pgt121 antibody engineering: enhanced infected cell killing and … · 2020. 6. 8. ·...

PGT121 Antibody Engineering: Enhanced Infected Cell Killing and Drug-Like Properties Introduction Methods © 2017 Gilead Sciences, Inc. All rights reserved. Gilead Sciences, Inc. 333 Lakeside Drive Foster City , CA 94404 Tel: (650) 574-3000 Fax: (650) 578-9264 1. Pancera et al. Nature 2014. 514(7523):455-461. 2. Bruner et al. Trends in Microbiology. 2015. 23(4):192-203. 3. Julien et al. PLoS Pathog. 2013. 9(5):e1003342. 4 Sok et al. PLoS Pathog. 2013. 9(11):e1003754. 5. Richman et al. PNAS. 2003. 100(7):4144-9. 6. Baker and Jones. Curr Opin Drug Devel. 2007. 10(2):219-227. 7. von Horsten et al. Glycobiology 2010. 20(12):1607-1618. 8. Barouch et al. Nature. 2013. 503(7475):224-228. . Poster # 329 Results Conclusions Acknowledgements References M Balakrishnan; ND Thomsen; CS Pace; X Zhang; M Hung; MR Nagel; BA Carr; YE Hu, H Yu, JA Corbin CROI, Seattle, Washington, Feb 13-16, 2017 Latency reversing agent Infected CD4+ T cell NK cells (ADCC) Ab - dependent cellular cytotoxicity Macrophages (ADCP) Ab-dependent cellular phagocytosis Complement (CDC) Complement-dependent cytotoxicity HIV provirus HIV Envelope (gp120/gp41) Anti-HIV Env bNAbs + Fc γRIIIA Fc γRIIA C1q PGT121v ∆Fuc A therapeutic agent that mediates the selective destruction of cells expressing latent HIV may sustainably suppress viremia in the absence of antiretroviral therapy (ART) and offers the potential for a sterilizing cure. Anti-HIV-1 envelope (Env) antibodies from elite neutralizers can neutralize a high percentage of viral strains across clades. Some of these broadly neutralizing antibodies (bNAbs) can recognize Env expressed on the surface of infected cells and mediate killing via effector function. A set of patient-derived bNAbs targeting different Env epitopes were tested for natural killer (NK) cell mediated killing of HIV-infected CD4+ T cells to identify a bNAb for further optimization. Fragment antigen-binding (Fab) variants were generated via genetic engineering, while enhancement of effector function was achieved through glycoengineering of the fragment crystallizable (Fc). 1 - Screen bNAbs in primary HIV infected ADCC assay 2 - In-silico and biophysical characterization of PGT121 3 - Design and screen ~30 PGT121 Fab point mutants 4 - Design and screen ~30 combinatorial variants 5 - Fc engineering 3 Fab Glycosylation Sites Multiple T-cell Epitopes No Fab glycosylation sites Fewer T-cell Epitopes Enhanced low pH stability Aggregation at low pH No Fab glycosylation sites Fewer T-cell Epitopes Enhanced low pH stability Enhanced ADCC Figure 3. PGT121 showed the highest ADCC activity among a panel of 28 mAbs targeting diverse epitopes. A panel of anti-Env antibodies was screened for ADCC activity using primary resting CD4+ T cells infected with four different HIV isolates. The mAbs exhibited significant differences in killing efficacy (Emax) and potency (not shown). All antibodies have an identical IgG1 framework. Figure 4. PGT121 contains numerous sequence liabilities. (a) Primary sequence analysis of PGT121 reveals multiple liabilities including poor match to the human germline (GL) precursor 4 , three consensus glycosylation sites in the heavy chain and multiple T-cell epitopes identified via a peptide mapping Epi-Screen (50 donors). (b) The liabilities in (a) mapped onto a structural model of the PGT121-gp120 complex based on PDB IDs 4TVP and 4JY4 1,4 . a b Figure 6. Top combinatorial variants have median potencies and breadth comparable to PGT121. PGT121 combinatorial variants were profiled for potency and breadth against a panel of 142 patient envelopes in the Monogram PhenoSense neutralization assay 5 as a high throughput and robust approach for detecting compromises in Env recognition. Most variants retained the Env recognition profile of PGT121. PGT121 IC50 = 0.0163 μg/ml, 66% breadth; IC50 of top variants ~0.01 μg/ml, 66-67% breadth. Figure 5. Several single point mutants retain activity comparable to PGT121. Single point mutants designed in the first phase of Fab engineering were screened for HIV neutralization activity against a set of sensitive isolates. Point mutants showing potencies within 2-fold of PGT121 (dashed lines) were next incorporated into a focused set of combinatorial variants. Figure 8. Afucosylation of PGT121v enhanced FcγR binding and ADCC activity. (a) Fc glycoengineering 7 increased apparent binding affinity to both alleles of FcγRIIIA ~10-fold in an FcγR ELISA. (b) Fc glycoengineered variant exhibited enhanced ADCC activity. Representative ADCC plots shown for cells infected with two different HIV patient isolates. Figure 7. Engineered PGT121 variants (PGT121v) have reduced risk of immunogenicity. Select PGT121 combinatorial variants were tested in a primary T-cell activation assay (50 donors) alongside PGT121 and control mAbs shown. Removal of T-cell epitopes reduced ex vivo T-cell activation rate from 32% (PGT121) to <15%, indicating a reduced potential for clinical immunogenicity. Antibodies with low clinical ADA rates were observed to have response rates of ~10% (dashed line) or lower in this assay 6 . PGT121 was selected as the lead bNAb for engineering on the basis of killing activity and previously demonstrated efficacy in SHIV infected monkeys 8 . Engineering of PGT121 enhanced its ability to kill HIV-infected cells and yielded a Fab variant with superior drug-like properties, while maintaining breadth of HIV Env recognition. Further validation is required to determine if this approach will be generally applicable to optimizing bNAbs for the selective elimination of cells harboring latent HIV. We thank the entire Gilead HIV Antibody R&D team, and in particular those additional individuals who supported the PGT121 engineering effort: Y Bai, K Brendza, B Dransart, W Fung, Y Gokarn, J Goldsmith, D Jin, M Kanwar, J Kaur, M Kenney, L Lagpacan, J Li, A Majka, A Medina, U Mehra, J Papalia, S Patrick, A Post, M Schenauer, L Serafini, S Sinha, A Snowden, M Sobczyk, G Stepan, H Stephenson, L Tong, R Wang, P Weissburg, and B Xia. 90 ° Figure 2. Targeting HIV Reservoirs with anti-Env antibodies. The primary barrier to HIV Cure is the presence of a latent and long lived reservoir of memory T cells containing integrated HIV provirus that is resistant to elimination using ART therapy alone 2 . The “kick and kill” strategy depicted here makes use of a latency reversal agent to stimulate production of viral antigens on infected cells. Anti-Env antibodies are capable of both recognizing the HIV envelop glycoprotein on the surface of infected cells, and recruiting host immune cells to kill and eliminate these cells. B al U S6 57 H T5 93 U S7 27 100 0 20 40 60 80 Killing Emax (%) PGT121 1 2 3 4 5 2 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 mAb rank (% Killing) PGT121v PGT121 Screen: gp120/Env binding HIV Neutralization ADCC Screen: gp120/Env binding HIV Neutralization ADCC Stability (Tm, low pH) Immunogenicity IC 50 PGT121/IC 50 Variant 0.01 0.1 1 10 92US727 92HT593 BaL 92US712 92US657 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 A25 A26 A27 A28 A29 A30 IC 50 PGT-121/IC 50 variant 0.01 0.1 1 10 Figure 1. Representative bNAbs and their epitopes on Env. HIV bNAbs are capable of cross clade HIV neutralization. Most bNAbs evolve over many years within infected individuals, developing the ability to both recognize rare conserved epitopes on Env and to become tolerant to changes in the ever evolving virus. Rendering of HIV Env based on PDB 4TVP 1 . FcgRIIIA 176F [Ab] nM A 450 0.01 1 100 10000 0 1 2 3 FcgRIIIA 176V [Ab] nM A 450 0.01 1 100 10000 0 1 2 3 PGT121v ∆Fuc PGT121 Isolate US657 [mAb] ug/mL 0.001 0.01 0.1 1 10 100 -20 -10 0 10 20 30 40 50 60 % Reduction in Infected Cells Isolate VS28 [mAb] ug/mL 0.001 0.01 0.1 1 10 100 -20 -10 0 10 20 30 40 50 60 % Reduction in Infected Cells Cell Membrane 3:1 2:1 - 3:1 3:1 3:1 3:1 1:1 1:1 - 3:1 CD4bs V1-V2 loops VRC01 NIH 45-46 3BNC117 N6 B12 F105 CH103 44-VRC13 PG16 PGT145 CH59 Glycan-V3 PGT121 10-1074 PGT128 PGT135 Glycans 2G12 gp120/gp41 PGT151 8ANC195 35022 MPER/gp41 10E8 4E10 2F5 -------SDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGIPERFSGSPD SYVLTQPPSV K T GNNI KS H QKP V V YDS NS QMQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSPGKGLEWIGYVHKSGDTNYSPSLKSR V T G SY QP IYY S N VNLSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMDVWGNGTQVTVSS TI V F K SSV TAV ---------------YYY Y K T SPFGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVL --- N SR QV SSDH- K 121LC: GL: 121LC: GL: 121HC: GL: 121HC: GL: a b CDR T-Cell Epitope Key: Glycosylation Site HC primary NK cells + HIV HIV-infected cells CD4+ T cells (primary or cell-line) gp120 gp120 LC co-culture ADCC Dose Response mAb Concentration mAb A mAb B mAb D mAb C % Reduction in infected cells + + A6 B4 B8 B11 B1 B7 B10 B3 B12 B5 B2 B9 B6 % Donor Response PGT121 B9 B3 B6 B4 VRC01 A33 KL H 0 10 20 30 40 50 70 90

pgt121 antibody engineering: enhanced infected cell killing and … · 2020. 6. 8. ·...

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