Adoptive therapy of cancer with genetically modified T cells: CARs and Armored CARs

(1)

Adoptive therapy of cancer with

genetically modified T cells: CARs

and Armored CARs

The 9th_{Dutch Hematology Congress}

Arnhem The Netherlands January 21, 2015 Renier Brentjens MD PhD Associate Member Leukemia Service

Chief, Cellular Therapeutics Center Department of Medicine

(2)

Renier Brentjens MD PhD

•Stockholder: Juno Therapeutics (scientific co-founder)

•Royalties: Juno Therapeutics •Honoraria: none

•Reserch Funding: Juno Therapeutics •Consultant fees: Juno Therapeutics

•Discussion of off-label drug use: Tocilizumab

(3)

Generation of a tumor targeted chimeric

antigen receptor (CAR)

V_H VL 5’ LTR α-tumor scFv CD8 ζ chain 3’ LTR ψ SD SA α-TAA mAb TCR complex

CAR retroviral vector α-TAA scFv—CD8-ζ

(4)

TAA CAR VH VL 5’ LTR αTAA scFv CD8 ζ chain 3’ LTR ψ SD SA

Generation of TAA-targeted

T cells for treatment of Cancer

αTAA scFv CD8 CD3 ζ 1. Construct a chimeric antigen receptor (CAR) SFG-CAR 2. Subclone CAR gene into a retroviral vector (SFG) 3. Transduce and expand patient T cells ex vivo 4. Infuse transduced T cells to eradicate TAA+_{tumor cells}

TAA

Native TCR

(5)

• HLA-independent antigen recognition,

therefore universal application

• Active in both CD4

+

and CD8

+

T cells

• Target antigens include proteins,

carbohydrates and glycolipids

• Rapid generation of tumor specific T cells

• Minimal risk of autoimmunity or GvHD

• A living drug, single infusion

Advantages of CAR T cell

therapy

(6)

Expression of CD19 and other B

cell markers on B lineage cells

preB-ALL

B cell lymphomas

and leukemias myelomas

Stem Cell pro B _{pre B} _{immature B} _{mature B} _{plasma cell}

CD19

CD22 CD20

(7)

(8)

0% 20% 40% 60% 80% 100% 0 20 40 60 80 100 120 140 19z1 (n=10) 19-28z (n=16) Pz1 (n=8) 200

Days since tumor injection

Surv

iv

al

P<0.003

Brentjens et al Clin Cancer Res. 2007 Sep 15;13(18 Pt 1):5426-35

(9)

Clinical trials using CD19

targeted T cells in relapsed B cell

ALL

(10)

Eligibility and Treatment Schema

• Adult patients are eligible (>18 year old).

• Patients must have B-ALL refractory, relapsed, MRD+, or in CR1. Ph+, extramedullary disease, prior history of CNS

leukemia, and/or

relapsed after prior allo- stem cell transplant are all eligible.

(11)

Sci Transl Med. 2013 Mar 20;5(177):177ra38

Patient characteristics and treatment

outcomes

(12)

Rapid tumor elimination and recovery

of normal bone marrow after 19-28z

CAR T cell therapy

(13)

Maude et al N Engl J Med. 2014, 371:1507-17

UPenn studies of relapsed B-ALL

• 25 pediatric and 5 adult relapsed or

refractory B-ALL patients treated

• 19-4-1BBz CAR design

• 90% CR

• 6 month EFS 67%

• 6 month OSR 78%

(14)

Lee et al Lancet. Published online October 13, 2014

NCI studies of relapsed B-ALL

• 20 pediatric and young adult relapsed or

refractory B-ALL patients treated.

• 19-28z CAR design

• 70% CR (14/20)

• 60% MRD- CR

(15)

Adverse Events: Cytokine Release

Syndrome

• Cytokine release syndrome (CRS)

– fevers, hypotension, hypoxia, malaise

• Neurologic changes*

– confusion, speech disorders, obtundation, seizure-like activities

• There is a strong correlation between sCRS and pre-T cell disease burden

• 0/10 sCRS in MRD patients

(16)

Sci Transl Med. 2014 Feb 19;6(224):224ra25

C-Reactive Protein (CRP) as a surrogate

marker for sCRS

(17)

Sci Transl Med. 2014 Feb 19;6(224):224ra25

(18)

Clinical trials using CD19

targeted T cells in relapsed B cell

ALL

(19)

CD19-Targeted 19-28z CAR Modified

Autologous

T Cells Induce High Rates of CR and Durable

Responses in Adult Patients with

Relapsed or Refractory B-ALL

(Abstract #382)

Jae H. Park, Isabelle Riviere, Xiuyan Wang, Yvette Bernal, Sarah Yoo, Terrence Purdon, Elizabeth Halton, Hilda Quintanilla, Kevin Curran, Craig Sauter, Marco Davila, Michel Sadelain, and Renier J. Brentjens

(20)

Patient and disease characteristics

Patient Characteristics (n= 21) No. of Patients %

Age (years) Median Range

54 23-74 Baseline bone marrow cytogenetics

Unfavorable Ph+ Intermediate 11 6 10 52 29 48 Prior Allo-SCT Yes No 5 16 24 76

Refractory to Pre-CAR T cell salvage therapy Yes No 18 3 86 14

B-ALL tumor burden in BM before CAR T cell infusion (n=20)*

MRD- MRD+

Overt residual disease

2 8 10 10 40 50

(21)

Summary of Clinical

Outcomes

Number of Patients, N=27

Overall CR Rate

MRD Negative CR Rate

24/27 (89%)

21/24 (88%)

(22)

Post-CAR T Cell Follow Up

• Median follow-up: 6 months (1-38 months)

– Data cutoff date: 11/14/2014

• 12 patients remain disease-free

– 7 patients w/o subsequent HSCT

– 7 patients with > 1 year follow up

• 10 patients proceeded to allogeneic HSCT

• 9 patients relapsed during follow-up (3-8

months)

– 2 relapses post-HSCT (1 pt with CD19 negative

blasts)

(23)

Overall Survival

Median Survival: 8.5 months

Survival Rate at 6 mo.: 57% (95% CI, 40-79)

Median Survival: 10.8 vs. 8.5 months Survival Rate at 6 mo.: 68% vs. 67%

0 12 24 36 0

50 100

Months After CAR T Cell Infusion

O v e ra ll S u rv iv a l

Survival proportions: Survival of Allo-SCT post-CAR Responders

Allo-SCT Post-CAR No SCT Post-CAR Post-CAR Allo-SCT vs. No SCT (N=24) P=0.5204 0 12 24 36 0 50 100

Months After CAR T Cell Infusion

O v e ra ll S u rv iv a l

Survival Curve for All Patients_{All Patients}

(24)

CRS & Neurological Toxicities

Subgroups Severe CRS* Grade 3/4 Neurotoxicity Grade 5 Toxicity Overall 5 (18%) 7 (25%) 2 (7%)¶

Pre-T cell Disease Burden Morphologic disease (n=15) MRD (n=13) 5 (33%) 0 (0%) 6 (40%) 1 (8%)

*Requiring vasopressors and/or mechanical ventilation for hypoxia

¶1 patient with ventricular arrhythmia (DNR) and 1 patient had seizure, but unknown cause of death

• No GvHD exacerbation was observed in patients with prior allo-HSCT

• CRS managed with IL-6R inhibitor (3 pts), steroid (2 pts), IL-6R inhibitor+steroid (6 pts)

(25)

Refractory CRS in Recipients of CAR T

Cells

(Abstract #2296)

Noelle Frey, Bruce Levine, Simon Lacey, Stephan Grupp, Shannon

Maude, Stephen Schsuter, Pamela Shaw, Wei-Ting Hwang, Mariusz Wasik, Amrom Obstfeld, Mimi Leung, Angela Shen,

Solveig Ericson, Jan Melenhorst, Carl June, David Porter

(26)

Study Design & Cell Product

• Phase I study in R/R adult ALL

• Cell Product:

– α-CD19 scFv-4-1BBz-ζ, lentivirally transduced

• Cell Dose:

– 4.2x10

7

CAR

+

T cells – 1x10

9

CAR

+

T cells

(27)

Study Population

• 12 adult ALL patients

– No baseline disease status information provided

• Conditioning chemotherapy

– Clofarabine 30mg/m

2

x5 days

– HyperCVAD A/B

– Cyclophosphamide 300mg/m

2

q12h x 6 (3.6g/m

2

(28)

Summary of Clinical Outcome

• Response Rates:

– 8/9 evaluable pts achieved CR (89%) + 1 PR

• 2 proceed to alloHSCT in CR

• 2 relapses at 3 months (

1 CD19 neg

)

(29)

Summary of Clinical Outcome (2)

• Adverse Events:

– Grade 3 CRS (low dose pressors): 8/12 pts (67%)

• 5 required toci (4 pts x2) during days 3-10

• 1 required methylprednisone at day 4

– Grade 5 CRS (fatal): 3/12 pts (25%)

• Deaths occurred on days 5-16

• T cell dose: 6-8x106_{CAR T cells/kg}

• All with concomitant infections (influenza, pseudomonas, stenotrophomonas)

• Managed with Toci x2-3, etanercept x2, siltuximab x2, high dose steroid

(30)

Therapy of B Cell Malignancies with

CD19-Specific CAR-Modified T Cells of Defined

Subset Composition

(Abstract #384)

Cameron J. Turtle, Daniel Sommermeyer, Carolina Berger, Michael Hudecek, David Shank, Natalia Steevens, Tanya Budiarto, Mandana

Karimi, Colette Chaney, Anna Marie DeVito, Shelly Heimfeld, Michael Jensen, Stanley Riddell, and David Maloney

(31)

Rationale for T Cell Subset Infusion

• CAR modified effector T cells derived from

purified T cell subsets have different in vivo

persistence

• Combination of CAR modified CD8 T

_CM

+ CD4 T

cells provides optimal antitumor activity in their

tumor xenograft models

(32)

Study Design & Cell Product

• Phase I/II study in R/R ALL, CLL, NHL

• Cell Product:

– FMC63 α-CD19 scFv-4-1BB-CD3ζ with EGFRt, lentiviral – CD8 T_CM and CD4 T cells separated from a leukapheresis

product  cryopreserved  independent stimulation with CD3/CD28 beads  formulated 1:1 CD4:CD8 CAR T cells

• Cell Dose (3 dose cohorts):

– 2x105_{EGFRt cells/kg}

– 2x106_{EGFRt cells/kg}

(33)

Study Population

• 20 patients: 9 ALL, 10 NHL, 1 CLL

• 5/20 fail to meet the required cell dose

– 3 due to low lymphocyte counts

– 2 due to suboptimal cell proliferation in culture

• Conditioning chemotherapy

(34)

Summary of Clinical Outcome

• Response Rates:

– ALL: 5/7 CR (71%)

– NHL: 1/9 CR (11%), 5/9 PR (56%)

• Adverse Events:

– sCRS only observed in ALL patients with high tumor

burden

–

1 death in ALL due to sCRS

–

1 status epilepticus managed with high dose dex

– CRS appears usually at days 6-8

– Subset of NHL patients developed immune response

to murine sequence of scFv

(35)

CHOP/UPENN (abstract 380)

• Patients Treated (all trials) n = 130 • Pediatric B-ALL Trial (?Adult) n = 39 • CR = 92% (36/39)

• Relapse n= 10 (CD19-neg n=5) – 27% (10/36) – Relapse%

• Median f/u - 6m (Range 1.5-31 months) • Post CAR T cells

– HSCT n=3 – DLI n=1 – MDS n=1

• Treated post-HSCT n=27 (69% post HSCT) • No GVHD

• 2/3 of patients have CAR T cells at 6 months • EFS at 6months = 70%

(36)

Grupp et al N Engl J Med. 2013 Apr 18;368(16):1509-18

Relapsed disease with loss of CD19

expression: UPenn trial

(37)

NCI (abstract 381)

• Patients Treated n = 21 (B-ALL and NHL)

• Full dose achieved n=19 (90%) (one pt with ½ dose obtained CR) • Median Age = 13 yrs (Range 5-27 years)

• Median disease burden at time of treatment = 28% (bone marrow) • CR = 67%

• CR for B-ALL = 70% (60% MRD negative CR in paper) • LFS = 79%

• OS (10m follow up) = 52% • Conditioning

– Flu 25mg/m2 x 3 last dose day -2 – Cy 900mg/kg x 1 dose day -2

• CSF CAR Detection = 11/17 (65%) – CNS3 or isolated CNS relapsed ok • Post infusion - effector memory

• Endogenous Immune Response against CAR T cells

– 2 pt developed anti CAR post – 4 had anti-CAR pre HSCT

(38)

(39)

Clinical trials using CD19

targeted T cells in low grade B

cell malignancies (CLL)

(40)

Kalos et al Sci Trans Med 2011

UPenn clinical trial results

Patient Prior Chemotherapy Conditioning

Chemotherapy Response 1 Fludarabine, Rituximab, Alemtuzumab, R-CVP, Lenolidomide, PCR Bendamustine CR (3+ years) 2 Alemtuzumab Bendamustine/Rituximab PR (7 months) 3 Rituximab/Fludarabine, Rituximab/Bendamustine. Alemtuzumab Pentostatin/Cytoxan CR (3+ years)

(41)

Updated UPenn Trials in CLL

(ASH 2013)

• Abstract 4162

– CD19 CAR T cells treating relapsed/refractory CLL

• Utilizing a 4-1BBz CAR construct, 14 CLL patients treated

• 3/14 patients obtained CR (21%), 5/14 patients obtained PR (36%), 6/14 patients with no response (43%)

• 6/14 patients with persistent detectable CAR T cells (5-35 months) • No CR patients with reported relapsed disease

• No dose response reported

• Abstract 873

– Dose randomized dose optimization trial of CLL patients with either high or low dose CAR T cell infusions

• Utilizing a 4-1BBz CAR construct, 27 CLL patients treated

• Patients randomized to either low dose (5 x 107_{CAR T cells) or high dose (5}

x 108_{CAR T cells)}

• No dose response benefit seen in these treated patients • Overall response rate (CR + PR) was 40%

(42)

MSKCC clinical trial results: CLL

Cyclophosphamide

No

Cyclophosphamide

(43)

CAR questions

• What is the etiology of differential

responses by CAR T cell therapy to

relapsed B-ALL versus CLL?

• What is the role of bulky disease and CAR

T cell anti-tumor efficacy?

• The role of the hostile tumor

micro-environment and CAR T cell function

• How to build a better T cell?

• How can we extrapolate this technology to

other (solid) tumors?

(44)

The hostile tumor microenvironment

The tumor microenvironment contains multiple

inhibitory factors designed to potentially

suppress effector T cells.

– CD4+_CD25hi_FoxP3+_{regulatory T cells (Tregs)}

– MDSCs – TAMs

– Expression of inhibitory ligands by tumor (PD-L1) – Tumor secretion of T cell suppressive cytokines

(45)

19z1

+

Tregs abrogate anti-tumor

efficacy of 19z1

+

effector T cells

A * 0 20 40 60 80 100 0 10 20 30 40 50 100 Time (d) %

Survival 1928z Teff alone_{1928z Teff + 19z1 Treg}

19z1 Treg alone Pz1 Teff alone p < 0.001 (n=10) (n=10) (n=10) 0 20 40 60 80 100 0 10 20 30 40 50 100 Time (d) % Survival 1:16 Tregs 1:8 Tregs 1928z Teff alone 1:1 Tregs 1:4 Tregs Pz1 Teff alone (n=4) B (n=10) (n=10) (n=10) (n=10) p < 0.001 (n=10) p = 0.02

(46)

The solution?

(47)

Moving Forward: Armored CARs

Version 1.0

Version 2.0

(48)

Armored CAR T cells

version 1.0:

IL-12 secreting CAR

T cells

(49)

IL-12

• A heterodimeric cytokine secreted by activated APCs, neutrophils and macrophages.

• Induces Th1 CD4+_{T cell response enhancing IL-2 and}

IFN-γ secretion

• Enhances T cell clonal expansion and effector function in concert with TCR signaling (signal 1) and CD28

co-stimulation (signal 2), serving as a signal 3. • Avoids/reverses T cell anergy

• May overcome Treg mediated effector T cell inhibition • Recruits and activates NK cells

• Clinical trials in cancer using systemic IL-12 therapy has been limited by severe inflammatory side effects

(50)

(51)

(52)

(53)

IL-12 secreting CAR T cells

in vivo

(54)

IL-12 secreting CAR T cells are

(55)

Syngeneic EL4(hCD19) tumor model

mCD19-/-_hCD19 +/-mCD19-/-_hCD19 +/-IV injection EL4(hCD19) Harvest splenocytes IV injection Retroviral transduction with chimeric receptor Assess T cell homing to tumor Assess T cell eradication of tumor Assess T cell proliferation in vivo Assess long- term survival of T cells

Assess the efficacy of suicide vectors Assess memory T cell response to rechallenge with tumor 53%

Determine the side effects of therapy

(56)

Pe

rcent S

urvival

Days since tumor cell injection

Lymphodepletion enhances anti-tumor

efficacy of 19z1

+

T cells

100 ₁₀1 ₁₀2 ₁₀3 ₁₀4 100 101 102 103 104 1Élive cells FL1-H: FL1-Height F L 2 -H : h C D 1 9 PE 24.3 0.11 0.063 75.5 24.3% 100 101 102 103 104 100 101 102 103 104 6Élive cells FL1-H: mCD19 F L 2 -H : h C D 1 9 2.07 0.02 0.29 97.6 2.07% 0 20 40 60 80 100 0 10 20 30 40 50 60 Time (d) % S u rv iv a l Cytoxan + 19z1 No Cytoxan + 19z1 Cytoxan Pz1

(57)

Pre-cytoxan Post-cytoxan 0 2 4 6 8 10 F o xP 3 + c e lls i n p e ri p h e ra l b lo o d ( % ) * A B

Cyclophosphamide lymphodepletion reduces

Tregs and induces IL-12 and IFNγ secretion

Pegram et al Blood 2012 0 20 40 60 80 100 120 140 0 1 2 5 7 9

Time post-cytoxan (days)

se ru m c o n ce n tr a ti o n ( pg/ m l) IL-12 IFNγ

(58)

19z1IRESIL-12 modified T cells secrete biologically active IL-12 and exhibit enhanced targeted cytotoxic function and resistance

to Tregs 0 10 20 30 40 19mzIRESIL-12 19mz E:T ratio S p e c if ic L y s is ( % ) 1:1 2.5:1 5:1 10:1 * * * * A B C _D Pegram et al Blood 2012

(59)

A B

Syngeneic IL-12 secreting CD19 targeted

T cells induce B cell aplasias and tumor

eradication

(60)

Complete eradication of ID8(MUC-CD) ovarian tumors in mice with MUC16 targeted T cells expressing IL-12

ID8(MUC-CD) lysis by CAR+ T cells

0 20 40 60 80 100 20 10 5 2.5 E : T Ratio Ly s is (% ) 19m28z 19m28zIresIL12 4H11m28z 4H11m28zIresIL12 A B C D 0 20 40 60 80 100 120 140 T N F -a ( p g /m l) ID8(MUC-CD) 19m28mz 19m28mzIresIL12 4H11delIresIL12 4H11mz 4H11m28mz 4H11m28mzIresIL12 0 10000 20000 30000 40000 50000 60000 70000 IF N -g ( p g /m l) ID8(MUC-CD) 19m28mz 19m28mzIresIL12 4H11delIresIL12 4H11mz 4H11m28mz 4H11m28mzIresIL12

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Enhanced CM phenotype, enhanced cytotoxicity, enhanced persistence Resistance to Treg and TGFβ inhibition NK cell Recruitment and activation IL-12 secretion IL-12 secretion Targeted tumor cytotoxicity Targeted tumor cytotoxicity Targeted tumor cytotoxicity Tumor cell NK cell Activated TIL Anergic TIL IL-12 secretion Reversal of anergy CAR-IRES IL-12

IL-12 genetically modified T cells:

Armored CAR T cells

(62)

Armored CAR T cells

version 2.0:

Constitutively

expressing CD40L

CAR T cells

(63)

Armored CARs v2.0: CD40L (CD154)

• Type II transmembrane protein (TNF gene

superfamily)

– Trimer (most active form)

• Expression on activated T cells

– Inflammation/Infection/Injury

• Rapid upregulation (peak 6 hours)

• Cleavage (sCD40L)

(64)

Ad-CD154 CLL vaccine Trial

1. Eleven Patients (n =11) 2. CLL cell phenotype

+Ad-154 CLL/Bystander

3. Cytokine Profile

IL-6, IL-12, IFN-γ

4. Increase T cell# 5. Decrease ALC and

lymph node size

Wierda W; et al. Blood 2000

Messmer D; Kipps T. Ann. N.Y. Acad. Sci. 2005

(65)

IL-12 CD40L+ CAR+_{T cell} DC T_reg Endogenous Effector T cell CD19+_Tumor NK cell Secreted CD40L 1 2 3 4 5 6

Proposed mechanism of CD40L immune stimulation within the tumor microenvironment. (1) CAR/CD40L modified T cells activate tumor cells directly to upregulate CD80/86, CD54, CD95, and MHC, while (2) inducing auto or trans-costimulation of the modified T cells. (3) Further CD40L and sCD40L may modify tumor and recruit/activate endogneous NK cells. (4) CD40L induces maturation of DCs which in turn have increased APC function to stimulate endogenous T cells, as well (5) release IL-12 which (6) inhibits Tregs and reverses TIL anergy, further enhancing the anti-tumor immune response.

CD40L genetically modified T cells:

Armored CAR T cells v2.0

(66)

CD40L+ Armored CAR T cells v2.0: Enhanced

proliferation and cytokine secretion

(67)

CD40L+ Armored CAR T cells v2.0:

Enhanced immunogenicity of tumor cells

CD40+ DoHH2

(68)

CD40L+ Armored CAR T cells v2.0: CD40L+

T cells modify autologous CLL tumor cells

(69)

CD40L+ Armored CAR T cells v2.0: CD40L+

T cells mature autologous DCs

(70)

CD40L+ Armored CD19 targeted CAR T

cells v2.0: Enhanced anti-tumor efficacy

(71)

CD40L+ Armored CD19 targeted CAR T cells

v2.0: Enhanced

in vivo

anti-tumor efficacy

(72)

Conclusions

• Autologous CD19 targeted CAR modified T cells have demonstrated very promising anti-tumor efficacy in B cell ALL with more modest responses in patients with low grade B cell malignancies.

• Etiologies of CAR T cell resistance may be related to the hostile tumor microenvironment.

• Application of CAR T cell therapy for low grade B cell malignancies as well as moving forward towards application to solid tumor malignancies requires “armored” CAR T cells designed to both overcome the hostile tumor microenvironment and exhibit enhanced anti-tumor efficacy and long term persistence.

• Variations of “armored CAR” T cells appear to have enhanced anti-tumor efficacy based on pre-clinical anti-tumor models.

• Future studies using “armored” CAR T cell technology will focus on translation of these armored CAR T cells to the clinical setting both in the context hematological as well as solid tumor malignancies.

(73)

Renier Brentjens Hollie Pegram Mythili Koneru Swarish Rafiq Swati Pendeharkar James Lee Yan Nikhamin Jae Park Kevin Curran Peter Chang Michel Sadelain Marco Davila Michael Gong

Jean Baptiste Latouche

Leukemia Service David Scheinberg Jae Park Mark Frattini Peter Maslak Mark Heaney Joe Jurcic Nicole Lamanna Marco Davila Dan Douer

Cell Therapy and Cell Engineering Facility

(Isabelle Riviere, Director)

R&D, Manufacturing Xiuyan Wang (Dan Hollyman) Jolanta Stefanski Malgorzata Olszewska Oriana Borquez-Ojeda Clare Taylor Teresa Wasielewska Jinrong Qu QA/QC Shirley Bartido (Mark Przybylowski) James Hosey Domenick Pirraglia Vanessa Capacio Clinical Research Yvette Bernal Funding

CA59350 (MS) ; P30 CA-008748 (CT); 3RO1CA138738-02S1(RJB); Alliance for Cancer Gene Therapy ; Terry Fox Run for Cancer Research; William H. Goodwin and Alice Goodwin, and the Commonwealth Cancer Foundation for Research and the ETC of MSKCC; Damon Runyon Clinical Investigator Award (RJB); William Lawrence & Blanche Hughes Foundation (RJB); CLL-Global Research Foundation (RJB)

Lymphoma Service Craig Moskowitz Ariela Noy GYN service Samith Sandadi Stephen Lee Roisin O’Clearbhail Adult BMT Service Sergio Geralt Craig Sauter Department of Clinical Laboratories Lillian Reich David Wuest Kathy Smith Biostatistics Glenn Heller

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