Polypeptide
11155596 · 2021-10-26
Assignee
Inventors
Cpc classification
A61K38/16
HUMAN NECESSITIES
A61K47/42
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07K14/705
CHEMISTRY; METALLURGY
A61K38/16
HUMAN NECESSITIES
A61K47/42
HUMAN NECESSITIES
Abstract
The present disclosure relates to a class of engineered polypeptides having a binding affinity for programmed death-ligand 1 (PD-L1), and provides a PD-L1 binding polypeptide comprising the sequence ERNX.sub.4AAX.sub.7EIL X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 WAFIWX.sub.26LX.sub.28D. The present disclosure also relates to the use of such a PD-L1 binding polypeptide as a therapeutic, prognostic and/or diagnostic agent.
Claims
1. A PD-L1 binding polypeptide, comprising a PD-L1 binding motif BM, which motif consists of the amino acid sequence selected from: TABLE-US-00042 i) (SEQ ID NO: 837) ERNX.sub.4AAX.sub.7EIL X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20WAFIWX.sub.26LX.sub.26D wherein, independently from each other, X.sub.4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and Y; X.sub.7 is selected from A, E, F, H, N, Q, S, T, V, W and Y; X.sub.11 is selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W and Y; X.sub.16 is selected from N and T; X.sub.17 is selected from A, H, K, N, Q, R and S; X.sub.18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X.sub.20 is selected from H, I, K, L, N, Q, R, T, V and Y; X.sub.26 is selected from K and S; and X.sub.28 is selected from A, D and E; and ii) the amino acid sequence which has at least 96% identity to the sequence defined in i).
2. The PD-L1 binding polypeptide according to claim 1, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-808.
3. The PD-L1 binding polypeptide according to claim 1, wherein said PD-L1 binding motif forms part of a three-helix bundle protein domain.
4. The PD-L1 binding polypeptide according to claim 1, which comprises a binding module BMod, the amino acid sequence of which is selected from: TABLE-US-00043 iii) (SEQ ID NO: 838) K-[BM]-DPSQSX.sub.aX.sub.bLLX.sub.c EAKKLX.sub.dX.sub.eX.sub.fQ; wherein [BM] is a PD-L1 binding motif as defined in claim 1; X.sub.a is selected from A and S; X.sub.b is selected from N and E; X.sub.c is selected from A, S and C; X.sub.d is selected from E, N and S; X.sub.e is selected from D, E and S; and X.sub.f is selected from A and S; iv) the amino acid sequence which has at least 93% identity to a sequence defined in iii).
5. The PD-L1 binding polypeptide according to claim 1, which comprises the amino acid sequence selected from: TABLE-US-00044 xvii) (SEQ ID NO: 874) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD-L1 binding motif as defined in claim 1; and xviii) the amino acid sequence which has at least 89% identity to the sequence defined in xvii).
6. The PD-L1 binding polypeptide according to claim 1, which comprises the amino acid sequence selected from: TABLE-US-00045 xxi) (SEQ ID NO: 854) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD-L1 binding motif as defined in claim 1; and xxii) the amino acid sequence which has at least 89% identity to the sequence defined in xxi).
7. The PD-L1 binding polypeptide according to claim 5 or 6, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-814.
8. The PD-L1 binding polypeptide according to claim 7, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 811-813.
9. The PD-L1 binding polypeptide according to claim 7, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1, 2, 4, 5, 21, 811 and 812.
10. The PD-L1 binding polypeptide according to claim 7, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1 and 2 or SEQ ID NO:811 and 812.
11. The PD-L1 binding polypeptide according to claim 1, which is capable of blocking PD-L1 dependent signaling.
12. The PD-L1 binding polypeptide according to claim 11, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 5×10.sup.−8 M.
13. The PD-L1 binding polypeptide according to claim 11, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 1×10.sup.−8 M.
14. The PD-L1 binding polypeptide according to claim 11, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 5×10.sup.−9 M.
15. The PD-L1 binding polypeptide according to claim 11, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 3.5×10.sup.−9 M.
16. The PD-L1 binding polypeptide according to claim 11, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 1×10.sup.−9 M.
17. The PD-L1 binding polypeptide according to claim 11, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 5×10.sup.−10 M.
18. The PD-L1 binding polypeptide according to claim 11, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 1×10.sup.−10 M.
19. The PD-L1 binding polypeptide according to claim 1, which is capable of blocking the interaction of PD-L1 with PD-1.
20. The PD-L1 binding polypeptide according to claim 1, which is capable of binding to PD-L1 such that the K.sub.D value of the interaction is at most 2×10.sup.−8 M.
21. The PD-L1 binding polypeptide according to claim 20, which is capable of binding to PD-L1 such that the K.sub.D value of the interaction is at most 1×10.sup.−8 M.
22. The PD-L1 binding polypeptide according to claim 20, which is capable of binding to PD-L1 such that the K.sub.D value of the interaction is at most 1×10.sup.−9 M.
23. The PD-L1 binding polypeptide according to claim 20, which is capable of binding to PD-L1 such that the K.sub.D value of the interaction is at most 5×10.sup.−10 M.
24. The PD-L1 binding polypeptide according to claim which is capable of binding to PD-L1 such that the K.sub.D value of the interaction is at most 3×10.sup.−10 M.
25. The PD-L1 binding polypeptide according to claim 1, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93.
26. The PD-L1 binding polypeptide according to claim 1, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-24.
27. The PD-L1 binding polypeptide according to claim 1, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NOs:1, 2, 4, 5, and 21.
28. The PD-L1 binding polypeptide according to claim 1, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1 and 2.
29. A Fusion protein or conjugate comprising a first moiety consisting of a PD-L1 binding polypeptide according to claim 1; and a second moiety consisting of a polypeptide having a desired biological activity.
30. A complex, comprising at least one PD-L1 binding polypeptide according to claim 1 and at least one antibody or an antigen binding fragment thereof.
31. A polynucleotide encoding a polypeptide according to claim 1.
32. A composition comprising a PD-L1 binding polypeptide-according to claim 1, and at least one pharmaceutically acceptable excipient or carrier.
33. A method of treating a PD-L1 related disorder, comprising administering to a subject in need thereof an effective amount of a PD-L1 binding polypeptide according to claim 1.
34. The method of claim 33, wherein said PD-L1 related disorder is selected from the group consisting of infectious disease and cancer.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Summary
(13) The following Examples disclose the development of novel Z variant molecules targeted to human programmed death-ligand 1 (PD-L1), also known as human B7 homolog 1 (B7-H1) and cluster of differentiation 274 (CD274), based on phage display technology. The PD-L1 binding polypeptides described herein were sequenced, and their amino acid sequences are listed in
Example 1
Selection and Screening of PD-L1 Binding Z Variants
(14) In this Example, human PD-L1 (hPD-L1) was used as target in phage display selections using a phage library of Z variants. Selected clones were DNA sequenced, produced in E. coli periplasmic fractions and assayed against PD-L1 in ELISA (enzyme-linked immunosorbent assay).
(15) Materials and Methods
(16) Biotinylation of target protein: hPD-L1 (human PD-L1 Fc Chimera, R&D Systems, cat. no. 156-B7-100) was biotinylated using No-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scentific, cat. no. 21327) at a 10× molar excess, according to the manufacturer's recommendations. The reaction was performed at room temperature (RT) for 40 min. Subsequent buffer exchange to PBS (10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) was performed using a Slide-a-lyzer dialysis cassette (10000 MWCO, Thermo Scientific, cat. no. 66383) according to the manufacturer's instructions.
(17) Phage display selection of PD-L1 binding Z variants: A library of random variants of protein Z displayed on bacteriophage, constructed in phagemid pAY02592 essentially as described in Grönwall et al. (2007) J Biotechnol, 128:162-183, was used to select PD-L1 binding Z variants. In this library, an albumin binding domain (ABD, GA3 of protein G from Streptococcus strain G148) is used as fusion partner to the Z variants. The library is denoted Zlib006Naive.II and has a size of 1.5×10.sup.10 library members (Z variants). E. coli RRIAM15 cells (Ruther et al., (1982) Nucleic Acids Res 10:5765-5772) from a glycerol stock containing the phagemid library Zlib006Naive.II were inoculated in 20 l of a defined proline free medium [3 g/l KH.sub.2PO.sub.4, 2 g/l K.sub.2HPO.sub.4, 0.02 g/l uracil, 6.7 g/l YNB (Difco™ Yeast Nitrogen Base w/o amino acids, Becton Dickinson), 5.5 g/l glucose monohydrate, 0.3 g/l L-alanine, 0.24 g/l L-arginine monohydrochloride, 0.11 g/l L-asparagine monohydrate, 0.1 g/l L-cysteine, 0.3 g/l L-glutamic acid, 0.1 g/l L-glutamine, 0.2 g/l glycine, 0.05 g/l L-histidine, 0.1 g/l L-isoleucine, 0.1 g/l L-leucine, 0.25 g/l L-lysine monohydrochloride, 0.1 g/l L-methionine, 0.2 g/l L-phenylalanine, 0.3 g/l L-serine, 0.2 g/l L-threonine, 0.1 g/l L-tryptophane, 0.05 g/l L-tyrosine, 0.1 g/l L-valine], supplemented with 100 μg/ml ampicillin. The cultivations were grown at 37° C. in a fermenter (Belach Bioteknik, BR20). When the cells reached an optical density at 600 nm (OD.sub.600) of 0.75, approximately 2.6 l of the cultivation was infected using a 10× molar excess of M13K07 helper phage (New England Biolabs, cat. no. N0315S). The cells were incubated for 30 min, whereupon the fermenter was filled up to 20 l with cultivation medium (2.5 g/l (NH.sub.4).sub.2SO.sub.4; 5.0 g/l Yeast Extract (Merck 1.03753.0500); 25 g/l Peptone (Scharlau 07-119); 2 g/l K.sub.2HPO.sub.4; 3 g/l KH.sub.2PO.sub.4; 1.25 g/l Na.sub.3C.sub.6H.sub.5O.sub.7.2H.sub.2O; 0.1 ml/l Breox FMT30 antifoaming agent) supplemented with 100 μM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for induction of expression and with 50 μg/ml ampicillin, 12.5 μg/ml carbenicillin, 25 μg/ml kanamycin, 35 ml/l of 1.217 M MgSO.sub.4 and 10 ml of a trace element solution [129 mM FeCl.sub.3; 36.7 mM ZnSO.sub.4; 10.6 mM CuSO.sub.4; 78.1 mM MnSO.sub.4; 94.1 mM CaCl.sub.2, dissolved in 1.2 M HCl]. A glucose-limited fed-batch cultivation was started where a 600 g/l glucose solution was fed to the reactor (15 g/h in the start, 40 g/h at the end of the fermentation after 17 h). pH was controlled at 7 through the automatic addition of 25% NH.sub.4OH, air was supplemented (10 l/min), and the stirrer was set to keep the dissolved oxygen level above 30%. The cells in the cultivation were removed by tangential flow filtration.
(18) The phage particles were precipitated from the supernatant twice in PEG/NaCl (polyethylene glycol/sodium chloride), filtered and dissolved in PBS and glycerol as described in Grönwall et al., supra. Phage stocks were stored at −80° C. before use.
(19) Selections against biotinylated hPD-L1 were performed in four cycles initially divided in two different tracks (1 and 2). As selection proceeded, the tracks were further divided according to target concentration and number and/or time of washes to finally end up in nine tracks in cycle 4. More precisely, the first track (1) was divided in the second to the fourth cycles, resulting in a total of two tracks (1-1 to 1-2) in cycle 2, four tracks (1-1-1 to 1-2-2) in cycle 3 and six tracks (1-1-1-1 to 1-2-2-1) in cycle 4. The second track (2) was divided in the third to the fourth cycles, resulting in a total of two tracks (2-1-1 to 2-1-2) in cycle 3, three tracks (2-1-1-1 to 2-1-2-1) in cycle 4. In track 1 with descendants, Dynabeads® M-280 Streptavidin (SA-beads, Invitrogen, cat. no. 11206D) were used to catch the hPD-L1:Z variant complexes. In track 2, Dynabeads® Protein A (SPA-beads, Invitrogen, cat. no. 10002D) were used instead to catch the hPD-L1:Z variant complexes by binding to the Fc part of the hPD-L1 Fc chimeric protein.
(20) Phage stock preparation, selection procedure and amplification of phage between selection cycles were performed essentially as described for selection against another biotinylated target in WO2009/077175 with the following exception: the selection buffer consisted of PBS supplemented with % Fetal Bovine Serum (FBS, Gibco, cat. no. 10108-165) and 0.1% Tween20 (Acros Organics, cat. no. 233362500).
(21) In order to reduce the amount of background binders, a pre-selection was performed in each cycle. In the pre-selection, the same types of beads were used as during the selection, i.e. SA-beads in track 1 and SPA-beads in track 2. In all tracks of cycle 1-4, pre-selections were performed using SA- or SPA-beads coated with biotinylated human IgG-Fc (Jackson ImmunoResearch Lab, cat. no. 009-060-008). Furthermore in cycle 1, track 1, the pre-selection was performed using SA-beads coated with a mix of hPD-L2 (human PD-L2 Fc Chimera; R&D Systems, cat. no. 1224-PL-100), hB7-H3 (human B7-H3 Fc Chimera; R&D Systems, cat. no. 1027-B3-100), hB7-H4 (human B7-H4; R&D Systems, cat. no. 6576-B7-50), biotinylated previously as described for hPD-L1. In cycle 1, track 2, the pre-selection was performed using SPA-beads coated with a mix of biotinylated PD-L2 and biotinylated B7-H3. During pre-selection the phage stock was incubated with coated beads end-over end for 30-90 min at RT. All tubes and beads used in the pre-selections or selection were pre-blocked with PBS supplemented with 3% Bovine Serum Albumin (BSA, Sigma A3059-100G) and 0.1% Tween20. Selection was performed in solution at RT and the time for selection was approximately 120 min followed by wash with PBS+0.1% Tween20 and catch of target-phage complexes on SA-beads or SPA-beads using 1 mg beads per 1.6 or 8.5 μg biotinylated hPD-L1, respectively.
(22) For amplification of phage particles between selection cycle 1 and 2, E. coli strain ER2738 cells (Lucigen, Middleton, Wis., USA) were used for infection and grown in medium supplemented with 20 μg/ml tetracycline. A 5× excess of M13K07 helper phage compared to bacteria were allowed to infect log phase bacteria.
(23) TABLE-US-00015 TABLE 2 Selection against biotinylated hPD-L1 Fc chimera Phage stock from library Target Number Duration Selection or selection Proteins used conc. of of last Cycle track track in pre-selection (nM) washes wash (h) 1 1 Zlib006Naive.II IgG-Fc, hPD-L2, 100 2 hB7-H3, hB7-H4 1 2 Zlib006Naive.II IgG-Fc, hPD-L2, 100 2 hB7-H3 2 1-1 1 IgG-Fc 66 4 2 1-2 1 IgG-Fc 10 4 2 2-1 2 IgG-Fc 66 4 3 1-1-1 1-1 IgG-Fc 44 6 3 1-1-2 1-1 IgG-Fc 10 6 3 1-2-1 1-2 IgG-Fc 1 6 3 1-2-2 1-2 IgG-Fc 0.5 10 3 2-1-1 2-1 IgG-Fc 44 6 3 2-1-2 2-1 IgG-Fc 10 6 4 1-1-1-1 1-1-1 IgG-Fc 30 10 4 1-1-1-2 1-1-1 IgG-Fc 10 31 1 4 1-1-2-1 1-1-2 IgG-Fc 10 31 15 4 1-2-1-1 1-2-1 IgG-Fc 0.5 10 4 1-2-1-2 1-2-1 IgG-Fc 0.2 31 64 [4° C.] 4 1-2-2-1 1-2-2 IgG-Fc 0.05 31 15 4 2-1-1-1 2-1-1 IgG-Fc 30 10 4 2-1-1-2 2-1-1 IgG-Fc 10 31 1 4 2-1-2-1 2-1-2 IgG-Fc 10 31 15
(24) The amplification of phage particles between selection cycles 2 and 4 was done by performing infection of bacteria in solution as follows. After infection of log phase E. coli ER2738 with phage particles, TSB supplemented with 2% glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin was added, followed by incubation with rotation for 30 min at 37° C. Thereafter, the bacteria were infected with M13K07 helper phage in 5× excess. The infected bacteria were pelleted by centrifugation, re-suspended in TSB-YE medium supplemented with 100 μM IPTG, 25 μg/ml kanamycin and 100 μg/ml ampicillin, and grown overnight at 30° C. The overnight cultures were centrifuged, and phage particles in the supernatant were precipitated twice with PEG/NaCl buffer. Finally, the phage particles were re-suspended in selection buffer before entering the next selection cycle.
(25) In the final selection cycle, log phase bacteria were infected with eluate and diluted before spreading onto TBAB plates (30 g/l tryptose blood agar base, Oxoid, cat. no. CM0233B) supplemented with 0.2 g/l ampicillin in order to form single colonies to be used in ELISA screening. An overview of the selection strategy, describing an increased stringency in subsequent cycles, using a lowered target concentration and an increased number of washes, is shown in Table 2. Washes were performed for 1 min, if nothing else is stated in Table 2, using PBST 0.1% (PBS supplemented with 0.1% Tween-20) and elution was carried out as described in WO2009/077175.
(26) Production of Z variants for ELISA: Z variants were produced by inoculating single colonies from the selections into 1 ml TSB-YE medium supplemented with 100 μg/ml ampicillin and 1 mM IPTG in deep-well plates (Nunc, cat. no. 278752). The plates were incubated with rotation for 24 h at 37° C. Cells were pelleted by centrifugation, re-suspended in 200 μl PBST 0.05% and frozen at −80° C. to release the periplasmic fraction of the cells. Frozen samples were thawed in a water bath and the freeze-thawing procedure was repeated eight times. 600 μl PBST 0.05% was added to the thawed samples and cells were pelleted by centrifugation.
(27) The final supernatant of the periplasmic extract contained the Z variants as fusions to ABD, expressed as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (SEQ ID NO:896) (Grönwall et al., supra). Z##### refers to individual, 58 amino acid residue Z variants.
(28) ELISA screening of Z variants: The binding of Z variants to hPD-L1 was analyzed in ELISA assays. Half-area 96-well ELISA plates (Costar, cat. no. 3690) were coated at 4° C. overnight with 2 μg/ml of an anti-ABD goat antibody (produced in-house) diluted in coating buffer (50 mM sodium carbonate, pH 9.6; Sigma, cat. no. C3041). The antibody solution was poured off and the wells were washed in water and blocked with 100 μl of PBSC (PBS supplemented with 0.5% casein; Sigma, cat. no. C8654) for 1 to 3 h at RT. The blocking solution was discarded and 50 μl periplasmic solutions, diluted 1:1 with PBST 0.05%, were added to the wells and incubated for 1.5 to 2.5 h at RT under slow agitation. As a blank control, PBST 0.05% was added instead of a periplasmic sample. The supernatants were poured off and the wells were washed 4 times with PBST 0.05%. Then, 50 μl of biotinylated hPD-L1 at a concentration of 0.32 nM in PBSC was added to each well. The plates were incubated for 1 h at RT followed by washes as described above. Streptavidin conjugated HRP (Thermo Scientific, cat. no. N100) diluted 1:30,000 in PBSC, was added to the wells and the plates were incubated for approximately 1 h. After washing as described above, 50 μl ImmunoPure TMB substrate (Thermo Scientific, cat. no. 34021) was added to the wells and the plates were treated according to the manufacturer's recommendations. The absorbance at 450 nm was measured using a multi-well plate reader, Victor.sup.3 (Perkin Elmer).
(29) Sequencing: In parallel with the ELISA screening, all clones were sequenced. PCR fragments were amplified from single colonies, sequenced and analyzed essentially as described in WO2009/077175.
(30) EC50 analysis of Z variants: A selection of PD-L1 binding Z variants was subjected to an analysis of the response against a dilution series of biotinylated hPD-L1 following the procedure described above. The Z variants were diluted 1:1 in PBST 0.05%. Biotinylated hPD-L1 was added at a concentration of 40 nM and diluted stepwise 1:4 down to 32 pM. As a background control, all Z variants were also assayed with no target protein added. Periplasm samples containing the PD-L1 binding Z variant Z13112 (SEQ ID.NO:777) were included on each plate and analyzed as positive control. Periplasm containing the ABD moiety only was used as negative control. In the same assay, the specificity of the Z variants was tested by incubating periplasm samples with the four different biotinylated control proteins hPD-L2, hB7-H3, hB7-H4 and IgGFc, respectively, added at a concentration of 8 nM. Data were analyzed using GraphPad Prism 5 and non-linear regression, and EC50 values (the half maximal effective concentration) were calculated.
(31) Results
(32) Phage display selection of PD-L1 binding Z variants: Individual clones were obtained after four cycles of phage display selections against biotinylated hPD-L1.
(33) ELISA screening of Z variants: The clones obtained after four cycles of selection were produced in 96-well plates and screened for hPD-L1 binding activity in ELISA. Several unique Z variants were found to give a response of 0.3 AU or higher (corresponding to at least 3× the blank control) against hPD-L1 at a concentration of 0.32 nM. The average response of the blank controls was 0.067 AU.
(34) Sequencing: Sequencing was performed for clones obtained after four cycles of selection. Each variant was given a unique identification number #####, and individual variants are referred to as Z#####. The amino acid sequences of the 58 amino acid residues long Z variants are listed in
(35) EC50 analysis of Z variants: A subset of Z variants having the highest ELISA values in the ELISA screening experiment described above was selected and subjected to a target titration in ELISA format. Periplasm samples were incubated with a serial dilution of biotinylated hPD-L1. A periplasm sample containing Z13112 (SEQ ID NO:777), confirmed to bind PD-L1 in the ELISA screen, was selected as a positive control and used to normalize different plates to each other. Obtained values were analyzed and their respective EC50 values were calculated (Table 3).
(36) No significant binding was detected to any of the included control proteins of the B7-family (hPD-L2, hB7-H3 and hB7-H4), nor to the control protein IgGFc (included here because Fc chimeric proteins were used in the selection and screening). These results indicate that the selected Z variants are specific to PD-L1.
(37) TABLE-US-00016 TABLE 3 Calculated EC50 values from ELISA titration analysis Z variant SEQ ID NO: EC50 (M) Z13080 774 2.8 × 10.sup.−10 Z13088 775 3.8 × 10.sup.−10 Z13091 776 2.2 × 10.sup.−10 Z13104 788 4.1 × 10.sup.−10 Z13112 777 2.2 × 10.sup.−10 Z13115 789 4.0 × 10.sup.−10 Z13117 790 2.9 × 10.sup.−10 Z13134 791 4.5 × 10.sup.−10 Z13147 779 2.8 × 10.sup.−10 Z13154 780 1.1 × 10.sup.−10 Z13158 782 2.5 × 10.sup.−10 Z13164 783 2.2 × 10.sup.−10 Z13165 784 2.4 × 10.sup.−10 Z13169 785 1.5 × 10.sup.−10 Z13186 792 4.7 × 10.sup.−10 Z13190 793 2.6 × 10.sup.−10 Z13198 786 1.6 × 10.sup.−10 Z13210 794 3.5 × 10.sup.−10 Z13304 787 3.2 × 10.sup.−10 Z13368 795 4.8 × 10.sup.−10 Z13447 796 2.9 × 10.sup.−10
Example 2
Subcloning and Production of a Subset of Primary PD-L1 Binding Z Variants
(38) Materials and Methods
(39) Subcloning of Z variants with a Hiss-tag: The DNA of 14 PD-L1 binding Z variants, Z13080 (SEQ ID NO:774), Z13088 (SEQ ID NO:775), Z13091 (SEQ ID NO:776), Z13112 (SEQ ID NO:777), Z13120 (SEQ ID NO:778), Z13147 (SEQ ID NO:779), Z13154 (SEQ ID NO:780), Z13156 (SEQ ID NO:781), Z13158 (SEQ ID NO:782), Z13164 (SEQ ID NO:783), Z13165 (SEQ ID NO:784), Z13169 (SEQ ID NO:785), Z13198 (SEQ ID NO:786) and Z13304 (SEQ ID NO:787) were amplified from the library vector pAY02592. A subcloning strategy for construction of monomeric Z variant molecules with N-terminal Hiss-tag was applied using standard molecular biology techniques (essentially as described in detail in WO2009/077175 for Z variants binding another target). The Z gene fragments were subcloned into the expression vector pAY01448 resulting in the encoded sequence MGSSHHHHHHLQ-[Z#####]-VD (seq ID NO:897.
(40) Subcloning of Z variants with a C-terminal Cys: Two Z variants, Z13091 (SEQ ID NO:776) and Z13156 (SEQ ID NO:781) were mutated to start with the N-terminal amino acids AE instead of VD and further subcloned with the C-terminal addition of the amino acids VDC (incorporating a unique cysteine in the polypeptide) using standard molecular biology techniques. The resulting encoding sequences are referred to as Z15168-Cys (SEQ ID NO:809) and Z15169-Cys (SEQ ID NO:810), respectively.
(41) Cultivation: E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragments of each respective PD-L1 binding Z variant and cultivated at 37° C. in 940 ml of TSB-YE medium supplemented with 50 μg/ml kanamycin. In order to induce protein expression, IPTG was added to a final concentration of 0.2 mM at OD.sub.600=2 and the cultivation was incubated at 37° C. for another 5 h. The cells were harvested by centrifugation.
(42) Purification of PD-L1 binding Z variants with a Hiss-tag: Approximately 1-2 g of each cell pellet was resuspended in 30 ml of binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) supplemented with Benzonase® (Merck, cat. no. 1.01654.0001) to a concentration of 15 U/ml. After cell disruption by sonication, cell debris was removed by centrifugation and each supernatant was applied on a 1 ml His GraviTrap IMAC column (GE Healthcare, cat. no. 11-0033-99). Contaminants were removed by washing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4) and the PD-L1 binding Z variants were subsequently eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). After the IMAC purification, the protein buffer was exchanged to PBS using PD-10 columns (GE Healthcare, cat. no. 17-0851-01).
(43) Purification of PD-L1 binding Z variants with a C-terminal Cys: The respective cell pellet was resuspended in 20 mM Tris-HCl, pH 8 (10 ml buffer/g cell pellet) and lysed by heat treatment in a water bath at 90° C. for 10 min, followed by cooling on ice to approximately 20° C. Benzonase® was added (1 μl/g cell pellet) and each cell lysate was incubated at RT for 30 min, before cell debris was removed by centrifugation. For reduction of disulfides, dithiothreitol (DTT; Acros organics, cat. no. 165680250) was added to a final concentration of 20 mM followed by incubation at RT for 1 h. Purification was performed by anion exchange followed by reverse phase chromatography (RPC). Buffer exchange to 20 mM HEPES, 1 mM EDTA, pH 7.2 was carried out using HiPrep 26/10 columns (GE Healthcare, cat. no. 17-5087-01). Finally, each Z variant was purified on EndoTrap® red columns (Hyglos, cat. no. 321063) to ensure low endotoxin content.
(44) For each protein purified by any method described above, the concentration was determined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer and the extinction coefficient of the protein. The purity was analyzed by SDS-PAGE stained with Coomassie Blue and the identity of each purified Z variant was confirmed using HPLC-MS analysis (HPLC-MS 1100; Agilent Technologies).
(45) Results
(46) Cultivation and purification: The PD-L1 binding Z variants with a Hiss-tag or a C-terminal Cys were expressed as soluble gene products in E. coli. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the PD-L1 binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.
Example 3
Characterization of Primary PD-L1 Binding Z Variants
(47) In this Example, a subset of Z variants was characterized in terms of stability and in vitro binding properties. The specificity and affinity for human PD-L1 of the Z variants were analyzed by SPR and binding to PD-L1 expressing cells was analyzed using Fluorescence Activated Cell Sorting (FACS). Furthermore, the ability of Z variants to block the binding of PD-L1 to its receptor PD1 was investigated using AlphaLISA.
(48) Materials and Methods
(49) Biacore kinetic and specificity analysis: Kinetic constants (k.sub.a and k.sub.d) and affinities (K.sub.D) for hPD-L1 were determined for 14 Hiss-tagged Z variants using a Biacore 2000 instrument (GE Healthcare). Some of the Z variants were also tested for binding against the sequence-related proteins hPD-L2, hB7-H3, hB7-H4 and mPD-L1 (mouse PD-L1 Fc Chimera, R&D Systems, cat. no. 1019-B7).
(50) hPD-L1, hPD-L2, hB7-H3, hB7-H4 and mPD-L1 were immobilized in separate flow cells on the carboxylated dextran layer of different CM5 chip surfaces (GE Healthcare, cat. no. BR100012). The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, GE Healthcare, cat. no. BR100188). The ligand immobilization levels on the surfaces were 468-894 RU for hPD-L1, 537-742 RU for hPD-L2, 383 RU for hB7-H3, 538-659 RU for hB7-H4 and 482 RU for mPD-L1. One flow cell surface on each chip was activated and deactivated for use as blank during analyte injections. In the kinetic experiment, HBS-EP was used as running buffer and the flow rate was 50 μl/min. The analytes, i.e. the Z variants, were each diluted in HBS-EP buffer within a concentration range of 1000 to 0.01 nM and injected for 5 min, followed by dissociation in running buffer for 15-25 min. After dissociation, the surfaces were regenerated with one or two injections of 0.1% SDS. Kinetic constants were calculated from the sensorgrams using the Langmuir 1:1 model of BiaEvaluation software 4.1 (GE Healthcare).
(51) AlphaLISA blocking assay: The potential of Z variants to inhibit binding of PD-L1 to PD-1 was analyzed by AlphaLISA and recordings in an EnSpire multiplate reader 2300 (Perkin Elmer). hPD-1 (human PD-1 Fc-chimera; R&D Systems, cat. no. 1086-PD-050) was immobilized on AlphaLISA Acceptor beads (Perkin Elmer, cat. no. 6772002) according to the manufacturer's recommendations. Stepwise serial dilutions 1:3 of Hiss-tagged Z variants to final concentrations of 250 nM to 12 pM were made in a 384 plate (Perkin Elmer, cat. no. 6005350) and incubated for 1 h with 10 nM biotinylated hPD-L1 in AlphaLISA buffer (Perkin Elmer, cat. no. AL000F). hPD-1-coated Acceptor beads were added to a final concentration of 10 μg/ml and incubated for 1 h. Finally, streptavidin coated Donor beads (Perkin Elmer, cat. no. 6772002) were added to a final concentration of 40 μg/ml and incubated for 30 min. All incubations were performed at RT in the dark. The plate was analyzed in the EnSpire instrument and the IC50 values were calculated using GraphPad Prism 5.
(52) Cell binding analysis by FACS: The potential of Z variants to bind PD-L1 expressing cells was investigated using Fluorescence Activated Cell Sorting (FACS). THP-1 cells, cultivated in RPMI (Lonza, cat. no. BE12-702F) containing 10% FBS were stimulated with 10 ng/ml IFNg (R&D Systems, cat. no. 285-IF-100) overnight which results in up-regulation of PD-L1. 150,000 stimulated and unstimulated cells were pipetted per well of a v-bottomed 96 well plate (Nunc, cat. no. 277143) and the cells in the plate were subsequently pelleted at 400 g for 3 min at RT. The supernatants were removed and the cells were resuspended in 100 μl PBS plus 2.5% FBS (staining buffer) containing 10 μg/ml of the different His-tagged Z variants. A mouse anti-PD-L1 antibody (R&D Systems, cat. no. MAB1561) at 1 μg/ml was used as a positive control. Cells incubated with buffer alone were used as negative controls. The cells were incubated for 1 h at 8° C. in the dark, washed twice with 100 μl staining buffer and resuspended in 100 μl of staining buffer containing a goat anti-Z antibody (produced in house) at a concentration of 5 μg/ml. Cells stained with the positive control were treated with buffer only. The cells were incubated for 1 h at 8° C. in the dark, washed twice with 100 μl staining buffer and resuspended in 100 μl of staining buffer containing an Alexa Fluor 647 chicken anti-goat IgG antibody (Life technologies, cat. no. A21469) or an Alexa Fluor 647 goat anti-mouse IgG antibody (Life technologies cat. no. A21236). The cells were once again incubated for 1 h at 8° C. in the dark, washed twice with 100 μl staining buffer and resuspended in 200 μl of staining buffer. Data from 10,000 cells were obtained using a FACS Calibur (Beckman Coulter) and the data was analyzed using Flowing software 2.5.0 (Turku University). Mean fluorescence intensity (MFI) was used as a read out of binding capacity.
(53) Circular dichroism (CD) spectroscopy analysis: Two purified Z variants with a C-terminal cysteine, Z15168-Cys (SEQ ID NO:809) and Z15169-Cys (SEQ ID NO:810), were diluted to 0.5 mg/ml in 20 mM HEPES, 1 mM EDTA, pH 7.2. For each diluted Z variant, a CD spectrum at 250-195 nm was obtained at 20° C. In addition, a variable temperature measurement (VTM) was performed to determine the melting temperature (Tm). In the VTM, the absorbance was measured at 221 nm while the temperature was raised from 20 to 90° C., with a temperature slope of 5° C./min. A new CD spectrum was obtained at 20° C. after the heating procedure in order to study the refolding ability of the Z variants. The CD measurements were performed on a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) using a cell with an optical path length of 1 mm.
(54) Results
(55) Biacore kinetic and specificity analysis: The interactions of 14 Hiss-tagged PD-L1-binding Z variants with hPD-L1 were analyzed in a Biacore instrument by injecting various concentrations of the Z variants over a surface containing immobilized hPD-L1. All tested Z variants showed binding to hPD-L1. A summary of the kinetic parameters (K.sub.D, k.sub.a and k.sub.d) for binding of the Z variants to hPD-L1 obtained using a 1:1 interaction model is given in Table 4. Typical resulting curves, where responses from a blank surface were substracted, are displayed in
(56) TABLE-US-00017 TABLE 4 Kinetic parameters for binding of Z variants to hPD-L1 hPD-L1 Z variant SEQ ID NO: k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) Z13080 774 1.4 × 10.sup.6 9.2 × 10.sup.−3 6.6 × 10.sup.−9 Z13088 775 1.4 × 10.sup.6 3.7 × 10.sup.−3 2.6 × 10.sup.−9 Z13091 776 3.1 × 10.sup.6 1.4 × 10.sup.−3 .sup. 4.6 × 10.sup.−10 Z13112 777 6.1 × 10.sup.5 1.5 × 10.sup.−3 2.5 × 10.sup.−9 Z13120 778 2.0 × 10.sup.6 1.2 × 10.sup.−2 6.0 × 10.sup.−9 Z13147 779 1.0 × 10.sup.6 1.7 × 10.sup.−3 1.6 × 10.sup.−9 Z13154 780 2.3 × 10.sup.6 3.1 × 10.sup.−3 1.4 × 10.sup.−9 Z13156 781 1.7 × 10.sup.6 2.5 × 10.sup.−3 1.5 × 10.sup.−9 Z13158 782 6.7 × 10.sup.5 5.6 × 10.sup.−3 8.3 × 10.sup.−9 Z13164 783 7.9 × 10.sup.5 8.7 × 10.sup.−3 1.1 × 10.sup.−8 Z13165 784 2.0 × 10.sup.6 1.3 × 10.sup.−3 .sup. 6.4 × 10.sup.−10 Z13169 785 2.4 × 10.sup.6 3.5 × 10.sup.−3 1.5 × 10.sup.−9 Z13198 786 5.8 × 10.sup.6 5.6 × 10.sup.−3 .sup. 9.7 × 10.sup.−10 Z13304 787 1.6 × 10.sup.6 6.4 × 10.sup.−3 4.1 × 10.sup.−9
(57) A subset of the Z variants was also tested for binding against four immobilized, sequence-related proteins: hPD-L2, hB7-H3, hB7-H4 and mPD-L1. No binding against hPD-L2, hB7-H3, hB7-H4 or mPD-L1 was detected at Z variant concentrations up to 50 nM. When injecting 1000 nM of a few selected Z variants (Z13088, Z13091, Z13112, Z13147, Z13154, Z13156, Z13165, Z13169, Z13198) some response against B7-H4 was observed for Z13156 and Z13165. The result of the binding specificity analysis is summarized in Table 5. Typical non-interacting traces from the SPR analysis against hPD-L2, hB7-H3 and hB7-H4 are shown in
(58) TABLE-US-00018 TABLE 5 Binding specificity against mPD-L1, hPD-L2, hB7-H3 and hB7-H4 Z variant SEQ ID NO: mPD-L1 hPD-L2 hB7-H3 hB7-H4 Z13080 774 n.a. n.d. n.d. n.a. Z13088 775 n.d. n.d. n.d. n.d. Z13091 776 n.d. n.d. n.d. n.d. Z13112 777 n.a. n.d. n.a. n.d. Z13120 778 n.a. n.d. n.d. n.a. Z13147 779 n.d. n.d. n.a. n.d. Z13154 780 n.d. n.d. n.d. n.d. Z13156 781 n.d. n.d. n.d. K.sub.D > 5 μM Z13158 782 n.a. n.d. n.d. n.a. Z13164 783 n.a. n.d. n.d. n.a. Z13165 784 n.d. n.d. n.d. K.sub.D > 5 μM Z13169 785 n.d. n.d. n.d. n.d. Z13198 786 n.d. n.d. n.d. n.d. Z13304 787 n.a. n.d. n.a. n.d. n.a. not assayed; n.d. no binding detected
(59) AlphaLISA blocking assay: The ability of 14 Hiss-tagged Z variants to inhibit hPD-L1 binding to hPD-1 was tested in an AlphaLISA blocking assay. Serial dilutions of the Z variants were incubated with biotinylated hPD-L1 and the blocking ability of each respective variant was measured after addition of hPD-1 coated Acceptor beads and subsequently streptavidin coated Donor beads. Inhibition could be measured as a decrease in AlphaLISA counts for positive Z variants. The calculated IC50 values for the 14 variants that were all shown to block PD-L1 binding to PD-1 in this assay are shown in Table 6.
(60) TABLE-US-00019 TABLE 6 IC50 values for Z variants inhibiting binding of PD-L1 to PD-1 Z variant SEQ ID NO: IC50 (M) Z13080 774 2.7 × 10.sup.−9 Z13088 775 .sup. 8.8 × 10.sup.−10 Z13091 776 6.9 × 10.sup.−9 Z13112 777 3.9 × 10.sup.−9 Z13120 778 4.2 × 10.sup.−9 Z13147 779 5.6 × 10.sup.−9 Z13154 780 1.1 × 10.sup.−9 Z13156 781 1.4 × 10.sup.−9 Z13158 782 2.4 × 10.sup.−9 Z13164 783 2.3 × 10.sup.−9 Z13165 784 1.3 × 10.sup.−9 Z13169 785 2.2 × 10.sup.−9 Z13198 786 1.3 × 10.sup.−9 Z13304 787 4.2 × 10.sup.−8
(61) TABLE-US-00020 TABLE 7 Normalized MFI for binding of Z variants to THP-1 cells Z variant SEQ ID NO: MFI (normalized) Z13080 774 1.06 Z13088 775 1.00 Z13091 776 1.00 Z13112 777 0.76 Z13120 778 0.71 Z13147 779 0.56 Z13154 780 1.04 Z13156 781 1.09 Z13158 782 0.91 Z13164 783 0.81 Z13165 784 1.24 Z13169 785 0.91 Z13198 786 0.89 Z13304 787 0.75 anti-PD-L1 antibody — 0.40
(62) Cell binding analysis by FACS: This experiment confirmed binding of the PD-L1 specific Z variants to PD-L1 expressing cells. THP-1 cells stimulated with IFNγ overnight, which increases the PD-L1 expression, were stained with 10 μg/ml of each of the Hiss-tagged Z variants. The analyses were performed at two different occasions and the MFI values, normalized against Z13091 included in both assays, are presented in Table 7.
(63) CD analysis: The CD spectra determined for two selected PD-L1 binding Z variants with a C-terminal cysteine, Z15168-Cys (SEQ ID NO:809) and Z15169-Cys (SEQ ID NO:810) showed that both variants had an α-helical structure at 20° C. based on the typical minima at 208 and 222 nm. Reversible folding was seen for both Z variants when overlaying spectra measured before and after heating to 90° C. (
(64) TABLE-US-00021 TABLE 8 Melting temperatures (Tm) Z variant SEQ ID NO: Tm (° C.) Z15168-Cys 809 50 Z15169-Cys 810 58
Example 4
Design and Construction of a Maturated Library of PD-L1 Binding Z Variants
(65) In this Example, a maturated library was constructed. The library was used for selections of PD-L1 binding Z variants. Selections from maturated libraries may result in binders with increased affinity (Orlova et al., (2006) Cancer Res 66(8):4339-48). In this study, randomized single stranded oligonucleotides are generated, using split-pool DNA synthesis enabling incorporation of defined codons in desired positions in the synthesis.
(66) Materials and Methods
(67) Library design: The library was based on the sequences of the PD-L1 binding Z variants identified and characterized as described in Example 1 and Example 3. In the new library, 13 variable positions in the Z molecule scaffold were biased towards certain amino acid residues, according to a strategy based on the Z variant sequences defined in SEQ ID NO:774-808. Two oligonucleotides, one forward and one reverse complementary, with complementary 3′-ends were generated using split-pool synthesis. The two oligonucleotides were annealed and extended by PCR, using outer primers, to yield one gene fragment covering 147 bp corresponding to partially randomized helix 1 and 2 of the amino acid sequence: 5′-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA CGT AAC NNN GCG GCT NNN GAG ATC CTG NNN CTG CCT AAC CTC ACC NNN NNN CAA NNN TGG GCC TTC ATC TGG AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3′ (SEQ ID NO:819; randomized codons are illustrated as NNN) flanked by restriction sites XhoI and SacI. The oligonucleotides were ordered from Ella Biotech GmbH (Martinsried Germany).
(68) TABLE-US-00022 TABLE 9 Design of maturated library Amino acid No of position in the Z Randomization (amino acid amino variant molecule abbreviations) acids Proportion 9 R 1 1/1 10 N 1 1/1 11 A, D, E, F, H, I, K, L, N, 16 1/16 Q, R, S, T, V, W, Y 13 A 1 1/1 14 A, D, E, F, H, I, K, L, N, 16 1/16 Q, R, S, T, V, W, Y 17 L 1 1/1 18 A, D, E, F, H, I, K, L, N, 16 1/16 Q R, S, T, V, W, Y 24 A, D, E, F, H, I, K, L, N, 16 1/16 Q R, S, T, V, W, Y 25 A, D, E, F, G, H, I, K, L, 17 1/17 N, Q, R, S, T, V, W, Y 27 A, D, E, F, H, I, K, L, N, 16 1/16 Q R, S, T, V, W, Y 28 W 1 1/1 32 W 1 1/1 35 50% D, 30% A, 20% E 3 .sup. 1/2 (D), .sup. 3/10 (A), 1/5 (E)
(69) The theoretical distributions of amino acid residues in the new library including 7 variable positions (11, 14, 18, 24, 25, 27 and 35) in the Z molecule scaffold are given in Table 9. The resulting theoretical library size is 5.3×10.sup.7 variants.
(70) Library construction: The library was amplified using AmpliTaq Gold polymerase (Life Technologies, cat. no. 4311816) during 12 cycles of PCR and pooled products were purified with QiAquick PCR Purification Kit (QIAGEN, cat. no. 28106) according to the supplier's recommendations. The purified pool of randomized library fragments was digested with restriction enzymes XhoI and SacI-HF (New England Biolabs, cat. no. R0146L, and cat. no. R3156M, respectively) and concentrated using a PCR Purification Kit. Subsequently, the product was run on a preparative 2.5% agarose (NuSieve GTG® Agarose, Lonza, cat. no. 50080) gel electrophoresis and purified using QIAGEN Gel Extraction Kit (QIAGEN, cat. no. 28706) according to the supplier's recommendations.
(71) The phagemid vector pAY02592 (essentially as pAffi1 described in Grönwall et al., supra) was restricted with the same enzymes and purified using phenol/chloroform extraction and ethanol precipitation. The restricted fragments and the restricted vector were ligated in a molar ratio of 5:1 with T4 DNA ligase (Thermo Scientific, cat. no. EL0011) for 2 h at RT, followed by overnight incubation at 4° C. The ligated DNA was recovered by phenol/chloroform extraction and ethanol precipitation, followed by dissolution in 10 mM Tris-HCl, pH 8.5. Thus, the resulting library in vector pAY02592 encoded Z variants each fused to an albumin binding domain (ABD) derived from streptococcal protein G.
(72) The ligation reactions (approximately 160 ng DNA/transformation) were electroporated into electrocompetent E. coli ER2738 cells (Lucigen, Middleton, Wis., USA, 50 μl). Immediately after electroporation, approximately 1 ml of recovery medium (supplied with E. coli ER2738 cells) was added. The transformed cells were incubated at 37° C. for 60 min. Samples were taken for titration and for determination of the number of transformants. The cells were thereafter pooled and cultivated overnight at 37° C. in 1 l of TSB-YE medium, supplemented with 2% glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin. The cells were pelleted for 15 min at 4,000 g and resuspended in a PBS/glycerol solution (approximately 40% glycerol). The cells were aliquoted and stored at −80° C. Clones from the library of Z variants were sequenced in order to verify the content and to evaluate the outcome of the constructed library vis-à-vis the library design. Sequencing was performed as described in Example 1 and the amino acid distribution was verified.
(73) Preparation of phage stock: Cells from a glycerol stock containing the phagemid library were inoculated in 3.5 l of TSB-YE supplemented with 1 g/l glucose, 100 mg/l ampicillin and 10 mg/l tetracycline. The cells were cultivated at 37° C. with orbital shaking (100 RPM). When the cells reached an optical density at 600 nm (OD600) of 0.59, approximately 620 ml of the cultivation was infected using a 5× molar excess of M13K07 helper phage. The cells were incubated for 30 min, whereupon the cells were pelleted by centrifugation at 3,000 g and resuspended in 3 l fresh TSB-YE supplemented with 100 mg/l ampicillin, 25 mg/l kanamycin, and 0.1 mM IPTG. The cultivation was split into 6×5 l shaker flasks at and incubated at 30° C. with orbital shaking and after ˜18 h the cells were pelleted by centrifugation at 4,700 g. The phage particles were precipitated from the supernatant twice in PEG/NaCl, filtered and dissolved in PBS and glycerol as described in Example 1. Phage stocks were stored at −80° C. until use in selection.
(74) Results
(75) Library construction: The new library was designed based on a set of PD-L1 binding Z variants with verified binding properties (Example 1 and 3). The theoretical size of the designed library was 5.3×10.sup.7 Z variants. The actual size of the library, determined by titration after transformation to E. coli. ER2738 cells, was 2.8×10.sup.9 transformants.
(76) The library quality was tested by sequencing of 116 transformants and by comparing their actual sequences with the theoretical design. The contents of the actual library compared to the designed library were shown to be satisfactory. A maturated library of potential binders to PD-L1 was thus successfully constructed.
Example 5
Selection, Screening and Characterization of Z Variants from a Maturated Library
(77) Materials and Methods
(78) Phage display selection of PD-L1 binding Z variants: The target protein PD-L1 was biotinylated as described in Example 1. Phage display selections, using the new library of Z variant molecules constructed as described in Example 4, were performed in four cycles against hPD-L1 essentially as described in Example 1, with the following exceptions: Exception 1: SA-beads were used to catch the PD-L1:Z variant complexes in all selection tracks. Exception 2: pre-selection was performed against SA-beads coated with biotinylated human IgG-Fc only before cycle 1 and 2. Furthermore in cycle 1, another pre-selection was performed against SA-beads coated with a mix of PD-L2, B7-H3, and B7-H4 as previously described in Example 1. Exception 3: selections against biotinylated human PD-L1 was performed in four cycles initially divided in two different tracks (1 and 2). As selection proceeded, the tracks were further divided according to target concentration and number and/or time of washes to finally end up in 11 tracks in cycle four. More precisely, the first track (1) was divided in the second to the fourth cycles, resulting in a total of 2 tracks (1-1 to 1-2) in cycle 2, four tracks (1-1-1 to 1-2-2) in cycle 3 and seven tracks (1-1-1-1 to 1-2-2-2) in cycle 4. The second track (2) was divided in the second to the fourth cycles, resulting in a total of 2 tracks (2-1 to 2-2) in cycle 2, four tracks (2-1-1 to 2-2-2) in cycle 3 and four tracks (2-1-1-1 to 2-2-2-1) in cycle four. Exception 4: during the 19 h washing step in selection cycle 1-1-2-3 a 20-fold molecular excess of non-biotinylated hPD-L1 was added to the wash buffer. An overview of the selection strategy, describing an increased stringency in subsequent cycles obtained by using a lowered target concentration and an increased number of washes, is shown in Table 10.
(79) TABLE-US-00023 TABLE 10 Selection against biotinylated hPD-L1 Fc using a maturated library Phage stock Addition from library Target Number Duration to last Selection or selection Proteins used conc. of of last wash Cycle track track in pre-selection (nM) washes wash (h) buffer 1 1 Zlib006PD-L1.I IgG-Fc, PD-L2, 50 2 B7-H3, B7-H4 1 2 Zlib006PD-L1.I IgG-Fc, PD-L2, 25 2 B7-H3, B7-H4 2 1-1 1 IgG-Fc 25 8 2 1-2 1 IgG-Fc 10 12 2 2-1 2 IgG-Fc 2.5 12 2 2-2 2 IgG-Fc 0.5 12 3 1-1-1 1-1 no pre-selection 5 20 3 1-1-2 1-1 no pre-selection 5 20 18 3 1-2-1 1-2 no pre-selection 2.5 20 3 1-2-2 1-2 no pre-selection 1 20 18 3 2-1-1 2-1 no pre-selection 0.5 20 3 2-1-2 2-1 no pre-selection 0.1 20 18 3 2-2-1 2-2 no pre-selection 0.05 20 18 3 2-2-2 2-2 no pre-selection 0.005 20 4 1-1-1-1 1-1-1 no pre-selection 2.5 20 4 1-1-2-1 1-1-2 no pre-selection 2.5 20 4 1-1-2-2 1-1-2 no pre-selection 2.5 20 19 4 1-1-2-3 1-1-2 no pre-selection 2.5 20 19 50 nM hPD-L1 4 1-2-1-1 1-2-1 no pre-selection 0.1 20 4 1-2-2-1 2-1-2 no pre-selection 0.5 20 4 1-2-2-2 1-2-2 no pre-selection 0.5 20 19 4 2-1-1-1 2-1-1 no pre-selection 0.05 20 4 2-1-2-1 2-1-2 no pre-selection 0.1 30 19 4 2-2-1-1 2-2-1 no pre-selection 0.01 30 19 4 2-2-2-1 2-2-2 no pre-selection 0.05 20 19
(80) Production of Z variants for ELISA: The Z variants were produced by inoculating single colonies from the selections into 1.2 ml TSB-YE medium supplemented with 100 μg/ml ampicillin and 1 mM IPTG in deep-well plates (Nunc, cat. no. 278752). The plates were incubated with rotation for 24 h at 37° C. Cells were pelleted by centrifugation at 3300 g and re-suspended in 150 μl PBST 0.05% and frozen at −80° C. to release the periplasmic fraction of the cells. Frozen samples were thawed in a water bath and the freeze-thawing procedure was repeated eight times before the periplasmic fraction was isolated in deep-well plates (Axygen, cat. no. 391-01-101) by filtration using filter plates (EMD Millipore, cat. no. MSNANLY50). The final supernatant of the periplasmic extract contained the Z variants as fusions to ABD, expressed as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (SEQ ID NO:896) (Grönwall et al., supra). Z##### refers to individual, 58 amino acid residue Z variants.
(81) ELISA screening of Z variants: The binding of Z variants to human PD-L1 was analyzed in ELISA assays as described previously in Example 1 with the following exceptions. Exception 1: The periplasmic fraction was diluted 1:8 with PBST 0.05% before added to the wells and incubated for 1.7 h. Exception 2: instead of a blank control a negative control of a periplasmic fraction containing the fusion protein ABD with no Z-fusion partner was used. Exception 3: periplasm samples containing the primary PD-L1 binding Z variant Z13091 (SEQ ID.NO:776) was included in duplicates on each plate and analyzed as positive controls. Exception 4: 50 μl of biotinylated hPD-L1 at a concentration of 40 pM in PBSC was added to each well and the plates were incubated for 1.8 h at RT.
(82) Sequencing: In parallel with the ELISA screening, all clones were sequenced as described in Example 1.
(83) ELISA EC50 analysis: A selection of PD-L1 binding Z variants was subjected to an analysis of the response against a dilution series of biotinylated human PD-L1 as described in Example 1 with the following exceptions. Exception 1: the Z variants were diluted 1:8 in PBST 0.05% before added to the wells. Exception 2: biotinylated human PD-L1 was added at a concentration of 15 nM and diluted stepwise 1:3 down to 0.25 pM. Exception 3: a periplasm sample containing the primary PD-L1 binding Z variant Z13091 (SEQ ID.NO:776) was included for comparison and analyzed together with the maturated Z variants.
(84) Results
(85) Phage display selection of PD-L1 binding Z variants: Individual clones were obtained after four cycles of phage display selection against biotinylated hPD-L1.
(86) ELISA screening of Z variants: The clones obtained after four cycles of selection were produced in 96-well plates and screened for hPD-L1 binding activity in ELISA. A majority of the unique Z variants were found to give a higher response than average response of the positive control Z13091 (average 0.264 AU) against hPD-L1 at a concentration of 40 pM. The average response of the negative controls was 0.051 AU.
(87) Sequencing: Sequencing was performed for clones obtained after four cycles of selection. Each variant was given a unique identification number, Z#####, as described in Example 1. The amino acid sequences of the 58 amino acid residues long Z variants are listed in
(88) EC50 analysis of Z variants: A subset of Z variants having the highest ELISA values in the ELISA screening experiment described above was selected and subjected to a target titration in ELISA format. Periplasm samples were incubated with a serial dilution of biotinylated hPD-L1. A periplasm sample containing Z13091 (SEQ ID NO:776), the isolated primary Z variant that showed the highest binding affinity to hPD-L1, was included as a positive control. Obtained values were analyzed and their respective EC50 values were calculated using GraphPad Prism 5 (Table 11). All maturated Z variants showed lower EC50 values than the top primary Z variant Z13091.
(89) TABLE-US-00024 TABLE 11 Calculated EC50 values of Z-ABD variants from maturation Z variant SEQ ID NO: EC50 (M) Z variant SEQ ID NO: EC50 (M) Z variant SEQ ID NO: EC50 (M) Z17746 8 6.7 × 10.sup.−11 Z18054 22 6.2 × 10.sup.−11 Z18135 67 8.5 × 10.sup.−11 Z17748 11 6.7 × 10.sup.−11 Z18060 44 8.3 × 10.sup.−11 Z18137 68 7.4 × 10.sup.−11 Z17756 7 6.7 × 10.sup.−11 Z18064 1 6.8 × 10.sup.−11 Z18138 69 7.4 × 10.sup.−11 Z17758 25 8.0 × 10.sup.−11 Z18065 45 7.1 × 10.sup.−11 Z18140 70 8.7 × 10.sup.−11 Z17772 26 8.8 × 10.sup.−11 Z18066 12 7.3 × 10.sup.−11 Z18143 71 7.8 × 10.sup.−11 Z17825 5 6.7 × 10.sup.−11 Z18069 46 7.8 × 10.sup.−11 Z18144 72 7.9 × 10.sup.−11 Z17843 27 8.6 × 10.sup.−11 Z18070 10 7.6 × 10.sup.−11 Z18148 73 7.0 × 10.sup.−11 Z17911 3 7.0 × 10.sup.−11 Z18074 6 7.0 × 10.sup.−11 Z18149 18 6.9 × 10.sup.−11 Z17928 28 8.3 × 10.sup.−11 Z18078 47 7.8 × 10.sup.−11 Z18150 74 7.6 × 10.sup.−11 Z17950 29 7.8 × 10.sup.−11 Z18090 17 8.8 × 10.sup.−11 Z18152 75 8.0 × 10.sup.−11 Z17964 2 7.7 × 10.sup.−11 Z18092 48 7.7 × 10.sup.−11 Z18153 76 8.0 × 10.sup.−11 Z17968 30 8.2 × 10.sup.−11 Z18095 49 7.3 × 10.sup.−11 Z18156 77 8.0 × 10.sup.−11 Z17972 19 7.4 × 10.sup.−11 Z18096 50 8.0 × 10.sup.−11 Z18158 78 8.7 × 10.sup.−11 Z17975 31 8.9 × 10.sup.−11 Z18099 51 8.7 × 10.sup.−11 Z18164 79 7.6 × 10.sup.−11 Z17978 13 6.5 × 10.sup.−11 Z18101 23 6.7 × 10.sup.−11 Z18167 80 8.4 × 10.sup.−11 Z17990 32 8.0 × 10.sup.−11 Z18104 52 7.5 × 10.sup.−11 Z18172 81 7.6 × 10.sup.−11 Z17995 33 8.4 × 10.sup.−11 Z18106 53 8.3 × 10.sup.−11 Z18174 82 8.4 × 10.sup.−11 Z17997 34 7.7 × 10.sup.−11 Z18108 54 7.8 × 10.sup.−11 Z18176 83 8.9 × 10.sup.−11 Z17999 35 7.5 × 10.sup.−11 Z18110 55 8.1 × 10.sup.−11 Z18179 84 8.7 × 10.sup.−11 Z18000 36 7.9 × 10.sup.−11 Z18111 56 7.8 × 10.sup.−11 Z18185 85 8.1 × 10.sup.−11 Z18005 37 7.8 × 10.sup.−11 Z18115 57 7.7 × 10.sup.−11 Z18220 86 8.4 × 10.sup.−11 Z18008 38 7.3 × 10.sup.−11 Z18116 58 7.4 × 10.sup.−11 Z18228 87 8.8 × 10.sup.−11 Z18021 39 8.8 × 10.sup.−11 Z18117 59 8.1 × 10.sup.−11 Z18233 21 8.6 × 10.sup.−11 Z18022 9 7.4 × 10.sup.−11 Z18118 60 7.9 × 10.sup.−11 Z18240 88 7.8 × 10.sup.−11 Z18027 40 8.0 × 10.sup.−11 Z18119 61 1.0 × 10.sup.−10 Z18243 89 8.1 × 10.sup.−11 Z18036 41 7.6 × 10.sup.−11 Z18124 62 8.4 × 10.sup.−11 Z18252 90 8.5 × 10.sup.−11 Z18037 42 7.7 × 10.sup.−11 Z18128 63 7.7 × 10.sup.−11 Z18268 91 8.9 × 10.sup.−11 Z18038 43 6.9 × 10.sup.−11 Z18129 16 6.2 × 10.sup.−11 Z18353 15 7.7 × 10.sup.−11 Z18039 20 6.9 × 10.sup.−11 Z18130 64 7.4 × 10.sup.−11 Z18374 92 1.0 × 10.sup.−10 Z18048 4 6.5 × 10.sup.−11 Z18131 65 8.7 × 10.sup.−11 Z18377 93 7.8 × 10.sup.−11 Z18052 14 7.1 × 10.sup.−11 Z18133 66 7.7 × 10.sup.−11 Z18418 24 8.0 × 10.sup.−11 Z13091 776 1.2 × 10.sup.−10
Example 6
Subcloning and Production of a Subset of Maturated PD-L1 Binding Z Variants
(90) Materials and Methods
(91) Subcloning of Z variants with a Hiss-tag: The DNA of 24 maturated PD-L1 binding Z variants, (Z17746 (SEQ ID NO:8), Z17748 (SEQ ID NO:11), Z17756 (SEQ ID NO:7), Z17825 (SEQ ID NO:5), Z17911 (SEQ ID NO:3), Z17964 (SEQ ID NO:2), Z17972 (SEQ ID NO:19), Z17978 (SEQ ID NO:13), Z18022 (SEQ ID NO:9), Z18039 (SEQ ID NO:20), Z18048 (SEQ ID NO:4), Z18052 (SEQ ID NO:14), Z18054 (SEQ ID NO:22), Z18064 (SEQ ID NO:1), Z18066 (SEQ ID NO:12), Z18070 (SEQ ID NO:10), Z18074 (SEQ ID NO:6), Z18090 (SEQ ID NO:17), Z18101 (SEQ ID NO:23), Z18129 (SEQ ID NO:16), Z18149 (SEQ ID NO:18), Z18233 (SEQ ID NO:21), Z18353 (SEQ ID NO:15) and Z18418 (SEQ ID NO:24)) were amplified from the library vector pAY02592 and subcloned with a Hiss-tag as described in Example 2 above.
(92) Subcloning of Z variants with a C-terminal Cys: Three Z variants, Z18064 (SEQ ID NO:1), Z17964 (SEQ ID NO:2) and Z18090 (SEQ ID NO:17) were mutated to start with the N-terminal amino acids AE instead of VD and further subcloned with the C-terminal addition of the amino acids VDC (incorporating a unique cysteine in the polypeptide) using standard molecular biology techniques. The resulting sequences are referred to as Z18608-Cys (SEQ ID NO:811), Z18609-Cys (SEQ ID NO:812) and Z18610-Cys (SEQ ID NO:813), respectively.
(93) Cultivation: Generally, E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragments of each respective PD-L1 binding Z variant and cultivated at 37° C. in approximately 940 ml of TSB-YE medium supplemented with 50 μg/ml kanamycin. In order to induce protein expression, IPTG was added to a final concentration of 0.2 mM at OD.sub.600=2 and the cultivation was incubated at 37° C. for another 5 h. The cells were harvested by centrifugation. Specifically, Z18608-Cys and Z18609-Cys were fed-batch cultivated at 37° C. in approximately 700 ml of defined mineral medium supplemented with 50 μg/ml kanamycin. In order to induce protein expression, IPTG was added to a final concentration of 0.5 mM at OD.sub.600=75 and the cultivation was incubated for another 7 h. The cells were harvested by centrifugation.
(94) Purification of PD-L1 binding Z variants with a Hiss-tag: IMAC purifications, buffer exchange to PBS and concentration determinations were performed essentially as described in Example 2.
(95) Purification of PD-L1 binding Z variants with a C-terminal Cys: The respective cell pellet was re-suspended in 20 mM Tris-HCl, 0.5 mM EDTA, 0.1% Tween 80, pH 7.5 (10 ml buffer/g cell pellet) and lysed by heat treatment in a water bath at 80° C. for 10 min, followed by cooling on ice to approximately 20° C. Benzonase® was added (1 μl/g cell pellet) and each cell lysate was incubated at RT for 30 min, before cell debris was removed by centrifugation. For reduction of disulfides, dithiothreitol (DTT; Acros Organics, cat. no. 165680250) was added to a final concentration of 10 mM followed by incubation at RT for 20 min. Thereafter, the lysate was filtered through a 0.45 μm syringe filter (Millipore). Purification was performed by anion exchange followed by reverse phase chromatography (RPC). Buffer exchange to 20 mM HEPES, 1 mM EDTA, pH 7.2 was carried out using Sephadex G-25 medium (GE Healthcare) packed in an XK-50 column.
(96) For any protein purified by either method described above, the concentration was determined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer and the extinction coefficient of the protein. The purity was analyzed by SDS-PAGE stained with Coomassie Blue, and the identity of each purified Z variant was confirmed using HPLC-MS analysis (HPLC-MS 1100; Agilent Technologies).
(97) Results
(98) Cultivation and purification: The PD-L1 binding Z variants were expressed as soluble gene products in E. coli. The amount of purified protein from approximately 2.0-2.4 g bacterial pellet was determined spectrophotometrically by measuring the absorbance at 280 nm and ranged from approximately 18 mg to 29 mg for the different Hiss-tagged PD-L1 binding Z variants. SDS-PAGE analysis of each final protein preparation showed that these predominantly contained the PD-L1 binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.
Example 7
Additional Characterization of a Subset of Primary PD-L1 Binding Z Variants
(99) In this Example, a subset of Z variants was characterized in terms of stability and various binding properties. The specificity and affinity for PD-L1 of the Z variants were analyzed by Biacore and the ability of Z variants to block the binding of PD-L1 to its receptor PD-1 was investigated using AlphaLISA.
(100) Materials and Methods
(101) Biacore kinetic and specificity analysis: Kinetic constants (k.sub.a and k.sub.d) and affinities (K.sub.D) for human PD-L1 and rhesus monkey PD-L1 (RhPD-L1; rhesus PD-L1/Fc Chimera, Sino Biological Inc., cat. no. 90251-C02H) were determined for 24 matured Hiss-tagged Z variants (specified in Example 6). The Z variants were also tested for binding against the sequence-related proteins hPD-L2, hB7-H3 and hB7-H4. The Biacore analyses were performed essentially as described in Example 3, however a flow rate of 30 μl/min was used. The ligand immobilization levels on the surfaces were 1030 RU for hPD-L1, 1060 RU for RhPD-L1, 1070 RU for hPD-L2, 1090 RU for hB7-H3, and 770 RU for hB7-H4. In a first binding kinetic analysis, the 24 Z variants were injected at concentrations of 5 and 50 nM over chips immobilized with hPD-L1 and RhPD-L1, respectively. The 12 maturated PD-L1 binding Z variants that showed the highest affinity to hPD-L1 in the first experiment were analyzed in more detail and injected at concentrations of 135, 45, 15, 5 and 1.67 nM over immobilized hPD-L1 and RhPD-L1. In the specificity test, i.e. the binding analysis against hPD-L2, hB7-H3 and hB7-H4, the 24 Z variants were injected at a concentration of 500 nM.
(102) AlphaLISA blocking assay: The potential of the Z variants to inhibit binding of PD-L1 to its natural ligand PD-1 was analyzed in an AlphaLISA assay as described in Example 3 with the following exceptions: Exception 1: stepwise serial dilutions 1:3 of Hiss-tagged Z variants to final concentrations of 250 nM to 4 pM were made in a 384SW plate (Perkin Elmer, cat. no. 6008350) and incubated for 45 min with 8 nM biotinylated hPD-L1 (R&D Systems) in AlphaLISA buffer (Perkin Elmer, cat. no. AL000F). Exception 2: hPD-1-coated Acceptor beads were added to a final concentration of 10 μg/ml and incubated for 50 min.
(103) Circular dichroism (CD) spectroscopy analysis: A subset of the purified Hiss-tagged Z variants were analyzed by CD spectroscopy as described in Example 3, but with the exceptions that the analysis buffer was PBS and that the temperature was raised to 80° C. in the VTM.
(104) Results
(105) Biacore kinetic and specificity analysis: The interactions of 24 maturated Hiss-tagged Z variants with human and rhesus monkey PD-L1, were analyzed in a Biacore instrument by injecting various concentrations of the Z variants over surfaces containing immobilized hPD-L1 and RhPD-L1, respectively. A first kinetic analysis was performed in order to rank the Z variants in terms of their affinity for hPD-L1 and RhPD-L1, as well as to compare their binding kinetics with the primary PD-L1 binding Z variant Z13091. A summary of the approximate affinity constants from the ranking experiment, which were obtained by using a 1:1 interaction model, is given in Table 12.
(106) The 12 maturated Z variants that showed the highest binding affinity to hPD-L1 were further analyzed and the more precisely determined kinetic parameters for these 12 Z variants are given in Table 13. Typical resulting curves, where responses from a blank surface were subtracted, are displayed for two selected variants in
(107) TABLE-US-00025 TABLE 12 Approximate affinity constants for binding of Z variants to hPD-L1 and RhPD-L1 SEQ ID NO hPD-L1 RhPD-L1 Z variant of Z variant K.sub.D (M) K.sub.D (M) Z13091 776 7.4 × 10.sup.−10 5.2 × 10.sup.−9 Z17746 8 3.8 × 10.sup.−10 .sup. 5.2 × 10.sup.−10 Z17748 11 4.5 × 10.sup.−10 .sup. 7.4 × 10.sup.−10 Z17756 7 2.4 × 10.sup.−10 2.1 × 10.sup.−9 Z17825 5 2.5 × 10.sup.−10 1.2 × 10.sup.−9 Z17911 3 3.6 × 10.sup.−10 2.2 × 10.sup.−9 Z17964 2 3.0 × 10.sup.−10 2.0 × 10.sup.−9 Z17972 19 5.4 × 10.sup.−10 2.7 × 10.sup.−9 Z17978 13 4.5 × 10.sup.−10 1.0 × 10.sup.−9 Z18022 9 3.4 × 10.sup.−10 1.4 × 10.sup.−9 Z18039 20 6.3 × 10.sup.−10 1.9 × 10.sup.−9 Z18048 4 3.5 × 10.sup.−10 1.8 × 10.sup.−9 Z18052 14 4.5 × 10.sup.−10 1.2 × 10.sup.−9 Z18054 22 7.6 × 10.sup.−10 1.2 × 10.sup.−9 Z18064 1 1.3 × 10.sup.−10 1.5 × 10.sup.−9 Z18066 12 4.3 × 10.sup.−10 2.1 × 10.sup.−9 Z18070 10 3.1 × 10.sup.−10 2.5 × 10.sup.−9 Z18074 6 3.6 × 10.sup.−10 2.9 × 10.sup.−9 Z18090 17 5.1 × 10.sup.−10 2.3 × 10.sup.−9 Z18101 23 9.3 × 10.sup.−10 2.4 × 10.sup.−9 Z18129 16 4.8 × 10.sup.−10 1.2 × 10.sup.−9 Z18149 18 5.3 × 10.sup.−10 .sup. 9.6 × 10.sup.−10 Z18233 21 7.2 × 10.sup.−10 4.4 × 10.sup.−9 Z18353 15 4.6 × 10.sup.−10 1.6 × 10.sup.−9 Z18418 24 1.9 × 10.sup.−9 4.1 × 10.sup.−9
(108) Furthermore, all 24 maturated Hiss-tagged Z variants were also tested for binding against the three sequence-related proteins, hPD-L2, hB7-H3, and hB7-H4. In line with the results in Example 3, no binding to either of the control proteins were detected at a Z variant concentration of 500 nM.
(109) TABLE-US-00026 TABLE 13 Kinetic parameters for binding of Z variants to hPD-L1 and RhPD-L1 hPD-L1 RhPD-L1 Z variant SEQ ID NO: k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) Z13091 776 1.8 × 10.sup.6 1.1 × 10.sup.−3 6.3 × 10.sup.−10 1.9 × 10.sup.6 1.0 × 10.sup.−3 5.4 × 10.sup.−10 Z17746 8 1.6 × 10.sup.6 4.6 × 10.sup.−4 2.8 × 10.sup.−10 1.4 × 10.sup.6 4.0 × 10.sup.−4 2.8 × 10.sup.−10 Z17748 11 1.7 × 10.sup.6 5.7 × 10.sup.−4 3.4 × 10.sup.−10 1.8 × 10.sup.6 5.1 × 10.sup.−4 2.9 × 10.sup.−10 Z17756 7 2.0 × 10.sup.6 5.4 × 10.sup.−4 2.8 × 10.sup.−10 2.7 × 10.sup.6 4.5 × 10.sup.−4 1.7 × 10.sup.−10 Z17825 5 2.0 × 10.sup.6 4.7 × 10.sup.−4 2.4 × 10.sup.−10 1.9 × 10.sup.6 3.9 × 10.sup.−4 2.1 × 10.sup.−10 Z17911 3 2.3 × 10.sup.6 4.9 × 10.sup.−4 2.1 × 10.sup.−10 2.1 × 10.sup.6 4.2 × 10.sup.−4 2.0 × 10.sup.−10 Z17964 2 2.1 × 10.sup.6 4.3 × 10.sup.−4 2.1 × 10.sup.−10 3.4 × 10.sup.6 3.8 × 10.sup.−4 1.1 × 10.sup.−10 Z18022 9 1.7 × 10.sup.6 4.9 × 10.sup.−4 2.8 × 10.sup.−10 1.6 × 10.sup.6 4.6 × 10.sup.−4 2.8 × 10.sup.−10 Z18048 4 1.9 × 10.sup.6 4.4 × 10.sup.−4 2.3 × 10.sup.−10 1.6 × 10.sup.6 3.8 × 10.sup.−4 2.4 × 10.sup.−10 Z18064 1 3.5 × 10.sup.6 4.3 × 10.sup.−4 1.3 × 10.sup.−10 3.5 × 10.sup.6 3.4 × 10.sup.−4 9.6 × 10.sup.−11 Z18066 12 1.5 × 10.sup.6 5.4 × 10.sup.−4 3.7 × 10.sup.−10 1.4 × 10.sup.6 4.7 × 10.sup.−4 3.5 × 10.sup.−10 Z18070 10 2.0 × 10.sup.6 5.7 × 10.sup.−4 2.9 × 10.sup.−10 1.7 × 10.sup.6 4.7 × 10.sup.−4 2.8 × 10.sup.−10 Z18074 6 2.0 × 10.sup.6 5.4 × 10.sup.−4 2.7 × 10.sup.−10 1.7 × 10.sup.6 4.6 × 10.sup.−4 2.7 × 10.sup.−10
(110) AlphaLISA blocking assay: The ability of 24 maturated Hiss-tagged monomeric Z variants to inhibit hPD-L1 binding to hPD-1 was tested in an AlphaLISA blocking assay. The primary Z variant Z13091 was included as a reference. Serial dilutions of the Z variants were incubated with biotinylated hPD-L1 and the blocking ability of each respective variant was measured after addition of hPD-1 coated Acceptor beads and subsequently streptavidin coated Donor beads. Inhibition could be measured as a decrease in AlphaLISA counts for positive Z variants. The calculated IC50 values for the variants that were shown to block PD-L1 binding to PD-1 in this assay are shown in Table 14.
(111) TABLE-US-00027 TABLE 14 IC50 values for Z variants blocking the PD-1/PD-L1 interaction Z variant SEQ ID NO: IC50 AlphaLISA (M) Z13091 776 1.1 × 10.sup.−9 Z17746 8 1.3 × 10.sup.−9 Z17748 11 1.1 × 10.sup.−9 Z17756 7 1.5 × 10.sup.−9 Z17825 5 1.6 × 10.sup.−10 Z17911 3 5.7 × 10.sup.−10 Z17964 2 8.9 × 10.sup.−9 Z17972 19 2.9 × 10.sup.−10 Z17978 13 6.2 × 10.sup.−10 Z18022 9 3.1 × 10.sup.−10 Z18039 20 2.9 × 10.sup.−10 Z18048 4 1.5 × 10.sup.−10 Z18052 14 3.4 × 10.sup.−10 Z18054 22 1.1 × 10.sup.−9 Z18064 1 3.9 × 10.sup.−10 Z18066 12 8.3 × 10.sup.−10 Z18070 10 8.6 × 10.sup.−10 Z18074 6 3.1 × 10.sup.−10 Z18090 17 6.7 × 10.sup.−10 Z18101 23 6.2 × 10.sup.−10 Z18129 16 6.0 × 10.sup.−10 Z18149 18 1.9 × 10.sup.−10 Z18233 21 1.5 × 10.sup.−10 Z18353 15 7.2 × 10.sup.−10 Z18418 24 5.7 × 10.sup.−10
(112) CD analysis: The CD spectra determined for 24 maturated PD-L1 binding Z variants with a His.sub.6 tag showed that each had an α-helical structure at 20° C. The melting temperatures (Tm) were determined using variable temperature measurements (Table 15). Reversible folding was observed for all PD-L1 binding Z variants when overlaying spectra measured at 20° C. before and after heating to 80° C., as shown for two selected Z variants in
(113) TABLE-US-00028 TABLE 15 Melting temperatures of maturated PD-L1 binding Z variants Z variant SEQ ID NO: Tm (° C.) Z17746 8 59 Z17748 11 63 Z17756 7 63 Z17825 5 60 Z17911 3 59 Z17964 2 62 Z17978 13 59 Z18022 9 59 Z18039 20 55 Z18048 4 60 Z18064 1 62 Z18066 12 58 Z18070 10 60 Z18074 6 62 Z18090 17 68 Z18129 16 63 Z18233 21 57
Example 8
Characterization of Anti-PD-L1/PD-1 and Anti-PD-L1/CTLA-4 Complexes
(114) Materials and Methods
(115) Production of complexes and control antibodies: Four different complexes targeting PD-L1 and PD-1, and four different complexes targeting PD-L1 and CTLA-4 were constructed, as well as a control antibody targeting PD-1. An antibody denoted “Lam”, having the same CDR sequences and specificity as the commercially available, PD-1 targeting monoclonal antibody pembrolizumab (formerly lambrolizumab), was constructed using the heavy chain (HC) and light chain (LC) sequences HC.sub.Lam (SEQ ID NO:815) and LC.sub.Lam (SEQ ID NO:816). An antibody denoted “Ipi”, having the same CDR sequences and specificity as the commercially available, CTLA-4 targeting monoclonal antibody ipilimumab, was constructed using the heavy chain (HC) and light chain (LC) sequences HC.sub.Ipi; (SEQ ID NO:817) and LC.sub.Ipi (SEQ ID NO:818). The PD-L1 targeting Z variant Z15170 (SEQ ID NO:814; identical to Z13165 (SEQ ID NO:784) but starting with the amino acid residues AE instead of VD) with a C-terminal VD sequence was genetically fused, via a flexible 15 residue (GGGGS).sub.3 linker, to the N-termini of HC.sub.Lam, LC.sub.Lam, HC.sub.Ipi and LC.sub.Ipi, respectively, resulting in the complexes Z15170-HC.sub.Lam, Z15170-LC.sub.Lam, Z15170-HC.sub.Ipi and Z15170-LC.sub.Ipi, respectively; or to the C-termini of the same chains, resulting in the complexes HC.sub.Lam-Z15170, LC.sub.Lam-Z15170, HC.sub.Ipi-Z15170 and LC.sub.Ipi-Z15170, respectively. Gene synthesis, cloning, production by transient gene expression in CHO cells as well as purification by Protein A chromatography and verification of constructs by gel electrophoresis were performed by Evitria AG (Switzerland).
(116) Biacore kinetic analyses: Kinetic constants (k.sub.a and k.sub.d) and affinities (K.sub.D) for hPD-L1, human PD-1 (hPD-1; R&D Systems cat. no. 1086-PD-050) and human CTLA-4 (hCTLA-4; R&D Systems cat. no. 325-CT-200) were determined for all eight complexes produced and using a Biacore 2000 instrument (GE Healthcare). The control antibody Lam was also analyzed for binding against PD-1. 5 μg/ml solutions of each of the proteins hPD-L1, hPD-1 and hCTLA-4 were prepared in 10 mM NaAc buffer (pH 5.0 for PD-L1, and pH 4.5 for PD-1 and CTLA-4) and used for immobilization in separate flow cells on the carboxylated dextran layer of different CM5 chip surfaces (GE Healthcare, cat. no. BR100012). The immobilization was performed using amine coupling chemistry according to the manufacturer's protocol and using HBS-EP with 500 mM NaCl as running buffer. Immobilization levels obtained were ˜110-140 RU. A series of 3.33, 10, 30, 90, 270 nM concentrations of the respective complex and Lam were injected and the responses recorded, except for analysis of binding to PD-L1 for constructs with Z15170 positioned on the C-terminus of the respective antibody, for which a concentration series of 30, 90, 270 and 900 nM was used.
(117) In a separate experiment, the dual binding specificity was evaluated by a capture assay using the Biacore 2000 instrument. The complexes Z15170-HC.sub.Lam, Z15170-LC.sub.Lam, Z15170-HC.sub.Ipi and Z15170-LC.sub.Ipi, Lam and ipilimumab (Yervoy®, Bristol-Myers Squibb/Astra Zeneca via Apoteket AB, cat. no. 065544, lot no. 4A85968), at a concentration of 300 nM, were injected over chip surfaces immobilized with PD-1 or CTLA-4 as described above. In all cases, the duration of the injection was 5 min at a flow rate of 30 μl/min with a wait/dissociation step of 5 min before a second injection (5 min) of 100 or 500 nM PD-L1 was made. HBS-EP with 500 mM NaCl was used as a running buffer and for protein dilutions.
(118) Cell binding analysis by FACS: The potential of the complexes to bind PD-L1 expressing cells was investigated using FACS. 150 000 cells of the breast cancer cell line MDA-MB-231, cultivated in DMEM (ATCC cat. no. 30-2002) containing 10% FBS, were pipetted per well of a v-bottomed 96-well plate (Nunc, cat. no. 277143) and the cells in the plate were subsequently pelleted at 400 g for 3 min at RT. The supernatants were removed and the cells were resuspended in 100 μl PBS plus 2.5% FBS (staining buffer) containing 0.625 μg/ml of the complexes Z15170-HC.sub.Lam, Z15170-LC.sub.Lam, HC.sub.Lam-Z15170, LC.sub.Lam-Z15170, Z15170-HC.sub.Ipi, Z15170-LC.sub.Ipi, HC.sub.Ipi-Z1 5170 and LC.sub.Ipi-Z15170, respectively, or 0.625 μg/ml of the antibodies Lam or ipilimumab. A mouse anti-PD-L1 antibody (RnD Systems, cat. no. MAB1561) at a concentration of 1 μg/ml was used as a positive control. Cells incubated with buffer alone were used as negative controls. The cells were incubated for 1 h at 8° C. in the dark, washed twice with 100 μl staining buffer, and resuspended in 100 μl of staining buffer containing 2.5 μg/ml of a goat-anti-human IgG-Alexa488 (Molecular Probes, cat. no. A11013) or, for cells stained with the positive control antibody, goat anti-mouse IgG-Alexa647 antibody (Life Technologies, cat. No. A21236). The cells were incubated for 1 h at 8° C. in the dark, washed twice with 100 μl staining buffer and resuspended in 300 μl of staining buffer. Data from 10,000 cells were obtained using a FACS Calibur (Beckman Coulter) and the data was analyzed using Flowing software 2.5.0 (Turku University). Mean fluorescence intensity (MFI) was used as a read out of binding capacity.
(119) Co-culture of MDA-MB-231 and PBMC: A mixed lymphocyte assay was used to analyze if the Ipi-based complexes could affect proliferation or the cytotoxic effect of T-cells and thereby increase the elimination of cancer cells. Herein, peripheral blood mononuclear cells (PBMC) and MDA-MB-231 cells were co-cultivated for six days and the number of T-cells and cancer cells were assessed. 20000 MDA-MB-231 cells, cultivated in DMEM containing 10% FBS, were pipetted per well of a flat-bottomed 96-well plate and were left to adhere to the bottom of the well by incubation at 37° C. in a humidified 5% CO.sub.2 atmosphere. Day 2 of the experiment, serial dilutions (200-0.064 nM) of the Ipi-based complexes were prepared in a separate plate using RPM11640 with L-glut (Lonza) supplemented with 10% FCS, and 1% Pen-Strep (Lonza, cat. no. DE17-603E). The DMEM medium was discarded from the MDA-MB-231 cells and 100 μl of the diluted complexes were added. PBMC were prepared form a buffy coat using Ficoll Paque PLUS (GE Healthcare, cat no. 17-1440-02). In brief, the buffy coat was diluted 2× in PBS. 10 ml of the diluted buffy coat were layered on the top of 5 ml Ficoll in 15 ml falcon tubes and centrifuged at RT for 30 min at 400 g. The lymphocyte layer was collected and the cells were washed twice in the supplemented RPM11640 medium described above. The cells were counted and adjusted to 1 million cells per ml in supplemented RPMI medium. 100 μl of the cell suspension were added to the plate with the MDA-MB-231 cells. The plates were incubated for 6 days at 37° C. in a humidified 5% CO.sub.2 atmosphere. At day 7 of the experiment, the number of MDA-MD-231 cells and CD3+ T-cells were counted by FACS. The PBMC were transferred to a v-bottom plate, washed two times with PBS containing 2% FBS (also used as staining buffer) and stained with a mouse anti-CD3 antibody (EXBIO Praha, cat no. 12-631-M001) at a concentration of 2 μg/ml for 1 h at 4° C. The MDA-MB231 cells were trypsinated (20 μl/well) and transferred to another v-bottom shaped plate washed two times with PBS containing 2% FBS and stained with a rabbit anti-EGFR antibody (Abcam, cat no. ab2430-1) at a concentration of 2 μg/ml for 1 h at 4° C. The cells were washed two times with PBS containing 2% FBS and an Alexa-fluor 488-goat-anti-rabbit antibody (Invitrogen, cat no. A11008) and Alexa-fluor 647-goat-anti-mouse antibody (Life technologies, cat no. A21236) were used as detection antibodies at a concentration of 1 μg/ml and incubated for 1 h at 4° C.
(120) Results
(121) Production of complex constructs: A schematic representation of the design of each of the four types of produced complexes is shown in
(122) Biacore kinetic analyses: The affinity to the target proteins PD-L1, PD-1 and CTLA-4, respectively, were determined for each relevant complex. The control antibody Lam was also analyzed against its target PD-1. The kinetic parameters for the interactions with PD-L1 are summarized in Table 16. The capability of the Z moiety of the complex to interact with PD-L1 was maintained, although the affinity was reduced as well as affected by the positioning of the Z moiety on the antibody. For comparison, the K.sub.D of the Hiss-Z13165 interaction with PD-L1 was 0.64 nM (as presented in Example 3) whereas the K.sub.D for complexes with N-terminally positioned Z moieties was 1.5-2.6 nM and the K.sub.D for C-terminally positioned Z moieties was 12-41 nM. Thus, N-terminal positioning of the Z moiety was superior to the C-terminal positioning, with approximately 10 times higher affinity. This effect was evident with both Lam- and Ipi-based constructs. Whether the fusions were made to the heavy or light chains of the antibodies were of less importance for the N-terminally positioned Z moiety, but had major impact on the C-terminally positioned Z moiety, where the light chain fusions had a K.sub.D of 12-18 nM compared to a K.sub.D of 29-41 nM for the heavy chain fusions.
(123) TABLE-US-00029 TABLE 16 Kinetic parameters for binding of indicated complexes to hPD-L1 Analyte k.sub.a (1/MS) k.sub.d (1/s) K.sub.D (M) HC.sub.Lam-Z15170 2.44 × 10.sup.4 7.17 × 10.sup.−4 2.9 × 10.sup.−8 LC.sub.Lam-Z15170 3.06 × 10.sup.4 3.58 × 10.sup.−4 1.2 × 10.sup.−8 Z15170-HC.sub.Lam 1.16 × 10.sup.5 2.84 × 10.sup.−4 2.4 × 10.sup.−9 Z15170-LC.sub.Lam 2.33 × 10.sup.5 6.01 × 10.sup.−4 2.6 × 10.sup.−9 HC.sub.lpi-Z15170 1.78 × 10.sup.4 7.27 × 10.sup.−4 4.1 × 10.sup.−8 LC.sub.lpi-Z15170 3.07 × 10.sup.4 5.63 × 10.sup.−4 1.8 × 10.sup.−8 Z15170-HC.sub.lpi 2.05 × 10.sup.5 4.83 × 10.sup.−4 2.4 × 10.sup.−9 Z15170-LC.sub.lpi 2.30 × 10.sup.5 3.49 × 10.sup.−4 1.5 × 10.sup.−9
(124) The interactions of the complexes with PD-1 and CTLA-4, respectively, followed a bivalent model. The K.sub.D1, K.sub.D2, k.sub.a1, k.sub.a2, k.sub.d1 and k.sub.d2 are summarized in Table 17 and Table 18 for PD-1 and CTLA-4, respectively. The affinity constant K.sub.D1 for the interaction of PD-1 with the produced Lam control antibody was determined to 18.6 nM. The affinity was stronger for all the Lam based complexes, with a K.sub.D1 range of 0.8-2.7 nM. A somewhat slower association and rate, k.sub.a1, was seen for Z15170-HC.sub.Lam, but generally the differences between the complexes were small, i.e. the positioning of the Z moiety on the antibody seems to have minor impact on the interaction between the antibody and PD-1.
(125) TABLE-US-00030 TABLE 17 Parameters for binding of indicated complexes and Lam to hPD-1 k.sub.a1 k.sub.d1 K.sub.D1 k.sub.a2 k.sub.d2 K.sub.D2 Analyte (1/Ms) (1/s) (M) (1/RUs) (1/s) (RU) Lam 4.35 × 10.sup.4 8.10 × 10.sup.−4 1.9 × 10.sup.−8 1.88 × 10.sup.−3 1.83 × 10.sup.−3 0.97 HC.sub.Lam-Z15170 1.17 × 10.sup.5 1.64 × 10.sup.−4 1.4 × 10.sup.−9 3.35 × 10.sup.0 1.65 × 10.sup.1 4.9 LC.sub.Lam-Z15170 1.78 × 10.sup.5 1.34 × 10.sup.−4 .sup. 7.5 × 10.sup.−10 2.92 × 10.sup.−1 7.89 × 10.sup.−1 2.7 Z15170-HC.sub.Lam 4.27 × 10.sup.4 1.17 × 10.sup.−4 2.7 × 10.sup.−9 1.93 × 10.sup.−2 7.87 × 10.sup.−2 4.1 Z15170-LC.sub.Lam 1.14 × 10.sup.5 1.56 × 10.sup.−4 1.4 × 10.sup.−9 2.22 × 10.sup.−2 1.72 × 10.sup.−1 7.7
(126) TABLE-US-00031 TABLE 18 Kinetic parameters for binding of indicated complexes to hCTLA-4 k.sub.a1 k.sub.d1 K.sub.D1 k.sub.a2 k.sub.d2 K.sub.D2 Analyte (1/Ms) (1/s) (M) (1/RUs) (1/s) (RU) HC.sub.Ipi-Z15170 6.79 × 10.sup.4 5.40 × 10.sup.−4 8.0 × 10.sup.−9 6.76 × 10.sup.−2 4.10 × 10.sup.−1 6.1 LC.sub.Ipi-Z15170 5.48 × 10.sup.4 4.41 × 10.sup.−4 8.0 × 10.sup.−9 4.04 × 10.sup.−2 2.88 × 10.sup.−1 7.1 Z15170-HC.sub.Ipi 3.33 × 10.sup.4 2.79 × 10.sup.−4 8.4 × 10.sup.−9 1.38 × 10.sup.−2 8.20 × 10.sup.−2 5.9 Z15170-LC.sub.Ipi 4.55 × 10.sup.4 4.49 × 10.sup.−4 9.9 × 10.sup.−9 9.74 × 10.sup.−3 4.22 × 10.sup.−2 4.3
(127) The affinity constant K.sub.D1 for the interaction of complexes with CTLA-4 was in the range of 8-10 nM and this is in line with the reported K.sub.D for ipilimumab (5.25±3.62 nM; European Medicines Agency's assessment report 2011: EMA/CHMP/557664/2011). The kinetic profiles were similar for all Ipi-based constructs, but with somewhat slower association and dissociation rates for Z15170-HC.sub.Ipi.
(128) The Biacore capture assay confirmed the dual binding specificity of all complexes included in the assay, i.e. Z15170-HC.sub.Lam, Z15170-LC.sub.Lam, Z15170-HC.sub.Ipi and Z15170-LC.sub.Ipi.
(129) Cell binding analysis by FACS: This experiment was performed to analyze whether the complexes could bind to PD-L1 expressing cells. MDA-MB-231 cells that naturally express PD-L1 were stained with 0.625 μg/ml of the respective complex. The MFI values are presented in Table 19 and shows that the complexes had the ability to bind PD-L1 expressing cells. For both the Ipi- and Lam-based complexes, the highest MFI values were obtained for N-terminal positioning of the Z moiety on the light chain of the antibody.
(130) TABLE-US-00032 TABLE 19 MFI for binding of complexes to PD-L1 expressing cells Analyte MFI Lam 62 Z15170-HC.sub.Lam 161 HC.sub.Lam-Z15170 274 Z15170-LC.sub.Lam 327 LC.sub.Lam-Z15170 145 Ipilimumab 64 Z15170-HC.sub.lpi 488 HC.sub.lpi-Z15170 264 Z15170-LC.sub.lpi 582 LC.sub.lpi-Z15170 192 Negative control 69 Anti-PD-L1 antibody 610
(131) Co-culture of MDA-MB-231 and PBMC: To assess whether the Ipi-based complexes could affect the inhibitory mechanisms caused by CTLA-4 and PD-L1, a mixed lymphocyte assay was used. Breast cancer cells MDA-MB-231 were co-cultivated with PBMC for six days and the number of cancer cells and T-cells were evaluated. The analysis revealed a concentration dependent effect of the complexes, with an increased amount of T-cells and a lowered number of cancer cells.
Example 9
Conjugation and Radiolabeling of PD-L1 Binding Z Variants
(132) This Example describes the conjugation and radiolabeling of Z15168-Cys (SEQ ID NO:809), Z18608-Cys (SEQ ID NO:811), Z18609-Cys (SEQ ID NO:812) and Z18610-Cys (SEQ ID NO:813), cloned and produced as described in Example 2 and Example 6, and further used for the in vivo imaging studies described in Example 10 and 11.
(133) Materials and Methods
(134) Reduction and NOTA conjugation: To 5 mg of Z variant in [20 mM HEPES, 1 mM EDTA, pH 7.2] was added three molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) in 0.5 ml of degassed 0.2 M ammonium acetate buffer (pH 7.0). The reaction was kept at RT for 60 min before being transferred to an Ultracel 3K Centrifugal Filter and centrifuged at 4000 rpm for 90 min. The flow-through was discarded and an additional 1 ml of 0.2 M ammonium acetate buffer added, and the process repeated. The reduced Z variant was then transferred to a second reaction vessel in 2 ml of oxygen free 0.2 M ammonium acetate buffer (pH 7.0). 4 mg of NOTA-maleimide (Macrocyclics) in 0.5 ml of 0.2 M ammonium acetate buffer (pH 7.0) was then added, and the reaction vessel purged with argon before heating to 40° C. for 3 h, at which point the reaction mixture was transferred to an Ultracel 3K Centrifugal Filter and centrifuged for 90 min at 4000 rpm. The flow-through was discarded and 2 ml milliQ water added. Centrifugation was performed for an additional 90 min and the flow-through discarded. Purified NOTA-conjugated Z variant was collected in 1 ml milliQ water, lyophilized and stored at −70° C. prior to use. Purity of the final product was determined by LC/MS.
(135) Radiolabeling: A cartridge containing [.sup.18F]-fluoride was first washed with 1.5 ml of ultrapure water, then [.sup.18F]-fluoride was eluted with 1.0 ml of 0.4 M KHCO.sub.3. 100 μl of the eluted [.sup.18F]-fluoride solution was added to a stem vial charged with 10 μl acetic acid, 50 μl AlCl.sub.3 (2 mM in 0.1 M NaOAc buffer, pH 4) and 125 μl 0.1 M NaOAc pH 4. The solution was incubated for 2 min at RT before 1 mg of NOTA-conjugated Z variant in 400 μl of a 1:1 solution of acetrontrile and 0.1 M NaOAc pH 4 was added, then heated to 100° C. for 15 min. After heating was complete, the sample was transferred to a vial containing 0.7 ml of 0.1% formic acid, mixed and purified by HPLC [Waters Xselect CSH C18 column (250×10 mm, 130 μm)] using a gradient of 10-30% MeCN over 15 min at a flow rate of 5 ml/min, the balance being 0.1% formic acid. The peak corresponding to [.sup.18F]AIF-NOTA-Z##### was collected, the MeCN was removed in vacuo, and transferred to a sterile vial using physiologic saline as a rinse to give [.sup.18F]AIF-NOTA-Z#####. Specific activity and radiochemical purity was determined via a Waters Acquity LC/MS system (Milford, Mass., USA) and a R-RAM Model 4 Radio-HPLC detector (IN/US Systems, Brandon, Fla., USA).
(136) Results
(137) The PD-L1 binding Z variants, Z15168-Cys (SEQ ID NO:809), Z18608-Cys (SEQ ID NO:811), Z18609-Cys (SEQ ID NO:812) and Z18610-Cys (SEQ ID NO:813), were site specifically conjugated with NOTA at their respective unique C-terminal cysteine residue. Subsequent radiolabeling with [.sup.18F]AIF typically resulted in radiochemical purities of 97-100% and specific activities of 14.6±6.5 GBq/mmol. The radiolabeled Z variants will be referred to as [.sup.18F]AIF-NOTA-Z[#####].
Example 10
In Vivo Imaging and Biodistribution in Tumor Bearing Mice
(138) Materials and Methods
(139) Animal models: Female SCID Beige mice (6-8 week old, Charles River Laboratories) were housed in a temperature and humidity controlled room and kept on a regular diet. LOXIMVI (human melanoma cell line, PD-L1 positive) or SUDHL-6 (PD-L1 negative) cells were cultured in complete growth medium containing RPMI 1640 medium with 10% fetal bovine serum at 37° C. with 5% CO.sub.2. The growth medium was changed 2 or 3 times per week and the cells subcultured at a ratio of 1:10 when needed. Tumors were implanted at the right shoulder by subcutaneous injection of 1×10.sup.6 LOXIMVI cells in 100 μl PBS or 10×10.sup.6 SUDHL-6 cells in 100 μl PBS+Growth Factor Reduced Matrigel (1:1). The mice were used for micro-PET and ex vivo studies about 5-7 days and 3 weeks after the injection of LOXIMVI and SUDHL-6 cells, respectively, when tumors reached a mass of 100-400 mg.
(140) PET data acquisition: Mice were anesthetized with isoflurane (4-5% induction, 1-3% maintenance), prepared with tail vein catheters, and placed in a dedicated small animal PET scanner (microPET Focus220, Siemens Preclinical Solutions). A 20 min transmission scan with .sup.57Co was obtained to correct for photon attenuation and scatter. Then, 0.2-0.6 MBq of the respective [.sup.18F]-labelled Z variant was administered via the tail vein catheters, and PET data were collected for 90 min. In a separate pre-blocking experiment, 400 μg non-labelled NOTA-conjugated Z15168-Cys was administered prior to administration of [.sup.18F]AIF-NOTA-Z15168.
(141) Ex vivo biodistribution measurements: Immediately after PET acquisition, mice were euthanized. Tumor, heart, lung, spleen, liver, kidneys, blood, plasma and muscle were collected and measured using a gamma counter (PerkinElmer). For each mouse, biodistribution measurements were converted into units of Standard Uptake Value (SUV). Regions of Interest (ROI) were drawn on all tumors that could be identified in PET images, and time activity curves (TACs) were calculated.
(142) Results
(143) Representative PET images following injection of [.sup.18F]-labelled Z variants into tumor-bearing mice showed the highest uptake in kidney and bladder. PD-L1 positive LOX tumors could be clearly seen in images, while PD-L1 negative SUDHL6 tumors were not visible. Representative PET images are shown in
Example 11
In Vivo Imaging in Rhesus Monkey
(144) Materials and Methods
(145) Fasted rhesus monkeys were sedated with Ketamine (10 mg/kg, intramuscular). An intrevenous catheter was inserted into the right and left saphenous veins and the animals were maintained on propofol anesthesia (5 mg/kg for induction and 0.45 mg/kg/min throughout the scanning procedure). Following the initial induction with propofol, the animal was intubated and maintained on ventilated oxygen/air gas mixture at approximately 10 cm.sup.3/kg/breath, and 23 respirations per minute. Animals were instrumented with a temperature probe, a pulse oximeter and an end tidal CO.sub.2 monitor. Body temperature was maintained using K-module heating pads. General fluid therapy was maintained with Lactated Ringer's solution (10 ml/kg/h i.v.) throughout the scanning procedure. 84-138 MBq of [.sup.18F]AIF-NOTA-Z15168 and 147-227 MBq of [.sup.18F]AIF-NOTA-Z18609, respectively, were administered as a 2 min infusion. Whole body dynamic scan was initiated at the start of the tracer injection and acquired for 180 min using a Siemens Biograph 64 TPTV PET/CT scanner. Whole body reconstruction was performed using the PET/CT scanner vendor supplied software. PET image analysis was performed using customized Matlab based software.
(146) Results
(147) Representative maximum intensity projection images of rhesus monkeys administered with[.sup.18F]AIF-NOTA-Z15168 and [.sup.18F]AIF-NOTA-Z18609, respectively, are shown in
ITEMIZED LIST OF EMBODIMENTS
(148) 1. PD-L1 binding polypeptide, comprising a PD-L1 binding motif BM, which motif consists of an amino acid sequence selected from:
(149) TABLE-US-00033 i) (SEQ ID NO: 837) ERNX.sub.4AAX.sub.7EIL X.sub.11LPNLX.sub.16X.sub.17X.sub.18QX.sub.20 WAFIWX.sub.26LX.sub.28D
wherein, independently from each other, X.sub.4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and Y; X.sub.7 is selected from A, E, F, H, N, Q, S, T, V, W and Y; X.sub.11 is selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W and Y; X.sub.16 is selected from N and T; X.sub.17 is selected from A, H, K, N, Q, R and S; X.sub.18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X.sub.20 is selected from H, I, K, L, N, Q, R, T, V and Y; X.sub.26 is selected from K and S; and X.sub.28 is selected from A, D and E;
and
ii) an amino acid sequence which has at least 96% identity to the sequence defined in i).
2. PD-L1 binding polypeptide according to item 1, wherein in sequence i) X.sub.4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and Y; X.sub.7 is selected from E, F, H, N, Q, S, T, V, W and Y; X.sub.11 is selected from A, D, H, L, Q, R, T, V, W and Y; X.sub.16 is selected from N and T; X.sub.17 is selected from A, H, K, N, Q, R and S; X.sub.18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X.sub.20 is selected from H, I, K, L, Q, R, T, V and Y; X.sub.26 is selected from K and S; and X.sub.28 is selected from A, D and E.
3. PD-L1 binding polypeptide according to item 1, wherein in sequence i) X.sub.4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and Y; X.sub.7 is selected from A, E, F, H, N, Q, S, T, V, W and Y; X.sub.11 is selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W and Y; X.sub.16 is selected from N and T; X.sub.17 is selected from A, H, K, N, Q, R and S; X.sub.18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X.sub.20 is selected from H, I, K, L, N, Q, R, T, V and Y; X.sub.26 is selected from K and S; and X.sub.28 is selected from A, D and E.
(150) 4. PD-L1 binding polypeptide according to item 2 or 3, wherein in sequence i) X.sub.4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T and V; X.sub.7 is selected from F, H, Q and Y; X.sub.11 is selected from H, Q, W and Y; X.sub.16 is selected from N and T; X.sub.17 is selected from A, H, K, N, Q and S; X.sub.18 is selected from A, E, G, H, K, L, N, Q, R, S, T, V and Y; X.sub.20 is selected from H, I, K, Q, R and V; X.sub.26 is selected from K and S; and X.sub.28 is selected from A and D.
(151) 5. PD-L1 binding polypeptide according to any one of item 1-4, wherein sequence i) fulfills at least four of the seven conditions I-VII: I. X.sub.7 is selected from F, H, Q and Y; II. X.sub.11 is selected from H and Y; III. X.sub.16 is T; IV. X.sub.17 is selected from N, Q and S; V. X.sub.20 is selected from H, I, K and R; VI. X.sub.26 is K; and VII. X.sub.28 is A or D.
(152) 6. PD-L1 binding polypeptide according to item 5, wherein sequence i) fulfills at least five of the seven conditions I-VII.
(153) 7. PD-L1 binding polypeptide according to item 6, wherein sequence i) fulfills at least six of the seven conditions I-VII.
(154) 8. PD-L1 binding polypeptide according to item 7, wherein sequence i) fulfills all of the seven conditions I-VII.
(155) 9. PD-L1 binding polypeptide according to any one of items 1-8, wherein X.sub.7X.sub.11X.sub.20 is selected from FYK and YYK.
(156) 10. PD-L1 binding polypeptide according to any one of items 1-9, wherein X.sub.11X.sub.17X.sub.20 is selected from YNK and YQK.
(157) 11. PD-L1 binding polypeptide according to any one of items 1-10, wherein X.sub.11X.sub.18X.sub.20 is YAK.
(158) 12. PD-L1 binding polypeptide according to any preceding item, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-808.
(159) 13. PD-L1 binding polypeptide according to item 12, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 774-796.
(160) 14. PD-L1 binding polypeptide according to item 13, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 774-787.
(161) 15. PD-L1 binding polypeptide according to item 14, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781 and 784-786, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784 and 786, such as the group consisting of SEQ ID NO:1-93, 776, 781 and 784, such as the group consisting of SEQ ID NO:1-93, 776 and 784 or the group consisting of SEQ ID NO:1-93, 776 and 781, for example the group consisting of SEQ ID NO:1-93 and 776 or the group consisting of SEQ ID NO:1-93 and 781 or the group consisting of SEQ ID NO:1-93 and 784.
(162) 16. PD-L1 binding polypeptide according to item 15, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775, 780-786, such as the group consisting of SEQ ID NO:1-93, 775, 780, 781, 784 and 786.
(163) 17. PD-L1 binding polypeptide according to any one of items 14-16, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93.
(164) 18. PD-L1 binding polypeptide according to item 17, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-24.
(165) 19. PD-L1 binding polypeptide according to item 18, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-12, 14 and 17-21.
(166) 20. PD-L1 binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-12 and 17, such as the group consisting of SEQ ID NO:1-5 and 17, such as the group consisting of SEQ ID NO:1, 2 and 17.
(167) 21. PD-L1 binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, 4, 5, 6, 9, 14 and 18-21, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the group consisting of SEQ ID NO:4, 5 and 21.
(168) 22. PD-L1 binding polypeptide according to item 20 or 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1.
(169) 23. PD-L1 binding polypeptide according to item 20 or 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:4.
(170) 24. PD-L1 binding polypeptide according to item 20 or 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:5.
(171) 25. PD-L1 binding polypeptide according to item 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:21.
(172) 26. PD-L1 binding polypeptide according to any preceding item, wherein said PD-L1 binding motif forms part of a three-helix bundle protein domain.
(173) 27. PD-L1 binding polypeptide according to item 26, wherein said PD-L1 binding motif essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.
(174) 28. PD-L1 binding polypeptide according to item 27, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
(175) 29. PD-L1 binding polypeptide according to item 28, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.
(176) 30. PD-L1 binding polypeptide according to any preceding item, which comprises a binding module BMod, the amino acid sequence of which is selected from:
(177) TABLE-US-00034 iii) (SEQ ID NO: 838) K-[BM]-DPSQSX.sub.aX.sub.bLLX.sub.c EAKKLX.sub.dX.sub.eX.sub.fQ;
wherein [BM] is a PD-L1 binding motif as defined in any one of items 1-25; X.sub.a is selected from A and S; X.sub.b is selected from N and E; X.sub.c is selected from A, S and C; X.sub.d is selected from E, N and S; X.sub.e is selected from D, E and S; and X.sub.f is selected from A and S; and
iv) an amino acid sequence which has at least 93% identity to a sequence defined in iii).
(178) 31. PD-L1 binding polypeptide according to any preceding item, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-808.
(179) 32. PD-L1 binding polypeptide according to item 31, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 774-796.
(180) 33. PD-L1 binding polypeptide according to item 32, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 774-787.
(181) 34. PD-L1 binding polypeptide according to item 33, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781 and 784-786, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784 and 786, such as the group consisting of SEQ ID NO:1-93, 776, 781 and 784, such as the group consisting of SEQ ID NO:1-93, 776 and 784 or the group consisting of SEQ ID NO:1-93, 776 and 781, for example the group consisting of SEQ ID NO:1-93 and 776 or the group consisting of SEQ ID NO:1-93 and 781 or the group consisting of SEQ ID NO:1-93 and 784.
(182) 35. PD-L1 binding polypeptide according to item 33, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775 and 780-786, such as the group consisting of SEQ ID NO:1-93, 775, 780, 781, 784 and 786.
(183) 36. PD-L1 binding polypeptide according to any one of items 33-35, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93.
(184) 37. PD-L1 binding polypeptide according to item 36, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-24.
(185) 38. PD-L1 binding polypeptide according to item 37, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-12, 14 and 17-21.
(186) 39. PD-L1 binding polypeptide according to item 38, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-12 and 17, such as the group consisting of SEQ ID NO:1-5 and 17, such as the group consisting of SEQ ID NO:1, 2 and 17.
(187) 40. PD-L1 binding polypeptide according to item 38, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, 4, 5, 6, 9, 14 and 18-21, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the group consisting of SEQ ID NO:4, 5 and 21.
(188) 41. PD-L1 binding polypeptide according to item 40 or 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1.
(189) 42. PD-L1 binding polypeptide according to item 40 or 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:4.
(190) 43. PD-L1 binding polypeptide according to item 40 or 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:5.
(191) 44. PD-L1 binding polypeptide according to item 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:21.
(192) 45. PD-L1 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
(193) TABLE-US-00035 v) (SEQ ID NO: 839) YA-[BMod]-AP;
wherein [BMod] is a PD-L1 binding module as defined in any one of items 30-44; and
(194) vi) an amino acid sequence which has at least 90% identity to a sequence defined in v).
(195) 46. PD-L1 binding polypeptide according to any one of items 1-44, which comprises an amino acid sequence selected from:
(196) TABLE-US-00036 vii) (SEQ ID NO: 840) FN-[BMod]-AP;
wherein [BMod] is a PD-L1 binding module as defined in any one of items 30-44; and
(197) viii) an amino acid sequence which has at least 90% identity to a sequence defined in vii).
(198) 47. PD-L1 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from:
(199) TABLE-US-00037 (SEQ ID NO: 845) ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK; (SEQ ID NO: 846) ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 847) ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK; (SEQ ID NO: 848) ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK; (SEQ ID NO: 849) AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK; (SEQ ID NO: 850) VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; (SEQ ID NO: 851) AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK; (SEQ ID NO: 852) VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; (SEQ ID NO: 853) VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK; (SEQ ID NO: 854) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 855) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP; (SEQ ID NO: 856) AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 857) AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP; (SEQ ID NO: 858) AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; (SEQ ID NO: 859) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 860) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP; (SEQ ID NO: 861) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 862) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP; (SEQ ID NO: 863) AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK; (SEQ ID NO: 864) AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; (SEQ ID NO: 865) AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 866) AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP; (SEQ ID NO: 867) AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK; (SEQ ID NO: 868) AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; (SEQ ID NO: 869) AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK; (SEQ ID NO: 870) AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP; (SEQ ID NO: 871) AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK; (SEQ ID NO: 872) AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK; (SEQ ID NO: 873) AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; (SEQ ID NO: 874) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; (SEQ ID NO: 875) VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK; (SEQ ID NO: 876) VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK; (SEQ ID NO: 877) VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK; (SEQ ID NO: 878) VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK; (SEQ ID NO: 879) VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; (SEQ ID NO: 880) VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK; (SEQ ID NO: 881) VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK; (SEQ ID NO: 882) VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK; (SEQ ID NO: 883) VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK; (SEQ ID NO: 884) VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK; (SEQ ID NO: 885) VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK; (SEQ ID NO: 886) VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; (SEQ ID NO: 887) AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK; and (SEQ ID NO: 888) ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a PD-L1 binding motif as defined in any one of items 1-25.
(200) 48. PD-L1 binding polypeptide according to any one of items 1-47, which comprises an amino acid sequence selected from:
(201) TABLE-US-00038 xvii) (SEQ ID NO: 874) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a PD-L1 binding motif as defined in any one of items 1-25; and
xviii) an amino acid sequence which has at least 89% identity to the sequence defined in xvii).
(202) 49. PD-L1 binding polypeptide according to any one of items 1-47, which comprises an amino acid sequence selected from:
(203) TABLE-US-00039 xix) (SEQ ID NO: 861) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK;
wherein [BM] is a PD-L1 binding motif as defined in any one of items 1-25; and
xx) an amino acid sequence which has at least 89% identity to the sequence defined in xix).
(204) 50. PD-L1 binding polypeptide according to any one of items 1-47, which comprises an amino acid sequence selected from:
(205) TABLE-US-00040 xxi) (SEQ ID NO: 854) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a PD-L1 binding motif as defined in any one of items 1-25; and
xxii) an amino acid sequence which has at least 89% identity to the sequence defined in xxi).
(206) 51. PD-L1 binding polypeptide according to any one of items 1-47, which comprises an amino acid sequence selected from:
(207) TABLE-US-00041 xxiii) (SEQ ID NO: 856) AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK;
wherein [BM] is a PD-L1 binding motif as defined in any one of items 1-25; and
xxiv) an amino acid sequence which has at least 89% identity to the sequence defined in xxiii).
(208) 52. PD-L1 binding polypeptide according to any preceding item, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-814.
(209) 53. PD-L1 binding polypeptide according to item 52, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774-796 and 809-814.
(210) 54. PD-L1 binding polypeptide according to item 53, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774-787 and 809-814.
(211) 55. PD-L1 binding polypeptide according to item 54, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781, 784-786 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784, 786 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776, 781, 784 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776, 784, 809 and 811-814 or the group consisting of SEQ ID NO:1-93, 776, 781, 809 and 811-814, for example the group consisting of SEQ ID NO:1-93, 776, 809 and 811-814 or the group consisting of SEQ ID NO:1-93, 781, 809 and 811-814 or the group consisting of SEQ ID NO:1-93, 784 and 811-814.
(212) 56. PD-L1 binding polypeptide according to item 55, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775, 780-786 and 810-814, such as the group consisting of SEQ ID NO:1-93, SEQ ID NO:775, 780, 781, 784, 786 and 810-814.
(213) 57. PD-L1 binding polypeptide according to any one of items 54-56, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 811-813.
(214) 58. PD-L1 binding polypeptide according to item 57, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-24 and 811-813.
(215) 59. PD-L1 binding polypeptide according to item 58, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-12, 14, 17-21 and SEQ ID NO:811-812.
(216) 60. PD-L1 binding polypeptide according to item 59, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-12, 17, 811 and 812, such as the group consisting of SEQ ID NO:1-5, 17, 811 and 812, such as the group consisting of SEQ ID NO:1, 2, 17, 811 and 812.
(217) 61. PD-L1 binding polypeptide according to item 58, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1, 4, 5, 6, 9, 14, 18, 19, 20, 21 and 811, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the group consisting of SEQ ID NO:4, 5 and 21.
(218) 62. PD-L1 binding polypeptide according to item 60 or 61, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:1 or 811.
(219) 63. PD-L1 binding polypeptide according to item 60, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:2 or 812.
(220) 64. PD-L1 binding polypeptide according to item 60 or 61, wherein sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:4.
(221) 65. PD-L1 binding polypeptide according to item 60 or 61, wherein sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:5.
(222) 66. PD-L1 binding polypeptide according to item 61, wherein sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:21.
(223) 67. PD-L1 binding polypeptide according to any preceding item, which is capable of blocking PD-L1 dependent signaling.
(224) 68. PD-L1 binding polypeptide according to item 67, wherein the half maximal inhibitory concentration (IC50) of the blocking is at most 5×10.sup.−8 M, such as at most 1×10.sup.−8 M, such as at most 5×10.sup.−9 M, such as at most 3.5×10.sup.−9 M, such as at most 1×10.sup.−9 M, such as at most 5×10.sup.−1° M, such as at most 1×10.sup.−10 M.
(225) 69. PD-L1 binding polypeptide according to any preceding item, which is capable of blocking the interaction of PD-L1 with PD-1.
(226) 70. PD-L1 binding polypeptide according to any preceding item, which is capable of binding to PD-L1 such that the K.sub.D value of the interaction is at most 2×10.sup.−8 M, such as at most 1×10.sup.−8 M, such as at most 1×10.sup.−9 M, such as at most 5×10.sup.−10 M, such as at most 3×10.sup.−10 M.
(227) 71. PD-L1 binding polypeptide according to any preceding item, which is capable of binding to PD-L1 such that the k.sub.d value of the interaction is at most 1×10.sup.−3 s.sup.−1. such as at most 6×10.sup.−4 s.sup.−1.
(228) 72. PD-L1 binding polypeptide according to any preceding item, which is capable of binding to PD-L1 such that the EC50 value of the interaction is at most 1×10.sup.−9 M, such as at most 1×10.sup.−1° M, such as at most 7×10.sup.−11 M.
(229) 73. PD-L1 binding polypeptide according to any preceding item, wherein said PD-L1 is human PD-L1.
(230) 74. PD-L1 binding polypeptide according to any preceding item which comprises additional amino acids at the C-terminal and/or N-terminal end.
(231) 75. PD-L1 binding polypeptide according to item 74, wherein said additional amino acid(s) improve(s) production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide.
(232) 76. PD-L1 binding polypeptide according to any preceding item in multimeric form, comprising at least two PD-L1 binding polypeptide monomer units, whose amino acid sequences may be the same or different.
(233) 77. PD-L1 binding polypeptide according to item 76, wherein said PD-L1 binding polypeptide monomer units are covalently coupled together.
(234) 78. PD-L1 binding polypeptide according to item 77, wherein the PD-L1 binding polypeptide monomer units are expressed as a fusion protein.
(235) 79. PD-L1 binding polypeptide according to any one of items 76-78, in dimeric form.
(236) 80. Fusion protein or conjugate comprising a first moiety consisting of a PD-L1 binding polypeptide according to any preceding item, and a second moiety consisting of a polypeptide having a desired biological activity.
(237) 81. Fusion protein or conjugate according to item 80, wherein said desired biological activity is a therapeutic activity.
(238) 82. Fusion protein or conjugate according to item 80, wherein said desired biological activity is a binding activity.
(239) 83. Fusion protein or conjugate according to item 80, wherein said desired biological activity is an enzymatic activity.
(240) 84. Fusion protein or conjugate according to item 82, wherein said binding activity is albumin binding activity which increases in vivo half-life of the fusion protein or conjugate.
(241) 85. Fusion protein or conjugate according to item 84, wherein said second moiety comprises the albumin binding domain of streptococcal protein G or a derivative thereof.
(242) 86. Fusion protein or conjugate according to item 82, wherein said binding activity acts to block a biological activity.
(243) 87. Fusion protein or conjugate according to item 81, wherein the second moiety is a therapeutically active polypeptide.
(244) 88. Fusion protein or conjugate according to item 87, wherein the second moiety is an immune response modifying agent.
(245) 89. Fusion protein or conjugate according to item 87, wherein the second moiety is an anti-cancer agent.
(246) 90. Fusion protein or conjugate according to any one of items 80-83 and 86-89, wherein the second moiety is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.
(247) 91. Fusion protein according to any one of items 80-91, wherein the second moiety further comprises a linker.
(248) 92. Complex, comprising at least one PD-L1 binding polypeptide according to any one of the preceding items and at least one antibody or an antigen binding fragment thereof.
(249) 93. Complex according to item 92, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments, Fab′ fragments, F(ab′).sub.2 fragments, Fc fragments, Fv fragments, single chain Fv (scFv) fragments, (scFv).sub.2 and domain antibodies.
(250) 94. Complex according to item 93, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments and scFv fragments.
(251) 95. Complex according to item 94, wherein said at least one antibody or antigen binding fragment thereof is a full-length antibody.
(252) 96. Complex according to any one of items 92-95, wherein said antibody or antigen binding fragment thereof is a monoclonal antibody or an antigen binding fragment thereof.
(253) 97. Complex according to any one of items 92-96, wherein said antibody or antigen binding fragment thereof is selected from the group consisting of human antibodies, humanized antibodies and chimeric antibodies, and antigen binding fragments thereof.
(254) 98. Complex according to item 97, wherein said antibody or antigen binding fragment thereof is a human or humanized antibody, or an antigen binding fragment thereof.
(255) 99. Complex according to any one of items 92-98, wherein said PD-L1 binding polypeptide is attached at either the C-terminus or the N-terminus of the heavy chain or the light chain of said antibody or antigen binding fragment thereof.
(256) 100. Complex according to any one of items 92-99, further comprising a linker.
(257) 101. Complex according to any one of items 92-100, wherein said antibody or antigen binding fragment thereof has affinity for an antigen, for example an antigen associated with an infectious disease, or an antigen associated with cancer.
(258) 102. Fusion protein or conjugate according to any one of items 79-90 or complex according to any one of items 92-101, wherein said second moiety or said antibody or antigen binding fragment thereof is an inhibitor selected from the group consisting of inhibitors of: PD-1, CTLA-4, T-cell immunoglobulin and mucin containing protein-3 (TIM-3), galectin-9 (GAL-9), lymphocyte activation gene-3 (LAG-3), PD-L2, B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), V-domain Ig suppressor of T-cell activation (VISTA), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), B and T lymphocyte attenuator (BTLA), colony stimulating factor 1 receptor (CSF1R), herpes virus entry mediator (HVEM), killer immunoglobulin receptor (KIR), adenosine, adenosine A2a receptor (A2aR), CD200-CD200R and T cell Ig and ITIM domain.
(259) 103. Fusion protein, conjugate or complex according to item 102, wherein said second moiety, antibody or antigen binding fragment thereof is an inhibitor of PD-1, such as an inhibitor selected from the group consisting of nivolumab, pidilizumab, BMS 936559, MPDL3280A and pembrolizumab, such as pembrolizumab.
(260) 104. Fusion protein, conjugate or complex according to item 102, wherein said second moiety, antibody or antigen binding fragment thereof is an inhibitor of CTLA-4, such as an inhibitor selected from the group consisting of belatacept, abatacept and ipilimumab, such as ipilimumab.
(261) 105. Fusion protein or conjugate according to any one of items 80-91 or complex according to any one of items 92-101, wherein said second moiety or antibody or antigen binding fragment thereof is an agonist selected from the group consisting of agonists of CD134, CD40, 4-11BB and glucocorticoid-induced TNFR-related protein (GITR).
(262) 106. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-105, further comprising a label.
(263) 107. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to item 106, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides, radioactive particles and pretargeting recognition tags.
(264) 108. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to item 107, comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the PD-L1 binding polypeptide via a thiol group of a cysteine residue or an amine group of a lysine residue.
(265) 109. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to item 106, which comprises a pretargeting recognition tag forming part of a complementary pair of pretargeting moieties, for example selected from stept(avidin)/biotin, oligonucleotide/complementary oligonucleotide such as DNA/complementary DNA, RNA/complementary RNA, phosphorothioate nucleic acid/complementary phosphorothioate nucleic acid and peptide nucleic acid/complementary peptide nucleic acid and morpholinos/complementary morpholinos.
(266) 110. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to item 109, wherein said pretargeting recognition tag is a peptide nucleic acid tag.
(267) 111. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of item 110, wherein said pretargeting recognition tag is a 10-20-mer peptide nucleic acid sequence, such as a 15-mer peptide nucleic acid sequence.
(268) 112. A polynucleotide encoding a polypeptide according to any one of items 1-105.
(269) 113. Expression vector comprising a polynucleotide according to item 112.
(270) 114. Host cell comprising an expression vector according to item 113.
(271) 115. Method of producing a polypeptide according to any one of items 1-105, comprising culturing a host cell according to item 114 under conditions permissive of expression of said polypeptide from said expression vector, and isolating said polypeptide.
(272) 116. Composition comprising a PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-111 and at least one pharmaceutically acceptable excipient or carrier.
(273) 117. Composition according to item 116, further comprising at least one additional active agent, such as an agent selected from an immune response modifying agent and an anti-cancer agent.
(274) 118. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-111 or a composition according to any one of items 116-117 for oral, topical, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration, such as for topical administration.
(275) 119. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-111 or a composition according to any one of items 116-117 for use as a medicament, a diagnostic agent and/or a prognostic agent.
(276) 120. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 119 as a medicament.
(277) 121. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 119 as a diagnostic agent and/or a prognostic agent.
(278) 122. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use as a medicament according to item 120, wherein said polypeptide, fusion protein, conjugate or composition modulates PD-L1 function in vivo.
(279) 123. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to any one of items 119-121 in the treatment, prognosis or diagnosis of a PD-L1 related disorder.
(280) 124. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 122, wherein said PD-L1 related disorder is selected from the group consisting of infectious diseases and cancers.
(281) 125. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 124, wherein said PD-L1 related disorder is an infectious disease, such as a chronic viral infection, for example selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV).
(282) 126. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 124, wherein said PD-L1 related disorder is cancer, such as a cancer selected from the group consisting of: cancers manifesting solid tumors, for example selected from the group consisting of skin cancer, such as melanoma and nonmelanoma skin cancer (NMSC); lung cancers, such as small cell lung cancer, non-small cell lung cancer (NSCLC); head and neck cancer; renal cell carcinoma (RCC); bladder cancer; breast cancer; colorectal cancer; gastric cancer; ovarian cancer; pancreatic cancer; prostate cancer; glioma; glioblastoma; liver carcinoma; gallbladder cancer; thyroid cancer; bone cancer; cervical cancer; uterine cancer; vulval cancer; endometrial cancer; testicular cancer; kidney cancer; esophageal carcinoma; brain/CNS cancers; neuronal cancers: mesothelioma; sarcomas; small bowel adenocarcinoma; and pediatric malignancies; and cancers manifesting non-solid tumors, for example leukaemia, acute myeloid leukaemia, acute lymphoblastic leukaemia and multiple myeloma.
(283) 127. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 126, wherein said cancer is selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC, bladder cancer, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer and prostate cancer, such as selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC and bladder cancer.
(284) 128. Method of treatment of a PD-L1 related disorder, comprising administering to a subject in need thereof an effective amount of a PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-111 or a composition according to any one of items 116-117.
(285) 129. Method according to item 128, wherein said PD-L1 related disorder is selected from the group consisting of infectious disease and cancer.
(286) 130. Method according to item 129, wherein said PD-L1 related disorder is an infectious disease, such as a chronic viral infection, for example selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV).
(287) 131. Method according to item 129, wherein said PD-L1 related disorder is cancer, such as a cancer selected from the group consisting of: cancers manifesting solid tumors, for example selected from the group consisting of skin cancer, such as melanoma and nonmelanoma skin cancer (NMSC); lung cancers, such as small cell lung cancer, non-small cell lung cancer (NSCLC); head and neck cancer; renal cell carcinoma (RCC); bladder cancer; breast cancer; colorectal cancer; gastric cancer; ovarian cancer; pancreatic cancer; prostate cancer; glioma; glioblastoma; liver carcinoma; gallbladder cancer; thyroid cancer; bone cancer; cervical cancer; uterine cancer; vulval cancer; endometrial cancer; testicular cancer; kidney cancer; esophageal carcinoma; brain/CNS cancers; neuronal cancers: mesothelioma; sarcomas; small bowel adenocarcinoma; and pediatric malignancies; and cancers manifesting non-solid tumors, for example leukaemia, acute myeloid leukaemia, acute lymphoblastic leukaemia and multiple myeloma.
(288) 132. Method according to item 131, in which said cancer is selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC, bladder cancer, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer and prostate cancer, such as selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC and bladder cancer.
(289) 133. Method according to any one of items 131-132, comprising the steps of: contacting the subject with a PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 109-111 comprising a pretargeting recognition tag, or with a composition comprising such a PD-L1 binding polypeptide, fusion protein, conjugate or complex, and contacting the subject with a complementary pretargeting moiety, comprising a radionuclide.
(290) 134. Method of detecting PD-L1, comprising providing a sample suspected to contain PD-L1, contacting said sample with a PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-111 or a composition according to any one of items 116-117, and detecting the binding of the PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition to indicate the presence of PD-L1 in the sample.
(291) 135. Method for determining the presence PD-L1 in a subject, comprising the steps of: a) contacting the subject, or a sample isolated from the subject, with a PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-111 or a composition according to any one of items 116-117, and b) obtaining a value corresponding to the amount of the PD-L1 binding polypeptide, fusion protein, conjugate or composition that has bound in said subject or to said sample.
(292) 136. Method according to item 135, in which said PD-L1 binding polypeptide, fusion protein, conjugate or complex is according to any one of items 109-111, or said composition comprises such a PD-L1 binding polypeptide, fusion protein, conjugate or complex, and step a) further comprises contacting the subject with a complementary pretargeting moiety labeled with a detectable label, such as a radionuclide label.
(293) 137. Method according to item 135 or 136, further comprising a step of comparing said value to a reference.
(294) 138. Method according to any one of items 134-137, wherein said subject is a mammalian subject, such as a human subject.
(295) 139. Method according to any one of items 134-138, wherein the method is performed in vivo.
(296) 140. Method according to item 139, which is a method for medical imaging in which step a) comprises the systemic administration of said PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition to a mammalian subject; said PD-L1 binding polypeptide, fusion protein, conjugate, complex, composition or pretargeting moiety comprises a radionuclide label suitable for medical imaging; and step b) comprises obtaining one or more images of at least a part of the subject's body using a medical imaging instrument, said image(s) indicating the presence of the radionuclide inside the body.