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