Cellular based fret assay for the determination of simultaneous binding
10718762 ยท 2020-07-21
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Abstract
Herein is reported a method for the determination of the simultaneous binding of a bispecific antibody to a first and a second antigen comprising the steps of a) incubating a cell expressing cell-membrane bound FRET-donor-tagged first antigen and FRET-acceptor-tagged second antigen with the bispecific antibody, and b) determining the simultaneous binding of the bispecific antibody by determining the energy transfer from the FRET-donor to the FRET-acceptor.
Claims
1. A method for the determination of the simultaneous binding of a bispecific antibody to a first and a second antigen comprising the following steps: a) incubating a cell expressing cell-membrane bound FRET-donor-tagged first antigen and a cell-membrane bound FRET-acceptor-tagged second antigen with the bispecific antibody, and b) determining the simultaneous binding of the bispecific antibody by determining the energy transfer from the FRET-donor to the FRET-acceptor.
2. The method according to claim 1, wherein the cell-membrane bound FRET-donor and the cell-membrane bound FRET-acceptor comprise in N- to C-terminal direction: H.sub.2N-antigenFRET-donor/acceptortransmembrane domain-COOH.
3. The method according to claim 1, wherein the FRET-donor and/or acceptor is a conjugate of a tag and a FRET-donor-fluorescent dye or a FRET-acceptor-fluorescent dye, respectively.
4. The method according to claim 3, wherein the conjugate is a non-covalent conjugate and the FRET-donor-fluorescent dye and/or the FRET-acceptor-fluorescent dye is a fluorescent-dye conjugated antibody.
5. The method according to claim 3, wherein the conjugate is a covalent conjugate, the tag is an enzyme and the FRET-donor-fluorescent dye and/or the FRET-acceptor-fluorescent dye are fluorescent dye labelled suicide enzyme substrates.
6. The method according to claim 1, wherein i) the FRET-donor or FRET-acceptor is a covalent complex of a SNAP-tag and a benzyl guanine, and ii) the FRET-acceptor or the FRET-donor is a covalent complex of a CLIP-tag and an O2-benzylcytosine, whereby the FRET-donor and the FRET-acceptor are different tags.
7. The method according to claim 1, wherein the bispecific antibody comprises one or two binding sites that specifically bind to the first antigen and one or two binding sites that specifically bind to the second antigen.
8. A method for determining for a bispecific antibody, which comprises a first binding site that specifically binds to a first antigen and a second binding site that specifically binds to a second antigen whereby the two binding sites bind with at least 10 fold difference in binding affinity to their respective antigens, the strength of the binding interaction of the binding site that specifically binds to its antigen with the higher K.sub.D-value comprising the following steps: a) incubating a cell expressing cell-membrane bound FRET-donor-tagged first antigen and FRET-acceptor-tagged second antigen with the bispecific antibody and determining the binding of the bispecific antibody by determining the energy transfer from the FRET-donor to the FRET-acceptor, b) repeating step a) in the presence of increasing concentrations of a monospecific antibody comprising the binding site of the bispecific antibody with the lower K.sub.D value, and c) determining the strength of the binding interaction by determining the concentration of the monospecific antibody at which the energy transfer from the FRET-donor to the FRET-acceptor is reduced.
Description
DESCRIPTION OF THE FIGURES
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MATERIALS & GENERAL METHODS
(9) General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Amino acids of antibody chains are numbered and referred to according to numbering according to Kabat (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
(10) Recombinant DNA Techniques
(11) Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturer's instructions.
(12) Gene Synthesis
(13) Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The long gene segments, which were flanked by singular restriction endonuclease cleavage sites, were assembled by annealing and ligating oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene synthesis fragments were ordered according to given specifications at Geneart (Regensburg, Germany).
(14) DNA Sequence Determination
(15) DNA sequences were determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).
(16) DNA and Protein Sequence Analysis and Sequence Data Management
(17) The GCG's (Genetics Computer Group, Madison, Wis.) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.
(18) Expression Vectors
(19) For the expression of the described bispecific antibodies, expression plasmids for transient expression (e.g. in HEK293 cells) based either on a cDNA organization with or without a CMV-intron A promoter or on a genomic organization with a CMV promoter can be applied.
(20) Beside the Antibody Expression Cassette the Vectors Contain:
(21) an origin of replication which allows replication of this plasmid in E. coli, and a -lactamase gene which confers ampicillin resistance in E. coli.
The Transcription Unit of the Antibody Gene is Composed of the Following Elements: unique restriction site(s) at the 5 end the immediate early enhancer and promoter from the human cytomegalovirus, the intron A sequence in the case of cDNA organization, a 5-untranslated region derived from a human antibody gene, an immunoglobulin heavy chain signal sequence, the respective antibody chain encoding nucleic acid either as cDNA or with genomic exon-intron organization, a 3 untranslated region with a polyadenylation signal sequence, and unique restriction site(s) at the 3 end.
(22) The fusion genes encoding the antibody chains are generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective vectors. The subcloned nucleic acid sequences are verified by DNA sequencing. For transient transfections larger quantities of the plasmids are prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).
(23) For all constructs knob-into-hole heterodimerization technology was used with a typical knob (T366W) substitution in the first CH3 domain and the corresponding hole substitutions (T366S, L368A and Y407V) in the second CH3 domain (as well as two additional introduced cysteine residues S354C/Y349C) (contained in the respective corresponding heavy chain (HC) sequences depicted above).
(24) Cell Culture Techniques
(25) Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc., are used.
(26) Transient Transfections in HEK293 System
(27) The bispecific antibodies are produced by transient expression. Therefore a transfection with the respective plasmids using the HEK293 system (Invitrogen) according to the manufacturer's instruction is done. Briefly, HEK293 cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle 293 expression medium (Invitrogen) are transfected with a mix of the respective expression plasmids and 293Fectin or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293 cells are seeded at a density of 1.0*10.sup.6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO.sub.2. On the next day the cells are transfected at a cell density of approx. 1.5*10.sup.6 cells/mL with approx. 42 mL of a mixture of A) 20 mL Opti-MEM medium (Invitrogen) comprising 600 g total plasmid DNA (1 g/mL) and B) 20 ml Opti-MEM medium supplemented with 1.2 mL 293 fectin or fectin (2 l/mL). According to the glucose consumption glucose solution is added during the course of the fermentation. The supernatant containing the secreted antibody is harvested after 5-10 days and antibodies are either directly purified from the supernatant or the supernatant is frozen and stored.
(28) The relative plasmid ratio of 1:1:1:1 for 1+1 CrossMab or 1:1:1 for 2+2 CrossMab was used for the co-transfection of LC, HC, crossed LC and crossed HC plasmids.
(29) Protein Determination
(30) The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et al., Protein Science 4 (1995) 2411-1423.
(31) Antibody Concentration Determination in Supernatants
(32) The concentration of antibodies and derivatives in cell culture supernatants was estimated by immunoprecipitation with protein A agarose-beads (Roche Diagnostics GmbH, Mannheim, Germany). Therefore, 60 L protein A Agarose beads were washed three times in TBS-NP40 (50 mM Tris buffer, pH 7.5, supplemented with 150 mM NaCl and 1% Nonidet-P40). Subsequently, 1-15 mL cell culture supernatant was applied to the protein A Agarose beads pre-equilibrated in TBS-NP40. After incubation for at 1 hour at room temperature the beads were washed on an Ultrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twice with 0.5 mL 2 phosphate buffered saline (2PBS, Roche Diagnostics GmbH, Mannheim, Germany) and briefly four times with 0.5 mL 100 mM Na-citrate buffer (pH 5.0). Bound antibody was eluted by addition of 35 l NuPAGE LDS sample buffer (Invitrogen). Half of the sample was combined with NuPAGE sample reducing agent or left unreduced, respectively, and heated for 10 min at 70 C. Consequently, 5-30 l were applied to a 4-12% NuPAGE Bis-Tris SDS-PAGE gel (Invitrogen) (with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie Blue.
(33) The concentration of the antibodies in cell culture supernatants was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies that bind to protein A were applied to an Applied Biosystems Poros A/20 column in 200 mM KH.sub.2PO.sub.4, 100 mM sodium citrate, pH 7.4 and eluted with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an Agilent HPLC 1100 system. The eluted antibody was quantified by UV absorbance and integration of peak areas. A purified standard IgG1 antibody served as a standard.
(34) Alternatively, the concentration of antibodies and derivatives in cell culture supernatants was measured by Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Streptavidin A-96 well microtiter plates (Roche Diagnostics GmbH, Mannheim, Germany) were coated with 100 L/well biotinylated anti-human IgG capture molecule F(ab)2<h-Fc>BI (Dianova) at 0.1 g/mL for 1 hour at room temperature or alternatively overnight at 4 C. and subsequently washed three times with 200 L/well PBS, 0.05% Tween (PBST, Sigma). Thereafter, 100 L/well of a dilution series in PBS (Sigma) of the respective antibody containing cell culture supernatants was added to the wells and incubated for 1-2 hour on a shaker at room temperature. The wells were washed three times with 200 L/well PBST and bound antibody was detected with 100 l F(ab)2<hFc>POD (Dianova) at 0.1 g/mL as the detection antibody by incubation for 1-2 hours on a shaker at room temperature. Unbound detection antibody was removed by washing three times with 200 L/well PBST. The bound detection antibody was detected by addition of 100 L ABTS/well followed by incubation. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).
(35) Preparative Antibody Purification
(36) Antibodies were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a protein A Sepharose column (GE healthcare) and washed with PBS. Elution of antibodies was achieved at pH 2.8 followed by immediate neutralization. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine buffer comprising 150 mM NaCl (pH 6.0). Monomeric antibody fractions were pooled, concentrated (if required) using e.g., a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at 20 C. or 80 C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.
(37) SDS-PAGE
(38) The NuPAGE Pre-Cast gel system (Invitrogen) was used according to the manufacturer's instruction. In particular, 10% or 4-12% NuPAGE Novex Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE MES (reduced gels, with NuPAGE antioxidant running buffer additive) or MOPS (non-reduced gels) running buffer was used.
(39) CE-SDS
(40) Purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (PerkinElmer, USA). Therefore, 5 l of antibody solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according manufacturer's instructions and analyzed on LabChip GXII system using a HT Protein Express Chip. Data were analyzed using LabChip GX Software.
(41) Analytical Size Exclusion Chromatography
(42) Size exclusion chromatography (SEC) for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 buffer (pH 7.5) on an Dionex Ultimate system (Thermo Fischer Scientific), or to a Superdex 200 column (GE Healthcare) in 2PBS on a Dionex HPLC-System. The eluted antibody was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard.
(43) Mass Spectrometry
(44) This section describes the characterization of the bispecific antibodies with emphasis on their correct assembly. The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact antibody and in special cases of the deglycosylated/limited LysC digested antibody.
(45) The antibodies were deglycosylated with N-Glycosidase F in a phosphate or Tris buffer at 37 C. for up to 17 h at a protein concentration of 1 mg/ml. The limited LysC (Roche Diagnostics GmbH, Mannheim, Germany) digestions were performed with 100 g deglycosylated antibody in a Tris buffer (pH 8) at room temperature for 120 hours, or at 37 C. for 40 min, respectively. Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).
Example 1
(46) Production and Expression of Multispecific Antibodies which Bind to PD1 and Tim3
(47) Multispecific (bispecific) antibodies which binds to human PD1 and human Tim3 were generated as described in the general methods section by classical molecular biology techniques and were expressed transiently in HEK 293 cells as described above.
(48) Bispecific Antibodies with VH/VL Domain Exchange/Replacement (CrossMAb.sup.VH-VL) in One Binding Arm (1+1 Bispecific Antibody Format)
(49) The used bispecific 1+1 antibody format is described also in WO 2009/080252. The bispecific antibodies were expressed using expression plasmids containing the nucleic acids encoding the amino acid sequences depicted in the following Table.
(50) TABLE-US-00001 TABLE Amino acid sequences of light chains (LC) and heavy chains (HC) of the bispecific antibodies (1 + 1 CrossMAb.sup.VH-VL). 1 + 1 antibody HC1 HC2 LC1 LC2 PD1-Tim3-0389 SEQ ID NO: 01 SEQ ID NO: 02 SEQ ID NO: 03 SEQ ID NO: 04 PD1-Tim3-0168 SEQ ID NO: 05 SEQ ID NO: 06 SEQ ID NO: 07 SEQ ID NO: 08 PD1-Tim3-0476 SEQ ID NO: 09 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 PD1-Tim3-0477 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 PD1-Tim3-0166 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20
(51) For all constructs knobs into holes heterodimerization technology was used with a typical knob (T366W) substitution in the first CH3 domain and the corresponding hole substitutions (T366S, L368A and Y410V) in the second CH3 domain (as well as two additional introduced cysteine residues S354C/Y349C) (contained in the respective corresponding heavy chain (HC) sequences depicted above).
(52) Bispecific Antibodies with VH/VL Domain Exchange/Replacement (2+2 CrossMAb.sup.VH-VL) in two binding arm (2+2 Bispecific Antibody Format)
(53) The used multispecific 2+2 antibody format is described also in WO 2010/145792. The bispecific antibodies were expressed using expression plasmids containing the nucleic acids encoding the amino acid sequences depicted in the following Table.
(54) TABLE-US-00002 TABLE Amino acid sequences of light chains (LC) and heavy chains (HC), of the bispecific antibody (2 + 2 CrossMAb.sup.VH-VL). 2 + 2 antibody HC LC1 LC2 PD1-Tim3- SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23 0358 PD1-Tim3- SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 0359 PD1-Tim3- SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 0321
Example 2
(55) Purification and Characterization of Multispecific Antibodies which Bind to PD1 and Tim3
(56) The bispecific antibodies expressed above were purified from the supernatant by a combination of preparative protein A affinity chromatography and preparative size exclusion chromatography. All bispecific antibodies can be produced in good yields and are stable. The obtained products were characterized for identity by mass spectrometry and analytical properties such as purity by SDS-PAGE, monomer content and stability.
(57) Mass Spectrometry
(58) The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the deglycosylated intact bispecific antibodies and deglycosylated/plasmin digested or alternatively deglycosylated/limited LysC digested bispecific antibody.
(59) The bispecific antibodies were deglycosylated with N-Glycosidase F in a phosphate or Tris buffer at 37 C. for up to 17 h at a protein concentration of 1 mg/ml. The plasmin or limited LysC (Roche Diagnostics GmbH, Mannheim, Germany) digestions were performed with 100 g deglycosylated bispecific antibody in a Tris buffer (pH 8) at room temperature for 120 hours or at 37 C. for 40 min, respectively. Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).
Example 3
(60) FRET Assay for Simultaneous Binding of Bispecific Antibodies to Recombinant Cells
(61) General
(62) In this example a cell-based TR-FRET assay to determine the simultaneous binding of bispecific antibody formats to two different targets, such as, e.g., receptors, presented on one cell's outer membrane (surface) is described. The Tag-Lite technology has been chosen as an exemplary FRET method but any other FRET method could be equally chosen. It is a combination of a classical TR-FRET (time-resolved fluorescence resonance energy transfer) and SNAP-tag technology (e.g. New England Biolabs, CISBIO), which allows antigens present on the cell surface to be labeled with a fluorescent donor or acceptor dye.
(63) With this assay format it is possible to demonstrate the simultaneous binding of bispecific antibodies to cells expressing both antigens of the bispecific antibody, in this case PD1 and Tim3 receptors. The antigens are presented as recombinant fusion proteins consisting of the extracellular domains (ECD) of the given receptor and a tag, to which a fluorescence dye can be added. In the presence of an anti-PD1/Tim3 bispecific antibody, which can bind both receptors and, thus, indirectly thereby the FRET-donor and the FRET-acceptor, upon simultaneous binding the two receptors and likewise the FRET-donor and the FRET-acceptor will come into close proximity to allow energy transfer (FRET) (see
(64) Using an intracellular approach with two bispecific antibodies that work in extracellular approach, resulted in no FRET induction. The background with membrane-permeable dyes, which have to be used in this approach, is pretty high (data not shown).
(65) Generation of Recombinant PD1.sup.+Tim3.sup.+HEK Cells
(66) Standard methods were used to generate DNA as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. For Cloning of PD1 and Tim3 variants SNAP or CLIP was inserted proximal to the respective transmembrane-region of the receptor and cytoplasmatic domain was removed except for 7 amino acid residues and replaced by a Flag-tag.
(67) Transient Transfection
(68) HEK293 cells were co-transfected with transfection reagent 293free (Novagen) and Opti-MEM I Reduced Serum Media (Life Technologies) in 30 ml culture volume using two plasmids at a time with 15 g total amount of DNA. Briefly, HEK293 cells were transiently transfected with the following plasmids encoding for a fusion protein consisting of PD1 or Tim3 ECDs, respectively, and a SNAP or CLIP tag, respectively, as described elsewhere:
(69) Plasmids:
(70) a) PD1-SNAP (in 5 to 3 direction): nucleic acid encoding human PD1-extracellular domain including the signal peptide (residue 1-170 of SEQ ID NO: 30), nucleic acid encoding GGGGS spacer (SEQ ID NO: 31), nucleic acid encoding SNAP from pSNAP-tag(T7)2 (without N-terminal methionine residue) which is a mutant form of the human gene for O6-alkylguanine-DNA-alkyltransferase (hAGT). (Compared to wild type hAGT, the SNAP-tag protein contains the mutations C26A, K125A, A127T, R128A, G131K, G132T, M134L, R135S, C150S, N157G, S159E, and is truncated after G182) (SEQ ID NO: 32), nucleic acid encoding GGGGS spacer (SEQ ID NO: 31), nucleic acid encoding human PD1-transmembrane and cytoplasmic domain (residue 171-191 of SEQ ID NO: 30), nucleic acid encoding GGGGS spacer (SEQ ID NO: 31), nucleic acid encoding the Flag-tag (DYKDDDDK; SEQ ID NO: 33). b) Tim3-CLIP (in 5 to 3 direction): nucleic acid encoding human Tim3-extracellular domain including the signal peptide (residue 1-202 of SEQ ID NO: 34) nucleic acid encoding GGGGS spacer (SEQ ID NO: 31), nucleic acid encoding CLIP from pCLIPf (without N-terminal methionine residue), which is a mutant form of the human gene for O6-alkylguanine-DNA-alkyltransferase (hAGT) (SEQ ID NO: 35), nucleic acid encoding GGGGS spacer (SEQ ID NO: 31), nucleic acid encoding human Tim3 transmembrane and cytoplasmic domain (residue 203-230 of SEQ ID NO: 34) nucleic acid encoding GGGGS spacer (SEQ ID NO: 31), nucleic acid encoding the Flag-tag (DYKDDDDK; SEQ ID NO: 33).
(71) The combination PD1-CLIP and Tim3-SNAP was also constructed and expressed, and resulted in only low but still detectable FRET signals (data not shown).
(72) Upon transfection, cells were incubated in shaker flasks until final usage for FACS (24-48 hrs. after transfection) or FRET experiments (after 48 hrs.).
(73) Confirmation of PD1 and Tim3 Expression on Transiently Transfected HEK293 Cells (by FACS)
(74) 24-48 hrs. after transient transfection of HEK293 cells, the cells were analyzed for PD1 and Tim3 expression: Usually, 1-310.sup.5 single or double transfected cells were stained for 30 min. on ice at 10 g/ml, washed two times with PBS/2% FCS and analyzed on a FacsCanto II using FITC-conjugated anti-human PD1 antibody (Biolegend, Cat. No. 329904, clone EH12.2H7) or phycoerythrin (PE)-conjugated anti-human PD-1 antibody (R&D Systems, Cat. No. FAB1086P), and/or phycoerythrin (PE)-conjugated anti-human Tim3 antibody (R&Dsystems, Cat. No. FAB2365P, clone 344823).
(75) The FACS results for the single transfected cells are shown in
(76) Description Cell Labeling and FRET Assay with Anti-PD1/Tim3 Bispecific Antibodies
(77) Transfected cells were sedimented and resuspended at a density of 110.sup.6 cells/ml in tag-Lite buffer (Cisbio). Thereafter, cells were stained with 100 nM SNAP-Lumi4-Tb (Cisbio) and 100 nM CLIP-Red (Cisbio) for one hour at 37 C. in tag-Lite buffer (Cisbio). After washing and re-suspending in PBS buffer comprising 2% FCS (v/v), about 50.000 cells (in 50 l volume) were seeded into 96-well flat-bottom white plates (Costar) bevor either control antibody (e.g. a bivalent, monospecific antibody or an isotype control) or bispecific antibody was added to the cells at a final concentration of 0.001-10 nM. After an incubation for one hour at 4 C. or room temperature, time-resolved fluorescence was measured as ratio of the emission signal at 665 nm and 620 nm (665/620 nm ratio) with a BMG Pherastar reader or Tecan Infinite M1000 Pro using standard settings provided by the respective vendor.
(78) Exemplary standard settings were as follows: Mode Fluorescence Intensity Top Excitation Wavelength 340 nm Emission Wavelength 620/665 nm Excitation Bandwidth 20 nm Emission Bandwidth 10 nm Gain 232 Optimal (100%) Number of Flashes 100 Flash Frequency 100 Hz Integration Time 500 s Lag Time 60 s Settle Time 0 ms
(79) Optionally, SNAP-Lumi4-Tb and 100 nM CLIP-Red labeled cells were stored at 80 C. or in liquid nitrogen and freshly thawed for FRET experiments.
(80) Alternatively in some cases cross-linking of the parenteral, monovalent or bivalent monospecific monoclonal antibodies via goat anti human Fc-region antibody (20 nM final concentration) could be possible.
(81) To show the specificity of the FRET reaction induced by simultaneous binding of the bispecific antibody, monoclonal IgGs of only one specificity were added for competition.
(82) Results
(83) PD1 and Tim3 expressing HEK cells were treated as described above to measure FRET signal upon simultaneous receptor binding via incubation with titrated amounts of different bispecific antibodies (0.12 nM-10 nM).
(84) All tested bispecific antibodies induced a FRET signal in PD1/Tim3-expressing cells in a dose-dependent manner. All tested bispecific antibody formats (1+1, 2+2 and 2+1 (data not shown)) were comparable. A comparison of the results obtained with 1+1 format bispecific antibodies (antibodies PD1-Tim3-0389 and PD1-Tim3-0168) and 2+2 format bispecific antibodies (antibodies PD1-Tim3-0358 and PD1-Tim3-0359) is shown in
(85) Likewise as shown in
(86) To show the specificity of the FRET signal induced by simultaneous binding of the bispecific antibody, monoclonal IgGs of only one specificity were added for competition. Therefore, PD1 and Tim3 expressing HEK cells as described before were labelled with 100 nM SNAP-Lumi4-Tb and 100 nM CLIP-Red. After washing, labelled cells were incubated with the bispecific anti-PD1/Tim3 antibody PD1-Tim3-0168 at the indicated concentrations of 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM and 40 nM for one hour at 4 C. before time-resolved fluorescence was measured at 665/620 nm with an BMG Pherastar reader (
Example 4
(87) FRET Assay for Determining the Binding Strength
(88) In order to discriminate between the contributions of the two binding sites of a bispecific antibody a competition experiment was performed as outlined below.
(89) To discriminate for the contribution of the PD1 binding site and the Tim3 binding site to the overall interaction a special setting was used. The PD1 binding site has a higher affinity than the Tim3 binding site. The different bispecific antibodies showed similar FRET signal intensity under standard conditions. A difference could be visualized when the PD1 interaction (i.e. the interaction with the higher affinity/lower K.sub.D-value) was blocked or at least reduced.
(90) Therefore a monospecific, bivalent anti-PD1 antibody was added at different concentrations (80 nM, 20 nM, 5 nM, 1.25 nM and 0.31 nM) to bispecific anti-PD1/Tim3 antibodies differing in the Tim3 binding site (antibodies PD1-Tim3-0168 and PD1-Tim3-0389). At full PD1 neutralization almost no FRET signal was measured, whereas at lower anti-PD1 antibody concentrations, the FRET signal was restored and a stronger signal was observed with the antibody PD1-Tim3-0168 in which the Tim3 binding site has the higher affinity for Tim3 (see