cll cell dynamics · 2019. 12. 12. · c calissano et al. mol med 7: 1374, 2011 . cxcr4dimcd5bright...
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CLL cell dynamics
Nicholas Chiorazzi
The Feinstein Institute for Medical Research Northwell Health Manhasset, NY
Feinstein Institute
Bradley Messmer Carlo Calissano Joy Yan Florencia Palacios Shih-Shih Chen Rajendra Damle Davide Bagnara Piers Patten
Northwell Health
Kanti R. Rai
Jacqueline Barrientos Steven L. Allen Jonathan E. Kolitz
UC Berkeley Marc Hellerstein
MD Anderson Cancer Center Jan Burger
NIH NHLBI Adrian Wiestner
UC San Francisco Elizabeth Murphy
Scripps Institute, Florida Christoph Rader
Outline of Presentation
1. Overview of published information gleaned from patients who drank deuterated “heavy” water (2H2O) 2. Explanation of why we should be interested and concerned with the proliferative fraction of a CLL clone 3. Methods to preferentially target the proliferative fraction of a CLL clone
Hydrogen Deuterium
2H2O
Gas chromatography/Mass spectrometry
CLL cells
DNA
DNA
In vivo “pulse-chase” study
Methodology of deuterated “heavy” water use
What can these studies tell us?
Birth and death/elimination rates of CLL clones
Means to indirectly identify cells that have most recently been born/divided in patients
CLL cells proliferate faster than originally appreciated ~0.1% - ~2% of the clone divides daily
Higher birth rates correlated with disease activity, and therefore appear to be a key factor in disease outcome Calculated in vivo deaths rates are often comparable or only slightly unbalanced. Thus it is the rate of growth – not necessarily the absolute lymphocyte change – that is the crucial variable in clinical course.
Initial in vivo findings
Messmer et al. J Clin Invest 115: 755, 2005 van Gent et al. Cancer Res 68: 10137, 2008 deFoiche et al. Br J Haematol: 143: 240, 2008
Faster birth rates correlate with markers predicting worse clinical outcomes
EJ Murphy et al. Leukemia 31, 1348, 2017
Kaplan-Meier curve of treatment-free survival stratified by IGHV mutation status and CLL-cell birth rate
EJ Murphy et al. Leukemia 31, 1348, 2017
What can these studies tell us?
Birth and death/elimination rates of CLL clones
Means to indirectly identify cells that have most recently been born/divided in patients
Intraclonal cell fractions containing more cells with 2H- labeled DNA are enriched for the most recently replicated/born cells
Hypotheses used to define the membrane phenotype of the “proliferative fraction (PF)”
1. Cells that were stimulated to divide will express “activation markers” on their cell surfaces. CD5 is an activation antigen on human B lymphocytes
2. Cells from the blood that have recently left a solid lymphoid niche will have a chemokine display that supports emigration. Low CXCR4 levels are on recent tissue emigrants
3. Therefore, the fraction of circulating CLL cells with a CD5Bright and a CXCR4Dim phenotype will be enriched in recently-divided and recently-emigrated cells.
CXCR4DimCD5Bright faction is most enriched in 2H-DNA labeled cells
10 - 45%
1 – 10%
0%
Calculated percentage of cells with 2H-DNA
C Calissano et al. Mol Med 7: 1374, 2011
CD5
CX
CR
4 CXCR4dimCD5brightCD19+ : Proliferative, PF
CXCR4brightCD5dimCD19+ : Resting, RF
CXCR4intCD5intCD19+ : Intermediate, IF
* * 0123456789
101112131415
1st
time point 2nd
time point
CX
CR
4d
imC
D5
br/C
XC
R4
brC
D5
dim *
*
* *
CXCR4DimCD5Bright fraction contains the majority of recently divided cells in CLL clones
2H-DNA Ki-67 + cells MCM6+ cells
C Calissano et al. Mol Med 7: 1374, 2011
CXCR4DimCD5Bright defines the proliferative fraction in blood, lymph node, and bone marrow
TM Herndon et al. Leukemia 31, 1340, 2017
Fraction of newly-born CLL cells is highest in the lymph node
TM Herndon et al. Leukemia 31, 1340, 2017
Stromal cell Nurse-like cell
SDF-1 (CXCL12)
Solid Tissue
Blood
Blo
od
BCR signaling TLR signaling
CD5
CLL
CXCR4
CD38
BCR
CXCR4
CD5 CD38
BCR CLL
CXCR4
CD5 CD38
BCR
CLL
CXCR4
CD5
CD38
BCR
CLL
CXCR4
CD5
CD38
BCR
CLL CXCR4
CD38
BCR
CLL
CXCR4
CD38
BCR
Death
Life
Proliferative fraction
Life cycle of a CLL cell
Resting, re-entry fraction
CD5
CLL
Intermediate Fraction
CD5 Survive and re-initiate or reside resting
CLL
IL-4
Release
Exit
Important concepts and questions:
The CXCR4DimCD5Bright “Proliferative Fraction” represents, also exclusively, post-mitotic/recently divided cells – NOT dividing cells This fraction resembles and differs in some respects from the cycling fraction in lymph nodes, e.g., not a dominant BCR-related gene expression pattern but more of a trafficking, post-replicative fraction Therefore, more work is needed to define more precisely in “Cycling Fraction” by isolating and characterizing the CXCR4DimCD5Bright fraction of lymph nodes
The fraction of dividing cells is ~ 0.1 – 4.0% of the clonal load per day
Why should we be concerned with
such a small fraction of the clone?
Stromal cell Nurse-like cell
SDF-1 (CXCL12)
Solid Tissue
Blood
Blo
od
BCR signaling TLR signaling
CD5
CLL
CXCR4
CD38
BCR
CXCR4
CD5 CD38
BCR CLL
CXCR4
CD5 CD38
BCR
CLL
CXCR4
CD5
CD38
BCR
CLL
CXCR4
CD5
CD38
BCR
CLL CXCR4
CD38
BCR
CLL
CXCR4
CD38
BCR
Death
Life
Proliferative fraction
Life cycle of a CLL cell
Resting, re-entry fraction
CD5
CLL
Intermediate Fraction
CD5 Survive and re-initiate or reside resting
Disease progression
Disease perpetuation
Immune evasion
CLL
IL-4
Release
Exit
Life cycle of a CLL cell
Cells in the Proliferative Fraction (PF) lead to disease progression because of the ability to:
interact with and activate T cells
cause a Th2 polarization bias, resulting in not only survival signals but also in dampening of anti-tumor cytolytic responses
produce activation-induced cytidine deaminase (AID) and reactive oxygen species (ROS) that can each cause DNA mutations and deletions and thereby lead to new mutations throughout the genome
Life cycle of a CLL cell
Cells in the Proliferative Fraction lead to disease progression because of the ability to:
interact with and activate T cells
27 Clones
M-CLL: 13 U-CLL: 14
Acquire microarray expression data
of fractions using
Illumina BeadChips
HumanWG6 and HT12
RNA isolation
Gene expression comparisons of the Proliferative (PF) vs. Resting (RF) fractions
Sort PROLIF, INT, REST fractions
Gene Set Enrichment Analysis indicates that
the PF most resembles myeloid cells and activated B cells
PF displays active B-cell and myeloid cell-signatures and may play a role as antigen-presenting cells in vivo
Flow cytometry confirms that the PF displays a myeloid cell phenotype
CLECL1
CLECL1
CD1d CD11c
CD1d CD1c
CD1c
CD11c
CD
5+C
D1
9+
PF
and
RF
PF is the most effective intraclonal fraction for antigen presentation: in vitro evidence
1. Allo-mixed lymphocyte reaction
PF is the most effective intraclonal fraction for antigen presentation: in vivo evidence
2. Xenografting CLL B and T cells into alymphoid mice
Growth of CLL cells in alymphoid NSG mice is T-cell dependent
Bagnara et al. Blood 117: 5463-5472, 2011
1. Isolate CD3+ cells from CLL PBMCs
6. Euthanize at various
time points
4. Inject CLL B and activated T
cells iv or ip
3. Expand 3-14 days in vitro
2. Activate with α-CD3/CD28 beads + IL-2
5. Bleed serially
Euthanasia
Formalin fixation and
paraffin embedding
Immuno- fluorescence microscopy
Light microscopy
Single cell suspension
mC
D4
5
hCD45
CD
5
CD19 CD5+CD19+
CFSE
CD
8
CD4
T-cells
A.
B.
Growth of primary CLL B and T cells in alymphoid (NSG) mice
PF stimulates resting T cells to divide and grow in alymphoid mice
CD20
CD3
T cells alone T + PF T + IF T + RF
CD19
C
D5
Life cycle of a CLL cell
Cells in the Proliferative Fraction lead to disease progression because of:
T-B interaction leads to a Th2 polarization bias, resulting not only in survival signals but also in dampening of anti-tumor cytolytic responses
The CXCR4DimCD5Bright proliferative fraction preferentially induces IL-4 production by naïve T cells
Life cycle of a CLL cell
Cells in the Proliferative Fraction lead to disease progression because of:
production of activation-induced cytidine deaminase (AID) and reactive oxygen species (ROS) that can cause mutations and DNA deletion and thereby lead to new mutations throughout the genome
AID mRNA is most expressed in the CXCR4DimCD5Bright proliferative fraction
P Patten et al. Blood 120: 4802-4811, 2012
0.0003 * 0.0177
-100
0
100
200
300
400
500 A
I D e
x p
r e s
s i o
n ( M
F I ) 0.02
0
100
200
300
400
A I D
e x
p r e
s s
i o n
( M
F I )
* 0.02
**0.0031
AID protein is most expressed in the CXCR4DimCD5Bright proliferative fraction
F Palacios et al. unpublished
Reactive oxygen species are most abundant in the CXCR4DimCD5Bright proliferative fraction
E Sturgill et al. unpublished
Reactive oxygen species are most abundant in the CXCR4DimCD5Bright proliferative fraction
E Sturgill et al. unpublished
RF IF
PF
0
1
2
3
4
6
8
10
12
ge
oM
FI R
ela
tiv
eto
RF
CELLROX
***
****
RF IF P
F
0
1
2
3
4
6
8
10
12
ge
oM
FI R
ela
tiv
eto
RF
CELLROX
***
****
1. Will elimination of the CXCR4DimCD5Bright Proliferative Fraction in patients impact on clonal evolution?
Unanswered questions
What are the effects of current and novel therapies on the CXCR4DimCD5Bright Proliferative Fraction?
CD5
CX
CR
4
Ibrutinib preferentially eliminates the CXCR4DimCD5Bright Proliferative Fraction
Pre-Ibr Post-Ibr Merge
Initiation of therapy
Ibrutinib rapidly prevents new cell growth as evidenced by the stability of the fraction
of 2H-DNA-labeled cells in the blood
Cease 2H2O drinking
Ibrutinib inhibits AID expression
t0
1-3m
onth
3 m
onth t0
1-3m
onth
3 m
onth
0
100
200
300
400
500
AID
ex
pre
ss
ion
(M
FI)
U-CLL M-CLL
t0
1-3m
onth
3 m
onth
0
100
200
300
400
500A
ID e
xp
res
sio
n (M
FI)
Novel approach to targeting the Proliferative Fraction
C L L P :R M -C L L P :R U -C L L P :R
0
1
2
3
4
<0.0001 <0.00010.00010.0001
PF
:RF
1 .5
The CD52 gene is over-expressed in the proliferative vs. resting fraction
*
A
B * P < 0.01
§ P < 0.05
CD52 surface membrane levels are greater between proliferative and resting fractions
Anti-CD52: Alemtuzumab/Campath
Highly effective at eliminating CLL B cells, even in the setting of disease relapse or therapeutic unresponsiveness
Also eliminates other, non-leukemic cells expressing CD52 necessary for immune function, leading to severe immune deficiency and infections
Removed from the CLL market because of these life-threatening side effects
Could the action and effectiveness of Alemtuzumab/Campath be persevered and its broad reactivity restricted to provide an efficient and safe therapy?
Could an effective and safe form of Alemtuzumab/Campath be used to eliminate those cells responsible for clonal evolution or disease relapse?
Question and approach
Could one engineer a therapeutic antibody with dual specificities for CD52 and for a B-cell restricted epitope (e.g., CD20) that would bind to and eliminate selectively B cells but not normal T cells and myeloid cells?
Would the B-cell reactivity be preferential for those cells responsible for clonal evolution and expanding during disease relapse?
nr r
70
50
30
190
115
80
kDa
VL VL
VH VH
CH
2
CH
3
CH
2
CH
3
atz rtx
A. B.
21.0
22.0
23.0
24.0
25.0 mAU
0.0 4.0 8.0 12.0 16.0 mL
12.59
IgG1
CD52 (Alemtuzumab/Campath®) x CD20 (Rituximab/Rituxan®)
scFv-Fc IgG1-like “knob-in-hole” bispecific antibody
atz x rtx biAb
J Qi et al. Methods 2018 in press
VL VL
VH VH
CH
2
CH
3
CH
2
CH
3
atz rtx
His HA
A
VL VL
VH VH
CH
2
CH
3
CH
2
CH
3
atz atz
His His
VL VL
VH VH
CH
2
CH
3
CH
2
CH
3
rtx rtx
His His
VL VL
VH VH
CH
2
CH
3
CH
2
CH
3
rtx rtx
HA HA
VL VL
VH VH
CH
2
CH
3
CH
2
CH
3
atz atz
HA HA
B
CD20
BSA
0.0
0.5
1.0
1.5 atz-His
rtx-His
atz-His x rtx-HA
Ab
so
rban
ce a
t 405 n
m
*** **
*** C
CD52
BSA
0.0
0.5
1.0
1.5 atz-HA
rtx-HA
atz-His x rtx-HA
Ab
so
rban
ce a
t 405 n
m
*** ***
* D
Binding specificities of (atz x rtx), (atz x atz), (rtx x rtx) IgG1-like bispecific antibodies (ELISA)
J Qi et al. Methods 2018 in press
Jurkat
(CD52+/CD20-)
MEC-1
(CD52+/CD20+)
atz x atz scFv-Fc rtx x rtx scFv-Fc atz x rtx biAb A
B
Fluorescence
Eve
nts
128
0
101 102 103 104
128
0
101 102 103 104
128
0
101 102 103 104
128
0
101 102 103 104
128
0
101 102 103 104
128
0
101 102 103 104
Binding specificities of (atz x rtx), (atz x atz), (rtx x rtx) IgG1-like bispecific antibodies (flow cytometry)
J Qi et al. Methods 2018 in press
C
0 . 1 1 1 0 1 0 00
1 0
2 0
3 0
4 0
5 0
C o n c e n t r a t io n ( g / m L )
CD
C (%
)
0.1 1 10 1000
10
20
30
40
50 atz scFv-Fc
rtx scFv-Fc
atz x rtx biAb
Concentration (g/mL)C
DC
(%
)
D
K562/
CD20
(CD52+
CD20+)
B rtx scFv-Fc atz scFv-Fc
K562
(CD52+
CD20-)
128
0
101 102 103 104
128
0
101 102 103 104
128
0
101 102 103 104
128
0 101 102 103 104
Effector function – CDC - of (atz x rtx), (atz x atz), (rtx x rtx) IgG1-like bispecific antibodies
0 . 1 1 1 0 1 0 00
2 0
4 0
6 0
8 0
C o n c e n t r a t io n ( g / m L )
CD
C (%
)
A
atz scFv-Fc
rtx scFv-Fc
atz x rtx scFv-Fc
atz scFv-Fc
rtx scFv-Fc
atz x rtx scFv-Fc
J Qi et al. Methods 2018 in press
K56
2/CD20
K56
2
0
5
10
15atz scFv-Fc
rtx scFv-Fc
atz x rtx biAb
AD
CC
(%
)
0
5
10
15
20
25
AD
CC
(%
)
A B
**
** **
**
Effector function - ADCC - of (atz x rtx), (atz x atz), (rtx x rtx) IgG1-like bispecific antibodies
MEC1
(CD52+CD20+)
J Qi et al. Methods 2018 in press
1. Isolate CD3+ cells from CLL PBMCs
6. Euthanize at various
time points
4. Inject CLL B and activated T
cells iv or ip
3. Expand 3-14 days in vitro
2. Activate with α-CD3/CD28 beads + IL-2
5. Bleed serially
Euthanasia
Formalin fixation and
paraffin embedding
Immuno- fluorescence microscopy
Light microscopy
Single cell suspension
mC
D4
5
hCD45
CD
5
CD19 CD5+CD19+
CFSE
CD
8
CD4
T-cells
A.
B.
Growth of primary CLL B and T cells in alymphoid (NSG) mice
atz x rtx bispecific antibody selectively eliminates CLL B cells but not T cells
atz x rtx bispecific antibody preferentially eliminates the CXCR4DimCD5Bright proliferative fraction
Summary
A CD52 x CD20 bispecific antibody (biAb) based on alemtuzumab and rituximab:
1. preferentially bound CD52+CD20+ B cells and not
CD52+CD20- T cells 2. mediated potent CDC and ADCC in vitro 3. selectively eliminated leukemic B cells and
preferentially eliminated the proliferative fraction of malignant B cells, in a patient-derived xenograft model
Major unanswered question
Would repetitive pulse therapies targeting dividing and recently divided cells be able to eliminate/markedly decrease the proliferative fraction and cycling fraction and thereby prevent clonal progression?
Would a combination of therapeutic approaches targeting each of the three CXCR4/CD5 fractions by valuable and cost effective?
Thank you
Feinstein Institute
Bradley Messmer Carlo Calissano Joy Yan Florencia Palacios Shih-Shih Chen Rajendra Damle Davide Bagnara Piers Patten
Northwell Health
Kanti R. Rai
Jacqueline Barrientos Steven L. Allen Jonathan E. Kolitz
UC Berkeley Marc Hellerstein
MD Anderson Cancer Center Jan Burger
NIH NHLBI Adrian Wiestner
UC San Francisco Elizabeth Murphy
Scripps Institute, Florida Christoph Rader
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