Beyond HER2 CAR T cells:

consider how far you have

fallen ……

Nabil Ahmed, MD
Associate Professor
Baylor College of Medicine
Houston, Texas
 Disclosure
Research agreement with Cellmedica … NKT cells
Consult for Adaptimmune … tgTCR T cells
ACEA Bioscience … pro bono
 Concepts
1. Empiric preclinical to clinical
development of HER2CAR T-cells
2. Rational development of novel CARs
1. Target Finding
2. Exodomain Design
3. Endodomain Fitting

3. Realistic in preclinical testing

4. Pragmatic pipeline of CAR
development
 Human Epidermal growth factor
Receptor 2
• Multiple malignancies
• Amplified: breast and ovarian
• Non-amplified: brain, melanoma & sarcomas

• Aggressive tumor cell behavior

• ↑ in stem cells of GBM & sarcoma
• Associated with ↓ outcome
• Protein ↓↓↓ postnatally

Ozaki et al. Neurosc Res ‘98; Zhang el al. Clin Can Res ‘07; Koka et al. AJCO ‘03
 The HER2 CAR
FRP5

Winfried Wels
V V
L H

FRP5
CD28
Steve Gottschalk

ζ
Herceptin

Wels et al. Biotech ’92; Ahmed et al. Can Res 2007 and Mol Ther 2009
 Autologous PDC’s
Patient 1 Patient 2 Patient 5

VL VH

CD28

HER2
SSC

60
CD133-PE
CAR
30 60

40 CD133 (+) x HER2 CAR T cells

% Cr release

20 40

CD133 (–) x HER2 CAR T cells

20 10 20
CD133 (+) x NT T cells
CD133 (–) x NT T cells
0 0 0
40:1 20:1 10:1 5:1 40:1 20:1 10:1 5:1 40:1 20:1 10:1 5:1
T cell : Tumor

Wels et al. Biotech ’92; Ahmed and Salsman et al. Clin Can Res 2009
 Autologous PDX’s
Day 6
BRAIN
OSTEO
LM7 LUNG MET

Kleinermann; Ahmed (unpublished)

 Vita Brawley
HER2 CAR T cells NT
T cells
day 6 day 9 day 15
MB/GBM/EPN
HER2.CD28.ζ T cells
OSTEO
SARC MET

Can Res 2007; Clin Can Res 2009; Mol Ther 2009
 Fatal Adverse Event
• Patient: 39 yr; F; colon cancer metastatic to the lungs
and liver
• Lymphodepletion: CTX and FLUD
• Autologous CAR T cells:
– CAR based on Trastuzumab (Herceptin®)
– CD28+4-1BB+CD3.ζ
– DOSE: 1x1010
• Adverse Event: severe dyspnea;
pulmonary edema; respiratory failure;
died day+5
• Cytokine storm
RAC webcast, 12.2009; Morgan et al. M ol Ther 2010
 A different risk profile

• DOSE: 1,000 fold less 1x108

• Trial design: mCRM escalation

• Exodomain: FRP5 FRP5

• CAR: CD28+CD3.ζ (no 41BB)

• No lympho-depletion Herceptin
 Specific Cytokine Release
4000 GM-CSF
IFN−γ
IL-2
3000
TNF-α
pg/mL

IL-10
2000
IL-4
IL-5
1000

HER2-CAR
HER2+ Normal HER-ve Normal

NT
Tumor tissue Tumor tissue

T cells
HER2-CAR T NT T cells
cells
 HER2 CAR Trials: evolution
2008 2009 2010 2011 2012 2013 2014 2015 2016

T cells HEROS
HER2 specific

CTL HERT-GBM
+ durable

CTL HER-TGFß
+ tumor resistant

CTL
+ stoppable …
 HER2

HEROS: complete CAR

T cells

• 43/55 (~80%) tumors HER2+

• Generated 28 cell lines; infused 19
• No SAE
• Clinically
2 PR; 4 SD (142->600+ days); 13 PD

DL6

PREINFUSION POSTINFUSION
Ahmed et al. JCO 2015
 HERT-GBM 17 patients treated
Progressive GBM
No SAE & No CRS
Attrition 40%
7 SD 2.5 to >30 mon
1 PR
Overall survival probability
1

8 PD 0.8

0.6
OS=24.8 mon
0.4

0.2 Age
<18
>=18
0
0 10 20 30 40 50 60
Months from diagnosis

HERT-GBM in review
T cells home but don’t expand

Ahmed et al. JCO 2015

 HEROS 2.0
Flu only (3 pts)
Flu + CTX (2 pts)
Lymphodepletion CART cell Expansion
100000
2000 8
F lu + 1 x 1 0 /m
2
100000 8
F lu + 1 x 1 0 /m
2 Flu/Cy + 1x108/m2
10000
10000

copies per mL
c o p ie s p e r m L

1500
1000
1000
ALC

1000
100
100

500 10
10

0 1 1
0 20 40 60 0 10 20 30 40 50 0 10 20 30 40 50
Days P o s t In fu s io n Days P o s t In fu s io n Days Post Infusion

HEROS 2.0 is currently recruiting

 HEROS 2.0
Toxicity:
• 3 patients: CRS grade 1 (fever + CRP 8.9)
• 1 patient: CRS grade 2 (fever + CRP 7.2)
– hypotension (low dose dopamine for ~ 36 hours)
– transient oxygen requirement
Responses (5 patients):
• 2 patient: PD
• 2 patients: SD 8-12 months
• 1 patient: CR

HEROS 2.0 is currently recruiting

 Why do tumors relapse?
NT
T cells
1.0 day 6 day 9 day 15
Autologous
HER2.CD28.ζ T cells (n=8)
MB/GBM

0.8
Probability
HER2.CD28.ζ T cells

0.6 1. Antigenic Heterogeneity

2. Limited T cell Persistence
OSTEO
Survival

0.4
3. Inadequate T cell Activation

p<0.001
4. Inhibition @ Tumor Site
0.2
LUNG MET

Tumor only (n=8)

NT T cells (n=8)
0.0
0 10 20 30 40 50 60
Survival Time

Can Res 2007; Clin Can Res 2009; Mol Ther 2009
 Meena Hegde
The Great Escape
Day 1 3 7 14
HER2

IL13Rα2
HER2 IL13Rα2
HER2 CAR

IL13Rα2 CAR

HER2 CAR T cell

Hegde et al. Mol Ther 2013; Bielamowicz et al. Frontiers in Oncology 2014
 GBM: intratumoral heterogeneity

~ 70%
IL13Rα2
~ 93%
~ 98%

20 GBM (surgical excision)

Flowcytometry on ≥100,000 cells
IFC on 5+ cuts; 6+ hpf; 200+ cells/hpf
Hegde et al. Mol Ther 2013
GBM: intertumoral heterogeneity

15 SAMPLES; MULTIPLE PIECES POOLED; 100,000-200,000 FLOW EVENTS

Bielamowicz
 Hypothesis

+ +

Possible advantages:
Circumvent tumor escape
- Heterogenous expression of target
- Down regulation of target
- Antigen loss variants
Improve T cell activation

Hegde et al. M ol Ther 2013

 Tumor Profile

Chris Man
ES MB OS
ROR1 ROR2 SFRP1 250 OS SETS
MUC1 PDPN ROR2
STEAP1 FCER2 PDPN
SFRP1 SFRP1 NCAM1
NCAM1 ROR1 MUC16
L1CAM NCAM1 L1CAM
IL11RA KDR KDR +
FOLH1 IL13RA2 IL13RA2
CLDN1 CLDN1 EPHA2
CD248 BCHE CSPG4
BCHE ALK CD248
ALK ADAM12 CA9
ADAM12 BCHE
ANTXR1
ADAM12

MINIMUM
UNIVERSAL
PROFILE
 Tending to 100%
Kevin B
Routine
Analysis P Value
H vs. H or I <.0001
H vs. H or E 0.0001
I vs. H or I 0.0001
100 I vs. I or E 0.0001
E vs. H or E 0.0001
E vs. I or E <.0001
H vs. H or I or E <.0001
75 I vs. H or I or E <.0001
E vs. H or I or E <.0001
P a tie n ts ( % )

H H or I vs. H or I or E 0.0001
H or E vs. H or I or E <.0001
I
50 I or E vs. H or I or E 0.0001
E

H or I

H or E
25
I or E

H or I or E

0
0 30 60 60 70 80 90 100
T u m o r c e lls in p a tie n ts (% o f to ta l)

Bielamowicz, Joseph and Ahmed

 5' UTR

UCAR T cells
Kevin B
AccI
10000

p 2000
HER2
U.CAR
00
Insert 4.6 kb

4000
IL13Rα2
6000

EphA2 Autologous GBM PDXs 1:20

Glioblastoma
Ependymoma
Medulloblastoma
Bielamowicz, Joseph and Ahmed
 Smart CARs
The Boolean Logic in Action

CAR T cell Multi-CAR Conditional Gated-CAR On/Off

T cell CAR T cell T cell CAR T cell
(iCAR) (GoCART)

OR AND NOT PRN

Navai and Ahmed. UK Biochemical Society Proceedings. 2016

Concept CARs
 in silico Design: the virtual world
Target 2

Zakaria Grada
Target 1

scFv1

Meena Hegde
CAR

T cell
Baker, National Center for Macromolecular Imaging
 in silico Design: the virtual world
Target 2

Zakaria Grada
Target 1

scFv2

scFv1

Meena Hegde
TanCAR

T cell
Baker, National Center for Macromolecular Imaging
 in silico Design: the virtual world
Target 2

Target 1

scFv2

scFv1

Matthew Baker
TanCAR

T cell
Baker, National Center for Macromolecular Imaging
 Malini Mukherjee
Imaging Toolbox: exodomain
Immune
CAR Exodomain Synapse
- Ligand Aggregates at IS
- CAR Aggregates at IS
- Synapse Properties
- F-Actin
- LFA-1
 bi valency

Malini Mukherjee
Phase Confocal

TanCAR
T cell

U373 TanCAR
TanCAR T cell
T cell
2.8µm

Confocal STED PLA FRET

<3000A° <1500A° <400A° <80A°

Mukherjee & Orange. Center for Human Immunobiology
Imaging Toolbox: endodomain
pLck

CD19.28ζ CAR GFP Imaris© Rendition

T cells

CAR Signaling
- pLck
- pZap70
CD45
CD4/CD8.
Imaging Toolbox: killing machinery
Target

Immunological
Synapse

Effector Effector

F-Actin, Perforin, Pericentrin

CD19.4bb.ζ CD19 CD28.ζ

Death Pathway
- Fas
Target Target
- Perforin CD19. CD19
Fas receptor Fas Ligand 4bb.ζ CD28.ζ
TanCAR behavior

Hegde et al. JCI 2016

Kristen Fousek
Endodomain“fitting”
60

U373-GBM
40

Truncated
1:20 20

0
40:1 20:1 10:1 5:1
NT
OX.BB.Z

BB.Z

OX.Z

BB.OX.Z

28.OX.Z

28.BB.Z

28.Z
Zeta

160 hr
... & now NON adherent cells
Daudi

NT (-ve)

Tumor Only (-ve)

CAR T cell
products

Triton
(+ve)

Time (Hours)
 of Surrealism & Realism
NT T cells
Pooled T cells

1.E+08 biCAR T cells

TanCAR T cells

1.E+07

**
photons/cm2/sec/sr

**
1.E+06

**
**

**
*
*
1.E+05

1.E+04
0 8 17 31 63 100
days

1:30 1:3

Hegde et al. JCI 2016

 Preclinical CAR Assembly Line

Function
Simulation

Time
Target(s)

Effort
Value
Look how far you have fallen THEN repent!
Revelation 2:5
1. Empiric preclinical to clinical
development of HER2CAR T-cells
2. Rational development of novel CARs
SILICO > VITRO > VIVO

3. Realistic in preclinical testing

4. Unresolved: modeling toxicity
 Acknowledgments
THE PATIENTS MD Anderson Cancer Center-UT (P Anderson,
E Kleinerman)
Center for Cell and Gene Therapy The Methodist Research Institute (Y. Kew, R.
Baylor College of Medicine Grossman, SZ Powell, D Baskin, J Zhang)
Children’s Mercy and PACT (Doug Meyers)
Stephen Gottschalk
Helen Heslop The National Cancer Institute (B St Croix)
Malcolm Brenner University of Florida (B Fletcher)
Georg Speyer Haus (J Koch; W Wels)
Baylor College of Medicine
Texas Children’s Hospital
The Methodist
 TEM8 in TNBC
Tiara Byrd

DFS probability
**

Tiara Byrd ….