Involvement of CD44, a molecule with thousand faces, in autoimmune diseasesNathalie Assayag-Asherie, Itshak Golan, Lola Weiss, Lora Eshkar Sebban, Shimon Slavin, Itamar Raz and David Naor

Supported by: D CureDiabetes care in Israel andThe Israel Academy of SciencesIsrael Science Foundation

The inflammatory process

GENOMIC STRUCTURE OF THE CD44 MOLECULE

CD44 LIGANDSECM CELL SURFACE * Hyaluronic acid * Mucosal vascular addressin * Collagen * Serglycin/gp600* Fibronectin * Osteopontin (OPN)* Fibrinogen * Class II invariant chain (li)* Laminin * E-selectin * Chondroitin Sulfate * L-selectin Galectin-8

In collaboration with Professor Yehiel Zick The Weizmann Institute

CD44 FUNCTIONS* Cell-cell and cell-matrix interactions * Support of cell migration * Signal delivery for cell growth and cell differentiation * Transmission of apoptotic signals * Signal delivery for cell survival * Signal delivery for production of cytokines and chemokines * Presentation of chemokines and growth factors to cells with corresponding receptors * Docking of proteolytic enzymes on the cell surface and their presentation to relevant substrates * Internalization and degradation of hyaluronic acid

The inflammatory process

Anti-CD44 mAbs reduce pathological activities in the following experimental diseases:

Dr Lola Weiss

Department of Bone Marrow Transplantation and Cancer Research Laboratory, Hadassah University Hospital, Jerusalem, Israel

Nathalie Assayag Asherie

The Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, Jerusalem, Israel

Anti-CD44 mAb reduces insulitis in NOD micehistopathological analysisPNAS,97:285,2000

Shlomo Nedvetzki

Day 0

Day 21

Type II

Collagen

Injection

Type II

Collagen

Injection

2

-

10 days

Onset of

disease

Antibody injection

every second day

Generation of

Collagen

-

Induced Arthritis (CIA) Model

Experimental Protocol

DBA

-

1 Mice

Joint Inflammation in Collagen-Induced Arthritis (CIA) Model

In-Vivo Efficacy in CIA model

The CD44-dependency of CIA

J.Autoimmunity,13:39,1999

Multiple sclerosis

Diabetes

Ulcerative Colitis

DISEASE-SPECIFIC CD44

Atherosclerosis

Normal

Dr Itshak Golan

CD44vRA: exon v4 TTTCAACCACACCACGGGCTTTTGACCACACAAAACAGAACCAGGACTGGACCCAGTGG S T T P R A F D H T K Q N Q D W T Q WCD44v3-10: exon v4 TTTCAACCACACCACGGGCTTTTGACCACACAAAACAGAACCAGGACTGGACCCAGTGG S T T P R A F D H T K Q N Q D W T Q WCD44vRA: AACCCAAGCCATTCAAATCCGGAAGTGCTACTTCAGACAACCACAAGGATGACTGCA N P S H S N P E V L L Q T T T R M T A CD44v3-10: AACCCAAGCCATTCAAATCCGGAAGTGCTACTTCAGACAACCACAAGGATGACTGAT N P S H S N P E V L L Q T T T R M T DCD44vRA: exon v5 GATGTAGACAGAAATGGCACCACTGCTTATGAAGGAAACTGGAACCCAGAAGCACAC D V D R N G T T A Y E G N W N P E A HCD44v3-10: exon v5 GTAGACAGAAATGGCACCACTGCTTATGAAGGAAACTGGAACCCAGAAGCACAC V D R N G T T A Y E G N W N P E A HCD44vRA: CCTCCCCTCATTCACCATGAGCATCATGAGGAAGAAGAGACCCCACATTCTACAAGCACAA P P L I H H E H H E E E E T P H S T S T CD44v3-10: CCTCCCCTCATTCACCATGAGCATCATGAGGAAGAAGAGACCCCACATTCTACAAGCACAA P P L I H H E H H E E E E T P H S T S T

Fig. 1

C.

B.

1 2 3 4 5 6 1 2 3 4 5 6 M N K C1 C2

1714

571

1111

Primershs5 / hs3

Primerspv3I / hs3

A.

Exon 1

Exon 20

hs 5

hs 3

pv 3I

C1 C2 C3 C4 C5 V2 V3 V4 V5 V6 V7 V8 V9 V10 C16 C17 C18 C20

GENOMIC STRUCTURE OF THE CD44 MOLECULE

Sequence of V4-V5 junction in CD44v3-v10 of Keratinocytes in normal donors VARIANT EXON 4AT GTA GACV DVARIANT EXON 5ATG ACT GM TDJ. Clin. Invest. 111:1211-1220 (2003)

Sequence of V4-V5 junction in CD44vRA of synovial fluid cells in Rheumatoid Arthritis patients

VARIANT EXON 4

AT GTA GAC V D

VARIANT EXON 5

ATG ACT G M T A

CA GD

33/41 patients

alanine

J. Clin. Invest. 111:1211-1220 (2003)

A.

B.

anti-pan-CD44

anti-CD44v6

anti-CD44v9

SYNOVIAL FLUID CELLS FROM PATIENT 117RA

HUMAN KERATINOCYTE DONOR M

CELL COUNTS

Fig. 4

MEAN FLUORESCENCE INTENSITY

HUMAN KERATINOCYTES (M)

HUMAN KERATINOCYTES (L)

SYNOVIALFLUID CELLS

II Ab

F8:334mg/ml

Selective binding of F8:33 anti-CD44 mAb to synovial fluid cells from RA patients

SYNOVIAL FLUID CELLS FROM PATIENT 143 (RA)

MEAN FLUORESCENCE INTENSITY

CELL COUNTS

SYNOVIAL FLUID CELLS FROM PATIENT 139 (OA)

CELL COUNTS

F8:33100mg/ml50mg/ml20mg/ml

F8:332mg/ml200ng/ml20ng/ml

Question

Is the binding of anti-CD44 mAb to cell surface CD44 delivers intracellular signaling leading to a functional outcome, such as apoptosis ?

Anti-CD44vRA mAb +Anti-CD44vRA mAb +

Anti-CD44vRA mAb Induces Selective Apoptosisin Synovial Fluid Cells of RA Patient in-vitro

0 1mg/ml 5mg/ml 10mg/ml 50mg/ml

SFCF8:33

PBLF8:33

SFCAnti-Pan-hCD44

PBLAnti-Pan-hCD44

97%

97%

95%

8%

90%

6%

92%

13%

86%

14%

77%

Propidium Iodide

Annexin V

0

5

10

15

20

25

30

35

40

45

50

1

2

3

4

5

6

7

8

Antibody Concentration (

m

g/ml)

% of Apoptosis

SFC / anti-hCD44vRA (F8:33)

SFC / anti-pan-hCD44

PBL / anti-hCD44vRA (F8:33)

PBL / anti-pan-hCD44

0 0.1 0.5 1 5

10 50 100

Selective Induction of Apoptosis with Anti-CD44vRA mAb in Synovial Fluid Cells from RA patients

Multiple sclerosis

Diabetes

Ulcerative Colitis

DISEASE-SPECIFIC CD44

Atherosclerosis

Normal