the BRIGTT research group. Lund University ... Dept of Neurosurgery, Lund University. Hospital during a 18-year ... tumor-draining lymph nodes. J. Clin. Invest.
The BRIGTT project BRain Immuno Gene Tumour Therapy
Leif G. Salford Bengt Widegren Dept of Neurosurgery, Neurosurgery Tumour Immunology the Rausing Laboratory and the BRIGTT research group
Lund University Sweden
BRIGTT 2005, WFNOII/EANOVI, Edinburgh 050505 BRain Immuno Gene Tumour Therapy Leif G. Salford Dept of Neurosurgery, Neurosurgery Tumour Immunology the Rausing Laboratory and
the BRIGTT research group Lund University Sweden
CD133+ (?)
Grows like an octopus. Sends migrating “ “guerilla-cells” ill ll ” into the surroundi normall ding brain
“Local therapy will not cure malignant gliomas”
Salford
Less than 2 per thousand are cured from a GBM! Out of more than 1100 p patients treated in the Dept of Neurosurgery, Lund University Hospital during a 18 18-year year period period, only 3 adults survived >10 years. They were 22, 32 and 38 years old at operation. operation One had a recurrence after 12 years and the other two live happily without ih tumour 42 andd 38 years later. l
Due to a strong immune system? Salford 02
1980 In a collaboration between depts of Neurosurgery and Tumour Immunology Lund Univ.
Human IFN- Daily local I j ti Injections for f 3 months 4 patients Salford, Strömblad Sjögren et. al.
One year later Encapsulated b t mortal but t l tumour
R Resection ti
Brain
The T-lymphocytes T lymphocytes must be activated in order to ppass the Blood-Brain Barrier
cavity
T lympho - cytes
“Guerilla” ill cell nests
Activation when T-lympho c tes ooutside cytes tside the brain are presented to tumour cells which produce immune stimulating products such as Interferon-
Effect of activated T-lymphocytes on esophageal cancer
CANCER RESEARCH 61, 3932–3936, May 15, 2001
Effect of infiltratingg T-lymphocytes y p y on ovarian cancer
From NEGL J MED 348:3, 2007, JAN 16 2003 F
From; David H. Munn, Madhav D. Sharma, Deyan Hou, Babak Baban, Jeffrey R. Lee, Scott J. Antonia, Jane L. Messina, Phillip Chandler, Pandelakis A. Koni, and Andrew L. Mellor. Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J. Clin. Invest. 114:280–290 (2004).
WHEELER AND BLACK pproteinextrakt direkt från tumör pperop. p blandas med patientens egna dendritiska celler från blod som tar upp proteinerna och presenterar peptider för T-lymfocyter i lymfkörtlarna Goda resultat jämförbara med BRIGTTs men yngre patienter pat e te inkluderade ude ade
SHIRRMACHER S C –N NDV V Newcastle Disease Virus Som BRIGTT men endast fågelvirusinfekterade (RNA virus, infekterar men replikeras inte) samma problem som vi med månaders odling före maligna celler dominerar Resultat på yngre patientgrupp än BRIGTT: Signifikant förlängd överlevnad
From: AdvHSV-tk Gene Therapy Th with Intravenous Ganciclovir Improves Survival in Human Malignant Glioma:A Randomised, Controlled Study Arto A t IImmonen, M Matti tti Vapalahti, V l hti Kristiina Tyynel, Heleena Hurskainen, Hurskainen Anu Sandmair, Ritva Vanninen, Gillian Langford,Neil Murray, and Seppo Ylä-Herttuala.
Immunisation against inoculated experimental brain tumours N32 brain tumour cells
Day 0
N32 brain tumour cells transfected with IFN-gamma genes
VACCINATION
Stereotaxic inoculation of 5000 cells
Day 11,8,15 8 15 or 3,10,17
Day 21
CURED!
DEAD I Immuneresp
BRain Immuno-Gene Tumour Therapy (BRIGTT)) Lund Universityy IFNgamma
Tumourresection > 80%
Major tumourportion transported in sterile nutrient solution to the Human cell culture lab
Transduction The glioma cells with human IFNgrown in sterile medium for months gamma gene construct adenoviral vector Cell irradiation
IMMUNISATION 3-4 months postoper.
Salford 00
BRIGTT Surgery >80% resect.
Resected cells 95% glioma 5% normal
Normal cells have stopped dividing Glioma cells continue and Normal cells divide 1/1 d d. dominate the Glioma cells 1/4 culture (50%)
IFNgamma
2-3 months Activated T-cells
Lymph nodes
Karyotyping regularly l l Irradiation IMMUNISATION 3-4 months postop.
(repeated every 3 weeks, 4 - 10 times) Salford
• Goal: to study whether vaccination with autologous tumour cells expressing genesequences for human interferon-gamma • 1. 1 is safe for the patients • 2. gives rise to an immunological response • 3. 3 adds any beneficial effect to conventional therapy (tumour growth, prolonged survival) • • • • • • •
Inclusion criteria: Astrocytoma y ggrade IV ((WHO)) Age 50-70 y Tumour operable, chance for > 80% reduction of tumour burden P ti t´ consentt Patient´s Karnofsky > 70 RT only y other treatment after surgery g y
The BRIGTT protocol Neurol x exam
x
x x xx x x
Neuro x psych. p y
x
x
Blood Sampl. x
x x xx
xx x
MRI, rCBV DTH
x
x
x
x
x
x
Skin Biopsy Tumor p Spec.
x
x x
x x
x x
xx
xx
xx
x
x x
xx
x
x
X
xx
x
x
xx
xx
x
x
x
xx
x
xx
x
x
x x
x
xx
x
xx
x
x
x
x
x
(if reoperation)
x (Autopsy)
Op. - - 1 2 3 4 Immunisation #
5
6
7
8
9
10
Intradermal immunisation
0h DTH Reaction 24 andd 48 hrs h post i immunisation i i
26h
48h
Skin biopsy Day 7
BRIGTT Immunohistochemistry in skin biopsies from the vaccination area
The composition of the cellular infiltration in the vaccination site (and brain tumour specimens) is studied by markers for: CD3 ((T lymphocytes) y p y ) CD4 (subset of T cells, helper cells) CD8 (subset of T cells, killer cells) C 16 ((NK, naturall killer CD16 kill cells) ll ) CD20 (B cells) granulocytes and microglia proliferation (PCNA, Ki67) cytolysis y y (perforin, (p , Granzyme y A,, B)) And expression of cytokines for functional Tcell subsets: IL-2, IL-4, IL-10, IL-12, IL-18, TNF alfa/beta, IFN-gamma, TGF-beta 1-3 Salford
GA-CD4-10-imm
Pat. AMN
Pat. GF
CD16+ cells (NK) 1 week after immunisation # 1, 3 and 5
CD8+ cells (CTL) 1 week after immunisation # 1, 3 and 5
Infiltration of T-cells at the vaccination site DZ48-CD3 10
8
6
AMN39-CD3
4
2
10
0
co
nt ro
l
8
6
GF34-CD3
4
2
10 0
co nt r
ol
8
6
4
2
co nt r
ol
0
BRIGTT
Analysis of peripheral blood
Co-culture blood + tumour cells (MLTC, mixed lymphocyte tumour culture) from the patient selects for T-cells that can recognize tumour-specific antigens. Measure: - proliferative response of immune cells to tumour cells - production of cytokines (IFN-TNF-IL-2, IL-4, IL-10) spontaneously or provoked by tumour cell exposure - subset composition of immune cells (B, NK, T-helper, cytolytic T) - ability to kill tumour cells in vitro Salford 00
19 -s ep -9 19 6 -o kt -9 19 6 -n ov -9 19 6 -d ec -9 19 6 -j an -9 19 7 -f eb -9 19 -m 7 ar -9 19 7 -a pr -9 19 -m 7 aj -9 19 7 -j un -9 7 19 -j ul -9 19 7 -a ug -9 7
Activatio A on (Ratio o R4/R1 1)
General lymphocyte activation Active gate/Resting gate
0,07
0,06
0,05
0,04
0,03
0,02
0,01
0,00
Blood Sample
Patient: DZ48
Brain tumour specimen, i during d i immunisation period T lymphocytes l h t brown b (CD3 antibody)
CD8-TC-before
CD8-TC-after immunization
Neuropsychological results: TESTS
Cognitive functions Emotional status Quality of Life
Improved results in tests for short-term memory during the immunisation period The patients experience a clear improvement in quality of life during the later part of the immunisation period
BRIGTT 2000-2004 Goals:
Results:
to study whether vaccination with autologous l tumour cells ll expressing i gene-sequences for human interferon -gamma 1. is safe for the patients 1 2. gives rise to an immunological
YES !
response 3. adds any beneficial effect to
YES !
conventional therapy (tumor growth, prolonged l d survival) i l)
?
BRIGTT
Status Jan 2005
# pat # immunis. age (op) survival after diagn 4 10 52 y 21 months DZ 67 y 12 GF 58 y 19,5 19 5 EK 64 y 24,5 IC 1 10 + 4 52 y 26,5 26 5 GA 3 8 62 y 10 AMN 67 y 13 BP 66 y 12 AO 1 6 65 y 10 KN Mean age 61,4y 61 4y Mean survival time 16 16,44 m (169%) (c.f. not imm. pats 60,3y 9,7 m)
BRIGTT Non Non-imm. imm. pats
Jan 2005
# pat # immunis. age (op) survival after diagn 11 0 62 8 69 6 59 5 56 6 57 17 age non-imm 51 17 >60y 6,8m 66 6 18m 66 5 0/11 62 9 56 14 Mean age 60,3y. 9,7 months
imm. 13,6 22,3
4/9
Patient Survival 100
65
90
58
80 Survival ((%) S
62
55
70
66
65
60
controls t l patients
65
65
69
50
61
67 62
40
57 55
30
52
50
20
63
59
10
52
57
0 0
100
200
Age in graph mean age controls = 59.6 age range controls = 50-69
300 400 500 Days after operation
600
mean age patients = 61 age range patients = 52-67
700
800
BRIGTT
Future
Continue BRIGTT, add 11 patients Include patients younger than 50 y. y Utilize h-TERT cell selection? Less delay Add other genes? TGF-2 antisense, IL-4 etc Allogeneic cells instead of autologous? No delay Tumour cells direct from resected material? C bi with Combine ith oncolytic l ti viruses i Combine with electropermeabilization Combine with BNCT py Combine with Stem Cell therapy Collaboration with other centres
BRIGTT The Brain Immuno Gene Tumour Therapy project The Rausing Laboratory, Lund University and Lund University Hospital, Lund, Sweden Neurosurgery and the R i llaboratory Rausing b
Leif G. Salford (PI), Peter Siesjö, Gunnar Skagerberg, C li Larsson, Carolina L Charlotte Ch l O Orre, Susanne S Strömblad, Catarina Blennow, Lena-Liebera-Jannert, Edward Visse, Shorena Janelidze, Anna Darabi, Maria L Larsson, K i Enell, Karin E ll Anita A it Nilsson, Nil Gunnar G Gunnarsson Tumour immunology and Bengt Widegren (Lab Chief), Hans Olov Sjögren, Genetics Xiaolong Fan, Fan Johan Rebetz, Rebetz Anna Edqvist Medical Radiation Phys. Bertil Persson, Catrin Bauréus Koch, Gustaf Grafström Clinical Genetics Nils Mandahl Neuropathology Elisabet Englund, Englund Annette Persson Persson, Arne Brun Diagnostic Neuroradiology Elna-Marie Larsson, Cecilia Petersen, Lars Stenberg Neurology Anna Rydelius, Eva Ask Neuropsychology Åsa Lilja, Christina Elfgren Oncology Per Malmström, Sara Kinhult
THANK YOU
Patient Survival 100
65
90
58
80 S Survival (%)
62
55 65
70 60
controls patients
65
66 65
69
50
61
67 62
40
57 55
30
52
50
20
63
59
10
52
57
0 0
100
200
Age in graph mean age controls = 59.6 age range controls = 50-69
300 400 500 Days after operation
600
mean age patients = 61 age range patients = 52-67
700
800
