells using Peptide-N-Glycosidase F (PNGase F) (New England Biolabs, Ipswich, Mass.). 5 μg protein were first denatured by adding 0.5% sodium dodecyl sulfate (SDS) and 40 mM dithiothreitol (DTT) for 10 min at 100° C. Subsequently, 50 mM Na.sub.3PO.sub.4 pH 7.5, 1% (v/v) NP-40 and 250 units of PNGase F were added, incubated for 1 h at 37° C. and subjected to SDS-PAGE for comparison with the untreated ectodomain (Fig. S7D).
[0310] Site-Specific Thiol Labelling of PASylated Lipocalin Variants Via Maleimide Chemistry
[0311] For site-specific labelling at the engineered C-terminal Cys side chain (
[0312] ESI Mass Spectrometry
[0313] Mass spectra of proteins were measured on a maXis mass spectrometer with an electrospray ionization (ESI) source (Bruker Daltonics, Bremen, Germany) in the positive ion mode. To measure the intact protein mass (under denaturing conditions), the purified protein was dialyzed against 10 mM ammonium acetate pH 6.6 followed by the addition of 50% (v/v) methanol and 0.1% (v/v) acetic acid and application to the mass spectrometer via a syringe pump operated at 180 μL/h. The following conditions for the ion-transfer were used: 3400 V capillary voltage, 500 V endplate offset, 4 L/min dry gas at 200° C. temperature, 0.3 bar nebulizer pressure and 3 eV collision energy. Raw spectra were collected and deconvoluted with the Bruker Compass Data Analysis Software using the MaxEnt algorithm.
EXAMPLE 2—RESULTS
[0314] Phage Display Selection Divulged a hCD98hcED-Specific Lcn2 Variant with Pico-Molar Affinity
[0315] The soluble, monomeric extracellular domain of human CD98hc (UniProt ID P08195-2, residues Glu105-Ala526) was produced fully glycosylated in eukaryotic human embryonic kidney (HEK) cells (CD98hcEDg) and unglycosylated in E. coli (CD98hcED) (
TABLE-US-00006 TABLE 2 Size determination of the glycosylated and unglycosylated m/hCD98hcED Theoretical Apparent Apparent mass mass mass Protein [kDa] (SEC)[kDa] increase [%].sup.a hCD98hcED 48.4 49.4 — mCD98hcED 49.3 51.6 — hCD98hcEDg 49.3 73.1 32.4 mCD98hcEDg 50.2 79.8 37.1 .sup.aApparent mass increase caused by the N-linked glycosylation of h/mCD98hcED after production in HEK cells compared to production in E. coli
[0316] This extensive glycosylation of hCD98hcED could impede in vitro phage display selection of cognate Anticalins due to poor sterical accessibility of potential surface epitopes, as previously seen in the selection of Anticalins against the prostate-specific membrane antigen [65]. To avoid the necessity of glycan removal, which either requires harsh reaction conditions or results in incomplete deglycosylation, here the unglycosylated ectodomain produced in E. coli was used as the initial molecular target for selection. N-terminal fusion of the biotin acceptor peptide (BAP) resulted in the intracellular enzymatic attachment of a single biotin group upon co-expression of the BirA ligase (verified via western blot analysis, data not shown) for both CD98hcEDg and CD98hcED, regardless of the expression system used [66]. This modification allowed immobilization of the ectodomain to beads or surfaces in an orientation that resembles the in vivo situation for the type II membrane protein, accessible for binding of lipocalin variants during phage display selection, ELISA screening and in vitro binding studies.
[0317] Anticalin candidates were selected from a Lcn2 random library with a combinatorial complexity of 1×10.sup.10, which also had served for the development of other hapten- and protein-specific Anticalins in the past [50]. After six selection cycles, three independent CD98hc-specific lipocalin variants were identified by ELISA screening, then expressed as soluble proteins in E. coli at the shake flask scale and purified to homogeneity, resulting in monomeric proteins with apparent molecular size similar to wild type (wt) Lcn2 (
TABLE-US-00007 TABLE 1 Affinities for hCD98hcED and thermal stabilities of engineered lipocalin variants Affinity Stability Protein K.sub.D [nM] k.sub.on [M.sup.−1 × s.sup.−1] k.sub.off [s.sup.−1] T½ [min] T.sub.m [° C.] ΔH.sub.m [kJ/mol] ΔG.sub.U [kJ/mol] P1E4 4.5.sup.a 2.5 × 10.sup.6 1.1 × 10.sup.−2 1.1 68.8 812 104.0 P3A12 2.8.sup.b 6.2 × 10.sup.4 1.7 × 10.sup.−4 67.7 64.1 635 73.6 P3D11 0.15.sup.b 2.2 × 10.sup.5 3.2 × 10.sup.−5 359.4 55.2 368 33.8 D11vs 0.05.sup.b 4.5 × 10.sup.5 2.6 × 10.sup.−5 442.3 63.5 646 73.9 .sup.a= multiple cycle kinetics .sup.b= single cycle kinetics
[0318] Notably, the lipocalin variant P3D11 revealed a very low K.sub.D value of 150 pM for hCD98hcED and a long complex half-life of 6 h, hence suitable for tumor targeting in vivo (
[0319] Protruding Loops Constitute the Major Epitope for hCD98hcED Recognition by the Engineered Lipocalin
[0320] To identify the epitope region targeted by these Anticalin candidates and to understand the structural mechanisms of the tight molecular interaction of P3D11 with the ectodomain, we crystallized P3D11 in complex with hCD98hcED (carrying a N-terminal Strep-tag II) after isolation of the 1:1 complex via SEC (
TABLE-US-00008 TABLE 5 Summary of the contact area and contacts provided by the lipocalin variant P3D11 BSA [Å.sup.2] BSA mutated [Å.sup.2] HB/SB Loop #1 486 156 5/2 Loop #2 175 169 2/— Loop #3 90 1.0 1/— Loop #4 97 77 1/— β-barrel 576 364 6/1 SUM 1424 767 15/3
[0321] In part the interaction is driven by electrostatics as P3D11 is positively charged, favoring interaction with the predominantly negatively charged epitope on hCD98hcED, which is reflected by the calculated pl values of 8.8 and 5.2 for P3D11 and hCD98hcED, respectively. The epitope of hCD98hcED essentially consists of two loops, L1 (residues 128-137) and L2 (374-404), which follow to the first and the eighth β-strand of the TIM barrel, respectively.
[0322] Almost 75% of the contact interface is contributed by the interaction with L2, which penetrates deep into the β-barrel of the engineered lipocalin, whereas L1 provides approximately 20% of the total BSA (Table 6)
TABLE-US-00009 TABLE 6 Summary of the contact area and contacts provided by hCD98hcED BSA [Å.sup.2] HB/SB hCD98hcED 1521 15/3 Loop L1 288 6/1 Loop L2 1111 6/1
[0323] Further to the direct protein.circle-solid.protein contacts, 16 water molecules mediate hydrogen bonds at the complex interface. Based on the distribution of hydrogen bond donors, acceptors as well as water molecules, the P3D11 interface with the hCD98hcED can be dissected into two distinct areas, a rather polar region around the lipocalin loops #1 and #2 including the cavity as well as a rather apolar region around loop #3 and #4 (
[0324] Interestingly, binding of P3D11 causes an induced fit of hCD98hcED. Superposition of the hCD98hcED Cα positions in complex with P3D11 and for hCD98hcED crystallized alone (PDB entry 2DH2) [55], excluding the epitope loops L1 and L2, revealed an RMSD value of 0.97 Å (for 375 equivalent Cα positions), while in this context loops 1 and 2 deviate by 1.6 and 1.8 Å, respectively (
[0325] Although the selection of P3D11 was performed with the unglycosylated hCD98hcED, its affinity for the glycosylated hCD98hcEDg was indistinguishable (Table 3).
TABLE-US-00010 TABLE 3 Affinities of selected and engineered lipocalin variants for hCD98hcEDg determined via SPR analysis Protein K.sub.D [nM] k.sub.on M.sup.−1 × s.sup.−1] k.sub.off [s.sup.−1] T.sub.1/2 [min] P1E4 162 5.6 × 10.sup.5 9.0 × 10.sup.−2 <1 P3A12 1.9 2.2 × 10.sup.4 4.0 × 10.sup.−4 29 P3D11 0.17 3.0 × 10.sup.5 5.0 × 10.sup.−5 230 D11vs 0.05 4.6 × 10.sup.5 2.2 × 10.sup.−5 523
[0326] Modeling of the glycan moieties suggests that the Anticalin binds the protruding epitope in close proximity to two of the oligosaccharides but is not sterically hindered by them (
[0327] Directed Evolution Results in an Anticalin with Improved Affinity and Stability
[0328] Despite its tight binding activity towards hCD98hcED, the lipocalin variant P3D11 suffered from low thermal stability, a caveat for further preclinical as well as clinical development (Table 1). To tackle this issue, stability engineering was performed via directed evolution using bacterial surface display (BSD) starting from an error-prone library with moderate amino acid mutation rate (with approximately two amino acid exchanges on average) (
[0329] To select variants of P3D11 with enhanced thermal stability, expression of the membrane-anchored protein was induced at 37° C. and only bacteria showing both strong hCD98hcED binding and high cell surface display level were gated in the FACS experiment (
[0330] CD thermal unfolding studies at physiological pH demonstrated a significant rise by 8.6° C. in the melting temperature (T.sub.m) for D11vs (T.sub.m=63.5° C.) compared to P3D11 (T.sub.m=55.2° C.) (
[0331] The enhanced stability and affinity of D11vs can be explained on the basis of the crystal structure solved for the hCD98hcED.circle-solid.P3D11 complex: residue Gly81 was introduced during the initial selection campaign, where it replaced Arg81 present in wtLcn2, thus creating space for tight target binding (
[0332] D11vs Binds Human CD98hc on Different Tumor Cell Lines
[0333] For in vivo studies in mice, a modified version of D11vs with moderately prolonged plasma half-life was designed using PASylation technology [70]. To this end, a structurally disordered polypeptide comprising in total 200 proline, alanine and serine residues (PAS200) was genetically fused to the C-terminus of the engineered lipocalin, thus increasing its hydrodynamic molecular volume which leads to a retarded kidney filtration (
[0334] The ability of D11vs-PAS200 to bind hCD98hc in a native cellular environment was investigated by cytofluorometry and immunofluorescence microscopy using human cancer cell lines of different origins exhibiting strong expression of this tumor marker. Flow cytometric analysis of the B-cell lymphoma cell lines SU-DHL-4, Raji and Ramos, the prostate carcinoma cells DU-145 and PC-3 and the colorectal adenocarcinoma cell line Caco-2 with Cy5.5-labeled D11vs-PAS200 revealed strong binding of the membrane-associated target protein (
[0335] D11vs Shows Strong and Specific Accumulation in Two Human Xenograft Models
[0336] The applicability of PASylated D11vs for in vivo targeting of hCD98hc was evaluated by a PET/CT imaging study with the .sup.89Zr-labeled protein in two murine xenograft tumor models. For this purpose, the human cancer cell lines Ramos (
[0337] Furthermore, the .sup.89Zr-labeled D11vs-PAS200 tracer allowed good visualization of the Ramos xenografts 24 h p.i., yet at a lower level compared to the PC-3 tumors. Of note, the Ramos xenografts were less well localized and fast growing, which led to blurred imaging signals. Consequently, the PC-3 xenografts were used for further experiments. After in vivo PET imaging, explanted PC-3 tumors were used for autoradiography of tissue cross sections. In this analysis, a homogeneous distribution of the radiotracer within the PC-3 tumor tissue was detected (
[0338] In order to confirm these imaging results and to obtain quantitative data from ex vivo biodistribution experiments at the optimal time point 24 h p.i., a second study using male CB17-SCID mice baring PC-3 tumors was conducted (
EXAMPLE 3—DISCUSSION
[0339] The pathophysiological significance of CD98hc expression in several solid and blood cancers is well established today. Even though also expressed weakly in healthy cells and tissues such as activated lymphocytes, proximal tubules, placenta or testis, strong overexpression of this membrane protein has been shown to correlate with tumor progression and aggressiveness, thus allowing the assessment of disease prognosis, treatment response as well as overall survival in oncologic diseases. Hence, CD98hc constitutes a highly attractive biomedical target for diagnostic as well as therapeutic purposes in oncology. However, no CD98hc-specific reagents for in vivo imaging and only few drug candidates addressing this target are available to date.
[0340] To meet this need, an Anticalin was developed with exquisite specificity and high (picomolar) affinity towards the extracellular domain of hCD98hc. The initial Anticalin candidates were selected from a random library based on human lipocalin 2 (Lcn2), an abundant siderophore-binding plasma protein. This previously designed library, which carries an optimally distributed set of randomized amino acid positions within the binding site of the protein scaffold, was successfully applied in previous studies to generate Anticalins against proteinaceous biomedical targets (ED-B, Hsp70, VEGFR-3 or PSMA) [50, 60, 71, 72], peptides (monomeric Aβ) [73] and hapten-type ligands (Colchicin, Y.sup.III-DTPA, Petrobactin) [50, 60, 71, 72]. The lipocalin variants selected towards hCD98hcED from this naïve Lcn2 library show K.sub.D values already in the pico- to single-digit nanomolar range combined with a stable monomeric behavior, which once again validates the utility of this library.
[0341] Based on its association and dissociation rate constants, with a remarkable complex dissociation half-life of ˜6 h, P3D11 was chosen as the lead candidate for further characterization and optimization. X-ray structural analysis of its complex with the CD98hc ectodomain revealed deep penetration of loop L2 within the membrane-distal part of CD98hc into the cup-shaped β-barrel of the Anticalin, with a buried total paratope surface area of 1424 Å.sup.2. Competition analysis for binding of glycosylated hCD98hc showed that all the selected Anticalins, including P3D11, recognize the same epitope region. P3D11 and P3A12 share a similar set of amino acid exchanges compared with wtLcn2, with 8 identical residues among the 20 randomized positions. Highly similar sequence stretches are seen for loop #1, loop #2 as well as their neighboring β-strands A/B and C/D (
[0342] While N-glycosylation has no influence on target affinity of P3D11 (or its improved version D11vs) and P3A12, binding of P1E4 is significantly diminished (˜100-fold). This behavior can be explained by minor variations in the mutual orientations between hCD98hcED and the different Anticalins, which could lead to varying sensitivity towards sterically demanding carbohydrate side chains in the neighborhood despite targeting of a similar epitope region (
[0343] Despite remarkable antigen affinity, which would be suitable for in vivo tumor targeting, especially the Anticalin candidate P3D11 suffered from lower thermal stability and showed pronounced aggregation after prolonged incubation at 37° C. To overcome this obstacle, directed evolution via moderate random mutagenesis was employed in combination with high throughput sorting using a recently developed system for the bacterial surface display of Anticalins [59]. By applying high temperature (37° C.) during protein expression selection of variants showing improved protein folding and stability was forced. With just two additional amino acid substitutions—Phe71Ser and Gly81Val (located in loop #2 and β-strand D, respectively)—the variant D11vs exhibited both increased affinity (approximately three-fold) as well as significantly increased melting temperature (by ˜8° C.; cf. Table 1 and
[0344] Small animal PET/CT imaging and biodistribution analysis with the plasma half-life optimized and .sup.89Zr-labeled D11vs radiotracer showed high accumulation in PC-3 xenograft tumors with good imaging contrast. Blocking of CD98hc binding by addition of unlabeled D11 vs-PAS200 reduced tracer accumulation by ˜40%. This is in line with in vitro cytofluorometry and immunofluorescence microscopy, where highly specific binding of D11vs on several CD98hc-positive human cancer cell lines, but not on cells with the blocked CD98hc epitope, was evident. Tracer uptake in kidney, liver, spleen and joints was as expected due to the known in vivo release of .sup.89Zr(IV) from the radiochelator [69, 74]. Superior .sup.89Zr chelating reagents with higher in vivo stability, as for example fusarinin C, a siderophore-based cyclic chelator derived from Aspergillus fumigatus [75, 76], may be an option for future experiments.
[0345] In conclusion, human CD98hcED-specific Anticalin was selected and engineered with high affinity and successfully applied it for CD98hc-targeted PET diagnostic imaging. This novel radiotracer showed high tumor uptake in prostate carcinoma as well as B-cell lymphoma xenograft models in mice with excellent tumor visualization. To our knowledge, this is the first in vivo imaging study of CD98hc overexpression on tumor cells described in the literature. Considering the clinical importance of elevated CD98hc expression in several human cancers, this Anticalin constitutes a promising new tool for preclinical and, potentially, clinical applications in oncology.
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