METHOD FOR GENERATING AVID-BINDING MULTISPECIFIC ANTIBODIES
20220098326 · 2022-03-31
Assignee
Inventors
- Ulrich Brinkmann (Weilheim, DE)
- Alexander Bujotzek (Munchen, DE)
- Guy Georges (Habach, DE)
- Anja SCHRADE (Penzberg, DE)
Cpc classification
C07K16/2863
CHEMISTRY; METALLURGY
C07K16/2896
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
International classification
Abstract
Herein is reported a method for increasing the (avid-)binding specificity of a bispecific antibody comprising a first mammalian or mammalianized binding site specifically binding to a first (cell-surface) antigen and a second binding site specifically binding to a second (cell-surface) antigen, wherein the first mammalian or mammalianized binding site is at least a pair of an immunoglobulin light chain variable domain and immunoglobulin heavy chain variable domain, by decreasing the binding affinity of the mammalian or mammalianized binding site to its antigen by mutating in the first mammalian or mammalianized binding site at least one amino acid residue at a position in the CDRs of the light chain variable domain or in the CDR1 or CDR2 of the heavy chain variable domain or in the two framework positions directly preceding the CDR3 in the heavy chain variable domain to an amino acid residue present at said position in a germline immunoglobulin amino acid sequence of the same mammalian species as that of the mammalian or mammalianized binding site.
Claims
1. A method for decreasing the binding affinity of a bispecific antibody comprising a first binding site specifically binding to a first cell-surface antigen and a second binding site specifically binding to a second cell-surface antigen, with said first and second antigen being on the same cell, wherein the first binding site is a mammalian or mammalianized binding site, wherein the first binding site is at least a pair of an immunoglobulin light chain variable domain and an immunoglobulin heavy chain variable domain, wherein the decreasing the binding affinity is a decreasing of the binding affinity of the first binding site to its antigen by mutating in the first binding site at least one amino acid residue at a position in the CDRs of the light chain variable domain or in the CDR1 or CDR2 of the heavy chain variable domain or in the two framework positions directly preceding the CDR3 in the heavy chain variable domain to an amino acid residue present at said position in a germline immunoglobulin amino acid sequence of the same mammalian species as that of the mammalian or mammalianized binding site.
2. The method according to claim 1, wherein the germline immunoglobulin amino acid sequence is said germline immunoglobulin amino acid sequence that has the highest identity in a sequence alignment of all germline immunoglobulin amino acid sequences of said mammalian species.
3. The method according to claim 1, wherein for the determination of the germline immunoglobulin amino acid sequence in case of the heavy chain variable domain only the sequence fragment encompassing the first residue of CDR1 of the heavy chain variable domain to the last residue of the CDR2 of the heavy chain variable domain is used.
4. The method according to claim 1, wherein the side-chains of the amino acid residues to be mutated are solvent accessible.
5. The method according to claim 1, wherein the side-chains of the amino acid residues to be mutated are not involved in a VH-VL interactions.
6. The method according to claim 1, wherein the side-chains of the amino acid residues to be mutated are involved in interactions with the antigen.
7. The method according to claim 1, wherein the mutated mammalian or mammalianized binding site has no polyreactivity.
8. The method according to claim 1, wherein the second binding site is at least a second pair of an immunoglobulin light chain variable domain and an immunoglobulin heavy chain variable domain, or the second binding site is a second mammalian or mammalianized binding site and the mutating is of at least one amino acid residue in the first and the second mammalian, or mammalianized binding site.
9. The method according to claim 1, wherein the mammal is a human.
10. The method according to claim 1, wherein the method is for increasing the avid binding by reducing the monovalent affine binding of at least one binding site of the bispecific antibody.
Description
DESCRIPTION OF THE FIGURES
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CITATIONS
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EXAMPLES
Materials and Methods
[0227] All structural analysis is performed with BIOVIA Discovery Studio (“Dassault Systèmes BIOVIA, Discovery Studio 2017 R2, San Diego: Dassault Systèmes” 2016) and Pipeline Pilot (“Dassault Systèmes BIOVIA, Discovery Studio 2017 R2, San Diego: Dassault Systèmes” 2016).
Example 1
Expression and Purification
[0228] Amino acid sequences of monovalent IgG antibodies against hCD138, hHer2/c-neu, and hEGFR/Her1 were identified. These sequences were used as master to generate affinity variants. All antibodies were expressed transiently in non-adherent HEK-293 cells and purified using protein-A affinity and size-exclusion chromatography using an Äkta system (GE Healthcare) (Grote et al. 2012; Thorey et al. 2016). To verify antibody identity electrospray ionization mass spectra were acquired on a maXis Q-TOF (Bruker Daltonics, Bremen, Germany) equipped with a TriVersa NanoMate (Advion, Ithaca, N.Y.).
TABLE-US-00008 specificity expression system variant Yield [mg/L] CD138 HEK293F Hw-Lw 47 (parent IgG) Hw-La 50 Hw-Lg 49 Ha-Lw 42 Expi293F Ha-La 216 Ha-Lg 220 HEK293F Hb-Lw 50 Hb-La 33 Hb-Lg 48 Hg-Lw 46 Hg-La 56 Hg-Lg 62 Her2/c-neu Expi293F Hw-Lw 47 (parent IgG) Hw-La 297 Hw-Lb 175 Hw-Lg 83 Ha-Lw 392 Hb-Lw 103 Hg-Lw 170 Hg-Lg 325 EGFR/Her1 HEK293F Hw-Lw 23 (parent IgG) Hw-La 50 Hw-Lb 38 Hw-Lg 72 Ha-Lw 22 Hb-Lw 23 Hg-Lw 22 Hg-Lg 46
[0229] None of the proteins behaved aberrantly and expression levels of affinity variants were comparable or higher as to their corresponding non-mutated WT IgG's. This indicates that the mutations introduced based on the method according to the invention are well tolerated and that there is no influence of individual or combined mutations on folding and antibody structure. There were no structural incompatibilities of H- and L-variants upon combination within the combination set and molecular mass was determined to be correct for each antibody.
TABLE-US-00009 specificity expression system variant yield in [mg/L] CD138 HEK293F Hala-Lala 114 Her 2 EXPI Hala-Lala 909 EGFR/Her1 HEK293F Hala-Lala 152
Example 2
Surface Plasmon Resonance to Determine Antibody Kinetics
[0230] The kinetics of all antibody affinity variants (anti-hCD138, anti-hHer2/c-neu, anti-hEGFR/Her1) to the extracellular matrix domain of the corresponding antigen was evaluated using a BIAcore T200 instrument (GE Healthcare) (see Schlothauer et al. 2016). Association and dissociation were determined both, in a monovalent (affinity-driven) and bivalent (avidity-driven) binding mode.
CD138 Affinity SPR
[0231] Solution of anti-CD138 antibody affinity variants 30 nM in HBS-P+ (GE Healthcare) were captured with anti-huFc antibody (GE Healthcare BR-1008-39) on a CM5 sensor chip for 60 sec. (capture Level ˜900 RU). Thereafter the interaction with hCD138 (R&D-S. 2780-TS) was analyzed in a dilution series of 66 nM to 600 nM using 150 sec. association time and 600 sec. dissociation time at a flow rate of 30 μl/min. All BIAcore T200 experiments were carried out in HBS-P+ (GE Healthcare) pH 7.4 running buffer and at 25° C. Binding curves were evaluated using T200 evaluation software and the for the calculation of binding properties 1:1 Langmuir binding model was used.
CD138 Avidity SPR
[0232] For the determination of avidity-driven binding kinetics to CD138 the antigen was immobilized to 240 response units (RU) on a CM5 chip using the amine coupling kit (GE Healthcare) at pH 4.5 and 1 μg/ml. Thereafter the interaction of CD138 on the CM5 surface and anti-CD138 antibody affinity variants was analyzed in a dilution series of 33 nM to 300 nM in HBS-P+ (GE Healthcare) using 120 sec. association time and 600 sec. dissociation time at a flow rate of 60 μl/min. The surface regeneration was performed by a 60 sec. washing step with a 3 M MgCl.sub.2. All BIAcore T200 experiments were carried out in HBS-P+ (GE Healthcare) pH 7.4 running buffer and at 25° C. Binding curves were evaluated using BIAcore T200 evaluation software and for the calculation of binding properties 1:1 Langmuir binding model was used. Further analysis of heterogonous interaction was performed using Interaction Map software (Ridgeview Instruments AB).
Her2/c-neu Affinity SPR
[0233] The anti-Her2/c-neu antibody affinity variants at 5 nM in HBS-P+ (GE Healthcare) were captured with anti-huFc antibody (GE Healthcare BR-1008-39) on a CM5 sensor chip for 30 sec. (capture Level ˜100 RU). Thereafter hHer2/c-neu ECD was injected at a concentration of 19 nM to 300 nM diluted in HBS-P+ (GE Healthcare) with 150 sec. association time and 720 sec. dissociation time. All BIAcore T200 experiments were carried out in HBS-P+ (GE Healthcare) pH 7.4 running buffer and at 25° C. Binding curves were evaluated using T200 evaluation software and the for the calculation of binding properties 1:1 Langmuir binding model was used.
Her2/c-neu Avidity SPR
[0234] For the determination of avidity-driven binding kinetics the hHer2/c-neu ECD was immobilized to 60 response units (RU) on a CM5 chip using the amine coupling kit (GE Healthcare) at pH 4.5 and 1 μl/min. Thereafter the interaction of hHer2/c-neu ECD on the surface and anti-hHer2/c-neu antibody variants was analyzed in a dilution series from 7 nM to 600 nM in HBS-P+ (GE Healthcare) using 150 sec. association time and 600 sec. dissociation time at a flow rate of 60 μl/min followed by regeneration of the surface by a 60 sec. washing step with 3 M MgCl.sub.2. All BIAcore T200 experiments were carried out in HBS-P+ (GE Healthcare) pH 7.4 running buffer and at 25° C. Binding curves were evaluated using BIAcore T200 evaluation software and for the calculation of binding properties 1:1 Langmuir binding model was used.
EGFR/Her1 Affinity SPR
[0235] The anti-EGFR/Her1 antibody affinity variants at 5 nM in HBS-P+ (GE Healthcare) were captured with anti-huFc antibody (GE Healthcare BR-1008-39) on a CM5 sensor chip for 30 sec. (capture Level ˜100 RU). Thereafter the interaction with hEGFR/Her1 ECD antigen was analyzed in a dilution series from 19 nM to 300 nM using 120 sec. association time and 600 sec. dissociation time at a flow rate of 60 μl/min. All BIAcore T200 experiments were carried out in HBS-P+ (GE Healthcare) pH 7.4 running buffer and at 25° C. Binding curves were evaluated using BIAcore T200 evaluation software and for the calculation of binding properties 1:1 Langmuir binding model was used.
EGFR/Her1 Avidity SPR
[0236] An SA-chip was coated with ˜200 response units (RU) of hEGFR/Her1 ECD antigen. Binding kinetics were determined using a dilution series from 1.1 nM to 90 nM anti-hEGFR/Her1 antibodies in HBS-P+ (GE Healthcare) and a flow rate of 60 μl/min with 120 sec. association time and 600 sec. dissociation time. The surface regeneration was performed by a 30 sec. injection of 10 mM NaOH. All BIAcore T200 experiments were carried out in HBS-P+ (GE Healthcare) pH 7.4 running buffer and at 25° C. Binding curves were evaluated using BIAcore T200 evaluation software and for the calculation of binding properties 1:1 Langmuir binding model was used. Further analysis of heterogonous interaction was performed using Interaction Map software (Ridgeview Instruments AB).
[0237] In the following Table are listed on-rates (k.sub.a) and off-rates (k.sub.d) as well as K.sub.D-values of antibodies and corresponding variants measured in a monovalent assay set up.
[0238] ‘no valid signal’: very small ‘on’ signals were observed for some CD138 binders which may point towards weak interactions, which however cannot be called true binding events based on these monovalent SPR analyses.
TABLE-US-00010 VH-VL specificity combination k.sub.a [1/Ms] k.sub.d [1/s] K.sub.D [M] CD138 Hw-Lw 7.7E+04 6.2E−03 8.0E−08 (parent IgG) Hw-La 8.7E+04 6.1E−03 7.1E−08 Hw-Lg 1.3E+04 2.5E−02 2.0E−07 Ha-Lw 7.6E+04 1.5E−02 1.9E−07 Ha-La 5.5E+04 1.3E−02 2.4E−07 Ha-Lg 1.2E+05 4.5E−02 3.8E−07 Hb-Lw 1.8E+05 2.7E−02 1.5E−07 Hb-La 1.7E+05 2.5E−02 1.4E−07 Hb-Lg 1.3E+05 7.9E−02 6.1E−07 Hg-Lw No valid No valid No valid result result result Hg-La No valid No valid No valid result result result Hg-Lg No valid No valid No valid result result result Her2/c-neu Hw-Lw 1.5E+05 2.8E−04 1.9E−09 (parent IgG) Hw-La 1.3E+05 2.5E−04 1.9E−09 Hw-Lb 1.5E+05 4.1E−04 2.7E−09 Hw-Lg 1.4E+05 2.6E−04 1.9E−09 Ha-Lw 1.5E+05 3.1E−04 2.1E−09 Hb-Lw 4.1E+04 1.6E−02 3.9E−07 Hg-Lw 6.5E+04 2.2E−02 3.4E−07 Hg-Lg 6.1E+04 2.4E−02 3.9E−07 EGFR/Her1 Hw-Lw 1.07E+06 6.4E−04 6.0E−10 (parent IgG) Hw-La 9.97E+05 3.5E−03 3.5E−09 Hw-Lb 8.90E+05 3.5E−03 3.9E−09 Hw-Lg 1.00E+06 8.5E−03 8.5E−09 Ha-Lw 1.06E+06 8.0E−04 7.3E−10 Hb-Lw 1.06E+06 7.0E−04 6.6E−10 Hg-Lw 1.13E+06 1.1E−03 9.7E−10 Hg-Lg 1.08E+06 1.3E−02 1.2E−08
[0239] In the following Table are listed on- and off-rates as well as K.sub.D-values of antibodies and corresponding variants measured in a bivalent assay set up.
[0240] ‘no valid result’: very small ‘on’ signals were observed for some CD138 binders which may point towards weak interactions, which however cannot be called true binding events based on these monovalent SPR analyses.
TABLE-US-00011 VH-VL specificity combination k.sub.a [1/Ms] k.sub.d [1/s] K.sub.D [M] CD138 Hw-Lw 3.3E+05 7.8E−05 2.4E−10 (parent IgG) Hw-La 3.6E+05 8.7E−05 2.4E−10 Hw-Lg 3.6E+05 1.3E−04 3.6E−10 Ha-Lw 1.7E+05 7.1E−05 4.2E−10 Ha-La 1.6E+05 6.4E−05 4.0E−10 Ha-Lg 1.6E+05 2.9E−04 8.3E−10 Hb-Lw 3.5E+05 1.1E−04 2.8E−10 Hb-La 4.0E+05 1.2E−04 3.1E−10 Hb-Lg 3.9E+05 5.0E−04 2.4E−09 Hg-Lw 2.1E+05 5.1E−02 2.3E−07 Hg-La 2.2E+05 3.8E−02 1.7E−07 Hg-Lg No valid No valid No valid result result result Her2/c-neu Hw-Lw 1.6E+05 7.0E−05 4.4E−10 (parent IgG) Hw-La 2.6E+05 7.1E−05 2.7E−10 Hw-Lb 1.7E+05 1.7E−04 1.0E−09 Hw-Lg 8.2E+05 1.3E−04 1.6E−10 Ha-Lw 1.6E+05 7.1E−05 4.4E−10 Hb-Lw 3.1E+04 2.1E−04 6.8E−09 Hg-Lw 8.7E+04 1.5E−02 1.7E−07 Hg-Lg 8.3E+04 1.5E−02 1.8E−07 EGFR/Her1 Hw-Lw 1.5E+5 9.4E−05 6.3E−10 (parent IgG) Hw-La 1.6E+5 1.3E−04 8.1E−10 Hw-Lb 1.5E+5 1.2E−04 8.0E−10 Hw-Lg 1.4E+5 1.4E−04 1.0E−10 Ha-Lw 1.5E+5 1.0E−04 6.7E−11 Hb-Lw 4.1E+4 9.3E−05 2.3E−10 Hg-Lw 6.5E+4 1.1E−04 1.7E−09 Hg-Lg 6.1E+4 1.7E−04 2.8E−08
Example 3
Polyreactivity Assays
[0241] ELISA based polyreactivity assays were performed using non-specific antigens and specific antigens as positive controls. The antigens were coated to 384-well Nunc-Maxi Sorp plates at concentrations between 0.1 and 2 μg/mL in PBS at 4° C. over night. Between individual steps plates were washed with PBST (1×PBS+0.1% Tween 20). Blocking was performed (2% BSA in PBS+0.2% Tween 20) for 1 hour at room temperature without agitation. Antibody samples (1 μg/mL in One Step ELISA buffer; 1×PBS+0.5% BSA+0.05% Tween 20) were incubated 1 hour at room temperature without agitation. Secondary antibody (anti-human IgG Fc-specific; Jackson #109-036-098; dilution 1:7000 in OSEP) was added and incubated for 1 hour at room temperature on a microplate shaker at 400 rpm. Detection substrate TMB was added (4 Min without agitation) and absorbance was measured at a wavelength of 370 nm and a reference wavelength at 492 nm using a Tecan Safire II ELISA reader. The raw measurement signals at 492 nm were subtracted from the respective raw measurement signals at 370 nm (absorbance correction). The absorbance corrected signals from the blank wells (secondary antibody only) were subtracted from the absorbance corrected signals on the respective antigens (Blank correction). All measurements were done in triplicates.