Bertil R.R. Persson PhD,

(1)

R I G T

R I G T T T R I G T R I G T T T Radiation

Radiation immunomodulatory immunomodulatory gene tumor therapy

gene tumor therapy gene tumor therapy gene tumor therapy

Bertil R.R. Persson PhD,

Bertil R.R. Persson PhD, MDh.c MDh.c..

Professor of

Professor of medical medical radiation radiation physics physics

隆德

隆德大学大学隆德

隆德大学大学

Lund University, Lund University, 221 85 LUND

221 85 LUND Sweden 85 U 85 U Sweden S S

(2)

“BRIGTT”

“BRIGTT” BRIGTT BRIGTT

脑免疫基因肿瘤治疗脑免疫基因肿瘤治疗脑免疫基因肿瘤治疗脑免疫基因肿瘤治疗

nao3 mian3 yi4 ji1 yin1 zhong3 liu2 zhi4 liao2 nao3 mian3 yi4 ji1 yin1 zhong3 liu2 zhi4 liao2

Brain

Brain Immuno Immuno Gene Gene Tumour Tumour Therapy Therapy Brain

Brain Immuno Immuno Gene Gene Tumour Tumour Therapy Therapy Immunization with

Immunization with autologous autologous interferon interferon-- Immunization with

Immunization with autologous autologous interferon interferon--

gamma secreting glioma cells in patients with gamma secreting glioma cells in patients with

Glioblastoma

Glioblastoma Multiforme Multiforme Glioblastoma

Glioblastoma Multiforme Multiforme

A h 1

A h 1 2 li i 2 li i l t i l l t i l A phase 1

A phase 1--2 clinical trial 2 clinical trial

Salford

Salford LG LG

^1,2*^1,2*

Siesjö Siesjö P P

^1,2*^1,2*

Skagerberg Skagerberg G G

^1,2*,^1,2*,

Rydelius Rydelius A A

^2,3^2,3

Salford

Salford LG LG

^,,

, , Siesjö Siesjö P P

^,,

, , Skagerberg Skagerberg G G

^,^, ^,^,

Rydelius Rydelius A A

^,3^,3

, , Blennow

Blennow C C

^1,2^1,2

, , Lilja Lilja Å Å

^1,4,^1,4,

Persson BRR Persson BRR

^2,4^2,4

, , Strömblad Strömblad S S

^1,2^1,2

,Visse E ,Visse E

1,2

and and Widegren Widegren B B

^2,5^2,5

(3)

Purpose: 目的p

^mu4 ^di4 :

Th i i f th BRIGTT t d t

The primary aims of the BRIGTT study were to ascertain

• Safety, ^安全 ^{an1 quan2}

• Feasibility ^可行性 and

k 3 i 2 i 4

• ke3 xing2 xing4

• Efficacy ^效力 ^{xiao4 li4}

of immunotherapy with autologous IFN- transfected

(4)

Grows with arms as an octopus

S d i i

Sends migrating

“guerilla-cells”

I t th

Into the sur- rounding brain

h th BBB where the BBB is intact

Salford

(5)

Timeline of immunization and Timeline of immunization and Timeline of immunization and Timeline of immunization and

monitoring procedures.

monitoring procedures. g p g p

(6)

BRIGTT St d d i

研究设计

BRIGTT Study design:

研究设计

yan2 jiu1 she4 ji4

A t l t ll lt d d t f t d ith

Autologous tumor cells were cultured and transfected with the human IFN- gene by the use of an adenoviral vector.

After irradiation with 100 Gy the cells were administered as intradermal immunizations in the upper arm every 3 ^rd week.

Endpoints

• for safet ere: records of to icit and ad erse e ents

• for safety were: records of toxicity and adverse events,

• for feasibility percent treated patients out of eligible patients and time to treatment and

patients and time to treatment and

• for clinical efficacy overall survival (OS) and progress free survival (PFS).

2010-11-13 6 Bertil_R.Persson@med.lu.se

(7)

Results

8/17 (47%) of eligible patients, aged between 50 and 69 years, were immunized between 8-14 times after surgery and radiotherapy.

• No adverse events or toxicity were recorded.

• There was no deterioration in neurological status of the

• There was no deterioration in neurological status of the patients during treatment.

i i ifi

• The treated patients had a significantly longer overall survival (p<0.05) than the control group of 9 patients (525 days 17 4 months vs 325 days 10 4 months)

(525 days, 17.4 months vs 325 days, 10.4 months).

The treated group and control groups did not differ in terms of age, extent of tumor resection or performance. p

The prolongation of survival was also significant when compared to

historical and published controls within the same age group. p g g p

(8)

Conclusions Conclusions

Immunizations with autologous, irradiated tumor cells transfected with the gene for IFN- in patients with

glioblastoma multiforme is

• Safe,

• Feasible in slightly less than 50% of eligible patients

• Show signs of clinical efficacy.

The small number of patients warrants further studies in larger cohorts.

2010-11-13 8 Bertil_R.Persson@med.lu.se

(9)

Kaplan

Kaplan--Maier graph showing overall Maier graph showing overall survival of survival of

i i d (i l d d) d t l ti t

i i d (i l d d) d t l ti t Th Th immunized (included) and control patients.

immunized (included) and control patients. The The survival

survival was was analyzed analyzed with the with the log log--rank rank test. test.

(10)

MRI (T1 with gadolinium) MRI (T1 with gadolinium) images from non

images from non--responding responding and responding patients

and responding patients preoperatively

preoperatively preoperatively, preoperatively, postoperatively

postoperatively and and postoperatively

postoperatively and and

at the 6th immunization at the 6th immunization. . The postoperative image of The postoperative image of the non

the non responding patient responding patient the non

the non--responding patient responding patient shows a dense area, which shows a dense area, which constituted a haemorrhage constituted a haemorrhage constituted a haemorrhage constituted a haemorrhage also seen on non

also seen on non--gadolinium gadolinium enhanced images (not

enhanced images (not shown).

shown).

2010-11-13 10 Bertil_R.Persson@med.lu.se

(11)

”RIGTT”

”RIGTT” RadiationRadiation ImmunoImmuno Gene Tumor

Gene Tumor TherapyTherapy



After the ”BRIGTT” After the ”BRIGTT” studystudy has has shownshown that that the

the immunotherapyimmunotherapy isis safesafe wewe wishwish toto

the

the immunotherapyimmunotherapy is is safesafe wewe wishwish to to improve

improve the the efficacyefficacy by by combiningcombining it it ith

ith di tidi ti thth with

with radiationradiation therapytherapy

,,



WeWe wishwish to to useuse a a singlesingle lowlow dosedose f ti

f ti ii dd t bt b blbl tt tt tt fraction

fraction in order to be in order to be ableable to to treattreat previously

previously irradiatedirradiated patients.patients.

(12)

Investigation of Rats with intracerebral implanted N29 Brain Tumors after Single implanted N29 Brain Tumors after Single fraction 5 or 15 Gy Radiotherapy combined with Immunotherapy

with Immunotherapy.

Bertil R.R. Persson ^1,4 ⁾ ^, ^,

In Collabration with

Catrin Bauréus K ch

^{2 4)}

Gustav Grafström

^{2 4)}

Catrin Bauréus Koch

^2,4),

Gustav Grafström

^2,4),

Crister Ceberg

^2,4),

Per Munck af Rosenschöld

^2,4)

, Bengt Widegren

^3,4)

and Leif G. Salford

^1,4

)

1)Dept. Neurosurgery, Lund University, SE-221 85 LUND, Sweden 2)Dept. Radiation Physics, Lund University, SE-221 85 LUND, Sweden

3)Dept Tumorimmunology Lund University BMC Box 7031 SE 220 07 Sweden;

3)Dept. Tumorimmunology, Lund University, BMC Box 7031, SE 220 07, Sweden;

4)Rausing Laboratory, Biomedical Centre, Lund University, 221 85 LUND, Sweden E-mail: bertil_r.persson@med.lu.se

2010-11-13 12 Bertil_R.Persson@med.lu.se

(13)

Groups of animals with various treatments in the various experiments with N29 and N32 tumors various experiments with N29 and N32 tumors.

text

(14)

Animals were given a single radiation treatment using a g ⁶⁰ Co radiotherapy unit py

text

2010-11-13 14 Bertil_R.Persson@med.lu.se

(15)

Inoculation at day 0 and treatments were performed at day 7 after inoculation

performed at day 7 after inoculation

text

Complete remissions 75 % Day 40-60

(16)

Number of Survivals versus total number of rats in each group with intra cerebral implanted N29 tumors treated with IFN-g cell immunization (IMU IFNg), single fraction radiation therapy (RT) and their combination at 7 days after inoculation. Immunization was repeated for at least two more times at days 21 and 35.

text

2010-11-13 16 Bertil_R.Persson@med.lu.se

(17)

Survival plot of intra cerebral implanted N29 tumors Controls (Lower panel). Immunization with syngeneic N29 tumor cells (2nd panel); radiation therapy (3rd panel) and a combination of

radiation therapy and immunization (upper panel).

text

(18)

In the lower panel is given the primary survival rate in %. The percentage of the challenged animals that didn’t develop any tumor. given in the middle panel multiplied with the fraction of primary survival gives the middle panel, multiplied with the fraction of primary survival gives the

percentage of cured animal. that is displayed in the upper panel.

text

2010-11-13 18 Bertil_R.Persson@med.lu.se

(19)

Conclusion

The most effective therapeutic regime for N29

t s i f ti f di ti th f 5 G tumors is one fraction of radiation therapy of 5 Gy combined with immunization.

The immunization repeated for at least two more The immunization repeated for at least two more times at days 21 and 35.

This regime resulted in a significant prolonged survival and 75% complete remissions (p<0.05).

Corresponding combination with 15 Gy RT resulted in 50% complete remissions Neither immune therapy 50% complete remissions. Neither immune therapy

nor radiation therapy alone with 5 or 15 Gy resulted in

any significant therapeutic effect.

(20)

Subcutaneously implanted N29 Brain Tumors

2010-11-13 20 Bertil_R.Persson@med.lu.se

(21)

N29 tumours inoculated N29 tumours inoculated

Subcutaneously on both sides Subcutaneously on both sides

lsls

50 000 cells50 000 cells 200 000 cel200 000 cel

Only the right tumour was

55 22

tumour was irradiated

(22)

Investigation of Rats with subcutaneously implanted N29 Brain Tumors after 4 implanted N29 Brain Tumors after 4 fractions 5 Gy Radiotherapy combined with Immunotherapy

Immunotherapy.

Bertil R.R. Persson ^1,4 ⁾ ^, ^,

In Collaboration with

Catrin Bauréus K ch

^{2 4)}

Gustav Grafström

^{2 4)}

Catrin Bauréus Koch

^2,4),

Gustav Grafström

^2,4),

Crister Ceberg

^2,4),

Henrietta Nittby

^1,4)

,

Bengt Widegren

^3,4)

and Leif G. Salford

^1,4

)

1)Dept. Neurosurgery, Lund University, SE-221 85 LUND, Sweden 2)Dept. Radiation Physics, Lund University, SE-221 85 LUND, Sweden

3)Dept Tumorimmunology Lund University BMC Box 7031 SE 220 07 Sweden;

3)Dept. Tumorimmunology, Lund University, BMC Box 7031, SE 220 07, Sweden;

4)Rausing Laboratory, Biomedical Centre, Lund University, 221 85 LUND, Sweden E-mail: bertil_r.persson@med.lu.se

2010-11-13 22 Bertil_R.Persson@med.lu.se

(23)

10000

Cotrol Group 11 Left side

Controls Controls

1000

t 5410

me / mm3

Controls Controls

100

rat 5410 rat 5411 rat 5412 rat 5413 rat 5414 rat 5415 rat 5416 rat 5417

Tumor Volum

0 10 20 30 40 50 60 70 80 90 100

10

rat 5417 Average Exp Growth fit r² 0.98

Time after inoculation / days Cotrol Group 11 Right side Time after inoculation / days

1000 10000

e / mm3

100

Tumor Volume

0 10 20 30 40 50 60 70 80 90 100

10

(24)

10000

Immunotherapy Left side IFN at days 15; 29; 43;

1000



rat 5334

ume / mm3

Immunotherapy Immunotherapy

100

Average

Tumor Volu

i.p injection of

0 10 20 30 40 50 60 70 80 90 100

10

g Exp growth r² 0.999

Time after inoculation / days

p j

IFN_ tumor cells

10000

Immunotherapy Right side IFN at days 15; 29; 43;

1000

IFN_ at days 15; 29; 43;

me / mm3

100

Tumor Volum

i.p injection of IFN_ tumor cells

0 10 20 30 40 50 60 70 80 90 100

10

RRS 2009 24 Bertil_R.Persson@rmed.lu.se

(25)

10000

RT Untreated Left side

Radiation therap Radiation therap

1000

rat 5327 t 5336

lume / mm3

Radiation therapy Radiation therapy

100

rat 5336 rat 5345 rat 5348 rat 5360 rat 5357 rat 5367 rat 5370 Average

Exp Growth fit

Tumor Vol

RT Rigth

side

0 10 20 30 40 50 60 70 80 90 100 110 10

Exp Growth fit r² 0.90

Time after inoculation / days 10000

RT Right side

treatment at days 29; 30; 32; 33;

1000

rat 5327 rat 5336

olume / mm3

100

rat 5345 rat 5348 rat 5360 rat 5357 rat 5367 rat 5370 Average

Tumor Vo

RT

0 20 40 60 80 100

10

Exp Growth fit r² 0.89

(26)

Radiation Radiation

and and

Immuno therapy + RT

and and

Immunotherapy Immunotherapy

1000

10000 Untreated Left side

IFN at days 27; 42; 47; 55;

/ mm3

py py

100 1000

rat 5328 rat 5371 rat 5331 rat 5335 rat 5339

mor Volume /

i p injection of

10

100 rat 5342

rat 5364 rat 5365 Average Exp Growth fit r² = 0.90

Tum i.p injection of

IFN_ tumor cells RT

right tumor

10000

Immuno therapy + RT Right side

IFN at days 27; 42; 47; 55;

0 10 20 30 40 50 60 70 80 90 100 110

1000

RT 29; 30; 32; 33

t 5328

me / mm3

100

Tumor Volu

i.p injection of IFN_ tumor cells

0 10 20 30 40 50 60 70 80 90 100 110

10

rat 5365 Average Exp Growth fit r² = 0.93

RT

RRS 2009 26 Bertil_R.Persson@rmed.lu.se

(27)

Exponential Tumour Growth Model

Tumour growth rate “TGR” is estimated

f th t l t b

from the tumour volume measurements by fitting the data of each individual tumour to a model of exponential growth

to a model of exponential growth

 

0 exp

TV t _t  TV 0  exp  TGR t  

TV TV TGR t

where

“TV

_t

” is Tumour volume at time t t is time after first treatment.

“TV

₀

” is Tumour volume at time t = 0,

“TGR” is tumour growth rate constant (% per day)

(28)

Tumour growth rate of subcutaneous N29

Tumour growth rate of subcutaneous N29 tumours tumours: : Controls and after treatment with

Controls and after treatment with Controls and after treatment with Controls and after treatment with RT, IFN

RT, IFN immunization or their combination immunization or their combination

ur

40 10

11 12

reated tumou

t

19

8 8 9 10

a-lateral untr ed tumour

pal effec t

15

5 6 7

r day; Contra r day; Treate

Absco p

2 3 4

TGR / % pe TGR / % pe

Controls RT

IFNg

FNg

+ RT --

0 1

2010-11-13 28 Bertil_R.Persson@rmed.lu.se

Co IFNg

Type of treatment

(29)

Tumour Growth rate ”TGR” of subcutaneous

tumours implanted on both Right and Left hind leg. p g g

= The right tumour was treated with radiation (RT).

Average of all experiments

Resultat LEFT SE Right SE N t ctrl L t Ctrl /R t R/L

Controls

9.1  0,3 8.5  0.3 40 NS

INF

9.2  0,8 7.6  0.6 19 NS NS NS

RT

6.1  0.4 4.5  0.3 15 <0.001 <0.001 <0.01

RT +INF

6.4  0.5 5.9  0.5 7 <0.001 <0.01 NS

(30)

Specific Therapeutic Effect “STE”

is defined as follow is defined as follow is defined as follow.

is defined as follow.

STE TGR ^C  TGR ^E

STE = ^C ^E ;

C

TGR TGR TGR

TGR E The average of the individual Tumour growth rate constant in the

f d t d

¹

TGR E

TGR

group of exposed rats. day

^-1

The average of the individual Tumour growth rate constant in

TGR C the group of control rats. day

^-1

RRS 2009 30 Bertil_R.Persson@rmed.lu.se

(31)

Th STE i l t 0

Th STE i l t 0 h h th th The STE is equal to 0

The STE is equal to 0 when the average when the average of tumour growth rate constant of the

of tumour growth rate constant of the

exposed group, is equal to the average of exposed group, is equal to the average of the tumour growth rate constant of the

the tumour growth rate constant of the the tumour growth rate constant of the the tumour growth rate constant of the control

control _..

The STE is equal to 1 when the average

h f h d

tumour growth rate constant of the exposed

group, is equal to 0.

(32)

Specific therapeutic effect of subcutaneous N29 tumors Specific therapeutic effect of subcutaneous N29 tumors

Type of treatment

after RT, Immunization with IFN

after RT, Immunization with IFN and their combination RT+ IFN and their combination RT+ IFN

8 8

Radiation + IFN Type of treatment



19

Left None Right Irradiated



19 19

IFN 

15 Radiation

p < 0.0001 p < 0.0001

0,0 0,1 0,2 0,3 0,4 0,5

Left Specific Abscopal Effect "SAE"

Right Specific Therapeutic Effect "STE"

2010-11-13 32 Bertil_R.Persson@rmed.lu.se

Right Specific Therapeutic Effect STE

(33)

CONCLUSION CONCLUSION::

Significant

AbscopalAbscopal effectpp effect

^was was confirmed confirmed on

on subcutaneously subcutaneously implanted implanted N N29 29 rat rat glioma glioma tumours

tumours,, from from contra contra--lateral lateral treatments treatments with with radiation

radiation therapy therapy alone alone and and in in combination combination with with immunization

immunization by by using y y using syngeneic g g syngeneic interferon y g y g interferon-- gamma

gamma secreting secreting tumor tumor cells cells..

(34)

Mechanism of Abscopal effect ?

Ionizing radiaation ???

ROS???

p

(35)

(36)

Conclusion:

Based on the findings that

immunization combined with 5 immunization combined with 5

Gy radiation therapy increased the y py survival time 87% (p=0.003) with

75% l t i i

75% complete remissions, new

regimes of glioma treatment

regimes of glioma treatment might be developed.

2010-11-13 36 Bertil_R.Persson@med.lu.se

(37)

In view of the finding that radiation enhance the

antitumor effect of antitumor effect of Immune-therapy, mm py,

h bi i

T

he combination

approach should be

studied further for

clinical translation.

(38)

Conclusion:

For example single fraction For example, single fraction

radiation therapy sessions with a

target absorbed dose in the order of 5 10 Gy combined with clinically 5 - 10 Gy combined with clinically proven immunotherapy.

2010-11-13 38 Bertil_R.Persson@med.lu.se

(39)

Conclusion:

Due to the moderate absorbed dose Due to the moderate absorbed dose, relapse patients previously treated to

f ll d i h b fi f i l

full dose might benefit from a single fraction radiation therapy of 6 Gy py y

combined with immunotherapy.

(40)

Conclusion:

Another benefit of the moderate

absorbed dose is that if the response absorbed dose is that, if the response in not complete after the first

ddi i l bi d treatment, additional combined treatments with single fraction g radiation therapy and

i th i ld b

immunotherapy sessions could be given with a few weeks interval.

2010-11-13 40 Bertil_R.Persson@med.lu.se

g

(41)

In view of the finding that radiation enhance the

antitumor effect of antitumor effect of Immune-therapy, mm py,

h bi i

T

he combination

approach should be

studied further for

clinical translation.

(42)

Conclusion:

Other alternati es than the presentl Other alternatives than the presently

used immunization by vaccination with the patient´s own tumor cells might be used, such as dendritic cell vaccines or , other clinically proven methods of

immunization immunization

^.

2010-11-13 42 Bertil_R.Persson@med.lu.se

(43)

RIGTT History ? RIGTT History ? RIGTT History ? RIGTT History ?

E i L d 2001 INF ll

• Experiment at Lund 2001: INF- cells

• Lumniczky Safrani 2002: y

GM-CSF cells

• Prins and Graf 2002: Autol. Tum cells

• Sandra De Maria 2005: Sandra De Maria 2005

CTLA-4 blockade CTLA 4 blockade

• Newcomb 2006: GM-CSF

• Sharp NCI 2007: AntiFAS mAb

• Teitz-Tennenbaum 2008:

Dendritic cells

N b 2010

^{i CD137 Th}

• Newcomb 2010:

Bertil R.R. Persson PhD,

R I G T

R I G T T T R I G T R I G T T T Radiation

Radiation immunomodulatory immunomodulatory gene tumor therapy

gene tumor therapy gene tumor therapy gene tumor therapy

Bertil R.R. Persson PhD,

Bertil R.R. Persson PhD, MDh.c MDh.c..

Professor of

Professor of medical medical radiation radiation physics physics

Lund University, Lund University, 221 85 LUND

221 85 LUND Sweden 85 U 85 U Sweden S S

“BRIGTT”

“BRIGTT” BRIGTT BRIGTT

nao3 mian3 yi4 ji1 yin1 zhong3 liu2 zhi4 liao2 nao3 mian3 yi4 ji1 yin1 zhong3 liu2 zhi4 liao2

Brain

Brain Immuno Immuno Gene Gene Tumour Tumour Therapy Therapy Brain

Brain Immuno Immuno Gene Gene Tumour Tumour Therapy Therapy Immunization with

Immunization with autologous autologous interferon interferon-- Immunization with

Immunization with autologous autologous interferon interferon--

gamma secreting glioma cells in patients with gamma secreting glioma cells in patients with

Glioblastoma

Glioblastoma Multiforme Multiforme Glioblastoma

Glioblastoma Multiforme Multiforme

A h 1

A h 1 2 li i 2 li i l t i l l t i l A phase 1

A phase 1--2 clinical trial 2 clinical trial

Salford

Salford LG LG

Siesjö Siesjö P P

Skagerberg Skagerberg G G

Rydelius Rydelius A A

Salford

Salford LG LG

, , Siesjö Siesjö P P

, , Skagerberg Skagerberg G G

Rydelius Rydelius A A

, , Blennow

Blennow C C

, , Lilja Lilja Å Å

Persson BRR Persson BRR

, , Strömblad Strömblad S S

,Visse E ,Visse E

and and Widegren Widegren B B

mu4 di4 :

Th i i f th BRIGTT t d t

The primary aims of the BRIGTT study were to ascertain

• Safety, 安全 an1 quan2

• Feasibility 可行性 and

k 3 i 2 i 4

• ke3 xing2 xing4

• Efficacy 效力 xiao4 li4

of immunotherapy with autologous IFN- transfected

Grows with arms as an octopus

S d i i

Sends migrating

“guerilla-cells”

I t th

Into the sur- rounding brain

h th BBB where the BBB is intact

Salford

Timeline of immunization and Timeline of immunization and Timeline of immunization and Timeline of immunization and

monitoring procedures.

monitoring procedures. g p g p

BRIGTT St d d i

BRIGTT Study design:

yan2 jiu1 she4 ji4

A t l t ll lt d d t f t d ith

Autologous tumor cells were cultured and transfected with the human IFN- gene by the use of an adenoviral vector.

After irradiation with 100 Gy the cells were administered as intradermal immunizations in the upper arm every 3 rd week.

Endpoints

• for safet ere: records of to icit and ad erse e ents

• for safety were: records of toxicity and adverse events,

• for feasibility percent treated patients out of eligible patients and time to treatment and

patients and time to treatment and

• for clinical efficacy overall survival (OS) and progress free survival (PFS).

2010-11-13 6 Bertil_R.Persson@med.lu.se

Results

8/17 (47%) of eligible patients, aged between 50 and 69 years, were immunized between 8-14 times after surgery and radiotherapy.

• No adverse events or toxicity were recorded.

• There was no deterioration in neurological status of the

^mu4 ^di4 :

• Safety, ^安全 ^{an1 quan2}

• Feasibility ^可行性 and

• Efficacy ^效力 ^{xiao4 li4}

After irradiation with 100 Gy the cells were administered as intradermal immunizations in the upper arm every 3 ^rd week.

Bertil R.R. Persson ^1,4 ⁾ ^, ^,

Animals were given a single radiation treatment using a g ⁶⁰ Co radiotherapy unit py