CAR T-Cells:

Next Generation Hematology Practice

Dr (Surg Cdr) Gaurav Narula MD, DNB

Professor Pediatric Oncology
Convener, Pediatric Hemato- Lymphoid Group, Tata Memorial Hospital
Chair, InPOG Committee on Histiocytic Disorders
19th Aug 2018
Editor- in- Chief PediatricYHOP
Hematology Oncology Journal
2018, Lucknow 1
 Dr (Surg Cdr) Gaurav Narula, MD DNB
Date: 19th Aug 2018

Disclosures

No Relevant Financial Relationships with Commercial Interest

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 ALL: Improving Outcomes every Decade

Hunger SP, Mullighan CG. Acute Lymphoblastic Leukemia in Children. NEJM 2015;373:1541-52.

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 Rising Numbers
Data from PHL DMG at TMH. Unpublished Data: Accepted IJC

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 Social Interventions INR 19 Cr
USD 2.9 mill (approx)
OP Education Program
• Extensive social support Outings/ Birthdays Financial
Support
Home away
from Home
program in place:
• Medical Social Work Education/ Accommodation
Department Vocation

• Volunteers ICNs
• Non Governmental MSWs
Screening
Organizations Counsellors
• Counsellors Psychosocial
Infection
prevention

• Data Entry Operators support

• Dieticians
• Pharmacologists Save-a-Life Mid-day
• Infection Control Nurses Donation Drives
Transfusion
Nutrition
Meals
Dietiicans
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 Impact of Social Support Interventions
Unpublished Data: Accepted IJC

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 Acute Lymphoblastic Leukaemia (ALL) is the most common cancer of childhood worldwide with the
exception of Sub-Saharan Africa. It is estimated that over 10,000 cases of childhood ALL occur in
India every year (1). The lack of a national registry (1), health inequalities, and changing population

No Advances- Just Getting Smarter!

demographics (2) suggests that this is probably an underestimate.

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 ICiCLe Pilot: Improving ALL Outcomes
Data presented at SIOP, 13th Oct 2017

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 Chemotherapy for ALL

• Advances in Oh To Catch a Tiger by the Tail…

Chemotherapy Preceded
understanding Disease
Biology!
• But it was Successful
• Now we can’t let go
• Newer advances must
beat 80- 90% outcomes

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 The Power of Lymphocytes
& Immunotherapy

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 Background

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 Background
• So… Can we combine the Two?
• The Target and Assassin Coexist
• Biologically programmed to avoid each other

• Need to Arrange an Introduction!

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 BiTE®
Bi-specific T-cell Engager
VH

VL
-CD3
CD3 T Cell
antibody

Blinatumomab
BiTE®

Redirected
Target antigen
lysis
VH CD19
Tumor
Cell
VL
-CD19
antibody

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 CAR T-cells: The Big Idea!

• Approach
– Recombinant anti-CD19
chimeric receptor fusion
protein
– Transferred as a gene into
isolated patient T cells
– Resulting autologous anti-
CD19-reactive T cells
In Girl’s Last Hope, Altered
reintroduced
Immune Cells Beat Leukemia
– Dramatic tumor responses

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 CARs- Structure

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 CARs: Structure
• Extracellular domain
• scFv – recognises target antigen
• Intrcellular domains
• CD3-ζ – intracellular signalling
domain
• 4-1BB/CD28 – Costimulatory
domains
• Spacer
• Transmembrane domain

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 CARs: Generations

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 Insertion into T-Cell
• Replication incompetent
retroviral/ lentiviral viruses
• Encodes CAR transgene
• Used for transduction
• Achieves stable integration into
the T cells genome
• Leads to long term expression of
CAR on the T cell

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 CARs: “Live Drug”
• ScFv region engages target Ag
• Intracellular domain promotes
proliferation of CAR-T cell
• Triggers effector function of the
CAR-T cell
• Eliminates the target cell
expressing the specific Ag

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 CARs- Engage, Destroy, Proliferate

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 CAR T-Cell Manufacture

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 CAR T-Cell Manufacture

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 Process Validation in cGMP
Start Culture
TexMACS GMP + 200 IU/mL IL2 +
3% AB Serum

DAY 0 DAY 0
Fresh Apheresis Cryopreserved Apheresis Lentigen CD19/CD22 Vector Day 1
N=2 healthy donors N=2 Patient samples MSCV-CAR1922-WPRE Transduction

Remove Sample*: Day 5

counts, viability Culture Wash
CD4 CD8 Cell labeling / Removal of
1 vial CD4 + 1 vial CD8 beads
unbound beads
Day 6
Remove Sample : Feed 50mL fresh media
CD4 CD8 Cell separation
FACS, counts
Remove Sample*: Day 7
FACS, counts, viability Media Exchange 125mL
Anti-CD3/Anti-CD28
TransAct Stimulation
Remove Sample: Day 9**
FACS, counts Harvest
FN assays: mycoplasma,
endotoxin, sterility, gram Infuse Fresh or Cryopreserve
stain, RCL PCR based, RCL
culture based, identity,
TD efficiency **Option to extend culture until
day 11

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 Or… One Stop Solution
• Closed system, Integrated CliniMACS Prodigy®
cell processing device
• Disposable tubing set
• Allows for the complete
manufacturing process of
cell products
• Could run multiple devices
simultaneously in one room
• Minimizes the use of costly
GMP space

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 Early Success

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 When ALL Hope was Gone…
• 2 children with relapsed /refractory pre B ALL
• Treated with CTL019 CAR T Cells
• Patient 1- RR B ALL 2nd Relapse
• Patient 2- RR B ALL Post HSCT, and Post Blinatumomab
(BiTE)
• Dose - 1.4×106 to 1.2×107 CTL019 cells/ kg

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 On Road & Off Road Capabilities

• CTL019 T cells expanded to more than 1000 times initial engraftment level
• Cells were identified in Bone Marrow
• Also observed in CSF; Persisted ~ 6 months

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 Durable?

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 The Speed-breakers- CRS & Immune
Escape
• Grade 3 or 4 adverse events: 2nd Patient
• Cytokine- Release Syndrome CRS, B Cell Aplasia
Relapse 2 months later, CD 19
• B-cell aplasia developed in both patients Negative Blasts
• 1st Patient-
• Severe CRS, ICU, multiple pressors
• IL-6 elevated levels: and other cytokines
• Etanercept and Tocilizumab – The RA
connection!
• Remains ALL free 9 years later

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 Dramatic Responses
Pediatric ALL phase I/IIa study (N = 59):
Population
• 2nd or greater relapse or refractory
• 2/3 relapsed post SCT
Outcomes
• 55/59 (93%) in complete remission at 1 month
• 18 patients in remission ≥1 year, 13 without further therapy
• Median follow-up 12 months, range 1-43 months
• 20 relapses, 7 CD19(+) and 13 CD19(-)
• 6 patients proceeded to SCT, 1 to DLI
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 Cytokine Release Syndrome
Organ System Clinical Features
Constitutional Fever + - Rigors, malaise, Fatigue, Anorexia, Myalgias, Arthralgias,
Nausea, Vomiting, Headache
Skin Rash
Gastro-intestinal Nausea, Vomiting, Diarrhoea
Respiratory Tachypnea, Hypoxemia
Cardiovascular Tachycardia, wide pulse pressure, hypotension, increased cardiac
output (early sign), diminished cardia output (late sign)
Coagulation Elevated D-dimer, Hypofibrinogenemia, + - Bleeding
Renal Azotemia
Hepatic Transamnitis, Hyperbilirubinemia
Neurologic Headache, Mental status changes, Confusion, Delirium, Word
finding difficulty or frank aphasia, Hallucinations, Tremors,
Dysmetria, Altered Gait, Seizures

Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M, et al. Blood. 2014;124(2):188-95.
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 Grading of CRS
Grade Toxicity
I No life-threatening symptoms, and only requiring symptomatic treatment, for eg.
Fever, nausea, fatigue, headache, myalgia, malaise
II Symptoms require, and respond to, moderate intervention, with
Oxygen requirement <40%, or
Hypotension responsive to fluids or low dose of 1 vasopressor, or
Grade 2 organ toxicity
III Symptoms require, and respond to, agrresive intervention, with
Oxygen requirement >40%, or
Hypotension requiring high dose of, or multiple vasopressors, or
Grade 3 organ toxicity or Grade 4 transamnitis
IV Life-threatening symptoms
Requirement for Ventilator support, or
Grade 4 Organ toxicity (excluding transamnitis)
V Death

Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M, et al. Blood. 2014;124(2):188-95.
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 CRS Treatment Algorithm

Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M, et al. Blood. 2014;124(2):188-95.
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 CARs: Road Testing
(Completed or Near Completion)

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 CAR: Models in Trials
2018
2012 2013 2014 2015 2016 2017
(Projected)
12-C-0012 12-C-0012 12-C-0012 12-C-0012 12-C-0012
mCD19 CAR mCD19 CAR mCD19 CAR mCD19 CAR mCD19 CAR
14-C-0059 14-C-0059 14-C-0059
GD2 CAR GD2 CAR GD2 CAR
14-C-0168 14-C-0168 14-C-0168 14-C-0168
BCMA CAR BCMA CAR BCMA CAR BCMA CAR
15-C-0029 15-C-0029 15-C-0029 15-C-0029
CD22 CAR CD22 CAR CD22 CAR CD22 CAR
16-C-0054 16-C-0054
# o f a p p r o v e d p r o t o c o ls

10 ( p ro je c te d ) hCD19 CAR hCD19 CAR

17-C-0048 17-C-0048
8
CD30 CAR CD30 CAR
6
10-C-0054 10-C-0054
mCD19 Tscm CAR mCD19 Tscm CAR
4
18-C-0059
CD19CD22 CAR
2
TSLPR CAR
CD33 CAR
0
2012 2013 2014 2015 2016 2017 2018 BCMA hc CAR
CCR4 CAR
FGFR4 CAR
Mesothelin rCAR
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 CARs: Responses

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 CARs: Responses

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 USFDA Approval
• Novartis: 30th Aug 2017
• UPenn Group
• Kymriah® (tisagenlecleucel) (CTL019)
• Relapsed B-ALL, Relapsed/ Refractory B-Cell Lymphomas
• “Certified” Centers, Open Presently only in the USA
• USD 475,000 (Approx- INR 3.1 Cr, Does not Include Other Costs)
• Money-back Guarantee!
• Yescarta® (axicabtagene ciloleucel): Adult Relapsed B-NHL failed two
lines of therapy

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 The Indian Story
Are We There Yet?

• No…

• But Getting Close!

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 An Indian CAR?

The Tata-IIT-B
CAR Project

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 Design, Development and Clinical
Validation of CAR- T Cell Therapy in India
for Children with Relapsed/ Refractory B-
ALL Ineligible for Stem Cell Transplant

A Collaborative Project of Tata Memorial Center

Indian Institute of Technology- Bombay at Mumbai, India
and National Cancer Institute, Bethesda, USA
 Collaborators
Gaurav Narula, Hasmukh Jain, Minal Poojary, SD Banavali
Tata Memorial Centre, Mumbai, India

Rahul Purwar, Alka Dwivedi

Department of Biosciences and Bioengineering, Indian Institute of Technology-
Bombay, Mumbai, India

Terry J Fry, Nirali Shah, Dave Stroncek, Steven Highfill

National Cancer Institute, Bethesda, USA

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 Doing the Math

• Current projected 3-yr EFS of 71%

• Adds 100 cases of relapse/ refractory disease annually
• < 20/ 100: combination of financial support of matched donor
• < 10 would bear the extra finances of a Haplo-SCT
• < 5 would sustain 2nd remission for 1 year (current wait-list for an SCT)
• > 90% of Relapses at our Center do not undergo Transplant

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 Crystal Ball
• OS is set to fall by 5 years
• Patients relapsing now and subsequently will have
more events due to lack of transplant
• Gains from conventional strategies have been
maximized
• Further improvements will be incremental

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 The Big Question?
• We have the oldest and one of the largest SCT centers
in India
• Will even trebling our SCT capacity make a dent in
matching the need?
• SCTs are infrastructure, capital and trained manpower
intensive with significant Treatment Related Mortality
(TRM)

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 More Questions

• New Technology like CAR T-cells at affordable rates is

urgently needed
• Will it come to LMICs soon enough at an affordable
cost?
• Can it replace or reduce the burden of SCT?

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 Proposal
• Indigenously developed CAR- T cell technology will
ultimately be more affordable for our country

• A 2nd generation CAR T-cell construct with 4-1BB co-

stimulatory domain can cause 82% response rates

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 Proposal
• A 2nd Generation 4-1BB CAR has potential to replace
Stem Cell Transplant in a significant select population

• Use earlier in the treatment timeline

• First early relapse (within 36 months of diagnosis) or
• Refractory disease (Flow cytometry defined MRD below
0.01% after 3 cycles of intensive chemotherapy)

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 Early Steps
• Department of Biosciences & Bio-engineering, IIT- B
• Pursuing pre-clinical development of CAR T-cell technology
• Anti-GD2 and Anti-CD19 targets
• Lentiviral vectors
• Last 5 years
• Pediatric Hemato-lymphoid Group TMC Mumbai
• Resolution to pursue CAR-T cell therapy as a potential solution to
our burgeoning relapse issue
• Feb-2015

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 Preclinical: CAR-T cell technology
development

Production of CD19 gene

Design, synthesis and delivery vehicle Genetic modification of
cloning of CAR construct (CD19 lentiviral vector) autologous T cell

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 CAR-T development:
Design CAR construct
o CD19+ CAR construct
ScFv fragment

o scFv
o FMC63 mAb (Nicholson et al.1997)

o Costimulatory domain
o CD28 (Gene bank : NM_006139)/41BB

o Signalling domain FMC63 CD19 antibody clone

o CD3-zeta (Donor antibody)

Murine anti CD19-CD28 CAR VL VH CD28 CD3ζ

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 Generation of CD19+CAR T cells
Methodology
T cell isolation Transduction CAR-T cell Protein L staining
& activation expansion 37%
Anti-CD19-CAR surface
GFP+ expression
Lentiviral
vector
GFP reporter

% frequency of cells
a- CD19 CAR
CD19+scFv

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 Generation of CD19+ CAR T cells
Transduction efficiency in multiple donors
(T cells of healthy subjects)

% transduction efficiency

No. of experiments
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 B-Cell Killing
Cytotoxicity assay: “Autologous” B cells or Raji cells co-cultured with CD19+CAR-T cells and CD19+B cells
were quantified by flow cytometry
Killing of Autologous CD19+B cells Killing of CD19+ lymphoblast
Mock (Raji cells/ Burkitt’s lymphoma)
a- CD19 CAR
% Cytotoxicity

% Cytotoxicity
0.25:1 0.5:1 1:1 0.25:1 0.5:1 1:1

Effector: Target ratio Effector: Target ratio

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 Work in Progress

Pre-clinical ✔ Translational Clinical

CAR-T cell Technology Manufacturing & Clinical trial: CAR T

development Testing in the field (in cell therapy of B-
patients) (ex vivo) ALL patient
Reproducibility study
Preliminary translational work
(inter and intra user):
Clinical trial registration of India (CTRI) Number: Trial
Ongoing
REF/2016/11/012660
Collaborations
Patent filing:
- IDF submitted October
- Indian Institute of Technology-B, Mumbai
2017 for four products & - Tata Memorial Centre, Mumbai
two processes Project approved from IECs of TMH and IITs
- National Cancer Institute, Bethesda
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 Collaboration
Tata Memorial Hospital

Advanced Centre for Treatment Research

& Education in Cancer Clinical Trial

Mentoring &
Translational & Concept & Planning Knowledge
Cell Therapy Center Partner

Pre-clinical Development

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Indian Institute of Technology- Bombay
 Road Ahead…

Jan 2020- Jun 2022: Clinical Trials & Analysis

Jun 2019- Dec 2019: Regulatory Approvals

Jan 2019-Jun 2019: Matching preclinical development in

clinical grade CTC, and meeting regulatory benchmarks
Aug 2018-Dec 2018: Upgradation of preclinical Lab at IIT-
B and setting up CAR T-Cell Centre (CTCC) at TMC (ACTREC)

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 Thank You for Your
Attention

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