How to Screen a Billion Drug Candidates?

Transcription

1 How to Screen a Billion Drug Candidates? Single Cell Functional Assays: Ultra-sensitive, Ultra-fast, Ultrahigh-throughput Next-Generation Drug Discovery The advantages of de novo protein engineering for the generation of functional fully human antibodies to GPCRs Michael Gallo President Innovative Targeting Solutions Antibody Engineering 2017

2 No GPCR Targeting Antibody has been Approved by the United States Food and Drug Administration and few are in Clinical Development

3 The GPCR Functional Challenge Problem Solution 1. Soluble antigen 2. Tolerance/immunogenicity Use full length functional receptor in its native confirmation on the cell surface In vitro system required 3. Challenging epitope (rare specificities) 4. Potency Maturation* * Primary binders will not have the potency required for a drug development Large CDR3 diversity De novo targeted diversity to CDRs 100s of millions of variants Ultra-high throughput functional screens

4 De Novo Protein Engineering 1. Large repertoires (>1 billion unique CDR3s) 2. Single cell functional screens (>1 billion cells)

5 V(D)J Recombination Nature s most powerful diversification system V segments D segments J segments V(D)J Recombination via the proteins RAG1, RAG2 and TdT * * Imprecise joining of segments adds to diversity

6 De Novo Protein Engineering: Expand, Induce, Screen Pre-recombination Engineered HEK293 based HuTARG cell Scale up >10 9 cells No cloning of libraries No transformation of libraries Tetracycline Induction of V(D)J recombination De novo engineering: the cell does all the work Each cell expresses a unique antibody Functional assays at the single cell level A billion cells screened for function in a day

7 High Homogeneous Antibody Expression with HuTARG Very high surface expression allows efficient selection of antigen-specific cells Libraries consist of all human V, (D) and J segments Antibodies expressed as full-length IgG (no reformatting) Complex screens can be built into the cell line for functional screening up front (receptor neutralization, species crossreactivity) 7

8 Rare Specificities: Generation of Fully Human Antibodies to Two Proteins with Minimal Homology Identical positions: 75 Identity: 44% Signal peptide XX---XXXXXXXXXXXX--XXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX ** ::*. *** *.* * *.* *: :.**. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX ::*:* * *.. * :.*******. **:**** : :*:**.******:*: XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX * ********* **:**: * **:***:**:********::***

9 Ultra-rare Specificities: Targeting MHC-Peptide Complexes Dr. Song Tan Penn State Specificity determined by a limited number of amino acids The majority of the epitopes represent the MHC heterodimer

10 De Novo Engineering: The Cell does all the Work 1. Large repertoires (>1 billion unique CDR3s) 2. Single cell functional screens (>1 billion cells).

11 Functional GPCR Agonists Directly from De Novo Libraries Cell expressing non-agonist antibody will not activate cell surface reporter marker Cells expressing an agonist antibody will activate the reporter allowing isolation of the cell with the agonist Un-recombined cell line Integration GPCR and camp reporter gene Induction of recombination to generate > 10^9 diversity

12 Library Screen to Identify GPCR Agonists Negative Control Library Peptide Graft Library #1 Peptide Graft Library #2 Reporter Activation Agonists Antibody Expression Cells producing agonist peptide grafted antibodies are visible as reporter positive cells following magnetic bead enrichment Isolation of reporter positive cells identified fully human antibodies that activate the GLP1 receptor

13 Characterization of Agonist Antibody to the GLP-1 Receptor camp (pmol/ml) ITS007-V129 (inactive mutant) ITS007-V125 GLP-1R negative GLP-1R positive Cell line Cells Expressing Surface Reporter 20% 15% 10% 5% 0% ITS007-V125 (ug/ml) Binding (AU) 1. Agonist activity of antibody is GLP-1 Receptor dependent 2. Agonist activity correlates with binding to GLP-1 receptor in a dose dependent manner

14 Specificity of Agonists for the GLP1 Receptor Glucagon-Fc Dulaglutide ITS007-V129 ITS007-V130 ITS007-V222 Glucagon Receptor GLP-1 Receptor GLP-1R and GCGR Agonist Assay 0.5ug/ml agonist X axis = IgG (binding) Y axis = Cell Surface Reporter GLP1-R agonists specifically activate the GLP-1 receptor and not the Glucagon receptor

15 Scalable 3.0 x 10^6 cells per ml 300 ml = approx. 1 billion cells = 1 day of FACS sorting 3000 ml = approx. 10 billion cells = 10 days of FACS sorting

16 Functional Activation of a Drug Selectable Marker Cell expressing non-agonist antibody will not express the gene for puromycin resistance Puromycin Cells expressing an agonist antibody will activate the reporter allowing selection with puromycin Un-recombined cell line Integration GPCR and camp reporter gene Induction of recombination to generate > 10^9 diversity

17 Functional Selection Utilizing a Puromycin Resistance Marker Add Puromycin to Culture Cell alone Cell with ligand Cell with agonist antibody Y Y Y Y Y Y Y Inactive Receptor Y Activated Receptor Activated Receptor TF binding site Minimal Promoter Puromycin Resistance TF binding Minimal site Promoter Puromycin Resistance TF binding site Minimal Promoter Puromycin Resistance No Transcription Sensitive to Puromycin Puromycin selection greatly improves the ease of library enrichment Repertoires can be processed at a much larger scale Active Transcription Resistant to Puromycin Active Transcription Resistant to Puromycin Y Antibody GPCR Transcription active Transcription inactive

18 Puromycin Selection of Agonist Antibodies No Agonist Agonist Antibody Day 1 Puromycin sensitive cells do not adhere Day 3 Puromycin resistant cells expand and pack in the plate Day 0: Cells trypsinized and plated 1:3 with or without agonist antibody (100ng/ml) and Puromycin 4ug/ml

19 De Novo In Situ Mutagenesis Potency Maturation

20 Tdt: Primary MoA is Intimately Coupled to Deletion/Replacement GATCGATCttt cgatcgatc CTAGCTAGaaa gctagctag Deletions GATCXXXX CTAGXXXX XXXXXXTC XXXXXXAG Tdt recruits factors which have 3 5 exonuclease activity to the RAG synapse Increased Tdt levels results in increased deletions There is a linkage between deletion and Tdt insertion (N nucleotide addition) Large deletion have correspondingly large N insertions Small deletions have correspondingly smaller N insertions Reaction can be tuned so that the end result is that final length of the products are approximately equivalent to original size

21 CDR1 Optimization Cassette Structure Example: CDR1 Sequence QASQDISNYLN RSS Insertion QASQD 12bp RSS 23bp RSS ISNYLN ~90% will be composed of variants within 3AA each side of the junction diversified QAXXXISNYLN QASXXISNYLN QASQDXXXYLN QAXQDIXXYLN QASXDISXYLN QASQXIXNYLN

22 Affinity Maturation of an Antibody Diversified at Light Chain CDR1 using RAG/TdT Mediated Recombination Antigen binding Antigen binding Antibody expression Light chain diversified at CDR1 using RAG/TdT Enrichment with antigen linked to magnetic beads Cells expressing antibodies of similar affinity cluster along diagonal lines Antibody expression Red: Original antibody Blue: Library of antibodies diversified at light chain CDR1 using RAG/TdT and then antigen enriched Green: High affinity variant isolated from library by flow cytometry

23 CDR Mutagenesis Design All 6 CDRs in parallel CDR1 CDR2 CDR3 Constant FR1 FR2 FR3 JK CDR1 CDR2 CDR3 Constant FR1 FR2 FR3 JK CDR1 CDR2 CDR3 Constant FR1 FR2 FR3 JK A RSS cassette is placed every other amino acid Each variant antibody has amino acid changes in only 1 CDR Mutations from different CDRs can be combined Heavy chain and light chain CDRs can be combined

24 In Situ Mutagenesis/Maturation: Optimization of 6 CDRs in Parallel Clone 1 Clone 2 HuTARG Clone 150pM High Affinity 15nM Parental Binding to Target Light Chain Optimization Target Binding Clone 1 Clone 2 Heavy Chain Optimization Target Binding Surface IgG Expression Surface IgG Expression Approximately 1 billion variants are screened for affinity and expression 100 to1000 fold affinity improvement has been observed in a single round

25 CDR Optimization to Increase Potency Directly Antibody under the control of a Tetracylcine induced promoter TET CRE V(D)J Variants Reporter Gene Tune promoter activity such that current lead does not activate reporter gene Generate a library of variants and sort out cells able to signal at this new potency threshold

26 Functional GPCR Agonists Directly from De Novo Libraries Cell expressing non-agonist antibody will not activate cell surface reporter marker Cells expressing an agonist antibody will activate the reporter allowing isolation of the cell with the agonist Un-recombined cell line Integration GPCR and camp reporter gene Induction of recombination to generate > 10^9 diversity

27 De Novo Protein Engineering: Expand, Induce, Screen Pre-recombination Engineered HEK293 based HuTARG cell Scale up >10 9 cells No cloning of libraries No transformation of libraries Tetracycline Induction of V(D)J recombination De novo engineering: the cell does all the work Each cell expresses a unique antibody Functional assays at the single cell level A billion cells screened for function in a day

28 Direct Potency Enrichments Post - Sort 1 Post - Sort 2 Post - Sort 3 camp Responsive Reporter Gene Expression After 3 rounds of sorting variants able to deliver an agonist signal at new threshold are clearly visible Initial sort was with >500 million variants

29 Potency of Assessment of Isolated Leads from CDR3 Potency Maturation Isolated clones from potency maturation libraries exhibit very large increases in activity

30 Traditional Screening Paradigm Isolate Antigen Specific Binders Antibody reformatting/production Screen for Function Develop Functional Assay Scale 384 well Number screened 400K (1000 plates)

31 The HuTARG Way Scale single-cell EXPAND Ultra high-throughput (up to a billion variants) Develop Reporter Based Functional Assay Within HuTARG Line INDUCE SORT Ability to directly mature based on function Ability to screen for increased potency

32 HuTARG : Generation and Engineering of Future Biologics Single Domains (HCAb), Bites, Darts Fully Human mabs Fully Human TCRs Peptide-grafted mabs GPCRs, Ion Channels Single Chain mabs (CARs) Function First Screening Affinity Maturation Potency Maturation Alternative Scaffolds (Adnectins, Darpins, Avimers, etc)

33 Acknowledgements Paul Kang Craig Pigott Abby Lin Falene Chai Christine Yao Zenlynn Tsang Marie-Christine Perry Katie Baillie