What are the fundamental properties of curative anti-

tumor T cells?

Nicholas P. RestifoSITC

November 6, 2015

NIH Center for Regenerative

Medicine

Khajah, Int J Oncol, 2015

Surgery, radiation, and chemotherapy / targeted therapy can rapidly kill tumor cells but these modalities can fail to cure in the setting of metastatic

solid tumors . . .

Cancer Immunotherapy: Delayed gratificationAll patients (n=25) in TIL + TBI 12 Gy + HD IL-2

Months

Months

Cancer Immunotherapy: Delayed gratificationAll patients (n=25) in TIL + TBI 12 Gy + HD IL-2

Tumor rejection can be slow . . .

. . . but it can be complete and durable

Patterns of response to Ipilumumab in 4 patients with melanoma

Wolchok, et al, Clin Cancer Res, 2009

% persistence of the infused cells in peripheral blood

* CR + PR vs. NR < 0.001

T cell persistence at 1 month is highly correlated with objective clinical response

Rosenberg SA et al. Clin Cancer Res, 2011

25

14

1.5

Complete tumordestruction

> 30% tumordestruction

< 30% tumordestruction

Patient response status

0 40 60 80 10020

0

50

100

150

200

250

300

350

400

0 20 40 60 80

Tum

or S

ize

(mm

2)

Days post ACT

NT

Pcis-/- aCD8 D38

Pcis-/- aIgG D38

Prolonged tumor regression is mediated by ongoing activity of living CD8+ T cells

Injection of α-CD8 antibody

(d 38 post-treatment)

Pmel-1 CishKO + α-CD8 mAb

No treatment

Pmel-1 CishKO + IgG

Palmer, et al, J Exp Med, Online ahead of print Nov 2, 2015

How do we achieve the persistence of memory?

Effector function/CytoxicityGlycolytic metabolism

Senescence

Anti-tumor activityProliferative capacityStemness/Persistence

Increasing cellular differentiation; decreasing anti-tumor effectiveness after ACT

Stem cell-like capacity for each persisting T cell clonotype

Adapted from Restifo, Blood, 2014; Gattinoni, et al, Nat Med, 2009 & 2011

Identification of human memory stem T cells (Tscm)

Measuring mitochondrial membrane potential (∆ψm) in individual living cells using tetramethyl rhodamine methyl ester (TMRM)

∆ψm

Sukumar, Cell Metabolism, In Press

Measuring mitochondrial membrane potential (∆ψm) in individual living cells using tetramethyl rhodamine methyl ester (TMRM)

A low mitochondrial membrane potential marks self-renewing hematopoietic stem cells

Long-term reconstitutionIn lethally irradiated hosts

LT-HSC (Lin - c-kit + Sca+) (0.75% of population)Congenically labeled with CD45.1

cKit

Sca1TMRM

CD45.2 (competitor total bone marrow cells)

CD

45.1

(so

rted

for ∆ψ

m)

39 5

51 91

Low ∆ψm High ∆ψm

Low mitochondrial membrane potential marks self-renewing hematopoietic stem cells

Transplantation into lethally irradiated recipients (CD45.2) using: -- 300 CD45.1 (low or high membrane potential) Lin- Sca1+ c-Kit+ cells along with

-- 3 X105 CD45.2 competitor total bone marrow cells (CD45.2)

Reconstitution of host lymphocytes

Adoptive transfer into irradiated C57BL/6

rVV hgp100 challenge

Pmel TCR transgenic

spleen

Hgp100peptide

Phenotype and function of

CD8+ T cells

High∆ψ

Low∆ψ

CD44 CD69CD27 IL7Rα KLRG1

CD8-Naive Low ∆ψ High ∆ψ

Characterization of CD8+ T cells sorted based on ∆Ψm

TMRM

RNA-seq ‘volcano plot’ of cells sorted based on mitochondrial membrane potential

CD

8+T

hy-1

.1+

T c

ells

(10

6 )

Time after transfer (days)

Bcl6 Tcf7 Lef1 Klf2

Prdm1 Eomes Prf1 GzmbE

xpre

ssio

n re

lativ

e to

Act

-b(1

03)

Low ∆ψ High ∆ψHigh ∆ψLow ∆ψ

0 5 10

0

4

8

12

******

**

Mitochondrial membrane potential (∆ψm) segregates short-lived effector from memory T cell precursors

Low ∆ψ High ∆ψ

% P

ersi

sten

ce C

D8+

Thy

1.1+

0.01

0.1

1

10

100 *

Thy

1.1

SSC

High ∆ψ

Low ∆ψ

Low ∆ψm CD8+ T cells demonstrate increased long-term in vivo persistence (300 days)

∆ψm segregates long-lived memory T cells from short-lived effectors in vivo after infection

CD

44

CD62L

TSCM

TCMTEM

Low ∆ψm cells identify ‘metabolically fit’ T cells within sorted populations of TCM and Tc17

0.4 0.1

0.299.2

4.0 3.5

8.084.4

6.4 15.7

33.244.8

IFN

-γ

IL-17A

SS

C

IFN-γ

IL17

-F

IL-17A

Low ∆ψm High ∆ψm

% IL

-17A

+

CD

8+ T

cel

ls%

IL-1

7A+

CD

4+ T

cel

ls%

IFN

-γ+

CD

4+ T

cel

ls

Unstim0.4 0.0

0.399.3

1.3 0.3

24.174.4

0.1 0.3

74.325.2

58.914.62.5

Tc17

Th1

Th17

Low ∆ψHigh ∆ψm

High-∆Ψm is associated with effector cytokine production in T cells

Analysis of metabolites using HLPC-Mass

Spectrometry

Low Δψ High Δψ

Low ∆ψm cells display increased fatty acid metabolites

carnitineacetyl

carnitinedeoxy

carnitine

palmitate palmitoleate margarate

linolenate caprylate laurateSca

led

Inte

nsity

Low Δψ

High Δψ

Low ∆ψm cells display a metabolic profile of memory CD8+ T cells

EC

AR

(m

M/p

H)

Exp

ress

ion

rela

tive

to A

ct-b

(10

3 )

Cpt1a Slc2a1

Basal ExtracellularAcidifcation Rate

Low ∆ψ High ∆ψ

Low ∆ψm cells display a metabolic profile of memory CD8 + T cells

Low ∆ψm CD8+ T cells control established tumor even when sorted from an established T cell culture

**

High ∆ψ

Low ∆ψ

No Treatment

Low ∆ψm CD8+ T cells demonstrate increased autoimmune vitiligo

Low Δψm High Δψ

Low ∆ψm cells:

1. Are more stem cell-like

2. They burn fats not glucose

3. They have more spare respiratory capacity

4. The persist longer

5. They control established tumor better

High ∆ψm cells make more cytokines, but why do they die?

CD8+ T cells with low ∆ψm have decreased checkpoint

CD8+ T cells with low ∆ψm have decreased levels of reactive oxygen species (ROS)

DCFDA is a cell-permeable non-fluorescent probe. 2′,7′-Dichlorofluorescin diacetate is de-esterified intracellularly and turns to highly fluorescent 2′,7′-dichlorofluorescein upon oxidation.

DC

FD

A

Bulk CD8 TCM Tc17 Th1 Th17 LT-HSC

Low ∆ψm High ∆ψm

High ∆ψm CD8+ T cells display increased DNA damage

Stain for γ-H2AX, a marker for dsDNA breaks

High ∆ψm CD8+ T cells elevated levels of biomarkers of ‘physiological age’

Cdkn1a Cdkn2a

Exp

ress

ion

rela

tive

to A

ct-b

(103

)

Low ∆ψm

High ∆ψm

CD8+ T cells with high ∆ψm die by apoptosis

� Low ∆Ψm CD8+ T cells demonstrate long-term in vivopersistence and superior anti-tumor activity

� Low ∆Ψm T cells display metabolic signature of memory CD8+ T cells

� High-∆Ψm is associated with effector cytokine production in T cells, followed by DNA damage, senescence and death.

� Low ∆Ψm identifies metabolically fit cells among HSC and CD8+ TCM subsets

SUMMARY

THE VISION: What is required to bring cell-based therapies to the many patients who need them?

1. Concerted commitment to basic science.2. Concerted effort to create vector and cell production

laboratories for patients.3. Robust technology transfer: open sourcing & industrial

partners.

Restifo Lab:Past and present

Madhu SukumarRahul RoychoudhuriDouglas C PalmerChristopher A. KlebanoffNick AcquavellaJoe CromptonNick KlemenTori YamamotoZhiya YuRobert EilJenny PanShashank PatelDavid CleverGautam MehtaRaul VizcardoLinda TranDevi Gurusamy

Toren Finkel:Jie Liu

Leonard Lab:Warren LeonardRosanne SpolksiPeng Li

David Stroncek:Franco MarincolaEna Wang

Richard Siegel:Madhu RamaswamyAnthony C. Cruz

Luca Gattinoni:Luca GattinoniYun Ji

NIH Pharmacy:George GrimesGopal Poti

Clinical Team:Udai KammulaRick SherryStephanie GoffPaul RobbinsSteve FeldmanRobert SomervilleSteve Rosenberg

James Yang:Ken-ichi HanadaQiong Wang

Rosenberg Lab:Eric TranAlena Gross

John O’Shea:Golnaz VahediVittorio Sartorelli

Francis Collins:Stephen C. J. Parker

Larry Samelson:Lakshmi Balagopalan