CD40 BINDING PROTEIN
20230167185 · 2023-06-01
Inventors
Cpc classification
C07K2317/64
CHEMISTRY; METALLURGY
C07K16/2878
CHEMISTRY; METALLURGY
A61K2039/507
HUMAN NECESSITIES
International classification
Abstract
Provided is a binding protein that binds CD40 and is an agonist thereof. In a particular embodiment the invention provides an agonistic anti-CD40 antibody. Also provided are bispecific conjugates comprising the binding protein, and complexes comprising the bispecific conjugates non-covalently bound to a tag construct comprising an antigen, for antigen delivery to immune cells. Medical uses of the binding proteins, conjugates and complexes of the invention are also provided.
Claims
1. A binding protein that specifically binds CD40, wherein said binding protein is an agonist of CD40 and comprises a binding domain of an antibody, the binding domain comprising a heavy chain variable domain and a light chain variable domain, each comprising three complementarity determining domains (CDRs), wherein: VLCDR1 has the sequence set forth in SEQ ID NO: 1; VLCDR2 has the sequence set forth in SEQ ID NO: 2; VLCDR3 has the sequence set forth in SEQ ID NO: 3; VHCDR1 has the sequence set forth in SEQ ID NO: 4; VHCDR2 has the sequence set forth in SEQ ID NO: 5; and VHCDR3 has the sequence set forth in SEQ ID NO: 6.
2. The binding protein of claim 1, wherein the binding protein binds, and is an agonist of, human CD40.
3. The binding protein of claim 1, wherein the binding protein is a monoclonal antibody, an antibody fragment or an scFv.
4. The binding protein of claim 3, wherein the antibody, antibody fragment or scFv is human.
5. The binding protein of claim 3, wherein the antibody fragment is a Fab or F(ab′)2 antibody fragment.
6. The binding protein of claim 3, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 80% sequence identity thereto; and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 80% sequence identity thereto.
7. The binding protein of claim 3, wherein said specific binding molecule is a monoclonal antibody of the IgG2 isotype.
8. The binding protein of claim 7, wherein the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 80% sequence identity thereto; and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80% sequence identity thereto.
9. A bispecific conjugate comprising: (i) at least one first binding protein of claim 1; and (ii) at least one second binding protein, which comprises a binding domain of an antibody and binds a peptide moiety; wherein the first and second binding proteins are covalently linked.
10. The bispecific conjugate of claim 9, wherein the first binding protein is a monoclonal antibody and the second binding protein is an scFv, preferably wherein the monoclonal antibody is of the IgG2 isotype.
11. The bispecific conjugate of claim 10, wherein the scFv is covalently linked to: (i) a C.sub.H3 domain of the antibody; or (ii) a C.sub.L domain of the antibody.
12. The bispecific conjugate of claim 11, wherein the conjugate comprises one monoclonal antibody and two scFvs, and: (i) one scFv is conjugated to the C.sub.H3 domain of each heavy chain of said antibody; or (ii) one scFv is conjugated to the C.sub.L domain of each light chain of said antibody.
13. The bispecific conjugate of claim 9, wherein the peptide moiety has a non-human amino acid sequence, preferably wherein the peptide moiety has an amino acid sequence derived from a micro-organism.
14. The bispecific conjugate of claim 13, wherein the peptide moiety has an amino acid sequence derived from a bacterial toxin, preferably wherein the peptide moiety has an amino acid sequence derived from tetanus toxin.
15. The bispecific conjugate of claim 14, wherein the peptide moiety comprises the amino acid sequence set forth in any one of SEQ ID NOs: 11-16 or 42-46, or an amino acid sequence having up to two amino acid substitutions relative to any one of SEQ ID NOs: 11-16 or 42-46.
16. A complex comprising a bispecific conjugate claim 9 and a tag construct, the tag construct comprising a peptide moiety covalently attached to an antigen; wherein the peptide moiety of said tag construct is non-covalently bound to the second binding protein of the bispecific conjugate.
17. The complex of claim 16, wherein said antigen is a cancer antigen.
18. The complex of claim 17, wherein said cancer antigen is a neoantigen, a tumour-associated antigen, or an antigen derived from an oncovirus.
19. The complex of claim 16, wherein said antigen is derived from a pathogen.
20. A pharmaceutical composition comprising: (i) a binding protein as defined in claim 1, or a bispecific conjugate or complex comprising the same; and at least one pharmaceutically acceptable carrier or excipient.
21. A kit comprising a bispecific conjugate as defined in claim 9.
22-23. (canceled)
24. A method of treating or preventing cancer or an infection, comprising administering to a subject a binding protein as defined in claim 1 or a bispecific conjugate or complex comprising the same, to a subject.
25. (canceled)
Description
FIGURE LEGENDS
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EXAMPLES
Example 1
Phage Display Selection on Human CD40 Using a Human scFv Library
[0260] Phage display selections were performed to enable isolation of scFv fragments with specificity for human CD40.
Materials and Methods
Phage Display Selection
[0261] Biopanning was performed using four selection rounds of enrichment employing two human synthetic scFv phage libraries, SciLifeLib1 and SciLifeLib2 (SciLifeLab, Stockholm, Sweden). SciLifeLib 1 and 2 are naive human synthetic scFv libraries, similar in design and construction to previously reported (Säll et al., Protein Eng Des Sel (2016) 29: 427-437). Briefly, human germline genes IGHV3-23 and IGKV1-39 were used as library scaffold and Kunkel mutagenesis was used to introduce diversity into four of the six CDRs, namely VHCDR1, VHCDR2, VHCDR3 and VLCDR3. The selection was performed using streptavidin-coated magnetic beads (Dynabeads M-280, ThermoFisher Scientific, #11206D) and Avi-tagged human CD40 extracellular domain (AcroBiosystems, #CD0-H82E8, amino acids 21-193), referred to as hCD40-avi. The Avi-tag allows site-specific incorporation of biotin. The selection pressure was increased by gradually decreasing the antigen amount (327 nM to 35 nM) and by increasing the number and intensity of washes between the different rounds.
[0262] In order to remove non-specific or streptavidin binders, pre-selection was performed by incubation of the phage stocks against empty streptavidin coated magnetic beads prior round 1 and 2. Also, 1% bovine serum albumin (BSA) was included as blocking agent throughout the selection procedure. Elution of antigen-bound phages was performed using a trypsin-aprotinin approach. The entire selection process, except the phage-target protein incubation step, was automated and performed with a Kingfisher Flex robot. Recovered phages were propagated in XL1 blue E. coli, either on agar plates at 37° C. overnight (Round 1 and 2) or in solution at 30° C. overnight (Rounds 3 and 4). Phage stocks were made by infecting with an excess of M13K07 helper phage (New England Biolabs, #N03155) and scFv expression induced by the addition of IPTG. The overnight cultures were PEG/NaCl-precipitated, resuspended in selection buffer and used for the next round of selection.
Re-Cloning and Expression of scFv
To allow production of soluble scFv, phagemid DNA from the third and fourth round of each selection track was isolated. In pools, the genes encoding the scFv fragments were restriction enzyme digested and sub-cloned into a screening vector, providing a signal for secretion of the scFv along with a triple-Flag tag and a hexahistidine (His) tag at the C-terminus. The constructs were subsequently transformed into TOP10 E. coli. Single colonies were picked, cultivated and IPTG-induced for soluble scFv expression in 96-well format. In total, 940 scFv clones present in bacterial supernatant were prepared for a primary ELISA screen.
ELISA Screen
[0263] Streptavidin was coated onto a 384-well ELISA plate at 1 μg/ml in PBS, 4° C. overnight, and hCD40-avi added after washing, diluted to 0.1 μg/ml in blocking buffer (PBS supplemented with 0.5% BSA+0.05% Tween20). Two negative control proteins, streptavidin and BSA, were also coated onto plates. Flag-tagged scFv clones present in bacterial supernatant were diluted 1:3 in blocking buffer and allowed to bind to the coated proteins. Detection of binding was enabled through an HRP-conjugated α-Flag M2 antibody (Sigma-Aldrich #A8592) followed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) ELISA substrate (Thermo Fisher Scientific #34029). The colorimetric-signal development was stopped by adding 1 M sulphuric acid and the plate was read at 450 nm. All samples were assayed in duplicate.
DNA Sequencing
[0264] 157 positive scFv clones showing binding to hCD40-avi were sent for Sanger DNA sequencing by GATC Biotech (Ebergsberg, Germany).
Results
[0265] Two selection tracks were carried out in parallel on human CD40 using SciLifeLib 1 and 2. Following re-cloning of selected scFv clones, a total of 940 colonies were picked from selection rounds 3 and 4. A primary ELISA screen resulted in 159 potential hits. DNA sequencing of these resulted in the identification of 59 sequence unique clones.
Example 2
Kinetic Screen of 59 Sequence Unique scFvs by SPR
[0266] The 59 sequence unique scFv clones from Example 1 were selected for further characterisation by surface plasmon resonance (SPR) in a kinetic screen-based approach to enable ranking of the different clones.
Materials and Methods
[0267] The kinetic screen was performed on a Biacore T200 instrument (GE Healthcare). An α-Flag M2 antibody (Sigma-Aldrich #F1804), functioning as a capture ligand, was immobilised onto all four surfaces of a CM5-S amine sensor chip according to the manufacturer's recommendations.
[0268] 59 Flag-tagged scFv-clones present in bacterial supernatant were injected and captured on the chip surfaces, followed by injection of 25 nM human CD40 extracellular domain-Fc fusion construct (R&D Systems, #1493-CDB, CD40 amino acids 21-193; hCD40-Fc). The surfaces were regenerated with 10 mM glycine-HCl pH 2.2. All experiments were performed at 25° C. in running buffer (HBS supplemented with 0.05% Tween20, pH 7.5).
[0269] By subtracting the response curve of a reference surface (an α-Flag M2 antibody immobilised surface), response curve sensorgrams were obtained. Data was analysed using the Biacore T200 Evaluation 3.1 software.
Results
[0270] An α-Flag M2 antibody was immobilised onto all four surfaces of a CM5-S chip and similar RU-levels of captured scFv clones were obtained. Following hCD40-Fc injection at 25 nM, each surface was successfully regenerated using a low pH acid solution.
[0271] Analysis of data was performed by visual inspection of the sensorgrams (not shown). 15 of the tested scFvs were considered promising based on binding to hCD40. In particular, a high binding response and favourable slow off-rate were considered.
Example 3
Conversion to Full Antibody Format
[0272] Twelve of the most promising scFvs from phage selection were selected for conversion to full-length human IgG2 antibody format. The rationale for including these particular clones was their performance in a panel of binding assays (see Examples 1 and 2) and sequence analysis. Two negative controls were similarly converted to human IgG2: G-Strep-1 (GS1, an in-house anti-streptavidin scFv) and B1-8 (an anti-4-hydroxy-3-nitrophenylacetyl (NP) scFv, Reth et al., European Journal of Immunology 8(6): 393-400, 1978) as well as two agonistic anti-CD40 antibodies found in the literature, namely 1150/1151 (WO 2015/091853) and CP-870,893 (US 2017/0342159). As a comparison, the latter two were also converted to human IgG1 format. 1150/1151 as hIgG1 and hIgG2 is referred to herein as X-SM083-Ab-1 and X-SM083-Ab-2, respectively; CP-870,893 as hIgG1 and hIgG2 is referred to herein as X-SM083-Ab-4 and X-SM083-Ab-5, respectively (Ab-1, Ab-2, Ab-4 and Ab-5, respectively, for short). The twelve IgG2 antibodies produced from the scFvs from phage selection are referred to as Y-SM083-A1, Y-SM083-A6, Y-SM083-A9, Y-SM083-B1, Y-SM083-B3, Y-SM083-B8, Y-SM083-C6, Y-SM083-E7, Y-SM083-E8, Y-SM083-F4, Y-SM083-F7 and Y-SM083-G2 (A1, A6, A9, B1, B3, B8, C6, E7, E8, F4, F7 and G2, respectively, for short).
Materials and Methods
[0273] In-Fusion Cloning, Transfection into HEK293, Expression and Purification
The V.sub.H and V.sub.L regions of the chosen scFvs were PCR amplified and inserted into the in house-constructed vector pHAT-hIgG2 using the In-Fusion HD Plus Cloning Kit (Clontech #638909). Transfection of plasmid DNA into expiHEK293 cells was performed using an ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific #A14525) in 80 ml cultures. The cultures were harvested after 5 days and antibodies purified by affinity chromatography using a HiTrap Protein A HP column (GE Healthcare) followed by buffer exchange to PBS using a HiTrap Desalting column (GE healthcare). Endotoxin levels were <1 EU/mg as determined by LAL chromogenic endotoxin assay. SDS-PAGE was performed to determine purity and integrity of the purified IgG and concentrations determined using an Implen NP80 UV-Vis Spectrophotometer. In addition, size exclusion chromatography was run on each of the purified antibodies (Agilent Bio SEC-3).
ELISA
[0274] Biotinylated antigens were immobilised in the wells of a 384-well ELISA plate via streptavidin (1 μg/ml) at a concentration of 0.1 μg/ml for hCD40-avi (AcroBiosystems, #CD0-H82E8) or 1 μg/ml for NP and nitrohydroxyiodophenylacetate (NIP, also recognised by the B1-8 scFv) in PBS. Purified antibodies were diluted in blocking buffer (PBS+0.5% BSA+0.05% Tween20) to a final concentration of 1, 0.2 or 0.04 μg/ml, and added to the wells. Detection of bound IgG was performed using a horseradish peroxidase (HRP)-labelled anti-human IgG kappa antibody, followed by incubation with the chromogen Ultra TMB-ELISA (Thermo Scientific Pierce, Rockford, Ill., USA). The signal development was stopped by the addition of 1 M sulphuric acid and the absorbance was measured at 450 nm.
Results
[0275] All CD40-binding antibodies and controls were successfully re-cloned to human IgG format, expressed in HEK293 cells and purified by Protein A-conjugated magnetic beads on a Kingfisher Flex instrument. All antibodies were of the expected molecular weight and demonstrated an acceptable level of purity, as evaluated by SDS-PAGE. ELISA confirmed retained antigen binding of all clones after conversion, except for Y-SM083-C6 (data not shown). This clone was therefore excluded from further analysis.
[0276] The V.sub.L and V.sub.H sequences of Y-SM083-A9 are set forth in SEQ ID NOs: 7 and 8, respectively; the V.sub.L and V.sub.H sequences of Y-SM083-B8 are forth in SEQ ID NOs: 61 and 62, respectively. The V.sub.L and V.sub.H sequences of 1150/1151 and CP-870,893 are known from the prior art (see above). The sequences of the other scFv clones converted to IgG2 format are not shown.
Example 4
Kinetic Measurements of Eleven Anti-CD40 hIgG2 Antibodies
[0277] The kinetic constants of 11 of the IgG2 antibodies against human CD40 (produced in Example 3) were determined by surface plasmon resonance (SPR) using a single cycle kinetic (SCK) approach. Also, cross-species binding was assessed to both mouse CD40 (mCD40) and cynomolgus CD40 (cCD40).
Materials and Methods
[0278] The kinetic constants were determined by SPR on a BIAcore T200 instrument (GE Healthcare) using single cycle kinetics (SCK) or a single injection approach.
[0279] An α-kappa antibody (GE Healthcare #28958325), functioning as a capture ligand, was immobilised by EDC/NHS chemistry onto all 4 surfaces of a CM5-S amine sensor chip according to the manufacturer's recommendations. Protein A-purified IgG2 antibodies (Example 3) were injected and captured on the chip surface, aiming for equal response units (RU) between clones.
[0280] A 3-fold dilution series of hCD40-avi and 4-fold dilution series of hCD40-Fc and cCD40-Fc, consisting of 5 concentrations ranging from 100 nM-1.2 nM and 80 nM-0.31 nM, respectively, were prepared in running buffer (HBS supplemented with 0.05% Tween20 at pH 7.5) and sequentially injected over the chip surfaces. Following a dissociation phase, the surfaces were regenerated with 10 mM glycine-HCl, pH 2.1. Single concentration injections at 80 nM were also made with mCD40. Details on the CD40 proteins used in this study are given in Table 1.
[0281] By subtracting the response curve of a reference surface (an α-kappa antibody immobilised surface), response unit sensorgrams for all antibodies were obtained. Reaction rate kinetics constants were calculated using the Biacore T200 Evaluation Software 3.1 and the 1:1 Langmuir binding model.
TABLE-US-00001 TABLE 1 CD40 reagents Short Name name Source Characteristics Bio- hCD40- Aero Produced in HEK293 cells; tinylated avi Biosystems Biotinylated on C-terminal avi-tag. human #CD0- CD40 amino acids: E21-R193 CD40 H82E8 (Accession# P25942-1) Human hCD40- RnD Produced in mouse myeloma cell CD40 Fc Systems line; Fc-chimera (disulfide- Fc (#1493- linked homodimer) chimera CDB) CD40 amino acids: E21-R193 (Accession# P25942-1) Cyno- cCD40- RnD Produced in HEK293 cells molgus Fc Systems Fc-chimera (disulphide- CD40 (#9660- linked homodimer) Fc CD) CD40 amino acids: E21-R193 chimera (Accession # XP 005569274.1) Mouse mCD40 Aero Produced in HEK293 cells. CD40 Biosystems CD40 amino acids: V24-R193 #TN5- (Accession# P27512-1) M52H8
Results
[0282] Equal capture levels (RU) were obtained for all antibodies. Following injection of analyte, chip surfaces were successfully regenerated leaving an active surface ready for the next antibody-capture cycle. See Table 2 for summary of determined kinetic constants and affinities.
[0283] It is important to remember that two of the antigens used here are Fc-fused and these will therefore combine and form homodimers. Binding strength is traditionally reported by the affinity constant (K.sub.D). However, this constant is used to describe the strength of a monovalent interaction and given that the Fc-fused antigens most likely interact with the antibody surface in a bivalent fashion, thereby potentially giving rise to synergy and an apparent increase in affinity, we instead report the apparent affinity (denoted as .sup.appK.sub.D). The avidity contribution of an interaction will not only depend on the antigen being a monomer or a dimer but also on the antibody itself. Binders specific for one and the same antigen may have very different avidity effects due to differences in binding kinetics. Despite the potential avidity contribution for some of the binders, we have chosen a Langmuir 1:1 binding model for calculation of .sup.appK.sub.D for the interaction with both hCD40-fc and cCD40-Fc This was considered to provide an average picture that allowed us, in a pragmatic way, to select the best binders to move forward with.
[0284] As expected, all antibodies showed binding towards the human CD40 proteins. Some clones however, only displayed binding towards hCD40-Fc and no binding towards hCD40-avi (or too low a binding response for kinetic measurements). The .sup.appK.sub.D for binding to hCD40-Fc were in the range of 5-20 nM for all Y-SM083 antibodies, whereas the corresponding values for hCD40-avi was considerable higher. This discrepancy can most likely be explained by the fact that hCD40-Fc is a dimer, which can potentially give rise to an avidity effect (accumulated strength of multiple affinities). All antibodies, except Y-SM083-B1 and -G2, also showed cross-binding to cynomolgus CD40-Fc. Binding to mouse CD40 could not be detected for any of the antibodies, which is somewhat expected given the relatively low sequence homology (58%) between human and mouse CD40.
TABLE-US-00002 TABLE 2 Antibody Kinetic Parameters Human CD40-avi Clone Name ka (1/Ms) kd (1/s) K.sub.D (M) Y-SM083-F4 ** ** ** Y-SM083-A9 1.7E+05 6.1E−03 3.7E−08 Y-SM083-B8 4.5E+09 6.9E+01 1.5E−08 Y-SM083-B3 ** ** ** Y-SM083-A1 ** ** ** Y-SM083-B1 ** ** ** Y-SM083-F7 ** ** ** Y-SM083-E7 2.5E+05 1.1E−02 4.4E−08 Y-SM083-G2 -- -- -- X-SM083-ab-2 7.5E+05 1.4E−01 1.8E−07 X-SM083-ab-5 6.2E+04 1.1E−03 1.8E−08 Human CD40-Fc* Clone Name ka (1/Ms) kd (1/s) .sup.appK.sub.D (M) Y-SM083-F4 5.3E+05 4.0E−03 7.6E−09 Y-SM083-A9 3.1E+05 1.6E−03 5.1E−09 Y-SM083-B8 1.2E+05 1.5E−03 1.3E−08 Y-SM083-B3 2.2E+09 4.4E+01 2.0E−08 Y-SM083-A1 8.1E+04 1.4E−03 1.7E−08 Y-SM083-B1 9.0E+04 1.8E−03 2.0E−08 Y-SM083-F7 1.0E+06 5.9E−03 5.8E−09 Y-SM083-E7 1.5E+05 9.1E−04 6.3E−09 Y-SM083-G2 6.5E+05 5.0E−03 7.8E−09 X-SM083-ab-2 3.2E+05 1.4E−03 4.6E−09 X-SM083-ab-5 6.6E+04 3.2E−05 4.8E−10 Cynomolgus CD40-Fc* Clone Name ka (1/Ms) kd (1/s) .sup.appK.sub.D (M) Y-SM083-F4 4.9E+05 3.9E−03 8.1E−09 Y-SM083-A9 5.0E+05 2.2E−03 4.5E−09 Y-SM083-B8 9.5E+09 1.3E+02 1.3E−08 Y-SM083-B3 ** ** ** Y-SM083-A1 ** ** ** Y-SM083-B1 -- -- -- Y-SM083-F7 ** ** ** Y-SM083-E7 1.5E+05 1.9E−03 1.3E−08 Y-SM083-G2 -- -- -- X-SM083-ab-2 4.9E+05 1.8E−03 3.8E−09 X-SM083-ab-5 9.1E+04 2.5E−05 2.8E−10 -- No binding was detected. *Avidity effects may contribute to kinetic values and therefore, .sup.appK.sub.D rather than K.sub.D is given for these antigens. ** Binding detected but too low a binding response (RU) and/or too fast on (ka) and off (kd) rates to determine kinetic parameters.
Example 5
Assessment of Binding to Canine CD40 by Surface Plasmon Resonance
[0285] In Example 4, cross-species binding for a set of novel anti-CD40 antibodies was assessed to both mouse CD40 (mCD40) and cynomolgus CD40 (cCD40). In this Example, a subset of the same antibodies was analyzed for binding to canine CD40 (caCD40) by surface plasmon resonance (SPR).
Materials and Methods
[0286] The SPR experiments were run on a BIAcore T200 instrument (GE Healthcare) using a single cycle kinetics (SCK) approach. An α-Fab antibody (GE Healthcare #28958325), functioning as capture ligand, was immobilized through EDC/NHS chemistry onto all four surfaces of a CM5-S amine sensor chip according to the manufacturer's recommendations. Seven Protein A purified IgG2 antibodies from Example 3 (Y-SM083-A1, Y-SM083-A9, Y-SM083-B1, Y-SM083-B8, Y-SM083-F7, X-SM083-Ab-2, X-SM083-Ab-5) were injected and captured onto the chip surface, aiming for equal response units (RU) between clones (400-500 RU). A five-fold dilution series of hCD40-Fc (Sino Biological, #10774-H38H) and caCD40-Fc (Sino Biological, #70105-D02H) at five concentrations ranging between 0.16-100 nM was prepared in running buffer (HBS supplemented with 0.05% Tween-20 at pH 7.5) and sequentially injected over the chip surfaces, using an association time of 120 seconds. Following a dissociation phase of 600 seconds, the surfaces were regenerated with 10 mM glycine-HCl, pH 2.1.
[0287] In order to get a more reliable determination of the kinetic constants for CP-870,893 (X-SM083-Ab-5), the experimental parameters were optimized in a follow-up experiment. Here, the antibody capture levels were decreased from approximately 400 to 200 RU, the antigen concentration range was decreased to 0.62-50 nM (five concentrations, three-fold dilution series), and the association and dissociation time increased to 240 and 1200 seconds, respectively. By subtracting the response curve of a reference surface having an α-kappa antibody immobilized thereto, response unit sensorgrams for all antibodies were obtained. Data was analysed using the software BIAeval v.3.1 (GE Healthcare).
Results
[0288] As expected, all analysed antibodies showed binding towards human CD40. The obtained sensorgrams (data not shown) looked similar to those obtained previously, and kinetic parameters were similar to those reported in Table 2. In contrast, only one of the antibodies, namely X-SM083-Ab-5 (CP-870,893), also exhibited binding to canine CD40. However, the affinity of Ab-5 for canine CD40 was approximately 10 times lower than for the human orthologue (0.2 vs 2.5 nM), which is mainly due to a faster dissociation time for the former interaction. In Table 3, the obtained kinetic parameters for Ab-5 (CP-870,893) towards human and canine CD40 are given. Both antigens are Fc fusions and hence dimeric, so will most likely give rise to kinetic constants with avidity effect contributions. Therefore, .sup.appK.sub.D rather than K.sub.D is given. Despite the potential avidity contribution, we selected a Langmuir 1:1 binding model for calculation of .sup.appK.sub.D for the interaction to both human and canine CD40.
TABLE-US-00003 TABLE 3 K.sub.a k.sub.d .sup.appK.sub.D (1/Ms) (1/s) (nM) Model Human CD40-FC 1.9E+05 3.6E−05 0.2 1:1 Binding Canine CD40-Fc 1.7E+05 4.1E−04 2.5 1:1 Binding
Conclusions
[0289] The difference in cross-species binding between the different anti-CD40 antibodies can most likely be explained by difference in epitope. The CD40 ligand inhibition experiment presented below in Example 6 grouped X-SM083-Ab-5 and Y-SM083-A9 together, as they were the only two antibody candidates that were seemingly unaffected by the presence of CD40 ligand. Nevertheless, the data presented here show that only Ab-5 (CP-870,893) binds canine CD40, which suggest that these antibodies bind to different epitopes on CD40. Surprisingly, and contradictory to the finding here, the inventors in US2006-0093600 state that CP-870,893 does not bind to canine (dog) CD40. The reason for the discrepancy between what is reported in US2006-0093600 and the results of this SPR study is currently not known.
Example 6
CD40 Ligand Inhibition
[0290] This experiment describes, using an SPR-based approach, blocking of/interference with the interaction between CD40 ligand (CD40L) and human CD40 by eleven anti-CD40 antibodies.
Materials and Methods
[0291] An α-human Fab antibody (GE Healthcare #28958325), functioning as a capture ligand, was immobilised onto all four surfaces of a CM5-S amine chip according to manufacturer's recommendations. Eleven anti-CD40 antibodies (listed in Table 2 in Example 4) were injected and captured on the chip surface, aiming for equal response units (RU) between clones. Human CD40-Fc (RnD systems #1493-CDB) was diluted to 20 nM in running buffer (HBS supplemented with 0.05% Tween20) and injected either alone or pre-incubated with 10 times molar excess of human CD40 ligand (RnD Systems #6420-CL). A single injection of 200 nM CD40 ligand alone was also performed. Following a dissociation phase, the chip surfaces were regenerated with 10 mM glycine-HCl, pH 2.1. The anti-NP antibody B1-8 (Example 3) was included as a negative control.
[0292] By subtracting the response curve of a reference surface (an α-human Fab antibody immobilised surface), response sensorgrams and binding levels (response units (RU)) for each antibody interacting with hCD40-Fc alone or pre-incubated with CD40L could be retrieved. Data was analysed using software BIAeval v.3.1 (GE Healthcare).
Results
[0293] An α-human Fab antibody mix was successfully immobilised on all four surfaces of the chip and similar capture levels could be obtained for all antibody clones. Following injection of analyte, chip surfaces could successfully be regenerated leaving an active surface ready for the next antibody-capture cycle.
[0294] Obtained binding units (RU) for each antibody towards CD40-Fc were compared with obtained binding units for CD40-Fc pre-incubated with CD40 ligand (
[0295] As expected, BI-8 hIgG2 did not display binding towards CD40-Fc, nor towards CD40-Fc pre-incubated with CD40 ligand or CD40 ligand alone (data not shown).
Conclusions
[0296] The data obtained here is largely consistent with what has been described in the literature. Yu et al. (Cancer Cell 33(4): 664-675, 2018) demonstrated that the agonistic antibody CP-870,893 i.e. the antibody referred to here as X-SM083-ab-5, binds the membrane-distal cysteine-rich domain 1 (CRD1) of CD40 without blocking CD40 ligand interaction. Similarly, the epitope of X-SM083-ab-2 (1150/1151) has been reported as located in this domain. No competition between this antibody and CD40 ligand was observed in previous studies (WO 2015/091853). In contrast, here, we do observe a small effect on antibody binding when ligand is present. The discrepancy could be explained by the different methods used.
Example 7
Agonistic Activity of Novel Binders
[0297] The agonistic activity of eleven CD40 binders selected for continued study was tested, as determined by their ability to activate dendritic cells. Dendritic cell activation was assessed based on the up-regulation of activation markers and IL-12 expression in response to antibody binding.
Materials and Methods
Human Monocyte-Derived DC (MoDC) Differentiation
[0298] Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats, donated by healthy volunteers, by Ficoll separation using SepMate (85450, Stemcell Technologies) together with cell density gradient Ficoll®-Paque Premium (17-5442-02, GE Healthcare) according to the manufacturer's protocol. Further separation of CD14.sup.Pos (CD14+) monocytes was performed by positive selection using MACS cell separation CD14+ beads according to the manufacturer's protocol.
[0299] Solutions and cells were kept on ice for the duration of the isolation with wash steps performed in PBS (0.5% BSA, 2 mM EDTA). Purity of the isolated CD14.sup.+ cell fraction was evaluated by flow cytometry (for CD14 expression). Isolated CD14+ monocytes were cultured with a density of 4×10.sup.5 cells/ml in ø10 cm tissue-treated culture plate. The cells were cultured for six days in RPMI medium supplemented with 10% FBS, 1% Pen-Strep, 1% HEPES and 0.2% β-mercaptoethanol supplemented with 150 ng/ml hGM-CSF (PreProtech) and 50 ng/ml IL-4 (PreProtech) at 37° C. in 5% CO.sub.2. Half of the medium was exchanged with complete medium supplemented with hGM-CSF (150 ng/ml) and IL-4 (50 ng/ml) at day 3 and day 5.
MoDC Activation
[0300] Following on from above, on day 6, cultured monocytes were harvested and their differentiation status evaluated by flow cytometry by staining for CD14 and CD1a. DC activation was performed in a 96-well TCT plate (Standard F, Sarsted) with 1×10.sup.5 immature MoDC seeded per well in complete medium supplemented with hGM-CSF and IL-4 (concentrations used as previously described) using a panel of different anti-CD40 antibodies. 1:2 serial dilutions were performed starting from 500 nM. Supernatant was collected after 48 h of culture for IL-12 ELISA and cells were analysed by flow cytometry for surface marker expression.
[0301] Flow cytometry analysis was performed by staining of extracellular expression of CD14-APC-Cy7 (HCD14), HLA-DR-PE-Cy5 (L243), CD86-BV421 (IT2.2), CD40-FITC (5C3), CD1a-PE (H1149) and CD83-APC (HB15e). All antibodies were purchased from BioLegend. The cells were incubated with the antibodies for 20 min at 4° C. and thereafter washed with PBS containing 3 mM EDTA and centrifuged for 5 min at 1500 rpm.
tSNE Analysis
T-Distributed Stochastic Neighbor Embedding (t-SNE) was performed on the flow cytometry data (Rtnse package in R, https://github.com/jkrijthe/Rtsne; van der Maaten and Hinton (2008), J Mach Learn Res 9:2579-2605; van der Maaten (2014), J Mach Learn Res 15:3221-3245) to identify antibodies with similar properties to previously validated Ab1 (X-SM083-Ab-1) and Ab2 (X-SM083-Ab-2). LPS was used as an additional positive control. MFI (mean fluorescence intensity) values from the CD14-APC-Cy7 (HCD14), HLA-DR-PE-Cy5 (L243), CD86-BV421 (IT2.2), CD40-FITC (5C3), CD1a-PE (H1149) and CD83-APC (HB15e) panel were used as variables when performing the analysis. Scaling was performed for each marker prior to t-SNE analysis. Perplexity was set to 2 in all analyses.
IL-12 ELISA
[0302] The ELISA was performed using the human IL-12p40 ELISA Maxi kit (430704, BioLegend). Costar high binding 96-well plates (Sarstedt) were coated with human IL-12p40 capture antibodies. Plates were blocked with 1× Assay Diluent A for 1 h on a shaking table (500 rpm). All incubations were at RT with shaking. Washing was performed 4 times with 300 μl/well of PBS containing 0.05% Tween-20 (Sigma-Aldrich). The washing procedure was repeated after each step if not stated otherwise. The samples were diluted in 1× Assay Diluent A, as for the standard. IL-12 standard was diluted 1:1 with a starting concentration of 4000 μg/ml. The samples and standard were added to the plate and incubated for 2 h. Subsequently, the plate was incubated with 1× detection antibody (100 μl/well) for 1 h. Lastly, 1× Avidin-HRP solution (100 μl/well) was added to all wells and incubated for 30 min at RT with shaking. Plates were washed 5 times, soaked for 30-60 s per wash. TMB solution (100 μl/well) was added and incubated for 15 min before reactions were stopped with an equal amount of 1 M H.sub.2SO.sub.4. Absorbance was measured at 450 nM with FLUOstar Omega (BMG Labtech).
Results
[0303] The result of the tSNE analysis is shown in
[0304] The results of the IL-12 ELISA are similar (
Example 8
Epitope Mapping by HDX-MS
[0305] In this Example hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed on the Y-SM083-A9 antibody together with human CD40 in order to map its epitope.
[0306] When proteins are dissolved in a buffer containing heavy water (D.sub.2O), their hydrogen atoms attached to heteroatoms (e.g. in the context of —OH, —NH or —SH groups) are replaced by deuterium. The degree of deuterium incorporation can be monitored by MS since each D atom is one mass unit heavier than an H atom. Additionally, when deuterium labelling is further combined with enzymatic proteolysis, the deuteration profile of different areas within the protein can be monitored. Binding of a ligand to a protein target produces local changes in hydrogen bonding, e.g. structure stabilisation or destabilisation, and thus the HDX-MS technique can be used to identify the binding interfaces of protein complexes.
Materials and Methods
[0307] 4 μl of a 0.92 mg/ml hCD40-Fc (RnD Systems, #1493-CDB) solution in PBS was mixed with 105.7 μl of 1 mg/ml Y-SM083-A9 for a 1:1 molar ratio. The CD40/antibody complex was concentrated to 36 μl using a 10K Centrifugal filter unit (Amicon Ultra, Merck). In parallel, a sample containing hCD40-Fc only, without the addition of antibody, was prepared analogously. The samples were analysed in an automated HDX-MS system (CTC PAL/Biomotif HDX) in which samples were automatically labelled, quenched, digested, cleaned and separated at 2° C. More specifically, samples were labelled by mixing 4 μl of hCD40-Fc (or hCD40-Fc/antibody complex) with 24 μl of deuterated PBS and incubated at 4° C. for three labelling time points: 10 min, 25 min and 60 min.
[0308] The labelling reaction was stopped/quenched by decreasing the pH to ˜2.3 and temperature to ˜4° C. through the addition of 25 μl of a solution containing 6 M Urea, 417 mM TCEP and 0.5% TFA. Samples were digested using an immobilised pepsin column (2.1 column (2.1×30 mm) at 60 μl/min for 2 min, followed by an on-line desalting step using a 2 mm I.D×10 mm length C-18 pre-column (ACE HPLC Columns, Aberdeen, UK) using 0.2% formic acid at 400 μl/min for 1 min. Peptic peptides were then separated by an 18 min 8-55% linear gradient of ACN in 0.1% formic acid using a 2 mm I.D×50 mm length HALO C18/1.8 μm analytical column operated at 60 μl/min. An Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific) operated at 70,000 resolution at m/z 400 was used for analysis. The software Mascot was used for peptide identification and HDExaminer (Sierra Analytics, USA) was used to process all HDX-MS data. Statistical analysis was performed using a 95% confidence interval.
Results
[0309] The deuteration kinetics of 25 peptides were followed by HDX-MS covering 50% of the protein construct. Deuterium labelling (10, 25 and 60 min) and differential deuteration uptake kinetics between CD40 alone and in the presence of Y-SM083-A9 were calculated. Peptides close to the N-terminus showed statistically lower deuterium uptake in the presence of Y-SM083-A9 antibody, mapping the epitope to this region (Table 4).
TABLE-US-00004 TABLE 4 CD40 Peptides with Lowered Deuterium Exchange Identified by HDX-MS Amino Acid Peptide Positions Sequence SEQ ID NO: 1 21-30 EPPTACREKQ 63 2 21-32 EPPTACREKQYL 64 3 58-67 ECLPCGESEF 65
Peptides 1 and 2 are overlapping and located in the N-terminus of CRD1 of CD40 and peptide 3 is located in the N-terminus of CRD2 (or in the bridging sequence between CRD1 and CRD2, depending on how this is defined). It is possible that these constitute two parts of a conformational epitope recognised by A9.
Example 9
Epitope Mapping by Comparison with the Anti-CD40 Antibody 5C3
[0310] In this Example, flow cytometry and competitive binding are used to compare the CD40 epitope bound by the novel antibody A9 with that of known antibody CP-870,893 (Ab-5), using the known CD40-binding antibody 503 with a fluorescent label as a reference. If 503 staining does not occur after cells have been treated with the A9 or Ab-5 antibody, it means that there is a steric hindrance which prevents simultaneous binding. In other words, if there is no 503 staining after exposure to a tested candidate antibody, it would indicate that the tested antibody and 503 share similar epitopes on the CD40 receptor.
Materials and Methods
[0311] PBMC were isolated as in Example 7. In this Example, the CD14 negative cell fraction was used for incubation with 25 nM-50 nM of the antibodies A9 and Ab-5 for 30 min on ice to let the antibodies bind their target without internalisation. After terminated incubation, the cells were washed once with cold PBS, and then stained with CD3-BV510 (clone: UCHT1), CD19-APC (clone: H1149) and CD40-FITC (clone: 5C3) as described in Example 7. The cells were analysed in a Cytoflex flow cytometer (Beckman Coulter), and then the CD19+ cells were gated out to investigate CD40 expression. The experiment was performed on two separate donors, both stained with the same labelled anti-CD40 clone (5C3) and using the same experimental setup.
Results
[0312] In
Conclusion
[0313] The results indicate that the known antibody Ab-5 (CP-870,893) and the novel antibody A9 do not share the same CD40 binding epitope, because Ab-5 blocks binding of the labelled 5C3 antibody, while A9 does not.
Example 10
Internalisation of the A9 Antibody
[0314] In the context of the present invention, internalisation of an agonistic CD40 antibody is of great importance for its ability to deliver a cargo in the bispecific format. This Example studies the ability of the novel A9 antibody to be internalised into dendritic cells (DCs).
Materials and Methods
[0315] MoDCs were isolated and cultured as described in Example 7. At day 6, the cells were harvested and re-plated into two 96-well tissue treated plates and incubated for 2 h at 37° C. with 5% CO.sub.2. The test antibodies Ab-2 and A9 were then added at a concentration of 125 nM and incubated on ice for 30 min to allow receptor binding. The cells were then washed three times with cold, serum-free RMPI and centrifuged at 250 g for 5 min at 4° C. The supernatant was discarded between washes. The cells were resuspended in warm complete medium for internalisation and in cold medium for use in a control plate. One plate was incubated at 4° C. the whole time and one plate was incubated at 37° C. with 5% CO.sub.2. The incubation was terminated at different time-points from 5 min to 4 h by addition of cold PBS. Antibodies bound to the surface of cells were then detected through staining with an anti-kappa-APC (cat no: 9230-11, Biolegend) antibody for 30 min at 4° C. before analysis with flow cytometry (Cytoflex). The degree of internalisation was calculated as follows:
Result
[0316] The internalisation of the A9 (monoclonal IgG2) antibody was compared to the Ab-2 antibody (1150 as monoclonal IgG2). As shown in
Example 11
Design and Generation of Bispecific Antibodies (Conjugates)
[0317] A number of bispecific antibodies (bispecific conjugates) based on A9 were generated. For comparison, bispecific antibodies based on B8, which displays moderate agonistic activity, were also generated. Details of the bispecific antibodies generated are set forth in Table 5.
TABLE-US-00005 TABLE 5 Bispecific Antibodies scFv Append- Anti-CD40 ing Link- IgG Subclass scFv Position er X-SM083-bi-21 Y-SM083-A9 IgG2 14GIIICII-b CH3 (G.sub.4S).sub.2 X-SM083-bi-22 Y-SM083-A9 IgG2 14GIIICII-b CL (G.sub.4S).sub.2 X-SM083-bi-23 Y-SM083-A9 IgG2 FITC8 CH3 (G.sub.4S).sub.2 X-SM083-bi-24 Y-SM083-B8 IgG2 14GIIICII-b CH3 (G.sub.4S).sub.2 X-SM083-bi-25 Y-SM083-B8 IgG2 14GIIICII-b CL (G.sub.4S).sub.2 X-SM083-bi-26 Y-SM083-B8 IgG2 FITC8 CH3 (G.sub.4S).sub.2 X-SM083-bi-28 Y-SM083-A9 IgG2 FITC8 CL (G.sub.4S).sub.2 X-SM083-bi-29 Y-SM083-A9 IgG2 FITC8 CH3 (G.sub.4S).sub.2 C127S X-SM083-bi-30 Y-SM083-A9 IgG2 FITC8 CL (G.sub.4S).sub.2 C127S X-SM083-bi-31 Y-SM083-A9 IgG2 FITC8 CH3 G.sub.4S C127S X-SM083-bi-32 Y-SM083-A9 IgG2 FITC8 CH3 (G.sub.4S).sub.4 C127S X-SM083-bi-33 Y-SM083-A9 IgG2 FITC8 CL (G.sub.4S).sub.2 C127S
[0318] The 14GIIICII-b scFv is described above, and binds a known B cell epitope derived from tetanus toxin and denoted MTTE. The FITC8 scFv binds FITC, and has the amino acid sequence set forth in SEQ ID NO: 66 (originally described in Söderlind et al, Nature Biotechnology 18(8): 852-856, 2000). As described above, the scFvs were encoded as a single polypeptide chain with either the heavy chain or the light chain of the anti-CD40 antibody. As set forth in the table, in each bispecific antibody the scFv was fused to the C-terminus of the relevant antibody chain, and thus was located either C-terminal to the heavy chain C.sub.H3 domain or to the light chain C.sub.L domain, as shown. As previously described, in each bispecific antibody the scFv was joined to the antibody by a linker as indicated in Table 5.
[0319] As previously described, bispecific antibodies were expressed in HEK293 cells, and purified by affinity chromatography using protein A followed by preparative size exclusion chromatography (SEC). Retained binding to hCD40 and MTTE or FITC was verified by ELISA.
Example 12
Activation of Dendritic Cells with Bispecific Antibodies
Materials and Methods
Dendritic Cell Microscopy
[0320] Pictures of cultured human moDCs were taken at 10× magnification using a Visiscope microscope with a Moticam 1080 camera.
IL-12 ELISA
[0321] ELISA experiments were performed as described above in Example 7, except the 1:2 serial dilutions were performed starting from 200 nM.
Flow Cytometry
[0322] Flow cytometry was performed as described above in Example 7, using the antibodies HLA-DR-PE-Cy5 (L243), CD86-BV421 (IT2.2) and CD83-APC (HB15e) to measure expression of their target cell surface activation markers.
Results
[0323] Upon activation, dendritic cells form characteristic clusters (as shown by the appearance of unactivated dendritic cells (
[0324] As expected for a moderate CD40 agonist, the B8 antibody induced some clustering, indicating a degree of dendritic cell activation, particularly at 200 nM (
[0325] When analysing dendritic cell activation by IL-12 production, the A9 clone induced substantial cytokine release, while the A9-based bispecific antibodies also induced cytokine release but not to the same levels. Bi-23 induced the highest levels of IL-12 among the bispecific antibodies derived from the A9 clone and Bi-21 the lowest levels (
[0326] Similarly, both A9 and B8 induced expression of the dendritic cell surface activation markers HLA-DR (an MHC-II component,
Conclusions
[0327] These results unexpectedly showed that B8, despite displaying a moderate degree of agonistic activity in the context of a standard monoclonal antibody, lost all agonistic activity in the bispecific context. This demonstrates that not all anti-CD40 antibodies are suitable for use in the bispecific antibodies of the invention. The A9 antibody has high agonistic activity, and although some degree of agonism is lost when A9 is converted to bispecific format, sufficient activity is generally retained for efficacy. Accordingly, it is clear that A9 is a particularly suitable antibody for use in the bispecific conjugates of the invention.
[0328] The complete loss of agonism in bispecific format is a phenomenon which, to our knowledge, has not been previously observed. The location of the B8 epitope has not been established, but the antibody is known to bind at a site close to or overlapping with the CD40L binding site, since interaction of CD40 with CD40L blocks binding of the antibody (
Example 13
Activation of Dendritic Cells by Additional Bispecific Conjugates
[0329] In this Example, additional bispecific constructs derived from the A9 clone and prepared as described in Example 11 were used to evaluate the impact of linker length together with the C127S mutation in the IgG2 constant region. The scFv in the tested bispecific conjugates was a human anti-FITC scFv, positioned either at C.sub.H3 or CL as indicated in Table 5. The Example was performed to investigate the potential steric hindrance by the scFv due to linker length and how it affects the agonistic capacity of the bispecific conjugate.
Materials and Methods
[0330] CD14+ cell isolation and differentiation were performed as described in Examples 7 and 12, with the exception that 75 ng/ml GM-CSF was used. MoDCs were treated with antibody A9 with IgG2 or IgG2 C127S constant regions or bispecific test conjugates Bi-23, Bi-28, Bi-29, Bi-30, Bi-31, Bi-32, Bi-33 at concentrations between 100-12.5 nM, or with 1 μg/ml of LPS as positive control, for 48 hours before supernatants were collected and IL-12 production was measured using IL-12p40 ELISA as described in Example 7.
Results
[0331] IL-12 production from MoDCs in a titration of A9 and the A9 based bispecific antibodies (Bi23, Bi28) and the A9 C127S and A9 C127S bispecific antibodies (Bi29, Bi30, Bi31, Bi32 and Bi33) was measured, and the results are presented in
Example 14
Design and Generation of Further Bispecific Conjugates
Materials and Methods
Design and Protein Expression in CHO Cells
[0332] Further variants of antibodies and bispecific conjugates according to the disclosure were designed. Briefly, the three different antibody isotypes were used: IgG1, IgG2, and a hybrid consisting of said IgG1 sequence with human IgG2 hinge and C.sub.H1 sequence grafted into the IgG1 architecture. In bispecific conjugates, a humanized tag-binding scFv sequence was linked C-terminally to the C.sub.H3 domain to either of the three isotype variants by either a flexible (G.sub.4S).sub.2 or rigid (EA.sub.3K).sub.2 linker. DNA encoding each variant was transfected into cells using the ExpiCHO™ transfection system (ThermoFisher) in 24 deep well plates. Transfection was performed in duplicates with a total of 2 μg plasmid DNA in 2.5 ml culture volumes. For scaled-up production, ExpiCHO™ cells were transfected as suggested by the manufacturer with a total of 20 μg plasmid DNA per transfection of each construct, in 25 ml culture volumes.
IgG Quantification
[0333] IgG concentrations in the supernatant of transfected CHO cells were determined by bio-layer interferometry measurements in an Octet® RED96e system (Fortébio Biologics by Molecular Devices, USA) with Dip and Read™ Protein A biosensors (Fortébio Biologics by Molecular Devices) according to the manufacturer's instructions. The supernatant samples from day 5 of cultivation were diluted 1:1 in 20 mM citric acid pH 4.0, 0.1% BSA (w/v), 0.1% Tween-20, 0.5 M NaCl. A standard curve was prepared from a sample IgG with concentrations from 700 to 1 μg/ml.
IgG Purification
[0334] For size exclusion chromatography analysis, the expressed constructs were purified by Protein A facilitated purification on an AktaSTART system (GE Healthcare) using mAbSelect SuRe columns (GE Healthcare). A 20 mM sodium phosphate, 0.15 M sodium chloride (pH 7.3) buffer was used as binding and wash buffer, 0.1 M glycine (pH 2.5) as elution buffer and 1 M Tris—HCl (pH 8.5) as neutralization buffer. Endotoxin levels were measured with LAL Cartridges and Endosafe Nextgen-PTS system (Charles River, Mass., USA) according to manufacturer's instructions.
SDS-PAGE
[0335] A total of 4 μg of each sample purified as above were mixed with 3× loading buffer (0.1 M Tris—HCl, 45% glycerol, 0.03% bromophenol blue, 0.3% SDS) for non-reducing conditions and, for the reducing analysis, mixed with 3× loading buffer containing 0.15 M Tris 2-carboxyethyl-phosphine hydrochloride and incubated at 95° C. for 7 min. The samples were run on a 4-20% Criterion™ TGX Stain-Free™ protein gel (Bio-Rad Laboratories) according to the company's protocol. The bands were visualized by staining the gel in GelCode™ Blue Safe protein stain (Thermo Fisher Scientific) for 1 h at room temperature and gentle shaking.
Size Exclusion Chromatography (SEC)
[0336] In total, 25 μg of each expressed conjugate in 100 μl were injected onto a Superdex Increase 200 10/30 GL gel filtration column (GE Healthcare) coupled to an Agilent 1200 series HPLC system (Agilent Technologies). SEC runs were performed at a 0.5 ml/min flow rate with PBS as a running buffer. Eluted protein fragments were detected by an online 280 nm absorption measurement. Data analysis and peak integrations were performed using GraphPad prism 8.0 (GraphPad Software).
Results
Expression and Characterisation of Novel Antibodies and Bispecific Conjugates
[0337] The designed constructs (Table 6) were successfully expressed and purified with endotoxin levels below the threshold limit of 0.5 EU/ml. A reducing SDS-PAGE on the purified samples exhibited bands at the expected sizes for the antibody heavy and light chains respectively.
Size Exclusion Chromatography
[0338] The SEC analysis showed no signs of significant amounts of heavy molecular weight aggregates in the purified constructs. Non-native populations in the form of lower molecular weight species were observed across the tested constructs to varying degrees.
Summary of Expressed Antibodies and Bispecific Conjugates
[0339] The antibodies and bispecific conjugates listed in Table 6 were successfully designed, expressed and purified. In all bispecific conjugates, a variant of the previously tested scFv:s was used as second binding protein.
TABLE-US-00006 TABLE 6 Further Antibodies and Bispecific Conjugates Anti- scFv CD40 Appending Designation IgG Subclass Position Linker SP2 A9 IgG1 CH3 (G.sub.4S).sub.2 SP3 A9 IgG1 CH3 (EA.sub.3K).sub.2 SP4 A9 IgG 1/2 hybrid No scFv None SP5 A9 IgG 1/2 hybrid CH3 (G.sub.4S).sub.2 SP6 A9 IgG 1/2 hybrid CH3 (EA.sub.3K).sub.2 SP7 A9 IgG2 CH3 (G.sub.4S).sub.2 SP8 A9 IgG1 No scFv None SP9 (=A9) A9 IgG2 No scFv None
Example 15
Activation of Dendritic Cells with Further Antibodies and Bispecific Conjugates
[0340] In this Example, the constructs prepared as described in Example 14 were analysed for their agonistic activity.
Materials and Methods
[0341] Isolation and differentiation of CD14+ cells were performed as described in Examples 7, 12 and 13 using 75 ng/ml of GM-CSF and 50 ng/ml IL-4. The MoDCs were treated with antibodies or bispecific conjugates with and without the tag peptide 0001 (SEQ ID NO: 79) for 24 h or 48 h, before supernatants were collected and IL-12 production was measured using IL-12p40 ELISA. For the 24 h incubation, cells were additionally stained for the previously described activation markers.
Results
[0342] The measured IL-12 production is summarized below in Table 7, giving the relative % activity after 48 h, compared to the A9 (herein named SP9) antibody.
TABLE-US-00007 TABLE 7 % agonistic activity of A9 Construct at 25 nM SP2 9 SP3 70 SP4 123 SP5 82 SP6 23 SP7 105 SP8 11 SP9 100 (reference)
Table 7 summarizes the agonistic activity of the tested constructs SP2-SP8 in comparison with the SP9 (A9) antibody. IgG1-based antibodies, i.e. the parental antibody SP8 and bispecific versions SP2 and SP3, had lower agonistic activities than the reference. The IgG1/2 hybrid versions, i.e. the parental antibody SP4 and bispecific versions SP5 and SP6, had an increased agonistic activity compared to the IgG1-based clones. SP5 (flexible linker) retained agonistic activity better than SP6 (rigid linker). Furthermore, SP7, the bispecific version of the reference A9 IgG2 antibody retained similar agonistic activity.
[0343] Also, the parental (Ab5) and bispecific version of the CP-870,893 antibody (Bi2) (see WO 2020/104690) were assessed side-by side with the different A9-based constructs. Bi2 carries the murine 14GIIICII-b clone as scFv. In this version, the agonistic activity of Bi2 was 43% of the parental antibody Ab-5 (CP-870,893). The A9 based bispecific antibody Bi23 carrying the FITC8 scFv retained 92% of the agonistic activity when compared with the SP9 (A9) parental version.
[0344] Further, the agonistic activities of the bispecific conjugate SP7 and parental antibody SP9 (A9) were assessed in the presence of the 0001 peptide (SEQ ID NO: 79). Binding of peptide to the bispecific antibody did not affect the agonistic activity in terms of upregulation of activation markers (
Example 16
In Vitro Assay of CMV-Specific T Cell Proliferation
[0345] Interaction with an activated dendritic cell (DC) is required for efficient activation of a T cell. A T cell receptor (TCR) will recognize a peptide loaded onto an MHC molecule on the DC, and then interact with the co-stimulatory molecules CD83/CD86 through the CD28 receptor. Cytokines like IL-12 will be produced by the activated DC that acts on the T cell. This Example is essentially a repetition of Example 4 on pages 64-65 of WO 2020/104690, performed in order to also compare the ability of a bispecific conjugate based on the novel A9 antibody to stimulate the expansion of a pathogen-specific population of T cells, in comparison with the bispecific conjugate denoted Bi-17 in WO 2020/104690, which is based on the known CD40 binding antibody CP-870,893. It is shown that the bispecific conjugate based on the A9 antibody induces activation through CD40 binding, and that it transports the tag/antigen peptide intracellularly through the interaction between the second binding protein (scFv) and the tag. The fluorescence of the tag provides a detectable signal.
Materials and Methods
[0346] An HLA*2A and CMV positive donor was used. CD14+ cell isolation and differentiation was performed essentially as described in Examples 7, 12, 13 and 15. At day 6, the MoDCs were stimulated with the bispecific conjugates Bi-23 (Table 5) and Bi-17 (Table 1 on page 53 or WO 2020/104690), each tested both alone and in complex with the peptide designated UU-44 (SEQ ID NO: 87), which comprises a FITC tag for interaction with the scFv part of the respective bispecific conjugate and an epitope from cytomegalovirus (CMV). In the complexes, the ratio between conjugate and peptide was 1:2 at a concentration of 50 nM of conjugate. The bispecific conjugates and complexes were incubated for 24 h at 37° C. with 5% CO.sub.2. Stimulated DCs were split into two replicates in a 6-well plate. The CD14 negative population from the same donor was thawed and mixed at a 1:10 ratio with the stimulated DCs. The co-culture was incubated for another 11 days before specific CMV CD8.sup.+ T cells were measured. Cells were stained as described in Example 7 with CMV tetramer-PE (MBL International; cat. no. TB-0010-1), CD3-BV510 (clone: UCHT1) and CD8-FITC (clone: SK1). Samples were pre-incubated with the CMV tetramer for 10 min at room temperature before staining with antibodies.
Results
[0347] The results are shown in
Example 17
Determination of the Minimal Epitope Recognised by 14GIIICII and IBIIICI
[0348] Mouse monoclonal antibodies 14GIIICII and IBIIICI were originally raised by immunisation of mice with the 18-mer MTTE peptide (FIGITELKKLESKINKVF, SEQ ID NO: 25). Conversion of these two antibodies to scFv-format and subsequent binding characterisation by SPR of binding to the MTTE is described in Example 10 of WO 2020/104690.
[0349] In the below example we demonstrate that C-terminal shortening of the MTTE sequence can be performed without loss of scFv binding. A shortening of the peptide can potentially have several advantages for production purposes, including more efficient protection of the whole tag construct (tag peptide plus tumour/pathogen antigen) from degradation by e.g. proteases and more efficient manufacture.
Materials and Methods
[0350] The peptides (Capra Science, Lund, Sweden) used in this example are listed in Table 8:
TABLE-US-00008 TABLE 8 Peptide Sequence Length (aa) MW (Da) Modification SEQ ID NO UU24 FIGITELKKLESKINKVF 18 2462.0 C-term K- 25 biotin UU70 FIGITELKKLESKINK 16 1861.3 none 29 UU71 FIGITELKKLESKIN 15 1733.1 none 28 UU72 FIGITELKKLESKI 14 1619.0 none 27 UU73 FIGITELKKLESK 13 1505.8 none 26 UU74 FIGITELKKLES 12 1377.6 none 16
Affinity measurements for the 14GIIICII and IBIIICI scFv clones were performed by SPR using a Biacore T200 platform (GE Healthcare) and a single cycle kinetics (SCK) approach. The α-FLAG M2 antibody (Sigma-Aldrich) was immobilised onto a CM5-S chip by primary amine coupling using NHS-EDC chemistry, allowing capture of the scFvs via their FLAG tags. A 5-fold dilution series comprising five different concentrations (0.16 nM to 100 nM) of the different peptides (Table 8) was sequentially injected over the flow cells, allowing binding to the captured scFvs. Following a dissociation phase, regeneration of the surface was accomplished under acidic conditions using 10 mM glycine-HCl at pH 2.2. By subtracting the response curve of a reference surface (an α-FLAG antibody immobilised surface), response unit sensorgrams for all peptides were obtained. Data was analysed using the software BIAeval v.3.1 (GE Healthcare).
Results
[0351] Similar capture levels (RU) could be obtained for all cycles. Following injection of analyte, chip surfaces could successfully be regenerated leaving an active surface ready for the next antibody-capture cycle.
[0352] Surprisingly, the binding of the IBIIICI scFv is seemingly unaffected by shortening of the peptide from 18 to 12 amino acids. Visual inspection of the sensorgrams (not shown) shows a very similar binding pattern to all of the tested peptides. This is illustrated in Table 9, where the kinetic parameters of the different peptides are given, and in
[0353] The results presented here suggest that the MTTE tag peptide can be shortened to 12 or 13 amino acids without considerable loss of binding to IBIIICI or 14GIIICII, respectively. The kinetic parameters reported here differ somewhat to those reported previously for the same scFv clones. This discrepancy could be explained by differences in experimental set-up, including different peptide designs. Also, the obtained k.sub.d values are very close to the limit of detection of the instrument (<10.sup.−5). This is particularly true for the different peptides binding to IBIIICI. In order to get a more accurate estimation of the off-rates a considerably longer dissociation time is recommended. Hence, the reported kinetic parameters should be viewed with care and should primarily be used to compare the binding of the different peptides to the two scFv rather than as providing accurate absolute kinetic parameters.
TABLE-US-00009 TABLE 9 IBIIICI scFv 14GIIICII scFv 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) UU24 1.9E+05 2.3E−05 1.2E−10 6.6E+05 2.0E−04 3.0E−10 UU70 2.3E+05 3.7E−05 1.6E−10 6.5E+05 1.9E−04 2.9E−10 UU71 2.3E+05 * n/a 5.4E+05 3.2E−04 6.0E−10 UU72 2.5E+05 * n/a 5.7E+05 3.4E−04 6.0E−10 UU73 2.0E+05 6.7E−05 3.4E−10 5.1E+05 4.4E−04 8.7E−10 UU74 2.8E+05 3.3E−05 1.2E−10 5.7E+05 9.5E−04 1.7E−09 Kinetic parameters for 14GIIICII and IBIIICI scFv clones towards the MTTE sequence and trimmed versions thereof. The dissociation rate constant (k.sub.d) values reported as “*” indicate that these are too low for, or very close to the detection limit of, instrument specification (<10.sup.−5).
SEQUENCE LISTING
[0354]
TABLE-US-00010 Sequences in upper case are protein sequences; those in lower case are nucleic acid sequences. SEQ ID NO: 1 QSISSY SEQ ID NO: 2 AAS SEQ ID NO: 3 QQGYPYPFT SEQ ID NO: 4 GFTFSSYA SEQ ID NO: 5 ISGYSGST SEQ ID NO: 6 ARYYSYYGYYYFDY SEQ ID NO: 7 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIK SEQ ID NO: 8 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTV SS SEQ ID NO: 9 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQGYPYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 10 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 11 GILFLQWVRDIIDDFTNESSQK SEQ ID NO: 12 DSDKDRFLQTMVKLFNRIKNN SEQ ID NO: 13 IKNDLYEKTLNDYKAIANKLSQV SEQ ID NO: 14 QDPALLLMHELIHVLHGLYGM SEQ ID NO: 15 DGILFLQWVRDIIDDFTNESSQKR SEQ ID NO: 16 FIGITELKKLES SEQ ID NO: 17 MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLP CGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCV LHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNK TDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSN TAAPVQETLHGCQPVTQEDGKESRISVQERQ SEQ ID NO: 18 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVV SVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 19 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20 ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVV SVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 21 GGGGS SEQ ID NO: 22 GGGGSGGGGS SEQ ID NO: 23 MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSS LIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKF DTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGF GSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIP SKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDI DSYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDP VKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPT NIRENLYNRTASLTDLGGELCIKIKNEDLTFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLD KIIVDYNLQSKITLPNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRI TMTNSVDDALINSTKIYSYFPSVISKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVS TIVPYIGPALNIVKQGYEGNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFL EKRYEKWIEVYKLVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQ IADEINNLKNKLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKA NSKFIGITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISG FNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSA SHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNA YLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKF RIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTN APSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGN AFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGND PNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND SEQ ID NO: 24 MLVRGYWSRKLFASILIGALLGIGAPPSAHAGADDWDSSKSFVMENFSSYHGTKPGYVDS IQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTK VLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINN WEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDK TKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFA GANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSS LMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWN TVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAI DGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNS KLSLFFEIKS SEQ ID NO: 25 FIGITELKKLESKINKVF SEQ ID NO: 26 FIGITELKKLESK SEQ ID NO: 27 FIGITELKKLESKI SEQ ID NO: 28 FIGITELKKLESKIN SEQ ID NO: 29 FIGITELKKLESKINK SEQ ID NO: 30 YIGITELKKLES SEQ ID NO: 31 FVGITELKKLES SEQ ID NO: 32 FIVITELKKLES SEQ ID NO: 33 FIGVTELKKLES SEQ ID NO: 34 FIGISELKKLES SEQ ID NO: 35 FIGITDLKKLES SEQ ID NO: 36 FIGITEVKKLES SEQ ID NO: 37 FIGITELVKLES SEQ ID NO: 38 FIGITELKVLES SEQ ID NO: 39 FIGITELKKVES SEQ ID NO: 40 FIGITELKKLDS SEQ ID NO: 41 FIGITELKKLET SEQ ID NO: 42 NDYKAIANKLS SEQ ID NO: 43 LMHELIHVLHGLY SEQ ID NO: 44 LMHELIHVLHGLYGM SEQ ID NO: 45 LIHVLHGLY SEQ ID NO: 46 PALLLMHELIHVLH SEQ ID NO: 47 QVQLQQPGAELVMPGASVNLSCKASGYTFTDYWMHWVKQRPGQGLEWIGEIDPSDNFSN LNQNFRGKATLTVDKSSRTAFLQLSSLTSEDSAVYYCAVEDYWGQGTTLTVSSGGGGSGG GGSGGGGSGGGGSDIVMTQATPSVLVTPGEAVSISCRASRSLLHSNGITYLYWFLQRPGQ SPQVLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEFPYTFGGGTK LEIK SEQ ID NO: 48 EVRLLQSGAALVRPGASVKLSCTASGFNIKDFNIHWVKQRPEQGLEWIGRIDPENGDAEYV PKFQVRATMTTDTSSNTVYLHLSSLTSGDTAVYYCTTGSYDLDVEYWGQGTTLTVSSGGG GSGGGGSGGGGSGGGGSELQMTQSPSSLSASLGDTVTITCHASQNINVWLSWYQQRPG NIPKLLIYKASTLHTGVPSRFRGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGAGTKL ELK SEQ ID NO: 49 GYTFTDYW SEQ ID NO: 50 IDPSDNFS SEQ ID NO: 51 AVEDY SEQ ID NO: 52 RSLLHSNGITY SEQ ID NO: 53 RMS SEQ ID NO: 54 MQHLEFPYT SEQ ID NO: 55 GFNIKDFN SEQ ID NO: 56 IDPENGDA SEQ ID NO: 57 TTGSYDLDVEY SEQ ID NO: 58 QNINVW SEQ ID NO: 59 KAS SEQ ID NO: 60 QQGQSYPLT SEQ ID NO: 61 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQGYGYPPFTFGQGTKLEIK SEQ ID NO: 62 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYVWGIDYWGQGTLVTVSS SEQ ID NO: 63 EPPTACREKQ SEQ ID NO: 64 EPPTACREKQYL SEQ ID NO: 65 ECLPCGESEF SEQ ID NO: 66 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVSGISGNGGYTY FADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDGSGWSFWGQGTLVTVSSG GGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQ QLPGTAPKLLIYGNNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGR VFGGGTKLTVL SEQ ID NO: 67 FIGITELKKLESKINKVFAVGALKVPRNQDWLGVPRQL SEQ ID NO: 68 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSSSGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGRWGYYFDYWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQISGPFTFGQGTKLEIK SEQ ID NO: 69 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTPPPSSSRSYLDYWGQGTLVT VSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPG KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYPLFTFGQGTKL EIK SEQ ID NO: 70 EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYYMYWVRQAPGKGLEWVSYIGYSGGGTG YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVPFGAFDYWGQGTLVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYGFPYTFGQGTKLEIK SEQ ID NO: 71 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPRPYSIFISIDYWGQGTLVTV SSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGTPFTFGQGTKL EIK SEQ ID NO: 72 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYSVPYSPYYSFDYWGQGTLVT VSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPG KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSPPTFGQGTKL EIK SEQ ID NO: 73 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYTYGGFPFSSFDYWGQGTLVT VSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPG KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRFLSTFGQGTKL EIK SEQ ID NO: 74 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTV SSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 75 gacatccagatgacccagtctccatcctccctgagcgcatctgtaggagaccgcgtcaccatcacctgcagggcaagtcagag cattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtg gggtcccatcacgtttcagtggcagtggaagcgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaactt attactgtcaacagggttacccgtacccgttcacttttggccaggggaccaagctggagatcaaa SEQ ID NO: 76 gaggtgcaattgttggagagcgggggaggcttggtacagcctggggggtccctgcgcctctcctgtgcagccagcggattcacc tttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaggtatttctggttacagtggttct acatactatgcagactccgtgaagggccggttcaccatctcccgtgacaattccaagaacacgctgtatctgcaaatgaacagc ctgcgtgccgaggacacggctgtatattattgtgcgcgctactactcttactacggttactactactttgactattggggccaaggaac cctggtcaccgtctcctca SEQ ID NO: 77 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 78 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 79 FIGITELKKLESKINKVFAGILARNLVPMVATVQGQNLKY SEQ ID NO: 80 EAAAK SEQ ID NO: 81 EAAAKEAAAK SEQ ID NO: 82 GGGSGGGSGGGS SEQ ID NO: 83 GGGSGGGSGGGSGGGS SEQ ID NO: 84 GGGSGGGSGGGSGGGSGGGS SEQ ID NO: 85 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 86 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGYSGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYYGYYYFDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 87 AGILARNLVPMVATVQ