the challenges of measuring the response to ...mm33 veh. + pembro. low il2: tils high il2: tils high...

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THE CHALLENGES OF MEASURING THE RESPONSE TO IMMUNOTHERAPIES IN VIVO

Jonas Nilsson, PhD, professorDirector of Sahlgrenska Cancer Center

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Jkstudios.tv

IL-2

IFNBCG

Anti-CTLA4

Anti-PD1

Anti-TIM3 etc

TILs

NK cellsCARs

Targeted antibodies

Dendritic cellsPeptides

Immunotherapies are group of treatments

www.gu.se

Anti-PD1/PDL1

Anti-CTLA4Robert C, Paris Melanoma Conference 2013

Checkpoint immunotherapies

www.gu.se

PD1/PDL1 inhibitors are tested in >1000 trials

https://www.cancerresearch.org/scientists/immuno-oncology-landscape/pd-1-pd-l1-landscape

Wolchok et al., 2017, NEJMLong et al., 2015, Lancet

How can we make the durable responses last and convert non-responders to responders?

Immunotherapy vs targeted therapy in melanoma

• Accurately models the human disease and develop fast enough to be used to pre-screen patients before trials (Einarsdottir et al., 2014 Oncotarget; Olofsson Bagge et al., 2018 JCO-PO)

• Pre-clinical drug evaluation & Biomarker discovery• Tumor immunology

Patient-derived melanoma xenograft biobank

• Accurately models the human disease and develop fast enough to be used to pre-screen patients before trials

• Pre-clinical drug evaluation & Biomarker discovery (Gad et al. Nature 2014; Warpman-Berglund Ann Oncol 2016; Einarsdottir et al., 2018 Cell Death Dis; Muralidharan et al 2017 CDD; Xue et al., Nature Medicine 2017; Lunavat et al., 2017 PNAS)

• Tumor immunology


• Accurately models the human disease and develop fast enough to be used to pre-screen patients before trials

• Pre-clinical drug evaluation & Biomarker discovery• Tumor immunology (Jespersen et al., Nat Commun 2017, Forsberg et al., Cancer

Res 2019 & Ny/Rizzo et al., Annals of Oncol 2020)


Tumor-infiltrating lymphocytes (TIL)

Cancer immune therapy in PDX models?

Adoptive T-cell transfer (ACT)

Adapted from: Rosenberg SA, Nature Rev Immunol 2012

Tumor sample

In vitro expansion

Lymphodepletionbefore TIL transfer

(chemotherapy)

• High response rate in MM (Ph II), (>50% tumor reduction in half of patients)

• Complete and durable responses(20%, 8 years of follow-up)

• Considerable toxicities

• Labour intensive

• Expensive

IL2

+ IL-2

TIL infusion(~ 1.109 cells)

T cells fully trained to kill the tumor

= TILs (Tumor Infiltrating Lymphocytes)

In vitro expansion

+PDX

RESPONSE?(Tumor growth)

TIL infusion

PXD v2.0= PDX v2.0

RESPONSE?(Tumor growth)

• Less toxic for mice (GVHD) ?• Correlation to ACT patient data?• Marker for response?• Model for combination therapy?• Genetic manipulation of T cells?

TIL infusion

0 3 6 9 18 24 36 48 66

-80

-40

0

40

Time since TIL infusion (months)

Chan

ge in

targ

et le

sion

s fro

m b

asel

ine

(%)

1124

05

29

4604Spider plot

https://www.taconic.com/taconic-insights/oncology-immuno-oncology/new-humanized-mouse-immunotherapy.html

https://www.taconic.com/taconic-insights/oncology-immuno-oncology/new-humanized-mouse-immunotherapy.html

Transgenic expression of human IL2 enables TIL-mediated tumor eradication in NOG mice

0 10 20 30 400

200

400

Days after transplantation

Tum

or v

olum

e (m

m3 ) MM33 Veh. + Pembro.

Low IL2: TILsHigh IL2: TILsHigh IL2: TILs + Pembro.

TILs n.s.

p=0,007

0 10 20 30 40 50 60 700

100

200

300

Days after transplantation

Tum

or v

olum

e (m

m3 )

20x106 - F3,710x106 - F3,05x106 - F6,01x106 - F3,40.1x106 - F3,40 - Veh.

TILs

MM33

Dose dependency in levels of IL2 and number of injected TILsAnti-PD1 does not enhance

0 20 40 60 80 100 120 140106

107

108

109

1010

Days after s.c. transplantation of the primary 33-luc tumour - IL2 control mouse, tumour removed around day 40- Relapse (metastasis chest + back) observed on day 76- TILs injected (17.106) on day 84

Radi

ance

(p

h/se

c/cm

2 /sr

)

Growth of MM909.33/luc in #2821 - IL2 mouse (BLI)

#2821 Metastasis CHEST#2821 Relapse tumour BACK

#2821 Primary s.c. tumour BACK

Resection of the primary tumour

Observation of a metastasis (chest) + tumour growing again at

the initial site (back)

Injection of cryopreseved TILs

Day 129

Day 40Day 76

Metastasis

• Can the PDXv2 model be predictive of ICI therapy?• Can we achieve additional effect by combination with

other therapies?• How applicable is the PDXv2.0 method to other diagnoses

than cutaneous melanoma?

Questions arising

Can PDX data be predictive of therapy responses

Tumor sample

NOG mouse

hIL2-NOG mouse

• T cell activation ?• Correlation to immunotherapy patient data?• Comparison to other predictive assays?• Model for combination therapy?

• New case presentingwith high tumor load

• Normally refractory to immune therapy withipi/nivo

• BRAF mutant• No TILs, only tumor

cells!!

PDX

TILs

Lars Ny, Lisa Nilsson

Case 1

• Dabrafenib/Trametinib• CR of all except very few

lesions• One subQ lesion biopsy –

TILs grew but hardly anytumor cells

• Switched to pembrolizumab• Last lesions disappeared

PDX

TILs

Post-therapy

Lars Ny, Lisa Nilsson

Case 1

Case 1

Lars Ny, Lisa Nilsson& Larissa Rizzo

A PDX-platform for prediction of therapy responses

0 20 40 600

200

400

600

800 M608B

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

200

400

600

800M612

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 80 1000

50

100

150 M503B

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 600

100

200

300 M111

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 800

200

400

600 M812

Time (Days)

Tum

or v

olum

e (m

m3 )

NOG hIL2-NOG

0 50 100 1500

200

400

600

Time (Days)

Tum

or v

olum

e (m

m3 ) M816

0 20 40 60 80 1000

200

400

600M315

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 80 1000

100

200

300

400

500M131

Time (Days)Tu

mor

vol

ume

(mm

3 )

0 20 40 60 800

100

200

300

400

500M817

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

50

100

150

200 M626B

Time (Days)

Tum

or v

olum

e (m

m3 )

250

0 50 100 150 200 2500

50

100

150

200

250

Time (Days)

Tum

or v

olum

e (m

m3 ) M626A

0 50 100 150 2000

100

200

300 M830

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

50

100

150

200

250 M214LC

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 200 2500

100

200

300

400 M128B

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 1500

100

200

300

400 M608A

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 800

200

400

600M919A

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 800

200

400

600 M212

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 800

200

400

600 M503A

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

200

400

600 M811

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

100

200

300

Time (Days)

Tum

or v

olum

e (m

m3 ) M216

Growth pattern in hIL-NOG: No growth No growth but toxicity Growth Growth but delayed Growth and toxicity

Valerio Belgrano, Lars Ny, Henrik Jespersen, Lisa Nilsson& Larissa Rizzo


• 11/20 patients had been treated withPD-1 therapies

• Fisher test comparing mouse responsesto survival (p=0.0152)

0 10 20 30 40 500

50

100

Survival time from start of anti-PD1

Months

Perc

ent s

urvi

val

Blue/orange

Green/black&grey

Median survivalBlue/orangeUndefined

Green/black&grey19.27

Log-rank (Mantel-Cox) testChi squaredfP valueP value summaryAre the survival curves sig different?

1.42610.2324nsNo


0 20 40 600

200

400

600

800 M608B

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

200

400

600

800M612

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 80 1000

50

100

150 M503B

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 600

100

200

300 M111

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 800

200

400

600 M812

Time (Days)

Tum

or v

olum

e (m

m3 )

NOG hIL2-NOG

0 50 100 1500

200

400

600

Time (Days)

Tum

or v

olum

e (m

m3 ) M816

0 20 40 60 80 1000

200

400

600M315

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 80 1000

100

200

300

400

500M131

Time (Days)Tu

mor

vol

ume

(mm

3 )

0 20 40 60 800

100

200

300

400

500M817

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

50

100

150

200 M626B

Time (Days)

Tum

or v

olum

e (m

m3 )

250

0 50 100 150 200 2500

50

100

150

200

250

Time (Days)

Tum

or v

olum

e (m

m3 ) M626A

0 50 100 150 2000

100

200

300 M830

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

50

100

150

200

250 M214LC

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 200 2500

100

200

300

400 M128B

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 1500

100

200

300

400 M608A

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 800

200

400

600M919A

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 800

200

400

600 M212

Time (Days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 800

200

400

600 M503A

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

200

400

600 M811

Time (Days)

Tum

or v

olum

e (m

m3 )

0 50 100 150 2000

100

200

300

Time (Days)

Tum

or v

olum

e (m

m3 ) M216

Growth pattern in hIL-NOG: No growth No growth but toxicity Growth Growth but delayed Growth and toxicity


Case 2

0 50 100 1500

200

400

600 NOGhIL2-NOG

Time point (days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 80 1000

200

400

600 NOGhIL2-NOG

Time point (days)

Tum

or v

olum

e (m

m3 )

0 20 40 600

200

400

600

Days post TIL infusion (i.v.)

Tum

or v

olum

e (m

m3 ) ****

0 20 40 60 80 1000

200

400

600

Time point (days)

Tum

or v

olum

e (m

m3 )

NOGhIL2-NOG

TILs

a b

c

0 14 28 42 56 70 84 980

100

200

300

400

500

600

Days post therapy

Tum

or v

olum

e (m

m3 )

BRAFi/MEKivehicleBRAFi/MEKi-long+TILsBRAFi/MEKi-short+TILsTILs

d

eBefore After anti-PD1 therapy

0 50 100 1500

200

400

600 NOGhIL2-NOG

Time point (days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 80 1000

200

400

600 NOGhIL2-NOG

Time point (days)

Tum

or v

olum

e (m

m3 )

0 20 40 600

200

400

600


Tum

or v

olum

e (m

m3 ) ****

0 20 40 60 80 1000

200

400

600

Time point (days)

Tum

or v

olum

e (m

m3 )

NOGhIL2-NOG

TILs

a b

c

0 14 28 42 56 70 84 980

100

200

300

400

500

600

Days post therapy

Tum

or v

olum

e (m

m3 )


d


0 50 100 1500

200

400

600 NOGhIL2-NOG

Time point (days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 80 1000

200

400

600 NOGhIL2-NOG

Time point (days)

Tum

or v

olum

e (m

m3 )

0 20 40 600

200

400

600


Tum

or v

olum

e (m

m3 ) ****

0 20 40 60 80 1000

200

400

600

Time point (days)

Tum

or v

olum

e (m

m3 )

NOGhIL2-NOG

TILs

a b

c

0 14 28 42 56 70 84 980

100

200

300

400

500

600

Days post therapy

Tum

or v

olum

e (m

m3 )


d



Case 2

0 50 100 1500

200

400

600 NOGhIL2-NOG

Time point (days)

Tum

or v

olum

e (m

m3 )

0 20 40 60 80 1000

200

400

600 NOGhIL2-NOG

Time point (days)

Tum

or v

olum

e (m

m3 )

0 20 40 600

200

400

600


Tum

or v

olum

e (m

m3 ) ****

0 20 40 60 80 1000

200

400

600

Time point (days)Tu

mor

vol

ume

(mm

3 )

NOGhIL2-NOG

TILs

a b

c

0 14 28 42 56 70 84 980

100

200

300

400

500

600

Days post therapy

Tum

or v

olum

e (m

m3 )


d


Ny/Rizzo et al., Ann Oncol In press

Jkstudios.tv

IL-2

IFNBCG

Anti-CTLA4

Anti-PD1

Anti-TIM3 etc

TILs

NK cellsCARs

Targeted antibodies

Dendritic cellsPeptides(e.g. gp100)

Potential combination immunotherapies

• Can the PDXv2 model be predictive of ICI therapy?• Can we achieve additional effect by combination with

other therapies?• How applicable is the PDXv2.0 method to other diagnoses

than cutaneous melanoma?

Questions arising

Sahlgrenska Cancer Center PDX biobank

Control TIL therapy CAR-T therapy CRISPR library

Breast cancer

Breast cancer biobank

ID TYPE Grade ER/PR HER2 LN Follow-up TILs25 IDC 3 ER+ - + Surgery (LN-) ++27 IDC 3 - - + Surgery (LN+) ++28 IDC 2 ER+PR+ + - -29 IDC (apocrine) 2 - + - -30 IDC (lobular) 2 ER+PR+ + - -32 IDC 2 - + + ++34 Skeletal met - +35 IDC 2 - - + +36 IDC 2 ER+ + - +37 DC (medullary) 3 - - - ++38 IDC 3 ER+PR+ - + +

Patients scheduled for neoadjuvant chemo for breast cancer

• PDX models can predict responses to ACT and ICI therapy• This can be useful in clinical and translational research

• Guide inclusion into trials• Inform on necessity of next-line therapy• Tools for testing new types of therapies

• Checkpoint inhibitors• Epigenetic therapies• Targeted therapies• Cell therapy (antigen-selected, engineered cells etc)• Stroma-cell directed therapies

• Challenges include• Take rate• Full immune humanization• Imaging

Summary & challenges

Solutions• Organoids• HSC transplantation• Nanoprobes

AcknowledgmentsSurgeons:Roger Olofsson Bagge, MD, PhD, assoc profPeter Naredi, MD, PhD, professor Caroline Vilhav, MD, PhD studentKian Chin, MDMedical Oncologists:Lars Ny, Assoc prof, MD, PhDHenrik Jespersen, MD, PhD student

Senior Research Fellows:Lisa Nilsson, PhD, Senior staff scientistSamuel Alsén, PhDJoakim Karlsson, PhDPost-graduate students:Elin Forsberg, MSc, PhD studentVasu Sah, MSc, PhD studentTechnical staff:Sofia Stenqvist, animal technician Carina Karlsson, research technician

Center for Cancer Immune Therapy, HerlevInge Marie Svane, MD, PhD, professorMarco Donia & Rikke Andersen, MDs/PhDs

the challenges of measuring the response to ...mm33 veh. + pembro. low il2: tils high il2: tils high...

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