S. Khleif - Ovarian cancer - General lecture on vaccine

Cancer Vaccine-
What is the Current State of the Art?

Samir N. Khleif
Chief, Cancer Vaccine Section, NCI
Professor of Medicine, Uniformed Services University
for Health Sciences

Vaccines

Prophylactic Therapeutic

preventive Treatment

Cancer Vaccines

• Prophylactic: prevention or elimination of the
causative agents

• Preventive: elimination of premalignant lesion
or prevention of recurrence

• Treatment: established tumor

Vaccines

Prophylactic Therapeutic

HPV preventive Treatment

Provenge

Infectious agents and Cancer

• HPV
• HBV
• HCV
• EBV
• HIV
• HHV8
• HTLV1

• Helicobactore

Cervical Cancer

• 2nd most common cancer worldwide
• ~ 510,000 new cases / year
• ~ 274,000 deaths / year
• Most common cancer in the developing world

What is HPV?
• Small DNA Tumor Virus

• 55 nm in diameter

• 8000-base double stranded
circular DNA

• Causes an array of benign and
malignant pathology

Woodman et al. Nature Reviews Cancer 7, 11–22 (January 2007) | doi:10.1038/nrc2050

HPV Infection

Transient Persistent
Infection Infection
• 1 year
Low Grade
Dysplasia
CIN 1
• 5 years
High Grade
Dysplasia
CIN 2/3
• Decades
Invasive
Cancer

HPV Infection

Infection Infection

Low Grade
Dysplasia
CIN 1

High Grade
Dysplasia
CIN 2/3

Invasive
Cancer

HPV Infection

Infection Infection

Low Grade
Dysplasia
CIN 1
Integration
High Grade
Dysplasia
CIN 2/3

Invasive
Cancer

Viral Proteins
Expressed
HPV Infection
L1, L2

Infection Infection

Low Grade
Dysplasia
CIN 1
Integration
High Grade
Dysplasia
CIN 2/3

Invasive
Cancer

Humoral Vaccines

• Killed virus (influenza)

• Live attenuated (MMR)

• Sub-units

Live Attenuated/killed Viruses Are Not Suitable
For an HPV Prophylactic Vaccine

• Papillomavirus cannot be efficiently grown in
cultured cells

• The viral genomes contain oncogenes

HPV L1 VLP Vaccine Synthesis

L1 gene Empty viral
on HPV capsids
DNA

Yeast cell DNA
Transcription

L1 gene inserted Capsid proteins
into genome of mRNA
yeast cell tRNA
Translation
rRNA

Yeast Cell

HPV L1 VLP Vaccine Synthesis

L1 gene Empty viral
on HPV capsids
DNA
Elicits
immune
response in
host
Yeast cell DNA
Transcription

L1 gene inserted Capsid proteins
into genome of mRNA
yeast cell tRNA
Translation
rRNA

Yeast Cell

Vaccine Available

companyName Vaccine Type

Merck: Gardasil Quadrivalent (yeast)

FDA Approved 2006

GSK: Cervarix Bivalent (baculovirus)

FDA Approved 2009

Clinical Trials: HPV Prophylactic Vaccine

Study Vaccine No of Subjects End points Efficacy

Vaccine Control % (confidence
limits)
Koutsky Gardasil 6087 6080 CIN 2/3 AIS 98 (86-100)

Garland Gardasil 2241 2258 CIN 2/3 AIS 100 (94-100)
2261 2279 GW VIN 100 (94-100)
VAIN
Joura Gardasil 7811 7785 VIN 2/3 100 (72-100)
VAIN 2/3
Harper Cervarix 481 470 CIN 100 (42-100)
CIN 2/3

Paavonen Cervarix 7788 7838 CIN 2/3 90 (53-99)
MITT Analysis

Duration of Follow up in
Phase II Studies

Harper DM, Expert Rev Vaccines. 2009

Reduction in LEEP for CIN 2/3

Harper DM, Expert Rev Vaccines. 2009

US Recommendations
• FDA approved the vaccine for girls and women ages 9 to 26 for the
prevention of Cervical, Vulvar and vaginal cancers and precancerous
lesions

• FDA approved the vaccine for men and women ages 9 to 26 for the
prevention of anal cancers and precancerous lesions

• FDA approved the vaccine for boys and men ages 9 to 26 to prevent genital
warts

• Federal Advisory Committee on Immunization Practices recommends that
girls routinely receive the vaccine between the ages of 11 and 12.

• The HPV vaccine is most effective when administered to girls and women
before the onset of sexual activity. Vaccination is also recommended for
women up to age 26, regardless of sexual activity.

Incidence of Cervical cancer/ 100,000 cases

• US : 7
• Jordan 2.6
• Egypt 2.7
• Israel (Arabs) 2.5
• Israel (Jews) 5.3
• Cyprus 3.7
• Turkey 4.76

Komodiki et al, MECC Monograph
Hatipoglu/Ozgul, MOH Turkey

Issues to be addressed

• Cross sub-types protection

• Duration of protection

• Production cost

• Time frame for public health impact

Dendritic Cells
B-cells
Macrophage

Viral Proteins
Expressed
HPV Infection
L1, L2

Infection Infection E1, E2, L1, L2

Low Grade
E1, E2
Dysplasia
CIN 1
Integration
High Grade
Dysplasia
CIN 2/3

Invasive
Cancer

• Prevention of HPV infection Viral Proteins
Expressed
• Elimination of HPV infection HPV Infection
L1, L2


Low Grade
• Elimination of low grade dysplasia E1, E2/ E6, E7
Dysplasia
CIN 1
Integration
High Grade
High Grade
E6, E7/ E1, E2
• Elimination of high grade dysplasia Dysplasia
CIN 2/3
CIN 3

• Preventions of recurrence Invasive E6, E7
Cancer
Treatment of invasive disease

•Prevention of HPV infection Viral Proteins
Expressed
•Elimination of HPV infection HPV Infection
L1, L2


Low Grade
•Elimination of low grade dysplasia E1, E2/ E6, E7
Dysplasia
CIN 1
Integration
High Grade
High Grade
E6, E7/ E1, E2
•Elimination of high grade dysplasia Dysplasia
CIN 2/3
CIN 3

•Preventions of recurrence Invasive E6, E7
Cancer
Treatment of invasive disease

Clinical Trials: Preventive Vaccine for CIN
Vaccine Type Phase Antigen Lesion Patient Number Immunologic Clinical response Authors
response
Protein-based II Fusion protein CIN 3 58 Increased 13/58 CR Einstein 2007
HPV-16 E7- inflammation in 32/58 PR
HSP65 responders 11/58 SD
2/58 PD
II Fusion protein High grade CIN 21 9/17 7/20 CR Roman 2007
HPV-16 E7- 1/20 PR
HSP65 11/20 SD
1/20 PD
Viral vector- I/II MVA E2 CIN 1, 2 and 3 36 100% Ab 34/36 CR Gutierez 2004
based particles induction 2/36 PR
100% CTL
II MVA E2 CIN 2 or 3 34 100% Ab 19/34 CR Garcia-
particles induction 15/34 PR Hernandez
CTL response 2006
(data not shown)
II Recombinant Anogenital (non- 29 NA 1/29 CR Fiander 2006
HPV-16 E6E7L2 cervical) 5/29 PR
protein (TA- neoplasia 3
CIN) and TA- (AGIN 3)
HPV
Chimeric virus- I/II HPV-16L1E7 CIN 2 or 3 39 100% Ab 9/23 PR Kaufmann
like particle induction (p>0.05) 2007
5/23 E7-specific
T cells
DNA I Plasmid CIN 2 or 3 15 Low immune 3/9 PR Trimble 2009
expressing a Sig- response (p>0.05)
E7(detox)-HSP
70 fusion protein

Preventive HPV E7 Vaccine / VIN

Kenter GG et al. NEJM 2009

A combined prophylactic
therapeutic vaccines

Targeting HPV16 E2 Using Chimeric VLPs and E2 Peptides
Chimeric VLP: HPV16 VLP-E7E2

L2

L1

E7

E2

HPV16 L1/L2-E7-E2
Chimeric VLP

Conclusion

• E2 vaccine induce CTL that lyse the tumor cell line expressing HPV 16 E2.

• Modified E2 sequence showed higher binding affinity , elicited strong IFN-
production and can, also, induce CTL to lyse B16-AAD-E2.

• Combination with VLP priming enhances the E2 CTL

• E2 vaccine has a potential for the treatment of CINs.

Prostate antigen: PAP
48 hours incubation with GM

Disease Behavior

Kantoff PW, N Engl J Med. 2010 Jul 29;363(5):411-22

Sipuleucel-T (Provenge)
• Sipuleucel-T (Provenge) is an autologous, dendritic cell-
based vaccine (CD541) that is pulsed with a selective
prostate antigen: prostatic acid phosphatase.

• the FDA has recently approved sipuleucel-T in patients with
asymptomatic or minimally symptomatic metastatic
hormone-refractory prostate cancer.

• Side effects include chills, fatigue, fever, and joint aches.

Framing the Problem
Fact I
• Cancer Vaccine article

Framing the Problem
Fact I
• Cancer Vaccine article 18,543

Framing the Problem
Fact I
• Cancer Vaccine article 18,543

• Drug Approved 1

Fact II

• Most of the people who received cancer the
Vaccine did not benefit

Effectors vs. Suppressors

Tumor

Vaccine CD8+

CTL
CTL
CTL
CTL
CTL

APC

CD4+

Ideal Antigenic target

• Expressed uniquely in cancer cells

• Important for the maintenance of the
malignant phenotype

• Immunogenic

Solid tumor with Ras mutation

Sequencing Ras

Mutant Ras peptide

Toubaji et al, 2008

Tumor Immune Modulation Network (TIMN)

Co-inhibitory-Ligands
Tumor
CD8+

CTL
CTL
CTL Cytokines/factors
APC CTL
CTL

Treg
Treg
Treg
CD4+
Treg

MDSC

Other inhibitory mechanism

TAM

PD-1/PD-L1/PD-L2

Blank, Cancer Immunol Immunother, 2005

PD-1/PD-L1 Engagement Suppresses
Effector T cells
PD-L1

PD-1
Tumor cell MDSC

Suppressed/Apoptotic
Activated
T cell
CD28

B7

TCR

MHC/ APC
Treg peptide

PD-1/PD-L1 Engagement Suppresses
Effector T cells
PD-L1

PD-1
Tumor cell MDSC

Activated
T cell
CD28

B7

Anti-PD-1
TCR

MHC/ APC
Treg peptide


CTLA4
CTL Tumor
CD8+

CTL
CTL Cytokines
APC

Treg
Treg
CTLA4
CTL Treg
CD4+
Treg

CTL
MDSC


TAM

Improved Overall Survival in a Phase II Randomized Controlled Trial
of a Poxviral-Based PSA-Targeted Immunotherapy in Metastatic Castration-Resistant
Prostate Cancer

Kantoff, et al. JCO, 2010


PD1-PDL1
CTL Tumor
CD8+

CTL
CTL Cytokines
APC

Treg
Treg
PD1-PDL1
CTL Treg
CD4+
Treg

CTL
MDSC


TAM

Humanized mAb
PD-L1 PD-L2 to PD-1

PD-1

tumor DCs

Activated
T cells
anti-PD-1 antiboby

Evaluation of therapeutic efficacy of vaccine in
combination with anti-PD1

Days 0 7 8 15 22 Monitoring of tumor
growth and survival
TC-1 E7+aPD1 E7+aPD1 E7+aPD1

Day 7
after implantation of
50,000 TC-1 cell

1.5 1.5 1.5

Tumor Growth
1.2 1.2 1.2
0.9 0.9 0.9
0.6 0.6 0.6
0.3 0.3 0.3
Non-immunized E7 a-PD1
0 0 0
5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38
1.5 1.5 1.5
E7+CPM
1.2 1.2 1.2

0.9 0.9 0.9

0.6 0.6 0.6

0.3 0.3 0.3
CPM a-PD1 +CPM
0 0 0
5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38
1.5 1.5
E7+aPD1
1.2
1.2
0.9

0.6 0.9
Tumor Volume, cm3

0.3 0.6
E7+CPM+aPD1
0
5 8 15 22 26 29 31 35 38
0.3

0
5 8 15 22 26 29 31 35 38
Days after tumor implantation

Enhancing T cell Enhancing Memory
response response

Enhancing Ag
presentation
+ +

Long Lasting
Ag I. R.
I. R.

Effective
I. R.

Enhancing T cell Enhancing Memory
response response

Enhancing Ag
presentation
+ +

Long Lasting
Ag I. R.
I. R.

_
Better Targets

Inhibiting negative Effective
regulators I. R.

A Pilot Study of CT-011+ Provenge (Sipuleucel-
T)+ Cyclophosphamide
in Patients with Metastatic Castrate Resistant
Prostate Cancer

Study Design

Part 1, Run In phase, up to 12 patients
CPM 250 mg/m2 (Day -1 only) + Sipuleucel –T (Day 0) >>> Q 2 wk X 3

CPM 125 mg/m2

Part 2, Randomized, total 45 patients

• Sipuleucel-T Q 2 wk X 3

• Sipuleucel-T Day 0 + CT-011 (3 mg/kg Day 2)

• CPM (Day -1 only) + Sipuleucel-T Day 0 + CT-011 (3 mg/kg Day 2)

CPM = Low Dose Cyclophosphamide
Apheresis 2-3 days prior to each dose of Sipuleucel-T for cell generation

Objectives

Primary endpoint Secondary endpoint

• Feasibility Provenge™+ • Progression-free
CPM survival

• Immune efficacy on • Overall survival
PA2024-specific IFN-γ
• Toxicity

Inclusion Criteria

• mCRPC with progression, testosterone < 50 ng/dL.

• PSA over nadir at least 2X, 3 weeks apart.

• No prior chemotherapy.

A Pilot Study to of Gemcitabine and
CT-011 in Resected Pancreatic Cancer

Phase I trial of escalating doses of CT-011 in
combination with p53 vaccine in adults with
advanced solid tumors

PD-1 and CTLA-4 combination

blockade expands infiltrating T cells and
reduces regulatory T and myeloid cells

(Curran MA et al, PNAS 2010)

Tumor Growth Pattern with Chemotherapy
and Vaccines

Fojo et.al. Clin Cancer Res, 2010

treatment


treatment Chemotherapy


treatment Chemotherapy Vaccines


Pt Age Cancer Pathology Mutation Pre-vaccination Stage on Disease extension
Arm A Patient Profile: Il-2 Therapy Enrollment on enrollment
Liver, Lung, Para-
1A
56 Colon Adenocarcinoma ASP Sx1, Cx3 4 aortic LN

2A
57 Colon Adenocarcinoma ASP Sx2, Cx2 4 Lung

3A
62 Colon Adenocarcinoma ASP Sx1, Cx3, Rx1 4 Lung
Pancreas, Liver,
Adrenals,
Omentum,
4A
Peritoneum,
Infradiaphragmati
50 Pancreatic Adenocarcinoma ASP Sx1, Cx2 4 c LN

5A
60 Lung Adenocarcinoma VAL Cx2, Rx1 4 Chest Wall

6A
59 Colon Adenocarcinoma VAL Cx2 4 Liver

7A
52 Colon Adenocarcinoma CYS Sx2, Cx2 NED NED
Lung, Hilar
8A
68 Lung NSCLC CYS Sx1, Cx5, Rx2 4 Adenopathy

9A
56 Colon Adenocarcinoma ASP Sx2, Cx3, Rx2 NED NED

10A
63 Colon Adenocarcinoma ASP Sx3, Cx3 4 Liver, Lung

11A
42 Pancreatic Adenocarcinoma ASP Sx2, Cx1 NED NED

12A
39 Colon Adenocarcinoma ASP Sx1, Cx1 NED NED

13A
67 Colon Adenocarcinoma CYS Sx1, Cx2 4 Liver

14A
51 Colon Adenocarcinoma ASP Sx2, Cx1 NED NED

15A
60 Pancreatic Adenocarcinoma VAL Sx1, Cx4, Rx1 4 Liver

16A

# Of Off-study reason/Disease
Pt
Cycles A Clinical Outcome:PFS (ms)#
Arm Status Il-2 OS (ms)*

1A 2 PD 0.5 5.5
2A 3 PD 3.9 16.8
3A 3 PD 5.8 21.5
4A 3 PD 3.6 6.2
5A 1 PD 1 2.8
6A 3 PD 3.5 8.9
7A 11 Completed 129+ 129+
8A 3 PD 3.6 13.1
9A 10 PPS 15.4 37.2
10A 3 PD 3.3 19.9
11A 6 PD 7.5 24.1
12A 3 PD 6.2 23
13A 3 PD 3.5 4.8
14A 3 PD 7.1 41.3
PPS/Lost to Follow-
15A
3 Up 2.7+ 5.3

Study Design

Re-staging

-7 1 8 15 22 1 8 15 22 1
For 6 cycles or
Days until disease progression or SE

Objectives

Primary endpoint Secondary endpoint

• Feasibility and safety • Clinical response

Inclusion Criteria

• Resectable pancreatic CA

• Eligible for Gemcitabine

• No previous treatment

Schema
•

•Staging •Staging •Staging
evaluation •evaluatio •evaluatio
n n

1(0) 2 (3) 3 (6) 4 (9) 5 (12) 6 (15) 7 (18)
Until
Cycles (Weeks) disease
p53 (264-272)
progression
or
CT-011
toxicity

Schema
•

Dose Escalation Schedule

Dose Level Dose of CT-011^

Level 1 0.3 mg/kg every 3 weeks X 2 *

Level 2 1.5 mg/kg every 3 weeks X 2 *

Level 3 3 mg/kg every 3 weeks X 2 *

Level 4 6 mg/kg every 3 weeks X 2 *

^Evaluation of toxicity to define DLT will be performed 3 weeks after the 1st dose (cycle 1).
*Evaluation of clinical status and tumor response will be performed after 2 cycles

Disease Behavior

Show the difference in vaccine vehcle and then
Here add the understanding of the TIRN and
Then show CTLA and potentially PD1 for use together

T regs in colon cancer patients
p<0.05
*

12
11
%CD4+CD25+FOXP3+

10
9
8
%CD4+CD25+FOXP3+

7
6
5
4
3
2
1
0
Normal
NORMAL Colon Cancer
COLON CANCER COLON CANCER

WIT H RAS MUT AT ION WIT HOUT RAS MUT AT Ion

Successful cancer immunotherapy requires simultaneous
targeting of both arms of immune system

Induction of immune Inhibition of
response suppression

Bhardwaj et al. 2007

Long Lasting
Ag I. R.
I. R.

Effective
I. R.

SC Low dose IL-2
and Vaccines

Enhancing effector Enhancing memory
T cell response response

Enhancing Ag
presentation
+ +

Long Lasting
Ag I. R.
I. R.

Effective
I. R.

Enhancing effector Enhancing memory
T cell response response

Enhancing Ag
presentation
+ +

Long Lasting
Ag I. R.
I. R.

_
Immune Evasion

Immune Negative Effective
regulation I. R.

Combination Immune Therapy

• Immune Enhancing

• Immune Modulating
• Targeting inhibitory cells
• Blocking inhibitory cytokines/factors (anti-
IL10, TGFb etc)
• Blocking co-inhibitory molecules (anti-PD1,
CTLA-4 etc)

Immune Combination Therapy

• Vaccine
• Immune modulating and enhancing agents
• Antibodies

• Targeted Therapy

• Chemotherapy
• Radiotherapy

Evaluation of therapeutic efficacy of vaccine in combination
with anti-PD1

Days 0 8 15 22 Monitoring of tumor
growth and survival
Tumor Volume, cm3

Non-treated E7
n=10 n=9

E7+
aPD1 aPD1
n=10 n=8


Evaluation of therapeutic efficacy of vaccine/CT-011
combination in TC-1 mouse model

Days 0 8 15 22 Monitoring of tumor
growth and survival

120

100

Percent Survival
80
Tumor Volume, cm3

Non-treated E7
n=10 n=9 60

40

20

0
E7+
CT-011 CT-011 10 15 20 25 30 35 40 45
n=10 n=8 Days after tumor implantation


Treg cell inhibitor-cyclophosphamide (CPM)

Low Dose CPM selectively targets Treg cells, leaving other T cell
populations intact (Lutsiak et al, Blood, 2005; Ikezawa et al, J Dermatol Sci, 2005).


PD-1 PD-L1
CTL Tumor
CD8+

CTL
CTL Cytokines
APC

Treg
PD-1 PD-L1 Treg

CTL Treg
CD4+
Treg

CTL
MDSC


TAM

Therapeutic efficacy of vaccine in combination with
anti-PD1 and CPM

CPM

Days 0 7 8 15 22 Monitoring of tumor
growth and survival

Day 7
after implantation of
50,000 TC-1 cell

1.5 1.5 1.5

Tumor Growth
1.2 1.2 1.2
0.9 0.9 0.9
0.6 0.6 0.6
0.3 0.3 0.3
Non-immunized E7 aPD1
0 0 0
5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38
1.5
E7+CPM
1.2

0.9

0.6

0.3
CPM
0
5 8 15 22 26 29 31 35 38

E7+CT-011

1.5 1.5 1.5

Tumor Growth
1.2 1.2 1.2
0.9 0.9 0.9
0.6 0.6 0.6
0.3 0.3 0.3
Non-immunized E7 aPD1
0 0 0
5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38
1.5 1.5 1.5
E7+CPM
1.2 1.2 1.2

0.9 0.9 0.9

0.6 0.6 0.6

0.3 0.3 0.3
CPM aPD1+CPM
0 0 0
5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38 5 8 15 22 26 29 31 35 38
1.5
E7+aPD1
1.2

0.9

0.6

0.3

0
5 8 15 22 26 29 31 35 38

Vaccine in combination with anti-PD1 and CPM
Synergize in tumor rejection
Non-treated (n=15)
E7 (n=14)
a-PD1 (n=15)
CPM (n=15)
E7+ a-PD1(n=15)
E7+CPM (n=14)
a-PD1 +CPM (n=15)
100
E7 + a-PD1 + CPM (n=20)
80
Percent Survival

60

40

20

0
8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76


induces potent antigen-specific immune responses in tumor
bearing mice
Days 0 7 8 15 21

TC-1 tumor CPM E7+a-PD1 TERMINATION

140
***
Number of IFN spots per 106

*** R2=0.8106
120 ***

Number of IFN spots per 106
120 P<0.001
100
splenocytes

splenocytes
80 80

60
40
40

20
0
0
E7 E7 E7 E7 a-PD1 NT 0.0 0.3 0.6 0.9 1.2 1.5
a-PD1 +CPM a-PD1 +CPM Tumor volume, cm3
***P<0.001 +CPM

increases the levels of tumor-infiltrated CD8+ T cells
9.E+03
8.E+03 *** CD8+ cells
*** *

Absolute numbers per
7.E+03

10e6 tumor cells
6.E+03
5.E+03
4.E+03
3.E+03
2.E+03
1.E+03
0.E+00
E7 E7 E7 E7+ a-PD1 NT
+CPM a-PD1 a-PD1 +CPM
+CPM
12
*** *
***
10 * *
CD8+/Treg Ratio

8

6

4

2

0
E7 E7 E7 E7+ a-PD1 NT
+CPM a-PD1 a-PD1 +CPM
+CPM
*P<0.05, **P<0.01 and ***P<0.001

Anti-PD1 partially overcomes tumor-induced suppression
of stimulated Tconv cell proliferation in vitro
Isotype
control anti-PD-L1

100
25 ***
25
Percent of maximum

***
20
15
10
5
0 +TC-1
Tconv# +TC-1 +TC-1 +TC-1
+Irr Ab + a-PD1 +PD-L1

***P<0.001

Anti-PD1 and CPM synergize to decrease the level
splenic and tumor infiltrated Treg cells
**
Day 21
40 *
Treg - % of CD4+ cells

35
30
25
20
15
10
5
2.5E+03
0 ** Treg cells
E7 E7 E7 E7 CT-011 NT *
+ a-PD1 +CPM +a-PD1 +CPM 2.0E+03 **
+CPM Absolute numbers per * *
10e6 tumor cells
1.5E+03
**

1.0E+03

5.0E+02

*P<0.05 and **P<0.01 0.0E+00
E7 E7 E7 E7+ aPD1 NT
+CPM + a-PD1 a-PD1 +CPM
+CPM

Anti-PD1/CPM synergize to decrease and maintain low level of
Tregs in periphery

25
% of Tregs within CD4+T cell population

20 NT
CPM
a-PD1
15 a-PD1 +CPM
NS NS

10
*
* **
5 **
**

0
Tumor (days) 7 8 11 14 18 21 26
CPM (days) 0 +1 +4 +7 +11 +14 +19

T cell subsets involved in tumor protection after
Combination of Vaccine with anti-PD1/CPM
Anti-CD8 Anti-CD8
400ug i.p. 400ug i.p.
TC-1

Days 0 5 7 8 15 17 22 24 Monitoring of tumor
growth and survival
Anti-CD4 Vaccine + Vaccine + Anti-CD4 Vaccine +
300ug i.p. a-PD1 a-PD1 300ug i.p. a-PD1

CPM

100
No treatment
Percent Survival

80 E7+a-PD1+CPM
E7+a-PD1+CPM+anti-CD8
60
/anti-CD8
40
20

0
8 13 18 23 28 33 38 43 48 53 58 63 68 73 78

Vaccine/anti-PD1/CPM: Mechanism of
Action

PD-1 PD-L1
CTL Tumor
CD8+
CTL

CTL CTL
CTLCTL TGF
APC

Treg
CD4+ PD-1 PD-L1 Treg
Treg
CD4+ CTL
CD4+
Treg

PD-1 PD-L1

Summary

• Combination of anti-PD1 with Treg cell inhibition (CPM) and
vaccine is a feasible strategy that results in tumor
eradication under challenging conditions

• Anit-PD1 enhances CD8+ tumor infiltration when combined
with vaccine and CPM

• Anti-PD1 synergize with CPM to decrease both peripheral
and tumor-infiltrated Treg cells

Current Clinical Trials

• A Pilot Study to of Gemcitabine and CT-011 in Resected
Pancreatic Cancer

• Phase I trial of escalating doses of CT-011 in
combination with p53 vaccine in adults with advanced
solid tumors

• A Pilot Study of CT-011 and Provenge (Sipuleucel-T) in
combination with low dose Cyclophosphamide in
Patients with Metastatic Castrate Resistant Prostate
Cancer

B7-DC-Ig

PD-L1 PD-L2

PD-1
Chimera of ECD of murine
PD-L2 and Fc portion of IgG

tumor DCs

Activated
T cells

Vaccine in combinations with B7-DC-Ig and CPM induce potent
antigen-specific immune responses in tumor bearing mice
Number of IFN spots per 10e6 splenocytes

***

E7 E7 E7 E7 B7DCIg NT
+AMP-224 +CPM +B7DCIg +CPM
+CPM

*P<0.05, ***P<0.001

Number of IFN spots per 10e6 splenocytes

*** 30

Percent of apoptotic TC-1 cells
E:T=10:1
25 *** *** E:T=25:1
* E:T=50:1
20
15
10
5
E7 E7 E7 E7 AMP-224 NT
0 E7 E7/ E7/ E7/ CPM/ NT
+ B7DCIg +CPM + B7DCIg +CPM B7DCIg CPM CPM/ AMP-224
+CPM B7DCIg

*P<0.05, ***P<0.001

120

100 Non-treated (n=15)
Percent Survival

B7DCIg (n=15)
80 E7 (n=15)
E7+ B7DCIg (n=13)
60
B7DCIg +CPM (n=15)
40
E7+CPM (n=15)
CPM (n=15)
20 E7 + AMP-224 + CPM (n=15)

0
10 15 20 25 30 35 40 45 50 55 60 65 70

120

100 Non-treated (n=15)
Percent Survival

B7DCIg (n=15)
80 E7 (n=15)
E7+ B7DCIg (n=13)
60
B7DCIg +CPM (n=15)
40
E7+CPM (n=15)
CPM (n=15)
20 E7 + AMP-224 + CPM (n=15)

0
10 15 20 25 30 35 40 45 50 55 60 65 70
100 No treatment
E7+B7DCIg+CPM
Percent Survival

80 E7+B7DCIg+CPM+anti-CD8

60

40

20

0
8 13 18 23 28 33 38 43 48 53 58 63 68 73 78

T regulatory Cells
Treg

Anti-PD1

** Treg cells
Nnumbers per 10e6 tumor

*
**
* *
**
cells

E7 E7 E7 E7+ +aPD1 NT
+CPM +aPD1 +aPD1 +CPM
+CPM

*P<0.05, **P<0.01, ***P<0.001

T regulatory Cells
Treg

Anti-PD1 B7-DC-Ig

** Treg cells *** Treg cells
Nnumbers per 10e6 tumor

* **
** **
* * *
** **
**
cells

**

E7 E7 E7 E7+ +aPD1 NT E7 E7+ E7+ E7+ B7DCIg + NT
+CPM +aPD1 +aPD1 +CPM CPM B7DCIg CPM+ CPM
+CPM B7DCIg

*P<0.05, **P<0.01, ***P<0.001

T Conventional Cells
Tconv

Anti-PD1
Numbers per 10e6 tumor cells

3500
*
3000
2500
2000 *
1500
1000
500
0 E7 E7 E7 E7+ aPD1 NT
+CPM +aPD1 aPD1 +CPM
+CPM

*P<0.05, ***P<0.001

T Conventional Cells
Tconv

Anti-PD1 B7-DC-Ig

3500 3500
* ***
3000 3000

2500 2500

2000 * 2000

1500 1500

1000 1000

500 500

0 E7 E7 E7 E7+ aPD1 0 E7 E7+ E7+ E7+ CPM+ NT
NT
+CPM +aPD1 aPD1 +CPM CPM B7DCIg CPM/ B7DCIg
+CPM B7DCIg

*P<0.05, ***P<0.001

CD8+ T Cells

CD8

Anti-PD1 AMP-224

*** CD8+ cells * CD8+ cells
***
Numbers per 10e6 tumor

* NS
***
cells

E7 E7+ E7+ E7+ AMP + NT
+CPM +aPD1 aPD1
CPM AMP CPM+ CPM
+CPM
+CPM AMP

*P<0.05, ***P<0.001

CD8+ T Cells

CD8

Anti-PD1 B7-DC-Ig

*** CD8+ cells * CD8+ cells
***
Numbers per 10e6 tumor

* NS
***
cells

E7 E7+ E7+ E7+ B7DCIg NT
+CPM +aPD1 aPD1
CPM B7DCIg CPM+ CPM
+CPM
+CPM B7DCIg

*P<0.05, ***P<0.001

B7-DC-Ig decreases the level of tumor infiltrated PD-1high
CD8+T cells
5000

4500 *
4000
NS
***
3500
3000
2500
2000
1500
1000
500
0
E7 E7+ E7+ E7+
CPM B7DCIg B7DCIg
+ CPM

CD8+T cells
5000
TI CD8+ PD-1high cells and CD8+ PD-1low cells

4500 5000
*
4000
NS 4500
***
3500 4000
3000 3500
2500 3000
2000 2500
1500 2000
1000 1500
500 1000
0 500
E7 E7+ E7+ E7+
0
CPM B7DCIg B7DCIg E7 E7+ E7+ E7+
+ CPM
CPM B7DCIg B7DCIg
CPM

GATED ON CD8+
PD-1high PD-1high

53.81% 25.75%

GATED ON CD8+ PD-1high PD-1high

67.82% 23.36%

CD8+T cells
5000
TI CD8+ PD-1high cells and CD8+ PD-1low cells

4500 5000
*
4000
NS 4500
***
3500 4000
3000 3500
2500 3000
2000 2500
1500 2000
1000 1500
500 1000
0 500
E7 E7+ E7+ E7+
0
CPM B7DCIg B7DCIg E7 E7+ E7+ E7+
TI CD8+ PD-1low cells + CPM
4000
CPM B7DCIg B7DCIg

3500
CPM
*

GATED ON CD8+
* PD-1high PD-1high
3000 *
2500 53.81% 25.75%
2000

1500

1000

500 GATED ON CD8+ PD-1high PD-1high
0
E7 E7+ E7+ E7+ 67.82% 23.36%
CPM B7DCIg B7DCIg
+ CPM

In contrast to anti-PD1, B7DCIg doesn’t overcome tumor-induced
suppression of stimulated Tconv cell proliferation
Percent of maximum

***
*** ***

Tconv +TC-1 +TC-1 +TC-1 +TC-1
+Irr Ab +CT-011 +AMP-224

Immune Modulating Agents

• Does it act as we want it to ?

• What else would it do within the context
of TIMN ?

Final Rankings of Agents with
High Potential for Use in Treating
Cancer

NCI Immunotherapy Agent Workshop, 2007

Houot R and Levy R, Blood, 2011

Combinations of anti- OX40, CTLA4, GITR, and 4
(FR4)) with Intratumoral CpG


Vaccine and Anti-Transforming Growth Factor-β

Anti–TGF-β (1D11) synergistically enhanced the efficacy of a CTL-
inducing peptide tumor vaccine consisting of HPV16 E749-57
peptide, emulsified in incomplete Freund's adjuvant together
with Terabe M et al, Clin Can Res 2009

Synergistic antitumor effect of anti-GITR and
anti-CTLA4

DTA-1 is an anti-GITR mAb, 4F10 is an anti-CTLA4 mAb
Ko et al, JEM, 2005

What did we learn?

• Lack of understanding of simple biology---
Simplistic approach, “single agent approach”

• Difference in tumor behavior--- “wrong
monitoring”

• Clinical trial design--- chemotherapy based
approach


• Science


• Science

• Toxicity


• Science

• Toxicity

• Regulatory/ Clinical Trial Design


• Science

• Toxicity


• Business/IP and Data Sharing


• Combination

• Should understand the agent


• Business/IP and Data Sharing

Dose determination and Phase I
need?

Clinical Trial Design

1. Do cancer vaccines have significant toxicity?

2. Does dose escalation effect toxicity?

3. Does dose escalation effect response and how
is it measured?

Phase 1 for Cancer Therapy
• 3+3 Cohort Expansion with Dose escalation
• Patients with Late Stage Disease
• Determine DLT and MTD
• Cancer vaccines
– Vaccines are inherently safe, if have toxicity seen in
long term
– Patients will have weak immune response b/c of
advanced disease

Do phase 1 trials for cancer vaccines have
significant toxicity?

• Reviewed phase 1 trials for therapeutic cancer
vaccines – 1990/2011
– 241 studies, 4,952 patients
– Excluding all combination therapies

Toxicity vs. patient number

# of Trials Patients Grade 3/4 rxns Percentage Related (%)

Autologous 88 1692 37 2.19% 1.36%
DC 58 922 9 0.98% 0.33%
Tumor 30 770 28 3.64% 2.60%

Allogeneic Cells 17 407 22 5.41% 1.23%
Synthetic 136 2853 108 3.79% 1.23%
Peptide 68 1333 40 3.00% 0.83%
DNA 17 311 1 0.32% 0.32%
RNA 2 36 0 0% 0%

Virus/Bacteria 36 761 50 6.57% 2.63%

Anti-idiotypic 10 362 15 4.14% 0%
Liposomal 3 45 2 4.44% 4.44%
TOTAL 241 4952 167 3.37% 1.25%

Toxicity vs Vaccine number

# of Trials Patients Vaccines Grade 3/4 rxns Percentage Related (%)

Autologous 73 1301 5722 20 0.35% 0.14%
DC 51 796 3424 9 0.26% 0.09%
Tumor 22 505 2298 11 0.48% 0.22%
Allogenic Cells 16 347 1874 22 1.17% 0.26%
Synthetic 117 2376 14239 78 0.55% 0.21%
Peptide 61 1183 7637 37 0.48% 0.12%
DNA 15 259 1388 1 0.07% 0.07%
RNA 2 36 335 0 0% 0%
Virus/Bacteria 30 594 2714 31 1.14% 0.66%
Anti-idiotypic 7 266 1938 7 0.36% 0%
Liposomal 2 38 227 2 0.88% 0.88%
TOTAL 206 4024 21835 120 0.55% 0.20%

Does dose escalation effect toxicity?

• Correlation between dose and toxicity
• Dose escalation trials – 127 trials
– Grade 3/4 Toxicities
# of Trials Patients Grade 3/4 rxns Trials w/DLT
Autologous 40 847 11 0
DC 27 466 0 0
Tumor 13 381 11 0
Allogenic Cells 5 130 20 1
Synthetic 83 2008 67 2
Peptide 36 852 10 0
DNA 12 208 1 0
Virus/Bacteria 26 592 47 2
Anti-idiotypic 8 339 7 0
Liposomal 1 17 2 0
TOTAL 127 2985 98 3

Does dose escalation effect response and how is it
measured?

• Trials that looked at dose immune response
relationship
– Phase 1 and non-phase 1 trials
Dose Related Immune Response
Vaccine trials # of Trials Statistically
Total Our Analysis Study Analysis
Significant
Autologous 32 7 3 4 0

Allogenic 4 3 2 1 0

Synthetic 70 19 13 5 1

Phase II trials 10 7 2 4 1

TOTAL 116 36 20 14 2

31.03%

Acknowledgment
Khleif’s Lab • Margaret Wojtowitz Collaborators:
• Laurent Ozborn • Sarah Bernstein • Jay Berzofsky
• Tammie Brent-Steel •
• Osama Rahma Masaki Terabe
• Cathy Maruffi
• Mikayel Mkritchian • Frank Grollman • Phil Dennis
• Namju Chong • Rajif Shanker • Michael Hamilton
• Callie Raulfs • Steve Rucker • Barrie Gause
• Stephanie Zdanov • Margaret Edison • John Schiller
• Marie Rice
• Geoffray Guittard • Bernie Fox
• Mercedes Giliom
• Zhison Chen • Sandy Chatfield • Theressa Whiteside
• Omar Dakheel • Hong Yang • Albert Dileo
• Anat Ohali • Magis Madapathil
• Toni Toubaji
• Raed Samara
• Ramy Ibrahim
• Maher Abdallah • Robert Behrens CureTech
• Yana Najjar • Vincent Herrin • Mike Schinkler
• Orna Nitzan • Ed Ashtar • Rinat Rotem
AmplImmune
• Sol Langerman

S. Khleif - Ovarian cancer - General lecture on vaccine

S. Khleif - Ovarian cancer - General lecture on vaccine

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