Adult Onset of Stills Disease AOSD

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					A novel therapeutic molecule of inflammatory
arthritis by targeting the Pre-Ligand Assembly
  Domain (PLAD) of Tumor Necrosis Factor
                    Receptors


            Guo-Min Deng MD, Ph.D
        Molecular Development Section,
      Laboratory of Immunology, NIAID, NIH
 Concept for novel therapy:
 Inhibition of PLAD by soluble p60 PLAD protein could
 prevent receptor oligomerization and block p60 TNFR
 signaling



                   PLAD
CRD1
                   Ligand
CRD2               binding
                               Soluble
CRD3                TNF        PLAD
CRD4                           protein




       Signaling                          Signaling
                                          blocked
       PLAD-GST fusion proteins are produced in E. coli

P60-GST - 294 a.a.         GST (26 κD)               PLAD (6 κD)
P80-GST - 256 a.a.



           GST P60   P80
 170                                           P60                  P80
 130
 100                                    0.4   0.2 0.04   0.02    0.4 0.2 0.04 0.02 (µg)
 72
 55
 40                            P60MAb
 33

 24                                     0.4   0.2 0.04 0.02     0.4 0.2   0.04   0.02 (µg)

 17
                               P80MAb
 11
    PLAD proteins inhibit TNFα-induced cell death in L929 cells
                     a   medium                                   b       hTNF                         c    hTNF+P60
A         10 4

          10 3

          10 2
    FL3




          10
                                               73.6                                    12.1                             71.9
          1
                 0       100                  200             0           100         200          0         100       200
                         FSC-H                                            FSC-H                              FSC-H

                                                          d
                                              100
                                              80
                                cell loss %




                                              60
                                              40
                                              20

                               P60 (ug)               -       -       7    35    49     7     35       49
                                 mTNF                 +       -       +     +     +     -      -        -
                                  hTNF                -       +       -     -     -     +     +         +
    P60 PLAD abrogated TNFα-induced arthritis

a             PBS                     α
                                   TNF-    b
                                                               Synovitis       Pannus        Erosion
                                                           3               3             3
                     C




                                           (mean + s.d.)
                                             Severity
         ST     JC                                         2               2             2

                     B   ST                                1       **      1             1
                                                                                   **            **
                                                           0               0             0
                                                               TNF P60         TNF P60       TNF P60

            α
         TNF- + P60              α
                              TNF- + P80   c                   Synovitis       Pannus        Erosion
                                                           3               3             3
                              ST
                                           (mean + s.d.)   2               2             2
                                             Severity

    ST
                                                           1               1             1

                                                           0               0             0
                                                               TNF P80         TNF P80       TNF P80
               P60 PLAD significantly reduced collagen-induced arthritis
                         in a double-blind experiment
a                             PBS                             P60                         b        PBS                                                 P60




c
                                                                                  4
                                                                                    4
                                                                                                                                  30




                                                                                                                Mean weight (g)
                          4                                   (mean + s.d., mm)
                               PBS                                                       PBS
                                                                Paw thickness
  Mean arthritis
   index + s.d.




                          3    P60                                                3.5    P60                                      20
                               P80                                                3      P80
                          2                                                                                                                                      PBS
                                                                                  2.5                                             10                             P60
                          1                                                       2                                                                              P80
                                                                                  1.5                                             0
                          0
                              0 3 5 6 7 8 9 1011                                        0 3 5 6 7 8 91011                              0 3 5 6 7 8 91011
                                                                                  Weeks after first immunization

d                                       e                                                                                                     f
                                                            Synovitis                    Pannus          Erosion
                    100                                 4                           4                4                                                60                              40
Percent incidence




                                        (mean + s.d.)




                                                                                                                                                      50


                                                                                                                                       IL-1 (pg/ml)




                                                                                                                                                                       IL-6 (pg/ml)
                     80                                 3                           3                3                                                                                30
                                          Severity




                                                                                                                                                      40                                       *
                     60
                                                        2                           2                2                                                30
                                                                                                                                                             *                        20
                     40                                                                                                                               20
                     20                                 1     *                     1      *         1      *                                                                         10
                                                                                                                                                      10
                      0                                 0                           0                0
                          PBS P60 P80                       P60 PBS                      P60 PBS         P60 PBS                                       PBS P60 P80                         PBS P60 P80
                          Effect of P60 PLAD compared to Enbrel, infliximab
                                  in Collagen-induced arthritis

                  P60 PLAD, Etanercept                                          Effect of P60 in established CIA
PBS




                  8                                                         5
                                                                                    PBS
                          PBS                                               4




                                                           Mean arthritis
                                                            index + s.d.
                                                                                    P60
 Mean arthritis
  index + s.d.




                  6       P60
                          etanercept                                        3
                  4                                                         2
                  2                                                         1

                  0                                                         0
                      0    3 4      5     6 7     8 9                           3      4         5   6      7        8
                          Weeks after first immunization                            Weeks after first immunization




            0               21d        28d           42d                                   70d
      I        II
  Primary     Booster                        treatment
  immunization
                 TNFR expression in arthritic joints
       TNFα transgenic mice (a, b)          CIA mice with PBS (c, d)
         TNFR1              TNFR2           TNFR1(CIA)                     TNFR2 (CIA)
   a                  b                 c                d




                                                                           TNFR1
       TNFR1 (CIA)        TNFR1 (LPS)       TNFR1(LPS)
  e                   f                 g                h                         r
                                                                         lining laye       pannus
                                                                     4                 4




                                                         mean+/-SD
                                                                     3                 3
                                                                     2         ** 2            **
                                                                     1                 1
                                                                     0                 0
                                                                          LPS P60          LPS P60

CIA mice + P60(e) LPS + PBS(f)                   LPS + P60 (g, h)
                        PLAD proteins bind to TNFR and inhibit
                             TNFα binding to TNFR
                                                PLAD inhibit TNFα binding to TNFR
                                       a                                    b
                             100                                 100
                                                      A3 cells                           A3 cells         Bio-TNFα
                             80                                   80                                      Bio-TNFα+P60(3 ug)
               Cell number




                             60                                   60                                      Bio-TNFα+P60(15 ug)
                             40                                   40                                      Bio-TNFα+P60(30 ug)
                                                                  20                                      hTNFα
                             20
                                                                                                          negative control
                              0                                     0
                                   1       10     102 103 104           1       10   102 103 104
                                                  FL1                                FL1



        PLAD do not bind to TNFα                                                             PLAD directly bind to TNFR
                                                                                                       0.1 1 10 10 GST (µg)
B kDa       etanercept P60                      P80       Testing protein
                                                                                         0.1 1 10 10                 P80 PLAD (µg)
          +    - +     - +                      - +       TNFα
                                                                                     +     + + + +     + +    + + Enbrel (0.1µg)
   15                                                     TNFα
             mIgG                  P60          P80       IP Ab
                              TNFα                        Western Ab
P60 PLAD inhibits TNFα-induced osteoclastogenesis

    TNFα-arthritis                 Collagen-arthritis              P60 in CIA




 medium                     TNFα                  TNFα + P60
                                                                                        TRAP staining
                                                                                  160
                                                                                  120




                                                                     osteoclast
                                                                                  80
                                                                                  40
                                                                          0
                                                                    P60 (ug)            -   -   8   32 32
  TRAP = Tartrate-resistant acid phosphatase (osteoclast marker)    TNFa                -   +   +    + -
  In vitro                      In vivo

 P60 PLAD
 P80 PLAD                stop      TNF-induced arthritis

stop          P60 PLAD
                                   Collagen-induced arthritis
 Cell death
                         Mechanism
  TNF
                                   TNF binding to TNFR
                         stop
              P60 PLAD
                                   Osteoclastogenesis

				
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