Stem Cell transplantation

And Cellular Therapies

Koen van Besien, MD

Stem Cell Transplant Program ,WCMC
As faculty of Weill Cornell Medical College, we are committed to
providing transparency for any and all external relationships prior to
giving an academic presentation.
I do not have a financial interest in commercial products or services
related to the subject of this lecture.

-Koen van Besien, MD

 Learning Objectives
• Understand Function of Hematopoietic Stem Cells
• Understand procedures to collect stem cells from
bone marrow, blood or umbilical cord.
• Understand autologous stem cell transplantation.
• Understand allogeneic stem cell transplantation
(conditioning regimens, complications including
GVHD and its prophylaxis.)
• Understand Graft vs Tumor effects
• HLA-typing and Donor selection.
 Agenda
• Hematopoietic Stem Cells
• Collection of Stem Cells
• Autologous Transplantation
• Allogeneic Transplantation
– GVH and GVL
• Alternative Donors
• CAR T cells
Normal bone Marrow
 Cell Maturation
Stem cells and progenitors express CD34

self renew
differentiate

Mature Cells Do not express CD34

 Functions of Blood Cells
Blood cell Function
Erythrocyte Oxygen transport

Platelets Blood clotting

Neutrophil Host defense against

bacteria and fungi

Granulocytes Eosinophil Host defense against

parasites and allergies

Basophil/ Inflammation
mast cell and allergies
Leukocytes
Monocyte/ Phagocytosis, immune
macrophage regulation, tumor cytolysis
B lymphocyte Antibody defenses
(precursor of plasma cell)

T lymphocyte Cellular immunity

Why is this so important?
stemcells from a donor can be
transplanted into a parent
can sort homatopoiesis
 Agenda
• Hematopoietic Stem Cells
• Collection of Stem Cells
• Autologous Transplantation
• Allogeneic Transplantation
– GVH and GVL
• Alternative Donors
• CAR T cells
 Sources of Hematopoietic Stem Cells
• Bone marrow
– Requires donor to undergo “harvest” with general
anesthesia
• Peripheral blood use of medications that mobalise
onto the peripheral blood
the stem cells

– Stem cells can be “mobilized” into the peripheral blood

using hematopoietic growth factors (rh-GCSF) or CXCR
antagonists (Plerixafor)
– Collected using apheresis machine
unlike adult blood new born blood has hematopoietic stem
• Umbilical Cord cells the umbilical cord is a rich source of stem cells
– Cord “banks” have been established
– Techniques to “expand” cord blood stem cells, combine
cord blood stem cell units to increase availability
 Bone Marrow Harvest
• Donor receives general
anesthesia
• Done in O.R. under
sterile conditions
• Collections done
through repeated
aspirations
• Target CD34+ cell
number
 Stem Cell Mobilization into peripheral blood

Adhesive
interactions G-CSF Mobilization
between HSC
and matrix 2
components in activates neutrophils
to releaseenzymes
the BM that interfere with
if the interaction is Hsc interaction 80
disrupted the HSCs are they are released
releasedinto PB into the blood

the Hsc stays in the

bone marrow because Plerixafor
there are interactions k
between the Hsc
disruptsthe interaction
the micro environment betweencxcRHEi
SDF l leads to
the release of
HSC

Cathepsin G (CG), chemokine receptor-4 (CXCR4), hematopoietic stem cell (HSC), hyaluronic acid (HA), interleukin 8 (IL-8), kit ligand (KL), matrix
metalloproteinase-9 (MMP-9), neutrophil elastase (NE), stromal cell derived factor-1 (SDF-1), vascular cell adhesion molecule-1 (VCAM-1), very late
antigen-4 (VLA-4), P-selectin glycoprotein ligand-1 (PSGL). Source: Nervi B, et al. J Cell Biochem. 2006;99:690-705.
 Peripheral CD34+ Cells Phase III NHL Study

G-CSF + Plerixafor G-CSF+ Placebo

P Value*
(n = 130) (n = 124)
Pre-mobilization†
Mean
0
Median 0
Post-mobilization‡
Mean 53.5 ± 47.5 cells/µL 19.2 ± 23.7 cells/µL
< 0.001
Median 36.2 cells/µL 13.0 cells/µL

Note: Includes all randomized patients receiving any fraction of study treatment with CD34+ cell data on study day 4
and at least 1 of day 5 through day 8.
*Analysis of variance with terms fitted for treatment and study center.
† Pre-mobilization = after G-CSF treatment and prior to plerixafor or placebo.
‡ Post-mobilization = 10 to 11 hours after plerixafor or placebo treatment.

DiPersio JF, et al. J Clin Oncol. 2009;27:4767-4773.

Why measure CD34?
How do we collect these cells?
 Apheresis Machine
• Blood cells filtered through
special series of membranes
• Mononuclear cell
component is collected
• CD34+ stem cells are
present in this fraction
• Remaining elements of
blood returned to patient
after passage through
machine
 Leukapheresis procedure
from donan
• Blood cells filtered through
special series of membranes
• Mononuclear cell
component is collected
• CD34+ stem cells are
present in this fraction
• Remaining elements of
blood returned to patient
after passage through
machine
 Apheresis
• Stem cell collection
performed in outpatient
setting
• Each apheresis takes place
over 3-4 hours
• May require more than one
collection
• Collected stem cells are
counted, and then
cryopreserved under sterile
conditions
Patient with her stem cells
• Stem cells are infused
by vein, similar to a
blood transfusion
Patient getting cells
How do we use these cells?
 Agenda
• Hematopoietic Stem Cells
• Collection of Stem Cells
• Autologous Transplantation the patient reccenes their own
HSCs

• Allogeneic Transplantation
– GVH and GVL
• Alternative Donors
• CAR T cells
Autologous Transplantation
malignant stem cells collected
with Hsc and re infused
y into the patient
In vitro-purging
PBPC PBPC (post-purging
cryopreservation)

Apheresis a
Hsc collected

G-CSF + Conditioning
Plerixafor Chemo
us they release Hsc is the pan entrecieves tafferteffsemeaeadesment
PB requiredtherapy
us highdose chemo that from the patient
are gives back
can damage the Hscs
 What Happens During an Autologous
Transplant?
• Ablate patient’s bone marrow
– High dose chemotherapy
– Sometimes combined with radiotherapy
• Infusion of Stem Cells
• Blood product, antibiotic support until bone
marrow engraftment, blood count recovery
WBC and platelets after transplantation
Chemo PBSC

Afterinfusion blood cell

count drop later the
counts start to recover
 Indications for Autologous Stem Cell
Transplant
• Support after intensive chemotherapy:
– Lymphoma, Myeloma
• Autoimmune Diseases: Investigational
– Multiple Sclerosis
– Rheumatoid Arthritis
– Lupus
• Target for gene therapy
 High-Dose Chemotherapy With ABMT
Is Effective in Relapsed Chemosensitive NHL

100 Event-free survival 100 Overall survival

80 80
Transplantation
higher cure
rate with 9
Transplantation
Survival 60 µ dose chemo (n = 49) 60
Hsctransplant
(%) 40
40
Conventional treatment
20 (n = 54) 20 Conventional treatment
P = 0.001 P = 0.038
0 0
0 15 30 45 60 75 90 0 15 30 45 60 75 90

Months after randomization

• 215 patients treated with 2 cycles of DHAP (dexamethasone, cisplatin, cytarabine)

• 109 patients showed CR or PR and were randomized to
– Conventional treatment: 4 more cycles of DHAP (n = 54)
– High-dose treatment: BEAC (carmustine, etoposide, cytarabine, cyclophosphamide, and mesna) +
ABMT (n = 55)

Philip T, et al. N Engl J Med. 1995;333:1540–1545.

 Original Article: Brief Report
Gene Therapy in a Patient with Sickle Cell Disease
• A boy with hydroxyurea-refractory sickle cell anemia
underwent bone marrow transplantation with autologous
hematopoietic stem cells transduced by a lentivirus to
express an antisickling β-globin variant.
• No sickle cell crises occurred in the following 15 months.

the Hscs collected are genetically manipulated

that are re infused into the patient
Ribeil et al N Engl J Med
376:848-855, 2017
 Agenda
• Hematopoietic Stem Cells
• Collection of Stem Cells
• Autologous Transplantation
• Allogeneic Transplantation
– GVH and GVL
• Alternative Donors
• CAR T cells
 pgivenfor H days
G-CSF 6 mcg/kg BID

d1 d2 d3
cells are collected
from the donor
DONOR
d4

d5 pheresis
> 4 x10E6 CD34

Cryopreservation

Chemo + TBI
Infusion
HSC from donor is
given to the patient

RECIPIENT
 1960’s: Importance of HLA type in Animal Models
HLAsystem is involved HLA matching is ideal
in antigenrecognition for HSCtransplants

a
immunosupressents

Survival of Dogs Given 1000 RAD TBI and a Marrow

Infusion from a Littermate Matched or Mismatched for
Dog Leucocyte Antigens (Epstein 1968, Storb 1971)

Thomas: A History of Haemopoietic Cell Transplantation,

Brit J Haematol, 1999, 105, 330-339
 HLA molecules are involved in
Antigen Presentation
f z they presentantigens
on all nucleated on Apc to immune cells
cells

they are icky

polymorphic
2
so everyperson was
a unique HLA molecule

Class I single chain,expressed on most cell types à CD8 T cells

Class II double chain, expressed on APC à CD4 T cells
HLA antigens determine success of
transplant are located on Chrom 6

DP DQ DR BC A

Class I Antigens HLA A, B, C

Class II Antigens HLA DRαβ, DQαβ, DPαβ

These antigens are highly polymorphic.

In each person, there are two of each

 MHC is the major determinant of GVHD:
The MHC complex is located on Chromosome 6

A B C DR DP DQ

we get one 14nAgenefrom

each parent

44siblings will be
HLA identicle
 What Happens During an Allogeneic
Transplant?
• Ablate patient’s bone marrow
to eradicate leukemia the
– High dose chemotherapy I used
immune system in the recepiest
– Sometimes combined with radiotherapy a
try to prevent
• Immunosuppression required post transplant rejection

– Allows for engraftment prevent it frombeing rejected

– Prevention of graft vs host disease
• Infusion of Stem Cells
• Blood product, antibiotic support until bone
marrow engraftment, blood count recovery
• GVHD Prophylaxis
 Stem Cell Organ
Transplantation Transplantation
transplant HSC AND immune cells 014 transplantorgan

• Donor Immune system

– Immune reconstitution
– Graft vs Host Disease rejection of the
host by thegraft
• Host Immune system Ablated • Host Immune system
– Tolerance (no life-long immune – Graft Rejection soprevented by
suppression) immunosuppression

• Desired effects:
• Desired effects:
– Hematopoietic reconstitution
– Reconstitution of organ
– Graft vs Leukemia effects function
• Donors • Donors
– Living related – Cadaveric
– Living unrelated – Living related (limited)
– Cord Blood
• Eligibility: HLA • Eligibility: ABO
WBC and platelets after transplantation
Allo PBSC
 Infection Prophylaxis
• Neutropenia, lymphopenia >
severely immunosuppressed
patient
• Infections may be life-
threatening
• Broad spectrum antibiotics
given for:
– Antibacterial
– Antiviral
– Antifungal
– PCP
• Medications continued
Normal Chest X-ray
several months after blood
counts recover
WBC and platelets after transplantation
Allo PBSC Back-up PBSC
% donor engraftment 88% 65% 0%
Donor graft
has been
rejected
2
UO donor hematopoiesis
• Hematopoietic Stem Cells
• Collection of Stem Cells
• Autologous Transplantation
• Allogeneic Transplantation
– GVH and GVL
• Alternative Donors
Cell Maturation
Donor T cells that
are ten similar are
given to the recepiest
Graft vs Host Disease
BUI they are still reactive to
recepient's antigens 2
• Donor T cells with allo-reactivity against host
antigens
• Immunosuppressive medications essential
– Tacrolimus, cylcosporine
– Glucocorticoids: prednisone
– Monoclonal antibodies ( campath )
• Acute and chronic forms
– Can result in significant morbidity, mortality
• Development of “tolerance” over time
– Immunosuppression withdrawn over time
– Differs from solid organ transplantation
 Graft vs Host Disease
Attack
gut
skin
liver
Graft vs Host Disease
 Chronic

Bronchiolitis obliterans
Ocular sicca

Loss of bile ducts

Oral ulcers

Nail dystrophy Fasciitis

Infections
All Images Are Copyright Protected

Skin sclerosis Disability Skin ulcers

Quality of life
Endocrine Spectrum of
Metabolism manifestations
Deep sclerosis
Nutrition in chronic GVHD
Pain
The donor T cells
attack recepteints
body tissues AND
Graft vs Cancer Effect
their cancer

• Donor allo-reactive T-cells recognize Tumor

antigens or Tumor associated antigens
• Mediate anti-tumor effect
• Has been demonstrated in leukemia,
lymphoma, multiple myeloma
• Immune reactivity is major benefit of
allogeneic stem cell transplantation
 GVL in myeloma
result in lower relapse
rates

2transplant
ortransplantagain no cueneo
needed

myeloma
improves

no myeloma

the patient will suffer

some graft vs host disease
Verdonck et al, Blood, 90, 4206, 1997
• Hematopoietic Stem Cells
• Collection of Stem Cells
• Autologous Transplantation
• Allogeneic Transplantation
– GVH and GVL
• Alternative Donors
HLA antigens determine success of
transplant are located on Chrom 6

DP DQ DR BC A

Class I Antigens HLA A, B, C

Class II Antigens HLA DRαβ, DQαβ, DPαβ

These antigens are highly polymorphic.

In each person, there are two of each

 MHC is the major determinant of GVHD:
The MHC complex is located on Chromosome 6

A B C DR DP DQ
registries of unrelated
donors to try to find
HLAmatchingpairs
NMDP International
Donor Centers Cooperative Registries
Germany (3) Australia Ireland
Israel Austria Italy
Canada Japan
The
Netherlands Czech Republic (2)Singapore
England Spain
Norway France Switzerland
Sweden Germany Taiwan
These 2,000,000 additional
registries add donors
962,000 available by searching
donors these registries.
 Total Growth of the
Be The Match Registry® 2009

Cord Blood Units

Adult Donors

54
 8/8 Allele, Available-Match Rates in
the Adult Donor Registry

more
nemogeneas
G mostdiverse
HLA

NATIONAL MARROW DONOR PROGRAM® 55

Entrusted to operate the C.W. Bill Young Cell Transplantation Program, including the Be The Match Registry®
 No Caption Found

contain Hsc
HLA is very
adaptable 2
does Not need to
be HLA matched

Steinbrook, R. N Engl J Med 2004;351:2255-2257

 Acute Grade 2-4 GVHD

100

80
Probability, %

60
BM matched (n=116), 50%

40
CB MM (n=454), 40%
mismatched cord has lower incidence of
aVHDtween matched Bill
20
CB matched (n=35), 20%

0
0 20 40 60 80 100

Days
Eapen et al: Lancet. 2007 Jun 9;369(9577):1947-54.
 UCB Characteristics
• Contains HSC
• Causes Relatively Little GVHD
• Less Stringent HLA matching required
• “Appropriate” Unit is available for most
• Low Cell Dose f takes longer to secret producing cells
• Delayed Hematopoietic Recovery
• Increased Risk for early TRM
Haplo-Identical Donor Transplantation
• Parents share 50% HLA use a partially matched
family member

• Children share 50% HLA

• Siblings often (50%) share 50% HLA
• Increasingly are used as Donors
 MHC is the major determinant of GVHD:
The MHC complex is located on Chromosome 6

A B C DR DP DQ
 Overall Survival by transplantation period

Time (years)

Donor Source
MRD
MUD
MMUD
Haplo-identical
I
was lowest survival BIT improving EBMT, ASH 2017
outcomes
 Agenda
• Hematopoietic Stem Cells
• Collection of Stem Cells
• Autologous Transplantation
• Allogeneic Transplantation
– GVH and GVL
• Alternative Donors
• CAR-T cells
 Chimeric Antigen Receptor
Geneticallymodify Tcells
express chimericantigen receptors
recognises certain
antigens
antibody 9

in fmaEeeor

• Patient T-cells are transfected with a combined B cell-T cell receptor

• The extracellular portion is a B-cell receptor directed against a
Leukemia antigen (typically CD19, expressed in ALL and CLL
• The intracellular portion signals to the T-cell to react, attack, divide
Davila, Oncoimmunology, 1, 1577,2012
CD19 directed CAR-T cells in Adult ALL

lots of leukemia CAR T no leukemia

cell cells

Brentjens Sci Transl Med 20 March 2013:Vol. 5, p177ra38

 Overview of Tisagenlecleucel in the
Clinic
1 Leukapheresis: Patient’s white blood
cells are collected, cryopreserved and
shipped to the manufacturing facility1-3

2 T cell activation/transduction: T cells

are genetically transduced ex vivo with a
lentiviral vector encoding the anti-CD19
CAR1,3

3 Modified T cell expansion:

Tisagenlecleucel cells undergo ex vivo
expansion on magnetic, antibody-coated
beads1-3

4 Chemotherapy: The patient may

receive a preparative lymphodepleting
regimen before T-cell infusion1-3
Copyright © Novartis
Corporation
5 Modified T cell infusion:
Receipt-to-Return Time: Tisagenlecleucel cells are cryopreserved
Time from the receipt of leukapheresis material by the and then shipped back to the clinic and
manufacturing facility to the time the product is shipped back infused into the patient1-3
to the treatment site is approximately 22 days.
Actual time may vary.
1. Porter DL et al. N Engl J Med. 2011;365(8):725-733. 2. Porter DL et al.
J Cancer. 2011;2:331-332. 3. Kalos M et al. Sci Transl Med. 2011;3(95):95ra73.
65
 Duration of Response:
73.5% Relapse-free at 6 Months
100

80
Probabilty of Event Free (%)

60

40

20 Events, n = 8
6 month relapses-free survival 73.5% (95% CI, 52-87%)
0
0 1 2 3 4 5 6 7 8 9 10 11 12
Patients still at risk Time (months)
n = 43 36 25 18 16 13 9 9 5 2 2 1 0
Efficacy analysis set = All patients who received a tisagenlecleucel infusion at least 3 months prior to data-cut date.

• Median DOR (95% CI, NE-NE) and OS (95% CI, 7%-NE) were not reached
• Most patients achieving CR at month 3 have remained in CR
• No patients proceeded to transplant while in response

66
 FDA Approved
• axicabtagene ciloleucel
– relapsed or refractory large B-cell lymphoma after
two or more lines of systemic therapy
• Tisagenlecleucel
– patients up to 25 years old who have acute
lymphoblastic leukemia (ALL) that has relapsed or
is refractory
 Agenda
• Hematopoietic Stem Cells
• Collection of Stem Cells
• Autologous Transplantation
• Allogeneic Transplantation
– GVH and GVL
• Alternative Donors
• CAR-T cells
 Summary
• Hematopoietic Stem Cells can be collected from
Bone Marrow or more commonly from Peripheral
Blood after Mobilization.
• They can be infused intravenously to a recipient
• Autologous SCT Serves as rescue after very
intensive chemotherapy which improves cure
rates in lymphoma and multiple myeloma
• Autologous SCT may also serve as tool for gene
therapy
 Summary (2)
• Allogeneic Transplantation has additional
therapeutic properties by virtue of its associated
GVL effects
• Common Problems: GVH, immunocompromise,
occasionally graft rejection
• The ideal donor is HLA-identical (Sibling, matched
unrelated)
• Allo Tx is Treatment of Choice in Many patients
with Leukemia, SAA etc
 Summary (3)
• Mismatched Transplantation (e.g. UCB
transplant or haplo-identical transplant) is
rapidly improving.
• Adoptive Therapy with Genetically modified
cells (CAR T cells) is FDA approved for patients
with B-ALL and DLBCL.