Novel strategies for antibody discovery and optimization

Transcription

1 Novel strategies for antibody discovery and optimization Rene Hoet, Biologics, Head of Antibody Lead Discovery Berlin, February 1, 2016

2 Agenda Biologics at Bayer Success and Future Challenges Therapeutic Antibodies Antibody Discovery Technologies at Bayer Need to Select for Functional Antibodies Phenotypic Antibody Screening Optimization of Therapeutic Antibodies in Bayer Page 2 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

3 Bayer HealthCare Pharmaceuticals Top 10 Products Sales 2014 Examples of Biologics products at Bayer Replacement factors such as Factor VIII (Kogenate) in Hemophilia A Supplements such as Interferon-β (Betaferon) in Multiple Sclerosis EYLEA, a VEGFR1/R2 Fc-fusion protein for patients with wet Age-related macular degeneration (wet AMD) Page 3 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

4 Page 4 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

5 Agenda Biologics at Bayer Success and Future Challenges Therapeutic Antibodies Antibody Discovery Technologies at Bayer Need to Select for Functional Antibodies Phenotypic Antibody Screening Optimization of Therapeutic Antibodies in Bayer Page 5 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

6 Antibody Humanization Technology has Driven Therapeutic mab Success Recent Expansion into new targets and new indication areas Currently 52 antibodies approved by FDA Table legend: Autoimmune / inflammatory Cardiovascular Hematol. Tumors Solid tumors Page 6 Ophthalmology Resp. disease Osteoporosis Hematology Bact. Infection FDA approved Abs Table updated Jan Murine Chimeric Humanized Human Orthoclone Reopro Simulect Zenapax Rituxan Synagis Remicade Herceptin Mylotarg* Zevalin Erbitux Campath* Humira PD Bexxar Raptiva* Xolair Tysabri Avastin Lucentis Vectibix Soliris Simponi Cimzia Arzerra Actemra Ilaris Sterara Prolia Adcertis Yervoy Benlysta PD Perjeta Abthrax Kadcyla Gazyva Lymphoseek 2014 Sylvant Keytruda Cyramza PD Entyvio BLINCYTO Opdivo Lemtrada 2015 Cosentyx Unituxin Praluent Praxbind Empliciti Darzalex Nucala Repatha Cinquil Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

7 8 Novel antibody therapeutics approved in 2015 in the US* Approvals extended to new disease areas (Cardiovascular & Respiratory Disease) INN Brand name Target; Molecular format Indication Status in US Daratumumab Darzalex CD38; Human IgG1 Multiple myeloma Approved Elotuzumab Empliciti SLAMF7; Humanized IgG1 Multiple myeloma Approved Dinutuximab Unituxin GD2; Chimeric IgG1 Neuroblastoma Approved Mepolizumab Nucala IL-5; Humanized IgG1 Severe eosinophilic asthma Approved Reslizumab Teva IL-5; Humanized IgG4 Asthma Approved Alirocumab Praluent PCSK9; Human IgG1 High cholesterol Approved Evolocumab Repatha PCSK9; Human IgG2 High cholesterol Approved Idarucizumab Praxbind Dabigatran; Humanized Fab Reversal of dabigatran-induced anticoagulation Approved * Modified from J. Reichert, Antibodies to Watch in 2016, Mabs, Epub Dec 2015 Page 7 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

8 Human Therapeutic Antibody Technologies In vitro or in vivo technology? In vivo In vitro It is all about antibody diversity to your target!!! Yeast Display Page 8 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

9 Challenges Future Therapeutic Antibodies Insights into human disease that leads to new mab targets that are disease relevant How to improve therapeutic efficacy of antibodies Page 9 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

10 Insights into human disease leads to new therapeutic targets Example: Immune modulation:targeting T cells Blocking Immune Checkpoint Regulators Immune system controls tumor growth ( immune surveillance ) Tumors escape from immune surveillance by coopting mechanisms which downregulate the immune system Aim of immunotherapy is to restore efficient antitumor immunity Current focus on antibodies as inhibitors of immune checkpoint regulators actla-4 apd1 and apdl1 actla-4 Ab ipilimumab/yervoy and apd1 Ab pembrolizumab/keytruda approved for metastatic melanoma apd1 Ab nivolumab/opdivo approved for metastatic melanoma and squamous NSLC Current focus on new combinations with actla-4 and apd-1(l) therapy Impressive results with combination therapy of actla-4 and apd-1 in melanoma (40% ORR, 31% w. tumor reduction >80% after 12w) (Wolchok, JD et al. (2013) NEJM) Page 10 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

11 Challenges Future Therapeutic Antibodies Insights into human disease that leads to new mab targets that are disease relevant How to improve therapeutic efficacy of antibodies Page 11 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

12 Improve therapeutic efficacy of antibodies Future Antibody Developments Antibody Drug Conjugates- Targeted delivery of toxins Antibody Drug Conjugates (ADCs) Arming antibodies to kill On the market Opportunity in Bayer: Biologics expertise + Medicinal chemistry Adcetris (SGN-35, Brentuximab vedotin, Anti-CD30-MMAE, Takeda with Seatle Genetics) FDA approved in August 2011 for Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) Kadcyla (ado-trastzumab emtansine, Genentech/Roche with ImmunoGen) FDA approved Febr for late stage metastatic breast cancer BAY Anti-Mesothelin ADC Fully human monoclonal antibody (K D = 15 nm) targeting human mesothelin Conjugated to maytansinoid-derivative DM4 (tubulin polymerization inhibitor) Phase I dose escalation study solid tumors finalized (PR in some patients) Phase IIa study initiated for Mesothelioma. Orphan drug designation for treatment of mesothelioma in USA and EU Collaboration partners, Morphosys & ImmunoGen Inc. Page 12 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

13 Improve therapeutic efficacy of antibodies Future Antibody Developments Bi/Multi-specifics present new therapeutic modalities Multispecifics present new therapeutic opportunities that include: - dual inhibition of cytokines - cross-linking of receptors - PK modulation - redirected T-cell lysis Redirected T-cell lysis of PSMA-expressing tumors (ex-micromet) collaboration, Bayer holds IND BAY Phase I On the Market Dec. 3, 2014, FDA approval blinatumomab (BLINCYTO, Amgen Inc.) for B-cell precursor acute lymphoblastic leukemia (R/R ALL). Page 13 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

14 Agenda Biologics at Bayer Success and Future Challenges Therapeutic Antibodies Antibody Discovery Technologies at Bayer Need to Select for Functional Antibodies Phenotypic Antibody Screening Optimization of Therapeutic Antibodies in Bayer Page 14 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

15 Antibody Fab Phage Display Technology & HTS at Bayer Data collection BDP (Genedata) Page 15 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

16 Agenda Biologics at Bayer Success and Future Challenges Therapeutic Antibodies Antibody Discovery Technologies at Bayer Need to Select for Functional Antibodies Phenotypic Antibody Screening Optimization of Therapeutic Antibodies in Bayer Page 16 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

17 Example: Human Antibodies to a Novel Receptor No Correlation between Affinity and Activity of Leads Characterisation Fabs Characterisation IgGs Selection for in vivo models Fab phage display selection Screening 10 4 soluble Fabs 1700 Hits Characterisation 147 Unique Fabs Antibody Lead Cell binding Off-rate Hu/Mo Crossreactivity Inhibition Phosphor- ylation Inhibition Proliferation 1 + >1.E E E E E E E E E In vitro assays In vivo models 17 IgGs E >1.E E > E E E E Page 17 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

18 How to Increase the Chance to Select Functional Active Antibodies? Select leads in final therapeutic format (IgG instead of Fab/scFv) Adopt library format: IgG libraries New technologies: yeast & mammalian display allow IgG display Hurdles new technologies: library size and robustness of technologies Screen earlier in biological relevant functional assay Combine Fab phage display and HTS of IgGs Page 18 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

19 Screening for IgGs with Biological Activity to Increase Chance for Success Workflow Fab screening # Clones Alternative workflow IgG screening Fab-phage display library 2-5 x 96 Fab on Phage Pre-screening 20, ,000 Screening soluble Fabs Characterization Unique Fabs Efficient IgG batch reformatting using Golden-Gate Cloning Screening 1,000-5,000 IgGs (~1µg) Individual IgG reformatting Fabs IgGs tested by in vitro biological assays (0.5-1 mg) IgGs tested in vivo (100 mg) Select for IgGs with biological activity Faster Discovery process Earlier Go-no go decision Page 19 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

20 Enzyme assay (RFU) HT Functional Assays Increase the Chance for Success Examples from Successful Early Functional HT Screening Fab functional enzyme assay Enzyme inhibitor HT screening (FXI, 50,000 Fabs) IgG Screening for Agonists: TweakR NF-kB reporter gene assay (3600 IgGs) Page 20 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

21 Future Antibody Discovery at Bayer: Selection for Functional Active IgGs using Yeast Display Antibody lead discovery: Conventional: Antibody fragment libraries Screening typically based on affinity binding of Ab fragments to an antigen Limited number of full-length antibodies can be tested on functionality Screening by Display Display AND expression of Yeast full-length antibody libraries High throughput clonal multi-parameter antibody sorting using quantitative selection criteria Uniform expression format for all process steps without time consuming reformatting processes Antibody screening based on biological functionality Yeast selects for Developability antibodies Time consuming reformatting Functional assays Yeast Display Enhance quality and speed for discovery and engineering of human therapeutic antibodies Page 21 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

22 Novel Large Human IgG yeast Library Opportunity to Screen earlier for functional activity Heavy Chain (HA tag) Complexity human IgG library 8x10 9 E.Coli IgG libraries Yeast IgG libraries IgG display validated Sequence diversity validated Light Chain (Myc tag) 22 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

23 Agenda Biologics at Bayer Success and Future Challenges Therapeutic Antibodies Antibody Discovery Technologies at Bayer Need to Select for Functional Antibodies Phenotypic Antibody Screening Optimization of Therapeutic Antibodies in Bayer Page 23 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

24 What can we Learn from Decades Small Molecule Lead Discovery? Small molecules are often selected based on functional (cell based) assays and only in a second step characterized for target specificity Antibodies are typically selected first specifically on a target and only in a second step these antibodies are tested in a functional assay This limits unexpected (innovative) findings for Antibodies Can we reverse this process in Antibody Lead Discovery to broaden the target space? Introduce first functional activity selection in Antibody Lead Discovery prior to target selection? Page 24 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

25 Why Phenotypic Selection for Antibody Discovery? Phenotypic drug discovery Conventional drug discovery Terstappen et al, Nat Rev Drug Disc, 2007 Unknown target on tumor cells Screening for biologic effects on cells or tissues Target deconvolution Known and validated target Identification of Antibodies specific to the target Screening for biologic effect on target of interest Confirmation of therapeutic relevance Advantages Phenotypic Screening: Native environment and confirmation of target antigen Exclusive binding to extracellular domains Binding of immunogenic and accessible epitopes Broaden Target Space: Potential to find novel functional epitopes on known targets or (new) targets not associated previously with this disease Validation provides immediately starting Lead Antibody 25 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

26 L u m in e s c e n c e (R L U ) G M e a n A P C lo g G M e a n P E Proof on Concept: Phenotypic selection for Specific Internalization on Acute Myeloid Leukemia (AML) Potential for ADC targets 560 unique scfv-clones identified in NGS Unique IgGs produced after bulk reformatting and NGS Three sequential selection rounds of phage library on AML target cells depletion on PBMCs T P P T P P T P P T P P T P P T P P T P P T P P T P P T P P T P P (Is o ty p ) A M L 1 A M L 2 P B M C A M L 1 A M L 2 P B M C A M L 1 A M L 2 P B M C A M L 1 A M L 2 P B M C A M L 1 A M L 2 P B M C A M L 1 A M L 2 P B M C A M L 1 A M L 2 P B M C A M L 1 A M L 2 P B M C A M L 1 A M L 2 P B M C 1 0 A M L 1 A M L 2 P B M C A M L 1 A M L 2 P B M C A M L cell viability c e lls Successful target deconvolution for >70% antibody lead clones A M L s e le c te d Ig G N C 70%clones showed positive internalization in HUMZAP assay A M L s e le c te d Ig G N C P C C D % clones showed specific binding to 2 AML cell lines No non-target cell binding nm µ g /m l Page 26 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

27 Agenda Biologics at Bayer Success and Future Challenges Therapeutic Antibodies Antibody Discovery Technologies at Bayer Need to Select for Functional Antibodies Phenotypic Antibody Screening Optimization of Therapeutic Antibodies in Bayer Page 27 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

28 Optimization of Human Antibody Drugs Today Current trends in antibody discovery towards proteins with drug-like properties Ranking of leads earlier in discovery for manufacturing (CMC) feasibility Sequence optimization in FRs and CDRs to reduce immunogenicity and potential CMC risks Deamidation sites Free disulfides Non-conserved Met Glycosylation sites Etc. Developability assessment based on Expression Aggregation Thermo-stability Solubility etc. Page 28 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

29 LC HC CL CH1 Antibody Optimization: Testing HT individual substitutions in all CDR residues (35) (49) (58) (36) (46) Page 29 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

30 Affinity Maturation and Lead Selection Process by functional screening Example: Optimization of an agonistic antibody to TweakR Non-optimized Lead: BAY1 Bay2: best single aa substitution of Bay1 Affinity and potency maturation Bay3: best 2nd round maturation variant M = Maturation G = Germlining BAY2 best single substitution 2 nd round maturation recombination (Bay3) Bay4: Final germlined Lead candidate CRC Germlining Optimized Lead BAY4 Bay1 Bay2 Bay4 Page 30 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

31 Engineering Antibodies for Success The full measure of lead optimization Potency: affinity; specificity; functional effects (agonism, antagonism, cell death); internalization rates; effector functions Immunogenicity: germlining and T-cell epitope reduction of framework and CDRs CMC feasibility: expression, stability, homogeneity, aggregation, solubility Germline Affinity Improvements in Antibody Optimizations 1.000x 40x 17x 150x 300x Cross reactivity: to animal models Speed: accommodating all four in timely lead optimization campaigns All of the above 1 Project Fab Germline dev. Crit. res. Temp. stab. Affin. [nm] Lead C 22 Opt. lead C 2 Page 31 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

32 Summary Earlier incorporation of functional assays with IgGs Phage selections on large Fab-phage libraries linked with HT IgG screening allow early functional testing Use of IgGs in primary functional assays Large Yeast IgG display library offers opportunity for earlier functional screening Phenotypic Antibody Lead Discovery Antibody Optimization beyond affinity maturation Potential to identify novel targets and novel functional epitopes of known targets Proof of concept study in AML identified Antibody Leads that internalize specifically (potential ADC targets) In >70% of Antibody Leads (novel and known) AML targets could be identified Opportunity to extend method to other functional screenings/ ONC indications Germlining antibodies in Frameworks and CDRs to reduce as much sequencebased immunogenicity Sequence optimization in FRs and CDRs to increase potency, reduce immunogenicity and potential CMC risks Combined analysis to find optimal characteristics Therapeutic Lead Antibody Page 32 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016

33 Acknowledgements Thanks to Biologics Research Cologne, Wuppertal & San Francisco Page 33 Novel Strategies for Antibody Discovery and Optimization, Rene Hoet, Bayer, Biologics & Biosimilars Congress, Febr. 1, 2016