De-risking Vaccine Formulation Design

Transcription

1 De-risking Vaccine Formulation Design Drug Delivery & Formulation Summit and Expo San Diego 13 June 2016 Roger H Brookes

2 Overview Introduction TB vaccine Proof-of-Concept for antigen (H4) specific immunomodulation with: TLR4 IC31 Application to formulation design Conclusion / Next steps

3 Introduction - Formulation Design HTP screening Physicochemical analysis A major gap is linkage to immune functionality Mouse immunogenicity We are developing a complementary strategy - a fresh human Whole Blood (hwb) approach A simple root methodology Compatible with technologies from Discovery Platforms Preemptive of more sophisticated approaches Systems Biology, Biomarkers, etc A B Roque et al. Mol Pharm. Feb 2015

4 A Simple Root Methodology Fresh human whole blood (hwb) approach * * A profound adjuvant-modulated antigen-specific response Brookes et al, Hum Vaccin Immunother. Jan 2014

5 Tuberculosis has killed more than any other infectious disease in history. Over a billion lives in the past two hundred years The most successful Pathogen on earth Stefan Kaufmann, Immunol Lett Tom Paulson, Nature 2013

6 TB vaccine candidate - H4:IC31 Antigen: H4 fusion protein (partnership with Statens Serum Institut, SSI) H4 (41.4 kda) = Ag85B Secreted by replicating bacteria, early stage of infection Mycolyl transferase activity (cell wall biogenesis) High affinity to fibronectin (host cell entry) Immunogenic in Mtb+ & BCG-vaccinated subjects A BCG-boosting vaccine TB10.4 Secreted by replicating bacteria, early stage of infection Member of ESAT-6 family, function unknown (modulation of early infection events?) Immunogenic in Mtb+ & BCG-vaccinated subjects Adjuvant: IC31 (Valneva) 2/27/2012 IC31 = Component 1 Anti - bacterial peptide (KLKL KLK 5 KLK) (KLKL KLK 5 KLK) Anti-bacterial peptide Depot effect Ag and ODN1a uptake by APC + Component 2 Immunostimulatory oligonucleotide (non-cpg) ODN1a APC stimulation via TLR9 signaling Th1 bias Ratio KLK:ODN1a 25:1 (molar ratio) Dosages Evaluated 500nM:20nM 100nM:4nM

7 Adjuvant Customization Examples Guy B. Nat Rev Microbiol. 2007

8 PoC for H4:TLR4 A TLR4 modulated H4 response

9 A Simple Root Methodology Immunofunctional Analysis hwba An adjuvant modulated antigen specific response can be monitored in vitro

10 A Simple Root Methodology Immunofunctional Analysis hwba An adjuvant modulated antigen specific response can be monitored in vitro

11 pg/ml PoC demonstrated with H4:TLR4-Adjuvant Adjuvant-modulated antigen-specific response H4 2ug/mL hwba-h4-ifng-day6-hd217-1/20 dilution H4 0.2ug/mL H4 0.02ug/mL H ug/mL H ug/mL H ug/mL no Adj 10ng/mL Adj 5ng/mL Adj 2.5ng/mL Adj Adj only rhsa 2ug/mL LPS PMA/Iono Brookes et al, Hum Vaccin Immunother. Jan 2014

12 PoC for H4:IC31 IC31 modulated an H4 response

13 PoC for H4:IC31 was quite different from TLR4 Unlike TLR4-Adjuvant : No pro-inflammatory cytokines were found And IFNγ response required a longer days of total culture IC31 MoA is distinct from TLR4

14 Unlike TLR4A the H4:IC31 IFNγ response is significantly higher after days of culture The H4:IC31 responses were amplified when cultures were extended to days (4 subjects, 8 x observations) IC31 modulates the H4 response at days (7 subjects) Aboutorabian et al, Hum Vaccin Immunother. Jun

15 Unlike TLR4A the H4:IC31 IFNγ response is significantly higher after days of culture The H4:IC31 responses were amplified when cultures were extended to days (4 subjects, 8 x observations) IC31 modulates the H4 response at days (7 subjects) Aboutorabian et al, Hum Vaccin Immunother. Jun

16 Application to formulation design The H4 : IC31 ratio

17 Early Publication Preclinical study at SSI.antigen doses of 5 and 15 μg did not induce significant protection against M.tb, reducing the dose to 0.5 μg selectively increased the number of polyfunctional T cells and induced a strong protection Conclusions/Significance: Small changes in the antigen dose can greatly influence the induction of specific T cell subpopulations (100nmol KLK) Aagaard, PLoS One. Jun 2009

18 Clinical trial (Aeras) Dose ranging study in South Africa Loss of response at highest H4 dose Geldenhuys et al. Vaccine Jun 2015

19 Adsorption isotherm and binding capacity studies - IC31:H4 Adsorption isotherm experiments showed an excess of IC31 is used to bind H4 in clinical doses (9-88 fold) It was questioned why IC31 was in such excess and whether it had been optimized or could be reduced Possibility to reduce potential safety concerns around use of a new adjuvant A mouse immunogenicity study was designed to address the question The IC31 binding capacity result was used to calculate a dosages of IC31 that was either in excess or limiting, relative to H4

20 Mouse immunogenicity demonstrates need for excess binding of IC31 IC31 excess H4:IC31 minimal binding IC31 limiting Aboutorabian et al, Hum Vaccin Immunother. Jun

21 Animal immunogenicity and translation to human? Whole body in vivo response But TLR expression is species specific Can the animal response replicate the response in humans? TB vaccine needs to restimulate BCG primed immunity Impractical for animal studies (BCG needs ~3 months rest period) Does the excess IC31 results in mouse translate to human? Same formulations used in mouse study were tested in hwb

22 The human Whole Blood (hwb) approach confirms need for an excess of IC31 (5 subject) H4 dose: 0.05 µg/ml IC31 fold excess Controls IC31 excess H4:IC31 minimal binding IC31 limiting Profound adjuvantmodulated antigenspecific response in 5 BCG primed subjects Aboutorabian et al, Hum Vaccin Immunother. Jun

23 Zeta potential on H4 formulated with excess limiting IC31 + At > 9 fold excess IC31 zeta potential switches from negative to positive charge - IC31 excess H4:IC31 minimal binding IC31 limiting Aboutorabian et al, Hum Vaccin Immunother.Jun

24 Conclusion & Discussion The hwb culture shows a profound adjuvant-modulated antigen-specific response May be particular to hwb (intact innate immunity) PoC demonstrated for H4:TLR4 and H4:IC31 activities are quite distinct Diverse approaches confirm need for binding an excess of IC31 Animal in vivo and hwb ex vivo Bridges mouse human / de-risking clinical progression A potential mechanism is suggested H4:IC31 particle changes negative positive when IC31 is > 9 fold excess Rational explanation for unexpected mouse and clinical trial data A potential critical quality attribute (pcqa)

25 Next Steps Many questions remain around the IC31-modulated H4-response Are other cytokines or inflammatory proteins involved? How does an IC31 (TLR9) modulated response differ from that of TLR4? Can we define an immunomodulation signature? Streamlined to a focused higher throughput screen Is the IC31 modulated memory response unique? Clinical data suggests memory may have remarkable durability (H1:IC31) van Dissel JT, Vaccine Can we define markers associated with this population? Application to innovative technologies Automation / Robotics Immune response evaluation Gene expression / Proteomics / Systems Biology Multiplexing

26 Unique adjuvant-modulated antigen-specific response The H4:IC31 responses were amplified when cultures were extended to days (4 subjects, 8 x observations) IC31 modulates the H4 response at days (7 subjects)?

27 De-risking Formulation Design

28 Acknowledgement Salvador F Ausar 1 Sepideh Aboutorabian 1, Florence Boudet 2, Roger H Brookes 1 Annie Dookie 1, Jalil Hakimi 1, Sandrine Montano 2, Nausheen Rahman 1, Cristopher Roque 1, 1 Bioprocess Research and Development, Toronto, Canada 2 Research, Marcy l Etoile, France TB Project Team SSI/Valneva R&D (Building 95) Toronto site

29 Screening vaccine formulations in hwb using a multi-well analysis Hakimi et al, Vaccine Adjuvants, Springer MMB, in press Adjuvant modulated antigen specific response F3 and F4 (but not F2) are significantly different from F1 (Wilcoxon-Mann-Whitney) F1 F2 p = , F1 F3 p= and F1 F4 Drug p= Delivery & Formulation Summit, San Diego 13 June 2016