DAR-901A polyantigenic, inactivated, whole-cell

mycobacterial vaccine for the prevention of tuberculosis

Ford von Reyn MDGeisel School of Medicine at Dartmouth

Clinical development plan

Goal: Develop a booster vaccine to prevent tuberculosis in adolescents and adults who have previously been immunized with BCG

Strategy: 1. Chose a polyantigenic reagent to mimic infections and

vaccines known to prevent TB in humans2. Conduct initial efficacy trials in HIV-infected adults in

setting of endemic disease– Represent population with the highest risk of TB– Permits smaller sample size for efficacy trial– A vaccine that is effective in HIV-infected persons,

is likely to be more effective in healthy subjects

2

Polyantigenic exposures induce protection against TB in humans

• Mycobacterial infections (natural)– M. tuberculosis (Heimbeck, Norway; Stead, US)– Non-tuberculous mycobacteria (NTM; Edwards, US)

• Mycobacterial vaccines– Single-dose, live organism

• M. bovis BCG• M. microti (vole bacillus)

– Multiple-dose, inactivated whole cell organisms • M. bovis/MAC/MTB combination (VIP, VDS, Italy)• M. bovis (Jamaica)

• Common features: – Whole organism, polyantigenic– Cross protection within genus

3

SRL 172: An Inactivated NTM vaccine

• Heat-inactivated, whole-cell preparation derived from rough variant of an environmental non-tuberculous mycobacterium (NTM)– designated M. vaccae (MV) by J. Stanford & G. Rook– originally targeted for therapy of TB

• Animal studies – immunogenic and effective in preventing TB

(Skinner, Hernandez-Pando, Abou-Zeid)

• GMP product manufactured by SR Pharma– Demonstrated safe and well-tolerated in humans– 0.1 mL intradermal dose contained estimated 109 CFU

4

DAR-901: Overview

• Heat-killed environmental NTM vaccine for prevention of TB

• SRL 172 (1994-2008)– Agar-grown vaccine, limited size of production lots– Studies conducted: Phase I, II, III – Sites used: US, Zambia, Finland, Tanzania (NIH)

• Broth-grown DAR-901 (2011- ) – Product development in collaboration with Aeras

• Established MCB from seed lot used for SRL 172 • Developed high yield, scalable GMP broth mfg process• Animal immunogenicity studies: nearing completion

– Phase I/IIa human studies: Q4 2013, US and Tanzania5

Dartmouth Phase I, II and III Trials with SRL 172

Phase Site SRL172 (N)

Control(N)

1

US HIV - 9 0

US HIV+ 23 12

US HIV+ 11 0

2Zambia 22 22

Finland 19 20

3 Tanzania

~1000 ~1000

• All studies investigator-initiated (funding: NIH, EGPAF, Sigrid Juselius Foundation)• All safety, immunogenicity and efficacy studies conducted by the Dartmouth group as

a new product – no support, involvement or reference to work by Stanford, Rook, SR Pharma, Silence Therapeutics, or Immodulon

• All results presented in peer-reviewed publications6

Finland Phase II: Immunogenicity in HIV-positive

* P = 0.001 §p = 0.02 † p = 0.008

7

Study population: HIV pos, BCG pos; 70% on ART

CV = control (hep B)MV = SRL 172

IFN-γ responses to SRL 172 sonicate

Vuola et al, AIDS, 2003

Finland Phase II: Immunogenicity in 10 HIV-negative

8

Study population: Healthy BCG positive adults

Response to SRL-172 sonicate:

Baseline vs dose 3: p = 0.04 Baseline vs dose 5: p = 0.01 Baseline vs 1 year: p = 0.001

Median LPA response to SRL-172 sonicate after 5 doses:

HIV pos: 12,560 cpm HIV neg: 22,547 cpm (p = 0.170)

Lymphocyte proliferation responses to SRL 172 sonicate

Media alone

SRL-172 sonicate

WCL Ag85

9

DarDar Study – Design

• Study design: – randomized (1:1), double-blind, placebo-controlled– eligibility: HIV with CD4≥200, BCG scar– follow-up Q3 mos– immune assays: baseline and post-dose 5

• Intervention:– 5 intradermal doses of SRL 172 (or placebo)

10

2000 subjects

randomize

(1:1)

SRL-172 (0, 2, 4, 6, 12 mo)Placebo

Median f/u = 3.3 years

von Reyn et al. AIDS 2010; 24:675-85

DarDar Study – Outcome

At year 7, DSMB recommended the trial be stopped based on efficacy in preventing Definite TB

11

DarDar Study – Vaccine-induced responses

Antigen: SRL-172 sonicate SRL-172 sonicate MTB lipoarabinomannin

Assay: IFN-γ lymphocyte proliferation antibody (ELISA)

12Lahey T et al. Vaccine 2010; 28:2652-8

DAR-901 Development Program

• Scalable production method– SRL-172 was agar-grown – a procedure that scales poorly– Aeras has developed a scalable, broth-grown production method– That product is now designated DAR-901.

• Master Cell Bank prepared– 268 cryovials (5 mL)

• Drug product release assays validated– Potency: IL-12 induction in human macrophage cell line – Quantitation: qPCR of 16S RNA

• Bulk product manufactured – Yield from 20L Fermenter: 4.5 L at 49.2 g/L– Achieved greater dispersion and higher consistency than SRL 172– Estimated to represent 2,000 doses of final product

13

Among control (saline) animals, median SPU/10^6 cells was 35 for DAR-901 (sonicate) and <10 for the other antigens

Protocol: intradermal DAR-901 at week 0, 2, 4; dose range 0.01 to 2.5 mgAssay: IFN-γ spot forming units from splenocytes stimulated in vitroResults shown for optimal dose = 0.3 mg

DAR-901: IFNγ dose-response in B57BL/6 mice

DAR-901 SummaryA multiple dose series of agar-grown SRL 172 was safe, immunogenic and 39%

effective in preventing definite TB in HIV-positive subjects primed with BCG.– It was also immunogenic in HIV-negative subjects primed with BCG

Protection from SRL 172 is consistent with classically demonstrated human protection against TB (natural infection and vaccines) induced by polyantigenic exposure (including secreted, cell wall and cytosolic Ags) – Whole cell vaccine like DAR-901 is likely to stimulate both innate and

acquired immunityHomologous broth-grown DAR-901 (Aeras) has greater bacterial dispersion and

consistency than agar-grown SRL 172 and has the potential for higher efficacy in both HIV-positive and HIV-negative subjects

Phase I and IIa human safety and immunogenicity studies to begin Q4 2013 in US and Tanzania

15

Acknowledgements

Dartmouth Richard WaddellTim LaheySue Tvaroha

Lisa AdamsTodd MackenzieWendy Wieland-Alter

Tanzania Lillian MteiKisali PallangyoMuhammad BakariMecky Matee

Isaac MaroSajida KimamboJohnson LyimoBetty Mchaki

Boston Robert D. Arbeit C. Robert Horsburgh

Finland Jenni VuolaJuhani Eskola

Tarja LounasvaaraHanna Soini

New York Barry Kreiswirth

Aeras Tom EvansEric TsaoLew Barker

Veerabadran DheenadhayalanDominick LaddyAnn Ginsberg

16

END

Thank you

17

DarDar Study: Safety

• Koch reactions: – none, including 312 MV subjects with TST>5 mm

• Vaccine site: – Induration: average 5-6 mm – Desquamation: 37-58%– Local drainage: 22-49%– Sterile abscesses: 3 (0.3% of subjects, 0.06% of doses)

• HIV safety substudy in 150 subjects – Viral load and CD4 after each dose– No pattern of adverse changes

18

DAR-901 – Nonclinical Animal Studies - I

• Mouse Dose-Ranging & Immunogenicity Study– Species: both C57BL/6 and BALB/c mice– Intradermal immunization with heat-killed DAR-901– Three dose schedule: 0, 2, 4 weeks– Doses (mg): 0, 0.01, 0.03, 0.1, 0.3, 1, 2.5

• Tolerability– observed physical / behavioral changes

• Immunogenicity– collect samples at weeks 2, 4 and 6– stimulate cells in vitro using several antigen preparations – assay cytokine responses: Elispot of cells; multiplex of

culture supernatants19

DAR-901 – Nonclinical Animal Studies - II

• GLP Repeat-dose Toxicology Study– Mouse strain and dose selected based on above– Note: mouse is only species in which immunogenicity has

been demonstrated

• Dosing schedule– Total 6 immunizations (one more than maximum proposed

human exposure)

• Expected timeline– ~6 months from first dose to delivery of final report

• Proposed subcontractor – Spring Valley Laboratories, Sykesville, MD

20

DAR-901 – Proposed Phase I/IIa Trials

• DarDar trial used 5 dose regimen – 4 doses over 6 months plus booster at 1 year – highly desirable to shorten and simplify as supported by

prior work

• Goals of Phase I/IIa Trials – confirm safety, tolerability, and immunogenicity of

regimen of 2 vs 3 doses over 2-4 months plus booster at 1 year

– expand immunologic assessments– include both HIV-neg and HIV-pos subjects

21

Regimen HIV-neg HIV-pos Month

N N 0 2 4 6 8 12 14

D901 10 10 MV MV MV MV

PLA 5 5 PLA PLA PLA PLA

Immunology (predose)

DAR-901: Phase I in US

22

Subjects BCG positive

Regulatory US FDA; Dartmouth IRBs

Vaccine site evaluation 7 and 14 days after each dose

Safety Laboratory Studies

CBC, Chemistries, CD4schedule: 0†, 2†, 6, 14 mo (†, pre-dose)

Immunology IFN-γ response to DAR-901 sonicate, MTB whole cell lysate, Ag 85, ESAT

schedule: 0†, 6†, 8, 12†, 14 mo (†, pre-dose)

Regimen HIV-neg HIV-pos Month

N N 0 2 4 6 8 12 14

D901 20 20 MV MV MV PLA MV

Dar5 0 20 MV MV MV MV MV

PLA 10 10 PLA PLA PLA PLA PLA

Immunology

DAR-901: Phase IIa in Tanzania

23

Subjects BCG positive

Regulatory Tanzanian FDA; Dartmouth and Muhimbili IRBs

Vaccine site evaluation 7 and 14 days after each dose

Safety Laboratory Studies

CBC, Chemistries, CD4schedule: 0†, 2†, 8, 14 mo (†, pre-dose)

Immunology IFN-γ response to DAR-901 sonicate, MTB whole cell lysate, Ag 85, ESAT

schedule: 0†, 6†, 8, 12†, 14 mo (†, pre-dose)

Immune assays in I/IIb

• Assays previously used:– IFNg to DAR-901 sonicate, MTB WCL, ESAT

• Additional assays under discussion– Mycobacterial growth inhibition (performed at Aeras)– Transcriptome (using stored PBMCs)

• Final panel to be determined thru discussions with– Aeras CSO – NIH Vaccine Trials Network– DAR-901 Scientific Advisory Committee

24

DAR-901 Scientific Advisory Committee

Juhani Eskola, MD Deputy Director GeneralNational Institute for Health, FinlandChair, Steering Committee, TBVI

Vaccine trialsTB vaccine trials

Daniel Hoft, MD, PhD Director, Division of Infectious Disease, Allergy and Immunology

St. Louis Univ. School of Medicine

TB vaccine trials and immunology

David Lewinsohn, MD, PhD Professor of MedicinePortland VA Medical Center

TB vaccine immunology

George Siber MD Executive Board Chairman Genocea Biosciences

Vaccine trials

Peter Wright, MD Professor of PediatricsGeisel School of Medicine at Dartmouth

Vaccine trials

25

DAR-901 Development Timeline - I

26

March 2013 animal testing complete

June 2013 submit IRB applications to Dartmouth and Muhimbili (Tanzania)

August 2013 submit IND to US FDA and to Tanzanian FDA

September 2013 Initiate 30 subject Phase I at Dartmouth (completion in 18 mos, Feb 2015)

November 2013 initiate 80 subject Phase IIa in Tanzania (completion in 18 mos, April 2015)

27

Characteristic MV, n=988 Placebo, n=987 p value

Age, mean (sd) 33 .4 (8.0) 33.1 (7.7) 0.48

Female sex 756 (77%) 749 (76%) 0.74

CD4 cells / uL, median (minimum, 25th – 75th percentile, maximum )

425(200, 316 – 572, 1645)

403(200, 298 – 578, 2000)

0.30

Log HIV viral load, median (range)

3.96 (1.70 - 5.70) 3.95 (1.70 - 5.70) 0.70

HIV antiviral therapy 25 (2.5%) 33 (3.3%) 0.28

Prior treatment for TB 88 (9%) 81 (8%) 0.58

Tuberculin skin test ≥ 5 mm

312 (32%) 319 (33%) 0.81

Baseline Subject Characteristics

28

Prevention of TB by 5 doses of inactivated M. bovis

TB disease rates vaccine: 11% (23/210) control: 19% (39/206) efficacy = 42% despite inability to segregate until immunization complete

Opie, Freund et al. 1939, psychiatric hospital in Jamaica

-Opie, 1939

Multiple dose inactive whole cell vaccines effective against other intracellular bacterial pathogens: plague, typhoid

29

Natural Protection Against TB

Risk of TB among student nurses, Oslo, 1924-26

PPD TST Status at

Enrollment Number Cases of TB (%)

Negative 185 62 (34)

Positive 152 3 (2)

Heimbeck, Br J Tuberc 1938;32:154-66.

30

Target product profile

• Indication and application– Booster vaccine for the prevention of tuberculosis in

adolescents and adults primed with childhood BCG– Safe, well-tolerated (heat-inactivated, no adjuvant)– Effective both pre- and post-infection– HIV-negative: booster for all adolescents and adults– HIV-positive: immunize at first diagnosis of HIV

31

Finland Phase II: Immunogenicity in HIV-positive

Study population: HIV pos, BCG pos; 70% on ART

CV = control (hep B)MV = SRL 172

* P <0.001 § P = 0.003 † P = 0.02

Lymphocyte proliferation responses to SRL 172 sonicate

32

DarDar Study 2001-2008 (Dartmouth / Dar es Salaam, Tanzania)

A prime-boost strategy to prevent TB in persons with HIV infection

• Hypothesis: – SRL 172 (boost) of childhood BCG (prime) will reduce

disseminated TB by 50% and definite TB by 50%• Enrollment criteria:

– HIV-positive, CD4 >200– BCG scar– no active TB (smear, culture, CXR – all negative)

• Sponsor: Division of AIDS (DAIDS), NIH• Cost: $8 million

33

34

DarDar Study – Immunologic Assessment

• Tuberculin skin testing – INH x 6 mos for subjects with TST >5 mm

• LPA and IFN-γ assays in response to– SRL-172 sonicate – ESAT-6– Ag85– MTB WCL (whole cell lysate)

• Ab to lipoarabinomannan (LAM)• Assays done at baseline and after dose 5 of vaccine• CD4 – annually

35

DarDar DSMB

Neal Halsey, MD (Chair)

Prof. of International HealthDirector, Institute for Vaccine SafetyJohns Hopkins Bloomberg School of Public Health

Kennth McIntosh, MD Prof. of Infectious Disease and ImmunologyHarvard School of Public Health

Clyde Crumpacker, MD Prof. of MedicineInfectious DiseaseHarvard Medical School

Mary Wilson, MD Assoc. Prof. of Global HealthHarvard School of Public Health

Paige Williams, PhD BiostatisticsHarvard School of Public Health

Andrew Swai, MD Muhimbili University of Health and Allied Sciences

36

DarDar Study: Compliance and Follow-up

• Doses administered– MV: 4616 (84% completed 5 doses)– Placebo: 4603 (83% completed 5 doses)

• All subjects seen every 3 months or if symptomatic– routine HIV care– active evaluation for new cases of TB

(sputum and blood cultures for TB, CXR)

• Loss to follow-up: 3% per year• Median follow-up: 3.3 years

37

DarDar Study

Endpoint Definitions and Outcomes

Clinical Endpoints in TB Vaccine Trials

Requirements for valid clinical endpoints•Pre-specified endpoint definitions•Blinded review of all potential endpoints by expert panel

39

DarDar Study – Endpoint Definitions

• Disseminated Tuberculosis– Positive blood culture

• Definite Tuberculosis– Positive sterile site culture (other than blood)– or 1 sputum culture with >10 cfu– or 2 sputum cultures each with 1-9 cfu– or 2 positive sputum smears

• Probable Tuberculosis– {1 positive sputum smear or 1 sputum culture with 1-9 cfu}

plus {a positive CSR or positive symptoms}– OR positive CXR plus symptoms plus clinical response to

treatment40von Reyn et al. AIDS 2010; 24:675-85

DarDar Study Endpoints

• Primary Endpoint– Time to Disseminated TB

• Secondary Endpoints– Time to first episode of Definite TB– Time to first episode of Probable TB– Time to first episode of Definite / Probable TB

41von Reyn et al. AIDS 2010; 24:675-85

DarDar Study – Disseminated TB (ITT)

Group N Events Censored

(1) M. vaccae 988 7 981 risk reduction = 48%

(2) Placebo 987 13 974 p-value = 0.16 42

DarDar Study – Definite TB (ITT)

43

Group N Events Censored

(1) M. vaccae 988 33 955 risk reduction = 39%

(2) Placebo 987 52 935 p-value = 0.0273

Molecular epidemiology (IS6110 typing)

• Clustering – represents patients with recently acquired TB infection– 25/74 (74%) vaccine recipients – 31/47 (66%) placebo recipients

• Polyclonal disease– represents patients with two different strains isolated– 15 subjects had isolates available from 2 different cultures– Overall, 7 (40%) had polyclonal disease

• 3/7 (43%) subjects with 2 sputum isolates• 3/8 (38%) subjects with 1 sputum and 1 blood isolate

– Evidence for concurrent reactivation and recent infection • 4/6 (67%) polyclonal cases had 1 clustered and 1 unique isolate

Adams, Kreiswirth, Arbeit et al. J Clin Micro 2012; 50:2645-50 44

DarDar Study Immunology

Vaccine responses and predictors of TB risk

DarDar Study: Baseline Immunology

Baseline positive responses among 888 DarDar subjects

46

IFN-γ, LPA (lymphocyte proliferation) to SRL-172 sonicate; LAM: antibody to lipoarabinomannin; MAS: M. avium sensitin

Matee M et al. J Infect Dis 2007;195:118-123

Lahey et al. J Infect Dis 2010; 205: 1265-1272

Immunologic Predictors of TB risk - I

Among Placebo subjects, those who developed TB had, at baseline, weaker IFN-γ and lymphocyte proliferation responses.

47

Immunologic Predictors of TB risk - II

Conversely, the risk of TB was decreased among the Placebo subjects who at baseline had positive IFN-γ responses to multiple antigen preparations (“polyantigenic” responses).

48

Antigen preparations: Ag85, ESAT-6 and WCL (whole cell lysate)

Lahey et al. PLoS ONE 2011; 6 (7): e22074

SRL-172: Phase II in HIV-positives with CD4>200

Study Design Assays Comment

Zambia1 5 doses (0, 2, 4 mo and 6, 12 mo)

N: 22 SRL, 22 PLABCG pos & BCG neg

LPA to MV Responses greater in BCG pos

Immunogenicity data limited to 5 doses

Finland2 5 dosesN: 19 SRL, 20 PLABCG pos only

LPA to MV LPA to MTBIFN to MV

Included 10 HIV-neg controls – had greater LPA responses

49

1. Waddell et al, Clin Infect Dis 2000; 30: S309-152. Vuola et al, AIDS 2003; 17: 2351-2355

Immunologic Predictors of TB risk - III

• Results are adjusted for age, baseline CD4 count, previous TB treatment and a positive TST. Results were similar among TST-pos and TST-neg, and across HIV viral loads (for those with data).

50Lahey et al PLoS ONE 2011; 6(7): e22074

Number of antigen preparations eliciting positive IFN-Υ responses

Moving beyond SRL 172

Dartmouth Studies of SRL-172: Summary

• An inactivated whole cell mycobacterial vaccine• Safe, well-tolerated

– Based on experience over 14 years with >1,000 subjects administered multiple doses intradermally

– Subjects include: HIV pos and neg; adults and children(most with prior BCG)

• Immunogenic– Induced increased immune responses to multiple different

antigen preparations• Effective

– Successful proof-of-concept study in HIV-pos, BCG-pos subjects– Demonstrated decreased risk of Definite Tuberculosis

52