TG1050, an HBV-targeted immunotherapeutics, efficiently decreases HBV viremia and antigenemia in a preclinical model; a meta-analysis and the determination of the involvement of CD4 and CD8 T cells. Roland Kratzer 1 , Alexei Evlachev 1 *, Karine Lélu-Santolaria 1 *, Benoît Sansas 2 *, Doris Schmitt 3 , Clarisse Dubois 1 , Yasmin Schlesinger 3 , Jean-Baptiste Marchand 3 , Michel Geist 3 , Renée Brandely 3 , Annie Findeli 3 , Thierry Menguy 3 , Nathalie Silvestre 3 , Marie-Louise Michel 4,5 , Geneviève Inchauspé 1 , Perrine Martin 1 *All these authors equally contributed to this work. 1 Transgene SA, Dept. of Infectious Diseases, Lyon, France - 2 Transgene SA, Smart Data Lab, Strasbourg, France - 3 Transgene SA, Smart Virus Lab, Strasbourg, France - 4 Institut Pasteur, Laboratoire de pathogénèse des virus de l’hépatite B, Paris, France - 5 INSERM U994, Paris, France INTRODUCTION Purpose: Current therapies (nucleos(t)ide analogs or peg-IFNa) for chronic hepatitis B virus (HBV) infection rarely achieve virus clearance. Cohort studies have shown the critical role of cellular immune responses to control HBV infection. We developed an HBV-targeted immunotherapeutic called TG1050 and have shown the induction of multispecific T cells in an HBV-persistent mouse model (AAV-HBV) together with antiviral properties (Martin, Gut 2015 Dec;64(12):1961). We report here a meta-analysis of 5 experiments performed with the AAV-HBV model. Furthermore, we have started to dissect in this HBV model TG1050- associated mechanism of action in particular the role of CD4 and CD8 T-cells. CONCLUSION META-ANALYSIS OF PRE-CLINICAL DATA FOR TG1050 IN AN HBV TOLERANT MOUSE MODEL SHOWS : ANTIVIRAL EFFECT: Significant treatment effect on viremia and HBsAg levels (p=0.015 and p<0.001 resp.) Decreasing (≥ 0.5 log) viremia and HBsAg levels in 55% and 62% of mice, resp. (75% and 64%, resp., in long experiments >16Wp1) Mice receiving TG1050 were found to have 19 or 10 times higher chance to present a DNA or HBsAg response, resp. using a logistic regression (not shown) Mice receiving TG1050 show a higher probability of early response (decline in viral parameters; time to response) HUMORAL IMMUNE RESPONSE: Anti-HBs seroconversion (clinical goal of HBV therapies) in 24% of mice (in long experiments, >16Wp1) ANALYSIS OF TG1050 MODE OF ACTION IN AN HBV TOLERANT MOUSE MODEL SHOWS : CD8 AND CD4 T CELLS INDUCED BY TG1050 ARE INVOLVED IN ANTIVIRAL EFFICACY. FURTHER STUDIES ARE ONGOING TO VALIDATE THESE PRELIMINARY DATA A PHASE 1/1B STUDY (FIRST-IN-MAN AND DOSE FINDING) IS UNDERWAY 3 MATERIAL & METHODS TG1050 treatment in AAV-HBV mice : C57BL/6 mice were injected intravenously with recombinant AAV-HBV, encoding 1.2 copies of the HBV genome. In this model HBV antigens and DNA replication intermediates (HBV mRNA incl. pgRNA) are detected within the liver as well as HBsAg, HBeAg and HBV infectious particles in the blood of injected mice, with a persistence of around 1 year 2 . In parallel no HBV-specific T cells or antibodies were detected in AAV-HBV injected mice whereas an increased number of regulatory T cells and IL-10 producing T cells are present in the liver. This model mimics to some extent the HBV chronic infection. Four to 5 weeks after AAV-HBV injection, TG1050 was injected multiple times by sub-cutaneous route at a dose of 2x10 9 vp/injection/mice and viral parameters were monitored at various time points. HBsAg in mouse sera was quantified using a BioRad kit (Monolisa HBsAg plus using a recombinant HBsAg protein for quantification). HBV DNA in mouse sera was quantified using a qPCR assay (limit of quantification : 100 copies/reaction). Anti-HBsAg/HBcAg antibodies were detected by an « in house » ELISA assay. Meta-analysis: For the meta-analysis 5 preclinical experiments, lasting in total between 11 weeks to 20 weeks, were considered for statistical analyses: Female mice, C57BL/6 strain, AAV only or AAV+TG1050 treatment (at 2x10 9 vp/injection/mice), at least 3 time points after TG1050 administration. Global Mixed model: A global mixed model was done with all experiments considering the following covariates: Time, Treatment, the interaction between Time and Treatment and the HBsAg value at baseline as fixed effects and the Experiment as random effect. Meta-Analysis for interaction term: The meta-analysis was done by estimating for all experiments the interaction term Day*Treatment and the associated standard error. Then a meta-analysis using a fixed effect model was done by weighting estimation with the inverse-variance. Percentage of responder: A mouse was considered as “Responder” if it presented an HBsAg/DNA decrease higher than 0.5 log to the baseline value for two or more time points during the study (consecutive or not). The percentage of responders was calculated for each experiment and then the mean average percentage of responders was calculated for each treatment group and compared with a non-parametric Wilcoxon-Mann-Whitney test. TTR : The Time-To-Response (TTR) was defined as the time between the first TG1050/AAV administration and the time of response (defined as the second time point presenting a decreased more than 0.5 log). If a mouse did not present a response, the TTR was censored at the last blood sample measurement. A Cox model was done to estimate the Hazard Ratio and the estimated confidence interval. TG1050 mode of action experiments : In similar experimental settings, CD4 and/or CD8 T cells were depleted (twice a week, 150 μg, starting at D34 before 3 TG1050 immunizations (D40, D47, D54) until the end of the experiment D77) or adoptively transferred (7.5x10 6 CD8+ or CD4+ cells purified by magnetic beads from HBV-free mice immunized by 1 TG1050 immunization 2 weeks before into AAV-HBV mice) to determine their involvement in antiviral efficacy of TG1050. Transferred CD8+ T cells contained ~1-2x10 5 HBV-specific (polymerase and core) multimer positive T cells. Livers have been sampled 4 weeks post transfer to analyze intracellular IFNg secretion by intrahepatic CD8 T cells. (Serum HBsAg and viremia has been analyzed as described before.) AASLD 2016 Liver Meeting – Boston, MA – November 2016 Based on non-replicative E1 and E3 deleted human adenovirus serotype 5 encoding a fusion protein comprising truncated HBV Core fused to a deleted and mutated HBV polymerase and 2 selected HBsAg domains (genotype D sequence) Develop a viral vector-based HBV-specific immunotherapeutic inducing potent, multispecific, sustained and cross reactive T cell responses displaying properties of immune responses detected in HBV resolving patients TG1050 OBJECTIVE TG1050 : OBJECTIVE AND DESCRIPTION 1 Core t Pol1 Pol2 Pol3* Env 2 Env 1 832 (37 aa) (29 aa) 1 148 1 Martin et al., Gut, Dec 2015; 64(12):1961-71 2 Dion et al., J Virol, May 2013; 87(10):5554-63 3 ClinicalTrials.gov: NCT02428400 REFERENCES DISCLOSURE All authors except MLM are or were employees of Transgene SA. HBV PERSISTENT MODEL HBeAg HBV HBsAg HBcAg HBV replication intermediates AAV-HBV 2 (i.v.) AAV2: ITR AAV8: Capsid HBV: 1.2x full length C57BL/6 Liver Blood Absence of detected HBV-specific immune responses Following TG1050 injection in AAV-HBV C57BL/6 mice: Induction of HBV-specific functional T cells in spleens and livers and sustained antiviral effects including anti- HBsAg seroconversion 1 HBV replication Corresponding author: R.Kratzer : [email protected] 1 2 3 4 5 1 2 3 4 5 RESULTS (I): META-ANALYSIS OF TG1050 ANTIVIRAL EFFECTS IN AAV-HBV MODEL A Linear regression for mean predicted from baseline HBsAg Viremia B Interaction term meta-analysis C Percentage of responders # # percentage of mice displaying at least a 0,5 log decrease or more on ≥ 2 time pts ** percentage of mice displaying detectable anti HBsAg Abs on ≥ 2 time pts D Percentage of responders** (anti-HBsAg antibodies) long experiments, 16-18 weeks post 1 st immunization, N=2 E Time to response HBsAg Viremia TG1050 administration leads to a persistent decrease in HBV viremia and HBsAg levels up to 110 days post immunization Meta-analysis shows efficacy of TG1050 to significantly decrease HBV viremia and HBsAg levels Mice receiving TG1050 show a higher probability of early response (HBV viremia and antigenemia decrease) % responders ** No treatment TG1050 0 20 40 60 80 anti-HBs 0.0 23.5 % responders # [SEM] No treatment TG1050 No treatment TG1050 No treatment TG1050 0 20 40 60 80 Viremia HBsAg Both * p=0.021 p=0.059 * p=0.011 8.8 55.3 22.0 62 5.3 42.7 HBsAg Viremia log log RESULTS (II): MODE OF ACTION OF TG1050 IN THE AAV-HBV MOUSE MODEL HBsAg levels w/ and w/o TG1050 treatment in AAV-HBV mice G DNA levels w/ and w/o TG1050 treatment in AAV-HBV mice F H HBsAg I Viremia T cell depletion experiment CD8 and/or CD4 depletion leads to an abolishment of antiviral activity of TG1050 confirming the expected mechanism of action. Residual antiviral activity is observed in mice escaping CD8 depletion (lower left graph F+G) Adoptive transfer of TG1050-educated T cells in AAV-HBV mice CD8 or CD4 transfer (7.5 x 10 6 splenocytes sampled at D14 post TG1050 immunization from HBV-free mice) into AAV-HBV mice 56 days post-AAV-HBV administration CD8 T cell transfer results in a decrease in viral titers in a subset of mice. This decrease is concomitant with the detection of IFNg+ CORE-specific CD8 T cells in the liver. Whereas antiviral activity due to direct injection of TG1050 is associated with the detection of IFNg+ POL-specific CD8 T cells. Dotted lines represent TG1050 immunizations
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TG1050, an HBV-targeted immunotherapeutics, efficiently decreases HBV viremia andantigenemia in a preclinical model; a meta-analysis and the determination of theinvolvement of CD4 and CD8 T cells.
Roland Kratzer1, Alexei Evlachev1*, Karine Lélu-Santolaria1*, Benoît Sansas2*, Doris Schmitt3, Clarisse Dubois1, Yasmin Schlesinger3, Jean-Baptiste Marchand3, Michel Geist3, Renée Brandely3, Annie Findeli3, Thierry Menguy3,
Nathalie Silvestre3, Marie-Louise Michel4,5, Geneviève Inchauspé1, Perrine Martin1 *All these authors equally contributed to this work.1Transgene SA, Dept. of Infectious Diseases, Lyon, France - 2Transgene SA, Smart Data Lab, Strasbourg, France - 3Transgene SA, Smart Virus Lab, Strasbourg, France - 4Institut Pasteur, Laboratoire de pathogénèse des virus del’hépatite B, Paris, France - 5INSERM U994, Paris, France
INTRODUCTION
Purpose: Current therapies (nucleos(t)ide analogs or peg-IFNa) for chronic hepatitis B virus (HBV) infection rarely achieve virusclearance. Cohort studies have shown the critical role of cellular immune responses to control HBV infection. We developed anHBV-targeted immunotherapeutic called TG1050 and have shown the induction of multispecific T cells in an HBV-persistentmouse model (AAV-HBV) together with antiviral properties (Martin, Gut 2015 Dec;64(12):1961). We report here a meta-analysisof 5 experiments performed with the AAV-HBV model. Furthermore, we have started to dissect in this HBV model TG1050-associated mechanism of action in particular the role of CD4 and CD8 T-cells.
CONCLUSION
META-ANALYSIS OF PRE-CLINICAL DATA FOR TG1050 IN AN HBV TOLERANT MOUSE MODEL SHOWS :
ANTIVIRAL EFFECT: Significant treatment effect on viremia and HBsAg levels (p=0.015 and p<0.001 resp.)
Decreasing (≥ 0.5 log) viremia and HBsAg levels in 55% and 62% of mice, resp. (75% and 64%, resp., in long experiments >16Wp1)
Mice receiving TG1050 were found to have 19 or 10 times higher chance to present a DNA or HBsAg response, resp. using a logistic regression (not shown)
Mice receiving TG1050 show a higher probability of early response (decline in viral parameters; time to response)
HUMORAL IMMUNE RESPONSE: Anti-HBs seroconversion (clinical goal of HBV therapies) in 24% of mice (in long experiments, >16Wp1)
ANALYSIS OF TG1050 MODE OF ACTION IN AN HBV TOLERANT MOUSE MODEL SHOWS :
CD8 AND CD4 T CELLS INDUCED BY TG1050 ARE INVOLVED IN ANTIVIRAL EFFICACY.
FURTHER STUDIES ARE ONGOING TO VALIDATE THESE PRELIMINARY DATA
A PHASE 1/1B STUDY (FIRST-IN-MAN AND DOSE FINDING) IS UNDERWAY3
MATERIAL & METHODS
TG1050 treatment in AAV-HBV mice : C57BL/6 mice were injected intravenously with recombinant AAV-HBV, encoding 1.2 copiesof the HBV genome. In this model HBV antigens and DNA replication intermediates (HBV mRNA incl. pgRNA) are detected withinthe liver as well as HBsAg, HBeAg and HBV infectious particles in the blood of injected mice, with a persistence of around 1 year2.In parallel no HBV-specific T cells or antibodies were detected in AAV-HBV injected mice whereas an increased number ofregulatory T cells and IL-10 producing T cells are present in the liver. This model mimics to some extent the HBV chronic infection.Four to 5 weeks after AAV-HBV injection, TG1050 was injected multiple times by sub-cutaneous route at a dose of 2x109
vp/injection/mice and viral parameters were monitored at various time points. HBsAg in mouse sera was quantified using aBioRad kit (Monolisa HBsAg plus using a recombinant HBsAg protein for quantification). HBV DNA in mouse sera was quantifiedusing a qPCR assay (limit of quantification : 100 copies/reaction). Anti-HBsAg/HBcAg antibodies were detected by an « in house »ELISA assay.
Meta-analysis: For the meta-analysis 5 preclinical experiments, lasting in total between 11 weeks to 20 weeks, were consideredfor statistical analyses: Female mice, C57BL/6 strain, AAV only or AAV+TG1050 treatment (at 2x109 vp/injection/mice), at least 3time points after TG1050 administration.Global Mixed model: A global mixed model was done with all experiments considering the following covariates: Time, Treatment,the interaction between Time and Treatment and the HBsAg value at baseline as fixed effects and the Experiment as randomeffect.Meta-Analysis for interaction term: The meta-analysis was done by estimating for all experiments the interaction termDay*Treatment and the associated standard error. Then a meta-analysis using a fixed effect model was done by weightingestimation with the inverse-variance.Percentage of responder: A mouse was considered as “Responder” if it presented an HBsAg/DNA decrease higher than 0.5 log tothe baseline value for two or more time points during the study (consecutive or not). The percentage of responders wascalculated for each experiment and then the mean average percentage of responders was calculated for each treatment groupand compared with a non-parametric Wilcoxon-Mann-Whitney test.TTR: The Time-To-Response (TTR) was defined as the time between the first TG1050/AAV administration and the time ofresponse (defined as the second time point presenting a decreased more than 0.5 log). If a mouse did not present a response,the TTR was censored at the last blood sample measurement. A Cox model was done to estimate the Hazard Ratio and theestimated confidence interval.
TG1050 mode of action experiments : In similar experimental settings, CD4 and/or CD8 T cells were depleted (twice a week, 150µg, starting at D34 before 3 TG1050 immunizations (D40, D47, D54) until the end of the experiment D77) or adoptivelytransferred (7.5x106 CD8+ or CD4+ cells purified by magnetic beads from HBV-free mice immunized by 1 TG1050 immunization 2weeks before into AAV-HBV mice) to determine their involvement in antiviral efficacy of TG1050. Transferred CD8+ T cellscontained ~1-2x105 HBV-specific (polymerase and core) multimer positive T cells. Livers have been sampled 4 weeks post transferto analyze intracellular IFNg secretion by intrahepatic CD8 T cells. (Serum HBsAg and viremia has been analyzed as describedbefore.) AASLD 2016 Liver Meeting – Boston, MA – November 2016
Based on non-replicative E1 and E3 deleted human adenovirusserotype 5 encoding a fusion protein comprising truncated HBVCore fused to a deleted and mutated HBV polymerase and 2selected HBsAg domains (genotype D sequence)
Develop a viral vector-based HBV-specificimmunotherapeutic inducing potent, multispecific,sustained and cross reactive T cell responsesdisplaying properties of immune responses detectedin HBV resolving patients
TG1050OBJECTIVE
TG1050 : OBJECTIVE AND DESCRIPTION
1Core t Pol1 Pol2 Pol3*Env 2Env 1
832
(37 aa) (29 aa)1 148
1 Martin et al., Gut, Dec 2015; 64(12):1961-712 Dion et al., J Virol, May 2013; 87(10):5554-63 3 ClinicalTrials.gov: NCT02428400
REFERENCES
DISCLOSURE
All authors except MLM are or were employees of Transgene SA.
HBV PERSISTENT MODEL
HBeAg
HBV
HBsAgHBcAg
HBV replication
intermediates
AAV-HBV2(i.v.)
AAV2: ITR
AAV8: Capsid
HBV: 1.2x full length
C57BL/6
LiverBlood
Absence of detected
HBV-specific immune
responses
Following TG1050 injection in AAV-HBV C57BL/6 mice:
Induction of HBV-specific functional T cells in spleens and livers and sustained antiviral effects including anti-HBsAg seroconversion1
HBV replication
Corresponding author: R.Kratzer : [email protected]
1
2
3
4
5
1
2
3
4
5
RESULTS (I): META-ANALYSIS OF TG1050 ANTIVIRAL EFFECTS IN AAV-HBV MODEL
A Linear regression for mean predicted from baselineHBsAgViremia
B Interaction term meta-analysis C Percentage of responders#
# percentage of mice displaying at least a 0,5 log decrease or more on ≥ 2 time pts
** percentage of mice displaying detectable anti HBsAg Abs on ≥ 2 time pts
D Percentage of responders**
(anti-HBsAg antibodies)long experiments, 16-18 weeks post 1st
immunization, N=2
E Time to response HBsAgViremia
TG1050 administration leads to a persistent decrease in HBV viremia and HBsAg levels up to 110 days post immunization
Meta-analysis shows efficacy of TG1050 to significantly decrease HBV viremia and HBsAg levels
Mice receiving TG1050 show a higher probability of early response(HBV viremia and antigenemia decrease)
% r
esp
on
der
s **
No treatm
ent
TG1050
0
20
40
60
80
anti-HBs
0.0
23.5
% r
esp
on
ders
# [
SE
M]
No tr
eatm
ent
TG10
50
No tr
eatm
ent
TG10
50
No tr
eatm
ent
TG10
50
0
20
40
60
80
Viremia HBsAg Both
*p=0.021 p=0.059
*p=0.011
8.8
55.3
22.0
62
5.3
42.7
HB
sAg
Vir
em
ia
log log
RESULTS (II): MODE OF ACTION OF TG1050 IN THE AAV-HBV MOUSE MODEL
HBsAg levels w/ and w/o TG1050 treatment in AAV-HBV
mice
G DNA levels w/ and w/o TG1050 treatment in AAV-HBV
mice
F
H HBsAg I Viremia
T cell depletion experiment
CD8 and/or CD4 depletion leads to an abolishment of antiviral activity of TG1050 confirming the expected mechanism of action.Residual antiviral activity is observed in mice escaping CD8 depletion (lower left graph F+G)
Adoptive transfer of TG1050-educated T cells in AAV-HBV miceCD8 or CD4 transfer (7.5 x 106 splenocytes sampled at D14 post TG1050 immunization from HBV-free mice)
into AAV-HBV mice 56 days post-AAV-HBV administration
CD8 T cell transfer results in a decrease in viral titers in a subset of mice.This decrease is concomitant with the detection of IFNg+ CORE-specific CD8 T cells in the liver.
Whereas antiviral activity due to direct injection of TG1050 is associated with the detection of IFNg+ POL-specific CD8 T cells.
Dotted lines
represent TG1050
immunizations
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