Anti-Citrulline Antibody as Novel Therapeutic Drug in Rheumatoid Arthritis
SAT0289
Renato G.S. Chirivi and Jos M.H. Raats
ModiQuest B.V., Nijmegen, The Netherlands
IN TRODUCTION
OBJECTIVES
RESULTS
CONCLUSIONS
We have developed a novel treatment for Rheumatoid Arthritis (RA).
Early treatment of RA patients with our novel antibody drug, just before,
or immediately after the first clinical indications of the disease, prevents
the onset and/or further exacerbation of the disease, and will prevent, or
strongly reduce joint damage, even in established Rheumatoid Arthritis
patients.
Development of a novel antibody treatment for RA, targeting disease specific DPE
targets to prevent onset or exacerbation of the disease in a very early phase.
We identified a subset of rCit-hMabs that were capable of preventing
the onset of inflammation in both CIA and CAIA models.
When mild inflammation was present, administering the rCit-hMabs
resulted in stabilisation of the inflammation and prevented further
increase of the inflammatory response. Histological analysis of the
inflamed joints revealed that bone damage was strongly prevented,
as compared to control animals.
To identify the epitopes recognized by the rCit-hMabs, we performed
IP, followed by MS analysis on post-lytically huPAD4 deiminated
human 293F cells. The main DPE recognized by the rCit-hMabs
(Histon2a) was used to generate new therapeutic Mabs by phage
display and hybridoma generation techniques. Introduction of these
novel rCit-human and mouse Mabs in the CAIA model proved them
to be potent inhibitors of the inflammatory response.
ModiQuest has developed a family of novel rCit-hMabs which have strong therapeutic
potential in regard to preventing: 1) the onset of the inflammation, 2) joint damage
during inflammation, 3) further increase of inflammation and swelling, 4) inflammation
relapse and tissue/joint damage to occur.
The availability of the previously developed diagnostic test for RA, detecting RA up to
10 years before onset of the disease (Immunoscan, Euro-diagnostica AB; DiastatTM,
Axis-Shield), makes this novel therapeutic approach of special interest for early stage
RA. In a more progressive form of RA a combination therapy might be possible with
existing biologicals that have different mechanisms of action.
METHODS
To test novel therapeutics for RA, we established an in house Collagen Antibody
Induced Arthritis (CAIA) mouse model. Upon injection of 8 anti-Collagen-II Mabs
and synchronisation with LPS, DBA mice develop a severe RA like inflammation in
their paws. In addition the Collagen Induced Arthritis (CIA) mouse model was also
used to test mouse and recombinant human monoclonal anti-citrulline antibodies
(rCit-hMabs) for their capacity to prevent the onset of arthritis. Histological
analyses on paws were performed to asses cartilage and bone damage
prevention. Immunoprecipitations (IP) followed by mass-spectrometric (MS)
analysis have been used to determine the therapeutic target epitope recognized
by the therapeutic rCit-hMabs.
CAIA mouse model
mAb induced arthritis
1-2 swollen toes
3 swollen toes
> 3 swollen toes
Swollen footpad or ankle with or without swollen toes
Swollen footpad + swollen ankle with or without swollen toes
: 0.25
: 0.50
: 0.75
: 1.00
: 2.00
Control
Mild
SO
IP-administration of CAIA Ab-mix
(2.6 mg MQ antibody mix per mouse (MQ18.101))
yields 100% arthritis induction within 12 days
BALB/c
DBA/1
*
HE
*
0.50
0.25
1.50
1.75
DBA/1 mice were treated i.p. with a mixture of 8 collagen specific monoclonal antibodies (2.3 mg total). An irrelevant control
antibody (CTL) has been used at the same concentration. LPS (25ug/mouse) was injected i.p. on day 7. Pictures are histological
preparations of hind paws after 25 days ifanti-collagen injection.
SO = Safranin O staining; Blue is bone, red is cartilage. HE = Haematoxylin staining; * = inflammatory infiltrate.
Mean Arthritis Score
6
Severe
Mean Arthris Score
No Ab
7
RhmAb2.101
5
Mean Arthris Score / Mouse
CAIA à Arthritis scoring method:
RhmAb2.102
RhmAb2.104
4
Irrelevant Ab
3
2
1
0
0
1
2
3
4
5
6
7
8
9
10
11
12
Mean Arthris Score
Mean Arthris Score / Mouse
7
No Ab & No DEX
DEX
DEX + RhmAb2.102
6
7)
Target validation
We tested ~1500 different deiminated peptides from various proteins identified by
nLC LTQ FTMS ULTRA analysis of human PAD2- and PAD4-deiminated HEK293
cell lysates for reactivity with therapeutic versus non-therapeutic antibodies.
Histon2a (and highly homologous histon 4) N-terminal peptide has been identified
as lead distinguishing epitope for therapeutic versus non-therapeutic compounds.
5
4
2nd generation anti-citrulline antibodies have been generated based on proteins
identified by MALDI-TOF/nLC LTC FTMS Ultra via Phage Display and
Hybridoma generation techniques.
3
2
1
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21
Days a!er CAIA mix injecon
Anti-coll-II
mix
LPS
Dexamethason (1mg/kg on day 7, 8 and 9)
RhmAb2.102 (1mg/mouse on day 7)
4
3
2
1
0
Purified 2nd generation anti-citruline mAbs were found to have more potent
anti-inflammatory potential in CAIA experiments compared to 1st generation
anti-citrulline mAbs.
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21
Days a!er CAIA mix injecon
LPS +
rCit-hMabs
LPS
Anti-coll-II
mix
Figure 5A shows that macroscopical inflammation in the right hind paws between experimental groups on day 35 of the experiment were similar. Most surprisingly however, all
known parameters for joint erosion were decreased. When scoring Inflammatory cell influx (D), Cartilage erosion (B), Cartilage PG depletion (E), Chondrocyte death (F) and Bone
erosion (C) a dramatic decrease is observed in the experimental group that has been treated on day 7 with RhmAb2.102, Indicating that RhmAb2.102 has a strong therapeutic
potential in regard to preventing joint damage during inflammation.
RhmAb2.102
8) 2nd generation anti-Cit Mabs identified
by Phage Display
9) 2nd generation anti-Cit Mabs identified
by hybridoma generation
Mean Arthritis Score
Mean Arthritis Score / Mouse
Combination therapy:
RhmAb2.102 and Dexamethason
5
0
13
3.2mg anti-Collagen-II antibody (day 0), together with LPS injection on day 3 with or without the “therapeutic” antibodies (1mg/mouse)(n=5) have
been injected i.p. in DBA/1 mice.
6)
No Ab
RhmAb2.102 day 3
RhmAb2.102 day 5
RhmAb2.102 day 6
RhmAb2.102 day 7
6
Days after CAIA mix injection
Anti-coll-II
mix
5) RhmAb2.102 prevents joint damage
during inflammation
4) Therapeutic RhmAb2.102 treatment
3) 1st generation rCit-hMabs in CAIA
CAIA Histology
2)
Mean Arthritis Score / Mouse
1)
REFERENCES
van Venrooij et al. Ann NY Acad Sci 2008; 1143:p268
van Venrooij & Pruijn: Artr Res Ther 2008;10:p117
Mean Arthritis Score
6
No Ab
7
Mean Arthritis Score / Mouse
For the development of this novel therapy, we focussed on more RA
specific targets like Deiminated Peptide-Epitopes (DPE). Deimination is
the posttranslational modification of arginine into citrulline residues
induced by peptidyl-arginine deiminases that are released in the
inflamed joints from dying cells. A growing number of studies indicated
that these modifications could be responsible for the initial triggering of
autoimmunity and the breaking of tolerance.
RhmAb2.102
RhmAb2.108
6
RhmAb2.109
5
RhmAb2.110
RhmAb2.111
4
RhmAb2.112
3
2
1
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Days after CAIA mix injection
Anti-coll-II
mix
LPS +
Cit-Mabs
3.2mg anti-Collagen-II antibody (day 0), together with LPS injection on day 3 with or without the “therapeutic” antibodies (1mg/mouse)(n=5) have been injected i.p. in DBA/1 mice.
No Ab
RmmAb22.101 (hybridoma mAb)
5
RhmAb2.102
4
3
2
1
0
0
1
2
3
4
5
6
7
8
9
10
Days a!er CAIA mix injecon
Anti-coll-II
mix
LPS +
Cit-MAbs
3.2mg anti-Collagen-II antibody (day 0), together with LPS injection on day 3 with or without the “therapeutic” antibodies (1mg/mouse)(n=5) have been injected i.p. in DBA/1 mice.