Adoptive cell therapy

John B.A.G. Haanen MD, PhD

ESMO I-O preceptorship Zürich November 2017

Disclosures

• I have provided consultation, attended advisory boards, and/or

provided lectures for: Pfizer, MSD, BMS, IPSEN,

Roche/Genentech, NEON Therapeutics, Novartis for which NKI

received honoraria

• Through my work NKI received grant support from BMS, MSD,

Novartis

• I declare no conflict of interest

Patient case

• Mr. X

– 48 years

– Aside obesitas no other comorbidities

– 2007: superficial spreading melanoma of the right

upper leg: resected

– 2010: Inguinal node metastasis: IND

– 2010: pulmonary metastases en in-transit in the

right upper leg

Patient case

• BRAF V600E mutation present

– Started treatment with vemurafenib

– Resolution of pulmonary metastases

– Disease progression after 6 months

• Started dacarbazine chemotherapy

– After 2 courses progressive disease (PD)

• Started anti-CTLA4 (ipilimumab)

– After 4 courses PD

Patient case

• No treatment options

– Participated in experimental trial with TIL

– One in-transit metastasis was removed from right

leg and TIL were isolated and cultured for 5 weeks

– He received TIL infusion in Oct 2012 preceded by

non-myeloablative chemotherapy and followed by

HD IL-2

Days -7 to -1: Nonmyeloablative chemotherapy with Cyclophosphamide and fludarabin

Day 0: 2 x 1011 TIL (unselected ‘young’ TIL)

Days 0 – 3: high dose bolus IL-2 (4 in total)

Tumor-infiltrating lymphocyte (TIL) therapy of melanoma

Prior to TIL 3 wks after TIL 20 wks after TIL8 wks after TIL

Clinical data N10TIL003 patient: CR at 20 weeks

Overview of presentation

• Adoptive cellular immunotherapy

– TILs

– TCR/CAR gene therapy

Immunosuppressive tumor microenvironment

Kerkar S P , Restifo N P Cancer Res 2012

TILs in melanoma

Taube et al. Science Transl Med 2012

Scheme for TIL therapy

Rosenberg & Restifo Science 2015

Scheme for ‘young’ TIL therapy

Rosenberg & Restifo Science 2015

Tumor digest

Rosenberg & Restifo Science 2015

Goff et al., J Clin Oncol 2016

Goff et al., J Clin Oncol 2016

Patient characteristics

Overall Survival

Goff et al., J Clin Oncol 2016

Progression Free Survival

Survival of patients receiving TIL treatment

Response to TIL treatment

24% of patients developed a CR, only 1 of these progressed. Median FU 40.9 months

NMA TIL NMA + 1200cGy TIL

Goff et al., J Clin Oncol 2016

• International, multicentre, open-label, randomized controlled phase III study

• Patients with unresectable stage IIIc/IV melanoma

• Zero to one prior treatment (now virtually always anti-PD-1)

• Health technology assessment

M14TIL (NCT02278887)

168 patients

84 TIL 84 ipilimumab

Herlev Hospital,Copenhagen

NKI-AvL, Amsterdam

Inclusions since start (2015)

Centre TIL Ipilimumab Screening Total

NKI 16 14 2 30

Herlev 7 6 - 13

Total 23 20 - 43

24 mm

14 mm

13 mm

16-9-15 28-12-15

Patient treated in the RCT with TIL after failure to pembrolizumab

Prior to TIL 3 months after TIL

- Strong correlation between immune cell infiltrates and various types of cancer

Rationale to develop adoptive T cell therapy:

Fridman et al., Nat Rev Canc 2012

• Strong correlation between immune cell infiltrates and various types of cancer

• Approaches to ‘non-specifically’ mobilize endogenous anti-tumor immune responses show clear efficacy• Anti-CTLA4• Anti-PD1/PD-L1• Combination I-O therapies

• With currently available technologies ACT is feasible at a large(r) scale

Rationale to develop adoptive T cell therapy:

TIL therapy: How does it work?

Patient pretreated withlymphodepletingchemotherapy

TIL are grown from melanoma

tumors

Infusion of TIL + IL-2

Rapid Expansion

Immunotherapy of melanoma: TIL therapy

TIL are grown from melanoma

tumors Rapid Expansion

1x1011 T cellsA few million T cells

Which (cytotoxic) T cells mediate cancer regression?

?

Patient pretreated withlymphodepletingchemotherapy

TIL are grown from melanoma

tumors

Infusion of TIL + IL-2

Rapid Expansion

TIL therapy: Key question

What could tumor-specific cytotoxic T cells detect on tumor cells?

1. Self antigens (to which tolerance is incomplete)

2. ‘Neo-antigens’, epitopes that are truly foreign

What could tumor-specific cytotoxic T cells detect on tumor cells?

1. Self antigens (to which tolerance is incomplete)

2. ‘Neo-antigens’, epitopes that are truly foreign

Three subclasses:

a). Melanocyte differentiation antigens (e.g. Mart-I)b). Cancer-Germline antigens (e.g. NY-ESO-1)c). Overexpressed antigens

What could tumor-specific cytotoxic T cells detect on tumor cells?

1. Self antigens (to which tolerance is incomplete)

2. ‘Neo-antigens’, epitopes that are the result of carcinogen (UV or smoking) induced DNA damage. These are most often patient specific and truly foreign to the immune system

Both types of antigens comprise 100-1000 of potential epitopes

Peptide 1 Peptide 2 Peptide 3

Disintegration

Rescue Peptides

Toebes et al. Nat. Med. 2006Bakker et al. PNAS 2008

Generation of pMHC multimers by UV-induced peptide exchange

Tools for high-throughput analysis of tumor-antigen specific CD8 T cell responses:

Allows analysis of T cell responses against 100s-1000s of (predicted) antigens

Generate fluorochrome conjugated MHC multimers

Mix to create a collectionof differentially encoded MHC multimers

Assembly of combinatorial codes on T cell surfaces

Analysis by flow cytometry

T cell T cell T cell

Self-assembling molecular codes

Allows detection of 47 T cell responses in parallel

Hadrup et al, Nat Methods 2009, Andersen et al, Nat Prot 2012.

Summary of analysis of T cell responses against self antigens in clinically infused TIL products

• TIL infusion products very often comprise self-antigen specific CD8 T cells

• MDA

• C/T

• Overexpressed antigens

• These self-Ag specific T cells make up only a small minority of tumor-reactive

T cells (1% or less) with the TIL product

• TIL therapy leads to a significant increase in the number of tumor-reactive T

cell responses against the group of self antigens: broadens the tumor-reactive

immune response

What are we missing?

Analysis of T cell responses against self antigens in clinically effective TIL products

� TIL therapy leads to a significant increase in the number of tumor-reactive T

cell responses against the group of self antigens

� These T cell responses are however of a surprisingly low magnitude

Explanation A). Few cells suffice (and things may even get better when we

induce stronger T cell responses against these antigens)

Explanation B). We may not be looking at (all) the right antigens: recognition

of mutated peptides (neo-antigens) may be much more important

Generate map of tumor-specific mutations (DNASeq: exome, whole genome)

Determine which mutatedgenes are expressed (RNASeq)

Predict epitopes for each mutation per HLA-allele in silico = ‘antigenome’

Screen for T cell recognitionof mutated epitope

MDLVLNELVISLIVESKLLEHLA-A2HLA-B7HLA-C2

T cell

Developing a strategy for analysis of neo-antigen-specific T cell responses

Analyzing the neo-antigen-specific T cell repertoire in human cancer?

Excise tumor

Isolate tumor cells Isolate tumor-infiltrating T cells

Identify tumor-specific mutations

Predict potential T cell epitopes

Screen for T cell reactivity

Pt 010: complete response upon TIL therapy

480 1 2 3.5 5 6 7 8 9 10 11 12 13 14 150

50

100

Months post infusion of TIL

Red

uct

ion

in tu

mo

r b

urd

en

Months post infusion of TIL

Tum

or

bu

rden

Strong neo-antigen specific T cell responses in the infusion product

TIL infusion productVARST>M MYLKG>V WDR1N>K LRP3T>S RBM12S>L

0.096 1.03 1.242 0.290 29

Strong neo-antigen specific T cell responses in the infusion product

TIL infusion productVARST>M MYLKG>V WDR1N>K LRP3T>S RBM12S>L

0.096 1.03 1.242 0.290 29

Profound effect of TIL therapy on the neo-antigen specific T cell pool

TIL infusion product

2 months

4.45

Pre therapy

0.02

6 months

15.06

34 months

2.20

12 months

8.78

VARST>M MYLKG>V WDR1N>K LRP3T>S RBM12S>L

0.096 1.03 1.242 0.290 29

>450 fold increase in neo-antigen specific T cell reactivity upon TIL therapy

Peripheral blood

Summary of neo-antigen analysis in CD8 T cells

12 patients analyzed, neo-antigen specific reactivity in 10

(Not all alleles covered, exome coverage incomplete, epitope predictions

imperfect)

Percentage neo-antigen reactivity in general higher than shared

antigens

CD8 neoantigen-specific T cells have cytolytic activity against

autologous melanoma in vivo (PDX model) and may be superior

over C/T specific T cells

CD8+ T cells frequently respond to the consequence of DNA

damage in human melanoma

Neo-antigen reactive CD4+ cells also detected (5 out of 6 patients)

Possible advantages:No requirement for in vitro expansion of autologous tumor-specific T cells

One set of ”good” TCRs could serve many

Alternatives to adoptive T cell therapy: transferring TCRs rather than T cells

Possible advantages:No requirement for in vitro expansion of autologous tumor-specific T cells

One set of ”good” TCRs could serve many

Alternatives to adoptive T cell therapy: transferring TCRs rather than T cells

Cancer patient

Removal of peripheralblood lymphocytes

Infusion of autologous TCR gene modified T cells

Ex vivo transduction process

Infusion of gene-modified T cells

Kershaw et al. Nat Rev Cancer 2013

Genetically modified peripheral blood lymphocytes

Mouse transgenic for human TCR gene locus and MHC cl I

Modified from: Restifo et al., Nature Rev Immunol (2012)

Leukapheresis Start chemotherapy:Cyclophosphamide + fludarabin for total of 7 days

-7 0

Infusion of transduced T cells

High dose IL-2

Monitoring response and survival

mnd 1, 2, 3, 6Patient:

Informed consent + screening

Preparation of gene modified T cells

Schedule of treatment

On-target toxicity of MART-1-specific TCR gene therapy

CT scan of liver metastasis in patienttreated with TCR-modified T cells

Feasibility of TCR gene transfer in the clinic

Clinical data with TCRs specific for melanocyte differentiation antigens(expression on melanoma and healthy melanocytes)

Clinical responses but also (acceptable) on-target toxicity

Feasibility of TCR gene transfer in the clinic

Clinical data with TCRs specific for melanocyte differentiation antigens(expression on melanoma and healthy melanocytes)

Clinical responses but also (acceptable) on-target toxicity

Pre 5 months

Feasibility of TCR gene transfer in the clinic

Clinical data with TCR specific for carcinoembryonic antigen(expression on colorectal cancer and healthy colonic epithelium)

Modest clinical responses with substantial on-target toxicity

Feasibility of TCR gene transfer in the clinic

Clinical data with TCR specific for NY-ESO-1(expression on cancer cells and -MHC-negative- spermatocytes)

Clear clinical responses (both melanoma and synovial sarcoma. No detectable toxicity

Platforms for gene modified T cells

Fesnak et al. Nat Rev Cancer 2016

CAR T cell concept

Ramos et al. Ann Rev Immunol 2016

CD19 CAR development and beyond

Fesnak et al. Nat Rev Cancer 2016

Success rate of CD19 CAR therapy in refractory ALL

Brentjens et al., Sci Transl Med 2013; Grupp et al., NEJM 2013; Davila et al., Sci Transl Med 2014; Maude et al., NEJM 2014; Lee et al., Lancet 2015; Frey et al., ASH 2014; Park et al., ASH 2014

Sadelain J Clin Invest 2015

Survival of refractory ALL patients treatedwith CD19 CAR T cells

Lee et al., Lancet 2015

Emily Whitehead story

• 2010: at 5 years: pre-B acute lymphoblasticleukemia: treated with standard chemotherapy (for6 months): CR

• 2011: at 6 years: recurrence of ALL: again treatedwith chemotherapy and scheduled for bonemarrow transplant in 2012

• 2012: at 7 years: before BMT, recurrence.• 2012: at 7 years: first child to receive CD19 CAR T

cells: developed CR.

Emily Whitehead story

Future of infusion of gene modified T cells

• CD19 CAR T cell therapy has now been approved in 2017 for refractory ALL and B-NHL

• Further development of CARs (3rd and 4th generation) with more potent efficacy (less toxicity?)

• Combination therapy a.o. with immune checkpoint blockade

• More CAR T cells targets for solid cancers– CEA, PSMA, HER2, GD2, MUC1, EGFRvIII, mesothelin

• Targets for TCR gene therapy: neo-antigens

• T cell responses against self antigens are enhanced by these T cell products but are nevertheless oftentimes weak

• It is now possible to also evaluate the contribution of T cell responsesagainst patient-specific neo-antigens

- We need to find out which T cells matter most

• Adoptive T cell therapy with patient-derived T cells can mediate cancer regression in melanoma patients

• Genetic engineering of tumor-specific T cell responses is feasible- Makes adoptive T cell therapy feasible for the (many) tumor types for which TIL can not be grown

- Early clinical data demonstrate feasibility and clinical potential- Antigen choice is critical- More advanced engineering strategies are in development- Will allow the design of T cells that carry out desired functions on command

Adoptive immunotherapy of cancer