1 2 4 3 1 2 1 3 1 2 3 4 Antigen- specific Background A 1 A ... · (A) Evolution of the bodyweight...
1
A live attenuated Salmonella Typhimurium oral T cell vaccine against PD-L1 protects 100% of animals from a leukemia challenge Sébastien Wieckowski 1 , Heiko Smetak 2 , Marco Springer 3 , Iris Kobl 3 , Amine A. Berkane 4 , Ming Wei 4 , Albrecht Meichle 3 , Klaus M. Breiner 1 , Philipp Beckhove 2 , Marc Mansour 1 , Matthias Schroff 1 , Heinz Lubenau 3 1 VAXIMM AG, Basel, Switzerland; 2 Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany; 3 VAXIMM GmbH, Mannheim, Germany; 4 CellVax S.A.S., Romainville, France. Background VAM VAXIMM's oral T-cell vaccine platform is based on the approved, live attenuated Salmonella Typhi strain Ty21a vaccine, which has been administered in millions of individuals for prophylactic vaccination against typhoid fever. This strain has been thoroughly studied, is safe and well tolerated. The bacteria are modified to deliver an eukaryotic expression plasmid, which encodes the genetic information of a specific target antigen 1 . Figure 2. (A) Schematic representation of VXM10 oral T-cell vaccine, and (B) domains of the murine PD-L1 protein encoded in VXM10 (orange) and VXM10a (brown) vaccines. VXM01 lead vaccine encodes vascular endothelium growth factor receptor 2 (VEGFR2) in order to evoke an immune response specifically directed against the tumor vasculature. It is currently in clinical development as a treatment for various solid cancer types. The murine analogue of VXM01 has shown consistent anti-angiogenic activity in different tumor models in several animal studies 2 . An increase in tumor immune cell infiltration was recently shown. A proposed mechanism of action of VXM01 is described in Figure 1. Figure 1. Intra-lymphatic delivery of Salmonella Typhi strain Ty21a T-cell vaccines via the oral route leading to target-specific T-cell activation. The current study summarizes the immunogenicity and preclinical anti-cancer efficacy for the Salmonella Typhimurium SL7207 murine vaccines VXM10 and VXM10a (Figure 2A), transformed with eukaryotic expression plasmids encoding the full-length murine programmed death-ligand 1 (PD-L1) protein and a truncated form of PD-L1, respectively (Figure 2B). Indeed, the deletion of the signal peptide (SP) prevents the proper localization of the native PD-L1 protein to the cell surface. The empty vector, i.e. without plasmid, was used as negative control throughout the study. Antibody response Figure 7. (A) Experimental design, and (B) anti-PD-L1 antibody response in sera collected 79 days after the final vaccination. The green dashed line represents the cut-off value (for 95% confidence). Soluble recombinant murine PD-L1 was used for immunization with CFA/IFA in the positive control group (blue). The systemic antibody response was evaluated by ELISA in the serum of animals vaccinated with either VXM10 or VXM10a, 79 days after the final vaccination (Figure 7A). Anti-PD-L1 antibodies were detected in a few animals vaccinated with VXM10 and VXM10a, and the response was more pronounced in the VXM10a/high-dose group, with 50% of the animals (3 out of 6) showing signal-to-background ratio above the cut-off value (Figure 7B). Antitumor efficacy We evaluated the prophylactic anti-cancer activity of VXM10 and VXM10a in the FBL-3 disseminated model of leukemia expressing PD-L1 3 (Figure 3A). Empty vector, VXM10 and VXM10a were given by oral gavage at ca. 10 8 CFU and 10 10 CFU, on days 1, 3, 5 and 7 as a prime vaccination, and on days 14 and 22 as boosts (Figure 3B). C57BL/6 mice (n=6 per group) then received 5×10 6 viable FBL-3 cells by intraperitoneal injection on day 20. All surviving animals were re- challenged with 5×10 6 viable FBL-3 cells by intraperitoneal injection on day 100. 1. Darji A. et al., Cell 1997; 91:765. 2. Niethammer AG. et al., Nature Medicine 2002; 8:1369. 3. Yamazaki T. et al., Journal of Immunology 2002; 169:5538. References Contact and Information Heinz Lubenau, Ph.D. VAXIMM GmbH Chief Operating Officer Office: +49 621 8359 687 10 Fax: +49 621 8359 687 99 [email protected] www.vaximm.com MAFINEX-Technologiezentrum Julius-Hatry-Straße 1 68163 Mannheim Germany Poster No. B057 presented during the Cancer Vaccines and Targets session at the Third CRI-CIMT-EATI-AACR International Cancer lmmunotherapy Conference on September 8 th 2017 in Mainz/Frankfurt, Germany. Figure 3. (A) Expression of PD-L1, but not PD-L2, by FBL-3 cell line 3 , as measured by flow cytometry (left inset) and RT-PCR (right), and (B) experimental design and treatment schedule in the prophylactic and re-challenge experiment. Figure 4. (A) Evolution of the mean bodyweight, and (B) overall survival in the indicated treatment groups and doses. The blue arrows represent the time points of leukemia challenge. Treatment-naive animals (yellow curves) were used as a control for the FBL-3 rechallenge and received the leukemia cells only day 100. We finally evaluated the therapeutic efficacy of VXM10 and VXM10a in the FBL-3 model. C57BL/6 mice (n=8 per group) received 5×10 6 viable FBL-3 cells by intraperitoneal injection on day 0. Empty vector, VXM10 and VXM10a were then administered by oral gavage at a dose of 10 9 CFU on days 1, 3, 5 and 7 as a prime vaccination, and on days 14 and 21 as boosts (Figure 5). Therapeutic vaccination with VXM10 and VXM10a was well tolerated (Figure 6A), and induced full leukemia control, with 100% (8 out of 8) of surviving animals 94 days after leukemia challenge (P<0.0001). In contrast, treatment with the empty vector control did not show any anti-cancer effect (Figure 6B). Conclusions ▪ Prophylactic and therapeutic vaccinations with VXM10 and VXM10a induced a strong and sustained anti-cancer activity in the FBL-3 model of leukemia. ▪ This study provides evidence that VAXIMM’s oral T-cell vaccination platform can be used to stimulate anti-tumor immunity against antigens of the immune checkpoint regulatory protein PD-L1. ▪ These data paved the way for advancing the clinical development of VXM10, in particular in leukemia. Figure 5. Experimental design of the therapeutic study. Figure 6. (A) Evolution of the bodyweight in each individual animal, and (B) overall survival in all treatment groups, in the therapeutic setting. The blue arrow represents the time point of FBL-3 challenge. D1,3,5,7 D14 D21 FBL-3 challenge D0 D94 Prophylactic vaccination with VXM10 and VXM10a was highly tolerated, as no deterioration in general status nor significant body weight loss were observed during the treatment (Figure 4A). It also generated a rapid and sustained anti- leukemia effect with 100% (6 out of 6) of surviving animals 80 days after leukemia challenge (P=0.0005) in the highest dose groups. In contrast, vaccination with the empty vector control did not show any anti-cancer activity, as the median survival reached 41 days, and 0% (0 out of 6) of cancer regression was observed (Figure 4B). Importantly, 100% of surviving mice in the high dose groups resisted re- challenge with FBL-3 cells for at least 100 days (P=0.0002), demonstrating that vaccination with VXM10 and VXM10a generated a potent memory T cell response against the leukemia (Figure 4B). A B 0 40 80 120 160 200 0 20 40 Days after initiation of treatment Mean % bodyweight change over baseline empty vector VXM10 10 8 CFU VXM10 10 10 CFU VXM10a 10 8 CFU VXM10a 10 10 CFU untreated (traitment-naive) 0 40 80 120 160 200 0 50 100 Days after tumor challenge Percent survival empty vector VXM10 10 8 CFU VXM10 10 10 CFU VXM10a 10 8 CFU VXM10a 10 10 CFU untreated (treatment-naive) *** * A B 0 20 40 60 80 100 0 10 20 30 Days after tumor challenge Percent bodyweight change over baseline empty vector VXM10 VXM10a 0 20 40 60 80 100 0 50 100 Days after tumor challenge Percent survival empty vector VXM10 VXM10a **** Oral administration of a suspension containing attenuated Salmonella bacteria carrying plasmids encoding for target antigens. Bacteria pass through M cells into Peyer’s patches and are taken up by macrophages. Bacteria die inside macrophages and release plasmids. Plasmids enter the nucleus and the encoded antigen is expressed. Bacterial infection induces macrophages to undergo apoptosis (cell death). Apoptotic vesicles contain antigen. Apoptotic vesicles are phagocytized by dendritic cells, processed, and antigen epitopes are presented on the surface via MHC Class I. Antigen-specific CD8+ T-cells are activated. CD8+ T-cells circulate through the body and bind to target cells expressing the specific antigen, initiating cell death. Salmonella bacterium Plasmid Small intestine Small intestinal lumen Macrophage M cell T-cell Salmonella bacteria carrying plasmids Peyer’s patch Phagocytosis of bacteria Bacteria carrying plasmids Antigen Plasmids in nucleus Macrophage Apoptotic macrophage Antigen Apoptotic vesicle Dendritic cell phagocytizing vesicle Antigen epitope MHC class I Antigen-specific CD8+ T-cells Activated dendritic cell Blood vessel CD8+ T-cell Target (tumor) cell MHC class I mounted antigen 7 6 5 4 3 2 1 D1,3,5,7 D14 D21 FBL-3 challenge D20 D205 FBL-3 rechallenge D100 A B Flow cytometry RT-PCR PD-L1 PD-L2 PD-L2 PD-L1 β-actin A B D1, 3, 5, 7 D14 D21 D100 serum OD (signal:background ratio) FBL-3 challenge D20 cut-off 0.5 1.0 1.5 2.0 2.5 3.0 immun. sPD-L1 negative control VXM10a 10 10 CFU VXM10a 10 8 CFU VXM10 10 10 CFU VXM10 10 8 CFU A B murine PD-L1 VXM10 VXM10a SP ECD TM ICD 1–18 240–260 290 261 239 19 Salmonella Typhimurium SL7207 carrier Eukaryotic expression plasmid encoding murine PD-L1 protein
Transcript of 1 2 4 3 1 2 1 3 1 2 3 4 Antigen- specific Background A 1 A ... · (A) Evolution of the bodyweight...
A live attenuated Salmonella Typhimurium oral T cell vaccine against PD-L1
protects 100% of animals from a leukemia challenge
Sébastien Wieckowski1, Heiko Smetak2, Marco Springer3, Iris Kobl3, Amine A. Berkane4, Ming Wei4, Albrecht Meichle3,
Klaus M. Breiner1, Philipp Beckhove2, Marc Mansour1, Matthias Schroff1, Heinz Lubenau3
1VAXIMM AG, Basel, Switzerland; 2Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany; 3VAXIMM GmbH, Mannheim, Germany; 4CellVax S.A.S., Romainville, France.
Background
VA M
VAXIMM's oral T-cell vaccine platform is based on the approved, live attenuatedSalmonella Typhi strain Ty21a vaccine, which has been administered in millions ofindividuals for prophylactic vaccination against typhoid fever. This strain has beenthoroughly studied, is safe and well tolerated. The bacteria are modified to deliveran eukaryotic expression plasmid, which encodes the genetic information of aspecific target antigen1.
Figure 2. (A) Schematic representation of VXM10 oral T-cell vaccine, and (B) domains of the murine PD-L1 protein encoded in VXM10 (orange) and VXM10a (brown) vaccines.
VXM01 lead vaccine encodes vascular endothelium growth factor receptor 2(VEGFR2) in order to evoke an immune response specifically directed against thetumor vasculature. It is currently in clinical development as a treatment forvarious solid cancer types. The murine analogue of VXM01 has shown consistentanti-angiogenic activity in different tumor models in several animal studies2. Anincrease in tumor immune cell infiltration was recently shown. A proposedmechanism of action of VXM01 is described in Figure 1.
Figure 1. Intra-lymphatic delivery of Salmonella Typhi strain Ty21a T-cell vaccines via the oral route leading to target-specific T-cell activation.
The current study summarizesthe immunogenicity andpreclinical anti-cancer efficacy forthe Salmonella TyphimuriumSL7207 murine vaccines VXM10and VXM10a (Figure 2A),transformed with eukaryoticexpression plasmids encoding thefull-length murine programmeddeath-ligand 1 (PD-L1) proteinand a truncated form of PD-L1,respectively (Figure 2B).Indeed, the deletion of the signalpeptide (SP) prevents the properlocalization of the native PD-L1protein to the cell surface. Theempty vector, i.e. withoutplasmid, was used as negativecontrol throughout the study.
Antibody response
Figure 7. (A) Experimental design, and (B) anti-PD-L1 antibody response in sera collected 79 days after the final vaccination. The green dashed line represents the cut-off value (for 95% confidence). Soluble recombinant murine PD-L1 was used for immunization with CFA/IFA in the positive control group (blue).
The systemic antibody response was evaluated by ELISA in the serum ofanimals vaccinated with either VXM10 or VXM10a, 79 days after the finalvaccination (Figure 7A). Anti-PD-L1 antibodies were detected in a few animalsvaccinated with VXM10 and VXM10a, and the response was more pronounced inthe VXM10a/high-dose group, with 50% of the animals (3 out of 6) showingsignal-to-background ratio above the cut-off value (Figure 7B).
Antitumor efficacy
We evaluated the prophylactic anti-cancer activity of VXM10 and VXM10a in theFBL-3 disseminated model of leukemia expressing PD-L13 (Figure 3A). Emptyvector, VXM10 and VXM10a were given by oral gavage at ca. 108 CFU and 1010
CFU, on days 1, 3, 5 and 7 as a prime vaccination, and on days 14 and 22 asboosts (Figure 3B). C57BL/6 mice (n=6 per group) then received 5×106 viableFBL-3 cells by intraperitoneal injection on day 20. All surviving animals were re-challenged with 5×106 viable FBL-3 cells by intraperitoneal injection on day 100.
1. Darji A. et al., Cell 1997; 91:765. 2. Niethammer AG. et al., Nature Medicine 2002; 8:1369.3. Yamazaki T. et al., Journal of Immunology 2002; 169:5538.
References
Contact and Information
Heinz Lubenau, Ph.D.
VAXIMM GmbH
Chief Operating Officer
Office: +49 621 8359 687 10
Fax: +49 621 8359 687 99
www.vaximm.com
MAFINEX-Technologiezentrum
Julius-Hatry-Straße 168163 Mannheim
Germany
Poster No. B057 presented during the Cancer Vaccines and Targets session at the Third CRI-CIMT-EATI-AACR International Cancer lmmunotherapy Conference on September 8th 2017 in Mainz/Frankfurt, Germany.
Figure 3. (A) Expression of PD-L1, but not PD-L2, by FBL-3 cell line3, as measured by flow cytometry (left inset) and RT-PCR (right), and (B) experimental design and treatment schedulein the prophylactic and re-challenge experiment.
Figure 4. (A) Evolution of the mean bodyweight, and (B) overall survival in the indicatedtreatment groups and doses. The blue arrows represent the time points of leukemiachallenge. Treatment-naive animals (yellow curves) were used as a control for the FBL-3 rechallenge and received the leukemia cells only day 100.
We finally evaluated the therapeutic efficacy ofVXM10 and VXM10a in the FBL-3 model. C57BL/6mice (n=8 per group) received 5×106 viable FBL-3cells by intraperitoneal injection on day 0. Emptyvector, VXM10 and VXM10a were thenadministered by oral gavage at a dose of 109 CFUon days 1, 3, 5 and 7 as a prime vaccination, andon days 14 and 21 as boosts (Figure 5).
Therapeutic vaccination with VXM10 and VXM10a was well tolerated (Figure6A), and induced full leukemia control, with 100% (8 out of 8) of survivinganimals 94 days after leukemia challenge (P<0.0001). In contrast, treatmentwith the empty vector control did not show any anti-cancer effect (Figure 6B).
Conclusions
▪ Prophylactic and therapeutic vaccinations with VXM10 and
VXM10a induced a strong and sustained anti-cancer activity in
the FBL-3 model of leukemia.
▪ This study provides evidence that VAXIMM’s oral T-cell
vaccination platform can be used to stimulate anti-tumor
immunity against antigens of the immune checkpoint regulatory
protein PD-L1.
▪ These data paved the way for advancing the clinical
development of VXM10, in particular in leukemia.
Figure 5. Experimental design of the therapeutic study.
Figure 6. (A) Evolution of the bodyweight in each individual animal, and (B) overall survivalin all treatment groups, in the therapeutic setting. The blue arrow represents the time point of FBL-3 challenge.
D1,3,5,7 D14 D21
FBL-3 challenge
D0
D94
Prophylactic vaccination with VXM10 and VXM10a was highly tolerated, as nodeterioration in general status nor significant body weight loss were observedduring the treatment (Figure 4A). It also generated a rapid and sustained anti-leukemia effect with 100% (6 out of 6) of surviving animals 80 days after leukemiachallenge (P=0.0005) in the highest dose groups. In contrast, vaccination with theempty vector control did not show any anti-cancer activity, as the median survivalreached 41 days, and 0% (0 out of 6) of cancer regression was observed (Figure4B). Importantly, 100% of surviving mice in the high dose groups resisted re-challenge with FBL-3 cells for at least 100 days (P=0.0002), demonstrating thatvaccination with VXM10 and VXM10a generated a potent memory T cell responseagainst the leukemia (Figure 4B).
A B
0 40 80 120 160 200
0
20
40
Days after initiation of treatment
Mean
% b
od
yw
eig
ht
ch
an
ge o
ver
baselin
e
empty vector
VXM10 108 CFU
VXM10 1010 CFU
VXM10a 108 CFU
VXM10a 1010 CFU
untreated (traitment-naive)
0 40 80 120 160 200
0
50
100
Days after tumor challenge
Perc
en
t su
rviv
al
empty vector
VXM10 108 CFU
VXM10 1010 CFU
VXM10a 108 CFU
VXM10a 1010 CFU
untreated(treatment-naive)
***
*
A B
0 20 40 60 80 100
0
10
20
30
Days after tumor challenge
Perc
en
t b
od
yw
eig
ht
ch
an
ge o
ver
baselin
e
empty vector
VXM10
VXM10a
0 20 40 60 80 100
0
50
100
Days after tumor challenge
Perc
en
t su
rviv
al
empty vector
VXM10
VXM10a
****
Oral administration of a suspension
containing attenuated Salmonella bacteria
carrying plasmids encoding for target antigens.
Bacteria pass through M cells into
Peyer’s patches and are taken up
by macrophages.
Bacteria die inside
macrophages and release
plasmids. Plasmids enter
the nucleus and the encoded
antigen is expressed.
Bacterial infection induces
macrophages to undergo
apoptosis (cell death).
Apoptotic vesicles
contain antigen.
Apoptotic vesicles are phagocytized
by dendritic cells, processed, and
antigen epitopes are presented on
the surface via MHC Class I.
Antigen-specific CD8+ T-cells
are activated.
CD8+ T-cells circulate through the
body and bind to target cells
expressing the specific
antigen, initiating
cell death.
Salmonella bacterium
Plasmid
Small intestine
Small intestinallumen
Macrophage
M cell
T-cell
Salmonella bacteriacarrying plasmids
Peyer’spatch
Phagocytosis
of bacteria
Bacteria
carrying
plasmids
Antigen
Plasmids in nucleusMacrophage
Apoptotic
macrophageAntigen
Apoptotic
vesicle
Dendritic cell
phagocytizing vesicle
Antigen
epitope
MHC
class I
Antigen-specific
CD8+ T-cells
Activated dendritic cell
Blood
vessel
CD8+ T-cell
Target (tumor) cell
MHC class Imounted antigen
7
6
54321
D1,3,5,7 D14 D21
FBL-3
challenge
D20
D205
FBL-3
rechallenge
D100
A B
Flow cytometry RT-PCR
PD
-L1
PD
-L2
PD-L2
PD-L1
β-actin
A B
D1, 3, 5, 7 D14 D21
D100
serum
OD (signal:background ratio)
FBL-3
challenge
D20
cut-off
0.5 1.0 1.5 2.0 2.5 3.0
immun. sPD-L1
negative control
VXM10a 1010
CFU
VXM10a 108
CFU
VXM10 1010
CFU
VXM10 108
CFU
A
B murine PD-L1
VXM10
VXM10a
SP ECD TM ICD
1–18 240–260
29026123919
Salmonella TyphimuriumSL7207 carrier
Eukaryotic expressionplasmid encodingmurine PD-L1 protein
![I.Einführung - Webis...2. a j = A[j] 3. i = j 1 4. WHILE i > 0 AND A[i] > a j DO 5. A[i+1] = A[i] 6. i = i 1 7. ENDDO 8. A[i+1] = a j 9. ENDDO Datenstruktur Array: q n gleich große](https://static.fdokument.com/doc/165x107/5f8aac8a29e0f77d616f2da7/ieinfhrung-webis-2-a-j-aj-3-i-j-1-4-while-i-0-and-ai-.jpg)
