Discovery and Humanization of Novel High Affinity Neutralizing Monoclonal Antibodies to Human IL-17A
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1 Discovery and Humanization of Novel High Affinity Neutralizing Monoclonal Antibodies to Human IL-17A Contacts: Marty Simonetti Kirby Alton Rick Shimkets Laboratories 111 Riverbend Road Athens, GA T- (706) Abeome Corporation Page 1 of 20
2 Background: Humanization Approach and Technology Abeome s high-throughput monoclonal antibody discovery process begins with the cloning of mouse variable regions recovered from single B cells into proprietary human constant region-containing vectors, thus generating chimeric antibodies which are tested for desirable properties. Ideally, multiple functional antibodies are selected to move forward into humanization, because in some cases it is not possible to design successful human framework grafts, while in other cases it is possible to obtain humanized antibodies with properties superior to the parent chimera. Abeome s approach to humanization comprises the generation of multiple heavy and light chain grafts, and pairwise testing to determine whether functional grafts can be immediately obtained. Structural models of each mouse fab are generated and compared to the designed grafted fab structure. As needed, back mutations are made in the grafted constructs in order to attempt to bring the structural models into alignment and maximize retention of parental affinity. Potential post-translational modification sites and other manufacturing challenges, such as non-canonical cysteine residues, are identified and engineered away. If additional affinity is required, mutagenesis of CDRs is undertaken using proprietary methods. Abeome Corporation Page 2 of 20
3 I. Abeome Antibody Discovery Platform: AbeoMouse TM We have developed a novel transgenic mouse system (AbeoMouse TM ) allowing for the direct selection of antigen-specific B-cells, paired with single-cell antibody gene cloning and screening. The AbeoMouse TM produces a 45-fold increase in surface immunoglobulin (Ig) positive antibody secreting cells and an accelerated immune response. Abeome s screening platform allows 1,000 times more affinity matured monoclonal antibodies to be isolated from a single AbeoMouse TM than by conventional technology. In contrast to other current antibody technologies, this platform allows for the enrichment and rapid cloning of specific, high-affinity chimeric antibodies against a target of interest. With this modular system, cloned variable regions (Vregions) may be swapped between multiple human Ig isotypes for empirical comparison of stability, affinity and functional potency, or to suit the specific therapeutic modality or effector function. Specifically, the transgenic AbeoMouse TM has been engineered to constitutively express multiple genes, including the Igα/Igβ B-cell receptor proteins, resulting in a hyper immune response and surface antibody expression during all stages of B cell differentiation (Fig.1). This enables the selection and sorting of antigen specific B-cells producing the most affinity matured antibodies, and this technology platform has been applied to obtain antibodies against a diverse set of antigens, including but not limited to whole cells, peptides, glycoproteins, viral envelope proteins and mouse proteins, typically producing chimeric leads with low picomolar dissociation constants. FIGURE 1. The transgenic AbeoMouse TM platform. A novel antibody discovery platform that generates mature B cells with high surface IgG expression, allowing for the direct selection and cloning of antigen-specific B cells Four strongly neutralizing chimeric lead antibodies were cloned from IL-17A reactive B cells from AbeoMice immunized with recombinant human IL-17A. Abeome Corporation Page 3 of 20
4 SDS-PAGE Bioactivity: HT-29 Cell Assay - Neutralization of Recombinant Human IL-17A Abeome Corporation Page 4 of 20
5 HT-29 Assay For Measuring IL-17A Neutralizing Antibodies Assay Buffer: HT-29 Growth Medium (McCoy s 5A Medium, supplemented with 10% fetal bovine serum and pen/strep) 1. For best results, only use inner rows/columns of 96-well plate (optional) 2. In assay buffer, dilute each antibody to 4X final concentration to be tested, and generate a dilution series. Make enough for 50uL for each replicate well (at least 3 replicates). 3. Dilute human IL-17 to 4X concentration (200ng/mL) in assay buffer 4. Combine IL-17A + test Ab (100uL total volume) in 96-well tissue culture plate, and include control wells for untreated cells and no antibody. Incubate for 37C 5. Harvest HT-29 cells and count 6. In assay buffer, dilute HT-29 cells to 20,000 cells per 100uL (200,000 cells/ml) 7. Add 100uL cells to IL-17A/ab mixes, for total volume of 200uL 8. Add 200uL PBS to outer wells of plate, or any empty wells 9. Cover & incubate for 48h at 37C, 5% CO2 CXCL1 ELISA: (R&D Systems, # DY275) 1. Coat ELISA plate with 100uL of capture Ab, seal & incubate overnight at RT 2. Centrifuge plates at 500g, and transfer supernatant to new plate 3. Wash ELISA plate 3x (PBS/tween) 4. Block ELISA with 300uL of reagent diluent/block 1hr, RT 5. Wash 3x 6. Add 100uL of HT29 supernatant and standards (in diluent), incubate 1-2hr 7. Wash 3x 8. Add 100uL of CXCL1 detection antibody, incubate 1hr, RT 9. Wash 3x 10. Add 100uL of Streptavidin-HRP, incubate 20min, RT 11. Wash 3x 12. Add detection reagent, incubate at RT 13. Read at 633nm at 5min Humanization Report- ABM59 Abeome Corporation Page 5 of 20
6 Germline Analysis: ABM59 is comprised of a heavy chain with two amino acid changes from mouse germline IGHV and a light chain with eight amino acid substitutions from mouse germline IGKV Structural Model: Based on amino acid similarity to known antibody fab structures, a structural model was obtained for comparison purposes to humanized constructs. Binding: By Surface Plasmon Resonance (SPR) using a Biacore T-100 with antibody immobilized directly to a CM5 chip, ABM59 has the following binding characteristics against recombinant human IL-17A: K on = 2.84 x 10 6 M -1 s -1 K off = 2.51 x 10-4 S -1 K D = 88 pm Activity: Abeome Corporation Page 6 of 20
7 ABM59 in the IgG4 isotype displays an IC50 of 3.6 g/ml: on HT29 cells in a neutralization assay of human IL-17A activity When tested as IgG1 or IgG2, ABM59 loses significant potency. Thus, humanization was initially conducted in the IgG4 backbone, with the idea to test potent humanized antibodies back in the IgG1 and IgG2 isotypes following successful humanization of the IgG4. Using modern grafting and DNA synthesis techniques, a variety of grafts were synthesized, cloned, transfected, purified, and tested for expression levels, binding to recombinant human IL-17A (rhil17a), and potency in the HT-29 assay (IC 50). Humanization Construct Pairs (Mouse Chimeras in Gray): Name Heavy Light Isotype K D-rhIL17A IC 50 Transient Expression Levels ABM59 59H 59L IgG pm 3.6 g/ml 59.9 mg/l ABM59G1 59H 59L IgG1 Not Tested >10 g/ml 36.1 mg/l ABM59G2 59H 59L IgG2 Not Tested >10 g/ml 33.0 mg/ml ABM H 59.1L IgG4 No Binding N/A Not Determined ABM59.X 59.2H 59.1L IgG4 No Binding N/A Not Determined ABM H 59.2L IgG4 No Binding N/A 24.3 mg/l ABM59. X 59.1H 59.2L IgG4 No Binding N/A Not Determined ABM H 59.2L IgG4 152 pm 7.25 g/ml 68 mg/l ABM H 59.3L IgG4 153 pm 7.45 g/ml 60 mg/l ABM H 59.3L IgG4 151 pm 5.45 g/ml 137 mg/l ABM H 59.4L IgG4 123 pm 1.7 g/ml 140 mg/l ABM59.6G1 59.3H 59.4L IgG1? pm 2.3 g/ml 68.6 mg/l ABM59.6G2 59.3H 59.4L IgG2? pm 2.3 g/ml 83.5 mg/l Expression and Post-Translational Modification Analysis: ABM59.3, 59.4 and 59.5 possessed putative N-glycosylation sites in the human framework residues, which was reflected in the slower migration of the light chain bands for those antibodies on denaturing gels. Abeome Corporation Page 7 of 20
8 ABM59.6 possesses an N-Q mutation which destroys the glycosylation site, improved expression, binding and potency. Abeome Corporation Page 8 of 20
9 Clone k on k off K D M -1 s -1 S -1 pm ABM x x ABM x x ABM x x ABM x x Abeome Corporation Page 9 of 20
10 ABM60 Germline Analysis: ABM60 is comprised of a heavy chain with three amino acid changes from mouse germline IGHV2-6 and a light chain with one amino acid substitution from mouse germline IGKV Structural Model: Based on amino acid similarity to known antibody fab structures, a structural model was obtained for comparison purposes to humanized constructs. Abeome Corporation Page 10 of 20
11 Activity: ABM60 was the most potent of the anti-il17a antibodies obtained in the discovery phase. A particular challenge to ABM60, however, is that the mouse frameworks for this antibody are very distant from human frameworks, making the design of grafts which are structurally similar to the parent ABM60 chimera very difficult. Using modern grafting and DNA synthesis techniques, a variety of grafts were synthesized, cloned, transfected, purified, and tested for expression levels, binding to recombinant human IL-17A (rhil17a), and potency in the HT-29 assay (IC 50). None of the humanized variants constructed showed significant binding to IL17A. Humanization Construct Pairs (Mouse Chimeras in Gray): Name Heavy Light Isotype K D-rhIL17A IC 50 Transient Expression Levels ABM60 60H 60L IgG4 188 pm 1.1 g/ml 55.9 mg/l ABM60G1 60H 60L IgG1 Not Tested 1.8 g/ml 28.6 mg/l ABM60G2 60H 60L IgG2 Not Tested 1.3 g/ml 50.0 mg/l ABM H 60.1L IgG4 No Binding N/A Not Determined ABM H 60.2L IgG4 No Binding N/A Not Determined ABM H 60.3L IgG4 No Binding N/A Not Determined ABM60.4* 60.4H 60.3L IgG4 No Binding >10 g/ml 18.7 mg/l ABM H 60.3L IgG2 No Binding >10 g/ml 4.2 mg/l ABM60.5H ABM60.6H ABM60.7H Abeome Corporation Page 11 of 20
12 Abeome Corporation Page 12 of 20
13 ABM64 Activity: ABM64 was the least potent of the four lead anti-il17a antibodies obtained in the discovery phase. Using modern grafting and DNA synthesis techniques, a variety of grafts were synthesized, cloned, transfected, purified, and tested for expression levels, binding to recombinant human IL-17A (rhil17a), and potency in the HT-29 assay (IC 50). A particular challenge to ABM64 was that the initial grafts were able to not only retain binding affinity, but in fact have superior binding kinetics to those of the mouse chimera, but with a loss of almost all potency in the cellular assay. It was thus theorized that the epitope of ABM64 must be on the edge of neutralization, and that minor conformational changes to the molecule outside the binding site may greatly influence the functional activity. It was further discovered that the ABM64 heavy chain, and the ABM64.1 humanized heavy chain, are able to bind IL17A absent their cognate light chain, or any light chain at all. This molecule may be able to be formatted in the future for a bispecific antibody. Germline Analysis: ABM64 is comprised of a heavy chain with eight amino acid changes from mouse germline IGHV1-67 and a light chain with no amino acid substitutions from mouse germline IGKV8-24. Structural Model: Based on amino acid similarity to known antibody fab structures, a structural model was obtained for comparison purposes to humanized constructs. Humanization Construct Pairs (Mouse Chimeras in Gray): Name Heavy Light Isotype K D-rhIL17A IC 50 Transient Expression Levels Abeome Corporation Page 13 of 20
14 ABM64 64H 64L IgG4 248 pm 9.6 g/ml 22.3 mg/l ABM64G1 64H 64L IgG1 Not Tested >10 g/ml 59.6 mg/l ABM64G2 64H 64L IgG2 Not Tested >10 g/ml? mg/ml ABM H 64.1L IgG4 127 pm >40 g/ml Not Determined ABM64.2G2 64.2H 64.2L IgG2 520 pm >40 g/ml 10.7 mg/l ABM H 64.2L IgG4 241 pm 36 g/ml 23.3 mg/l ABM H 64.2L IgG4? pm 15.1 g/ml mg/l Clone k on k off K D M -1 s -1 S -1 pm ABM 64.2 IgG x x ABM 64.2 IgG x x ABM x x ABM x x Abeome Corporation Page 14 of 20
15 Abeome Corporation Page 15 of 20
16 ABM67 Germline Analysis: ABM67 is comprised of a heavy chain with no amino acid changes from mouse germline IGHV5-9 and a light chain with three amino acid substitutions from mouse germline IGKV5-48. Structural Model: Based on amino acid similarity to known antibody fab structures, a structural model was obtained for comparison purposes to humanized constructs. Humanization Construct Pairs (Mouse Chimeras in Gray): Name Heavy Light Isotype K D-rhIL17A IC 50 Transient Expression Levels ABM67 67H 67L IgG4 248 pm 1.7 g/ml 48 mg/l ABM67G1 67H 67L IgG1 Not Tested 4.3 g/ml? mg/l ABM67G2 67H 67L IgG2 Not Tested 4.1 g/ml? mg/ml ABM H 67.1L IgG2 Not Tested N/A Not Determined ABM67.2G2 67.2H 67.2L IgG2 278 pm 2.5 g/ml 34.5 mg/l ABM H 67.2L IgG2? pm 7.2 g/ml? mg/l ABM H 67.2L IgG2? pm 2.8 g/ml? mg/l ABM H 67.2L IgG2 453 pm 0.87 g/ml 40.9 mg/l ABM H 67.3L IgG2 503 pm 1.22 g/ml mg/l ABM H 67.4L IgG2? pm 1.42 g/ml 62.1 mg/l ABM H 67.2L IgG1? pm? g/ml 20.1 mg/l ABM H 67.2L IgG4? pm? g/ml 31.3 mg/l ABM H 67.2L IgG2 425 pm 1.42 g/ml 55.8 mg/l ABM H 67.3L IgG2 400 pm 1.34 g/ml 190 mg/l ABM H 67.4L IgG2? pm 1.08 g/ml 44.9 mg/l ABM H 67.2L IgG1? pm? g/ml 29.8 mg/l ABM H 67.2L IgG4? pm? g/ml 48.4 mg/l ABM H 67.2L IgG2? pm 3.2 g/ml? mg/l ABM H 67.2L IgG2? pm 4.5 g/ml? mg/l Abeome Corporation Page 16 of 20
17 mutation IC50 ( g/ml) habm67.2-g2-2.5 habm g2 tbd 2.5 habm g2 A103V 7.2 habm g2 V105A 2.8 habm g2 Y106S 0.89 habm g2 Y106I 1.5 habm g2 Y106A 3.2 habm g2 Y106K 4.5 Clone k on k off K D M -1 s -1 S -1 pm ABM x x ABM x x ABM x x ABM x x Abeome Corporation Page 17 of 20
18 Abeome Corporation Page 18 of 20
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