Aridis Pharmaceuticals Inc. · OTC:ARDS
Aridis Pharmaceuticals — PEP Talk Conference 2020
39 slides from Aridis Pharmaceuticals's conference presentation, Jan 24, 2020
Slide 1: APEX Antibody Discovery and Production Technology
TM APEX Antibody Discovery and Production Technology Vu L. Truong PEP Talk Conf. San Diego 24Jan20 Aridis Pharmaceuticals 1
Slide 2: Outline
Outline ▪ Single cell cloning & mAb discovery ▪ B-cell immortalization using cell fusion ▪ Enhanced mAb production of established cell lines • CRISPR-enabled activation of transcription regulatory genes ▪ mAb production CHO cell line technology Aridis Pharmaceuticals 2
Slide 3: Outline
Outline ▪ Single cell cloning & mAb discovery ▪ B-cell immortalization using cell fusion ▪ Enhanced mAb production of established cell lines • CRISPR-enabled activation of transcription regulatory genes ▪ mAb production CHO cell line technology ▪ COMING SOON: Transposons-mediated gene amplification
Slide 4: Antibody Discovery and Production Challenges
Antibody Discovery and Production Challenges Rapid functional Immunized or screening of entire Rapid production of Convalescent repertoire for potent, mAb at high titer rare B-cell Size of B-cell repertoire ~1x 106 or more unique cells Immunized Aridis Pharmaceuticals 4
Slide 5: Single Cell Screening & Clonal Selection Using NanoArrays
Single Cell Screening & Clonal Selection Using NanoArrays Key Features ▪ Enables comprehensive, functional screening at single cell level of 106-107 cells simultaneously ▪ ~100,000x minimization of tissue culture wells. 0.5 to 3 million wells can be accommodated in a 96-well plate surface area ▪ Inc
Slide 6: Fabrication of Nanoculture Arrays
Fabrication of Nanoculture Arrays Peel UV Add polymer to master mold Key Features ▪ Simple, reproducible fabrication process Add photoresist layer & mask ▪ Scalable, cost effective, no specialized companion equipment ▪ Variety of dimensions, geometries, biocompatible polymer materials with surface c
Slide 7: B-cell Repertoire Screening, Single Cell Analysis & Single Cell Cloning with APEX Nanoarrays
B-cell Repertoire Screening, Single Cell Analysis & Single Cell Cloning with APEXTM Nanoarrays Fluorescently labeled A) B) Antigen Ag detection antibody C) (proteins, viruses, bacteria, etc. Antibody 75µm 75µm 100µm B-cells or CHO 30µm 30µm APEXTM Nanoarrays: A) Schematic top view of honeycomb shape
Slide 8: Versatile On-chip Assay Format for Single Cell Analysis
Versatile On-chip Assay Format for Single Cell Analysis Top view of Nanoculture wells (A-D). Wells are embedded with antigen as indicated, followed by detection with fluorescence labeled secondary mAb A B C D Anti-IgG Live B-cell secreting Anti-RSV F- Antigen= Human AR-105 mAb Antigen= adsorbed whol
Slide 9: APEX Technology Suite
TM APEX Technology Suite ▪ Single cell cloning & mAb discovery ▪ B-cell immortalization using cell fusion ▪ Enhanced mAb production of established cell lines • CRISPR-enabled activation of transcription regulatory genes ▪ mAb production CHO cell line technology Aridis Pharmaceuticals 9
Slide 10: Current Practice: Recombinant approaches to mAb discovery & production requires 1.5 - 2 years
Current Practice: Recombinant approaches to mAb discovery & production requires 1.5 - 2 years from discovery to drug in vials Expression Upstream & Vector Downstream Construction, Process Clonal Development, Cloned CDRs or Selection Scale Up GMP H&L chains production of IgG’s Mouse immunization Mous
Slide 11: Bypassing Recombinant Approaches Using B-cell Fusion & Immortalization
Bypassing Recombinant Approaches Using B-cell Fusion & Immortalization Selected Fusion with Immortalized Nanoculture GMP B-cell Fusion Partner Hybridoma Clonal Select production Aridis Pharmaceuticals 11
Slide 12: Engineering of the Fusion Partner Cell Line
Genetically engineered, non-secreting IgG mouse heteromyeloma cell line developed with high cell fusion Engineering of efficiency the Fusion Genetic modifications introduced to activate master transcription factors associated with increased transcription Partner Cell Line and translation of H & L ch
Slide 13: B-cell Screening & Cell Fusion Proof-of-Concept Study: RSV mAb Discovery
B-cell Screening & Cell Fusion Proof-of-Concept Study: RSV mAb Discovery RSV-F antigen used for PoC study ▪ Availability of primary RSV-F+ B-cells from biobanks ▪ Well described structure of RSV-F protein ▪ Antigen specific tools, assays and reagents are available Aridis Pharmaceuticals 13
Slide 14: Repertoire Screening & Cell Fusion Work Flow
Repertoire Screening & Cell Fusion Work Flow IMMORTALIZED MAB ISOLATE PBMCS B-CELL EXPANSION SECRETING PROGENY CLONAL SELECTION PRODUCTION Ag-Selective B-cell expansion and enrichment Fusion B cell B cell Partner PBMC isolation from B Cell Fusion with B Cell fused with Stable & high titer Nanocultur
Slide 15: RSV-F Ag-specific Human B Cell Activation
RSV-F Ag-specific Human B Cell Activation Day 7 Ag-specific Human B-cell Activation 5 % Memory B cells Pre-sorted Memory B Post-Sorted Memory B Post-Sorted 4 RSVF+ Memory B 3 2 1 4% 90% 97% 0 0 5 10 15 Day RSV-F memory B-cell secretion 8000 IgG Titer (ng/ml) Day 7 PBMCs Day 7 RSVF B-cell 6000 activa
Slide 16: Engineered Fusion Partner Exhibits High Fusion Efficiency
Engineered Fusion Partner Exhibits High Fusion Efficiency On-chip Single Cell Fusion: 10%-20% Fusion Efficiency [i.e. 10-20% of progenies that fused, survived, and are producing mAb] After Fusion Distribution of secreted IgG by fused hybridoma clones Mean Fluorescent Intensity 16000 12000 8000 * 400
Slide 17: Rapid identification of high-producing RSV-F specific hybridoma
Rapid identification of high-producing RSV-F specific hybridoma 15000 A B Mean Intensity Units 10000 ** Top producers are rare (less than 0.05% of the population) 5000 n = 62,500 0 5 yb 10 H R F -A - SV O H R C ti- An A. Panel of nanowells of hybridoma producing Human anti-RSVF mAb. B. Distribution
Slide 18: High content screening allows for rapid identification of rare high-producing hybridoma clones
High content screening allows for rapid identification of rare high-producing hybridoma clones Fused cells B C D Top 50 Lead Candidates A p<0.0001 ✱✱✱ 15000 Mean Intensity Units 10000 CHO-AR301 5000 E 0 a 1 30 om R rid -A yb O H H C X Ig ab M (A) Hybridomas generated from cell fusion compared to CHO
Slide 19: Stable Hybridoma Productivity Comparable to CHO over 60 Generations
Stable Hybridoma Productivity Comparable to CHO over 60- Generations Endpoint IgG Titer (g/ml) 4 G6 G24 G60 3 2 1 ▪ Endpoint titer of 3 hybridoma clones producing human 0 IgG demonstrates consistent productivity at each time point -1 12 1 b 30 8 -1 - -1 -4 R F2 ▪ Titer at generations 6, 24, and 60
Slide 20: Cell Fusion to Create Hybridoma: Advantages & Disadvantages
Cell Fusion to Create Hybridoma: Advantages & Disadvantages Advantages: ▪ Well established process Current Disadvantages: ▪ Up to ~1 year longer than the hybridoma approach Advantages: ▪ Convenient B-cell starting point ▪ Bypasses recombinant steps Aridis ▪ Little to no process development needed ▪
Slide 21: Outline
Outline ▪ Single cell cloning & mAb discovery ▪ B-cell immortalization using cell fusion ▪ Enhanced mAb production of established cell lines • CRISPR-enabled activation of transcription regulatory genes ▪ mAb production CHO cell line technology Aridis Pharmaceuticals 21
Slide 22: Key Master Transcription Factor Genes of Interests
Key Master Transcription Factor Genes of Interests FR5 Drm2 Bxp2 BPRs ▪ Transcription factor genes selected for CRISPR mediated addition & LiE2 activation • Drm2 • Fr5 Ig Ig Ig • Bxp2 mRNA TRANSPORT TRANSCRIPTION TRANSLATION ▪ Promotor activation is universal and can be applied to any host cell irre
Slide 23: CRISPR-Cas Targeted Gene Activation
Precise editing Reconstituted NHEJ CRISPR sgRNA DSB CRISPR-Cas Targeted Gene Activation assembly Precise deletion cEdit-X TM / replacement HDR RNAP TSS TSS dCas9 D10A CRISPRi Binding RNAP H84A RNAP Block Transcription inhibition Cas dCas9 TSS TSS mRNA activator RNAP CRISPRa RNAP Binding CRISPRa Recr
Slide 24: Multiplex CRISPR Guided Activation (CRISPRa)
Multiplex CRISPR Guided Activation (CRISPRa) 1. In traditional activation, open reading frames (ORF) of genes need to cloned under a ubiquitous promoter. The size of ORF MPH is a rate-limiting step as only 1-2 genes can be activated 2. Short gRNA (20 nt) for transcriptional activation allows for mul
Slide 25: Multiplex TF Activation by CRISPRa
Current Challenges ▪ gRNA that allow for potent transcriptional activation Multiplex TF needs to be empirically be determined ▪ Not all gRNAs will activate gene transcription Activation ▪ Multiple transfection and transduction are required for a single gene by CRISPRa ▪ Current CRISPRa technologies
Slide 26: Potent synergistic activation of transcription factors in Fusion Partner by multiplex guided RNAs
Potent synergistic activation of transcription factors in Fusion Partner by multiplex guided RNAs Fold Change in mRNA Expression Synergistic activation of master transcription factor genes • Predicted synergistic activation of targeted transcription factors with single gRNA • Multiplex approach show
Slide 27: Hypothesis: CHO production cell lines have similar master transcription regulatory elements
Hypothesis: CHO production cell lines have similar master transcription regulatory elements, but are not comparably activated or utilized as in B-cells Question: Can B-cell MTREs Enhance Productivity of Existing mAb Production CHO Cell Lines? Aridis Pharmaceuticals 27
Slide 28: CRISPR-Activation of Endogenous Master TF Regulatory Elements Increases Productivity of a CHO Cell Line
CRISPR-Activation of Endogenous Master TF Regulatory Elements Increases Productivity a CHO Production Cell Line CHO-AR1 CHO-AR105 CHO-Aer 15000 *** 10 CHO-Aer CHO-AR105 Fold Change Expression Mean Intensity Units 8 CHO-Aer CHO-AR105+CRISPRa 10000 Activated 6 4 5000 2 0 0 CHO-AR10 CHO-Aer CHO-Aer CHO
Slide 29: Case Study #2: CRISPR-Activation of CHO-Sal Cell Line
Case Study #2: CRISPR-Activation of Endogenous Master Transcription Regulatory Elements of CHO-Sal Cell Line Top 100 (Average of top 100 clones)* 20000 16.0 Relative Units 14.0 Ring Intensity Units 15000 Relative Units 12.0 Relative Units 10.0 96-well T-flask 10000 Shake flask 8.0 6.0 5000 4.0 2.0 0
Slide 30: Outline
Outline ▪ Single cell cloning & mAb discovery ▪ B-cell immortalization using cell fusion ▪ Enhanced mAb production of established cell lines • CRISPR-enabled activation of transcription regulatory genes ▪ mAb production CHO cell line technology Aridis Pharmaceuticals 30
Slide 31: Current State of the Art in mAb Manufacturing
Current State of the Art in mAb Manufacturing mAb mAb gene Random integration Single cell screening & Cell clone-specific GMP expression vector into genome Clonal selection manufacturing process development Production 5-8 months 8-10 months 4-6 months ~$0.5m ~$1m $2m-$4m Months Aridis Pharmaceutical
Slide 32: Product Concept
Product Concept: ▪ Engineered CHO cell lines with CRISPR-mediated transcription activation harboring a full length mAb H/L genes that is designed to be swapped out for the CDR or H/L of interest • Integration sites and H/L gene copy numbers are known and unchanged ▪ CDR or H/L can be switched from t
Slide 33: APEX's BREATH CHO Master Cell Line: Development of a Manufacturing Process Template
APEX’s BREATHTM CHO Master Cell Line: Development of a Manufacturing Process Template Master Cell Line Harboring IgG1 Ready for IgG1 Gene Swap √ MTRE activated √ Manufacturing Process √ Scalability to By CRISPR to be Developed GMP Manufacturing to be demonstrated B.R.E.A.T.HTM B-cell Regulatory Elem
Slide 34: APEX CHO Cell Line: CDR or H/L Chain Swappable CHO Host
APEXTM CHO Cell Line: CDR or H/L Chain Swappable CHO Host Site-specific CRISPR guided integration Selected Cloned CDRs B.R.E.A.T.HTM Nanoculture GMP B-cell or H&L CHO Clonal Select production of IgG’s B.R.E.A.T.HTM B-cell Regulatory Elements Assisted Transcription Host Aridis Pharmaceuticals 34
Slide 35: APEX Technology Solutions
APEXTM Technology Solutions APEX mAb Nanoarrays mAb gene Random integration Single cell screening & Cell clone-specific GMP expression vector into genome Clonal selection manufacturing process development Production APEX: Discovery, Development, and Manuf. ~12-15 months time saving Months ~$0.8m - $
Slide 36: CRISPR-Cas Targeted Gene Replacement
CRISPR-Cas Targeted Gene Replacement Random deletions Native NHEJ Cas Cas9 endonuclease TSS TSS Precise editing Reconstituted NHEJ CRISPR sgRNA DSB assembly Precise deletion cEdit-X TM / replacement HDR RNAP TSS TSS dCas9 D10A CRISPRi Binding RNAP H84A RNAP Block Transcription inhibition mRNA dCas9
Slide 37: Engineering of CHO with B-cell Master Transcription Factors (B.R.E.A.T.H CHO)
Engineering of CHO with B-cell Master Transcription Factors (B.R.E.A.T.HTM CHO) Genetically engineered transfection-ready production CHO cell line harboring H &L chains of a human IgG1 and B-cell master transcription factor regulatory elements Proprietary CRISPR-Mediated Activation Genetic modificat
Slide 38: Target Specific Insertion of Heavy and Light Chain CDR3 Genes
Target Specific Insertion of Heavy and Light Chain CDR3 Genes BREATHTM CHO Master Cell Line producing Sal Site-specific CRISPR guided CDR3 gene swap Sal GFP mAb mAb Aridis’ approach technology utilizes CRISPR/Cas to achieve site specific integration of antibody heavy and light chain genes for maximu
Slide 39: APEX Technology Suite
TM APEX Technology Suite B-cell Repertoire Screening: APEXTM NanoArrays B-cell immortalization using cell fusion mAb Productivity Enhancement: APEXTM CRISPR-assisted TF activation BREATHTM CHO Master cell line designed for CDR swapping Aridis Pharmaceuticals 39