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Immunobiology of
chronic lymphocytic leukemia

         Nicholas Chiorazzi


Departments of Cell Biology and of Medicine
    Albert Einstein College of Medicine
                     and
The Feinstein Institute for Medical Research
     North Shore – LIJ Health System
Ig V gene sequencing      Autoantigen reactivity
  Rajendra Damle             Charles Chu
  Joy Yan                    Rosa Catera
  Bradley T. Messmer         Manuela Woelfle
  Emilia Albesiano           Katerina Hatzi
  Angelo Valetto             Zev Sthoeger
  Fabio Ghiotto              Eric Meffre
  Franco Fais                Maxime Herve
                             Gregg Silverman

                Collaborators
               Kanti R. Rai
               Steven L. Allen
               Jonathan E. Kolitz
               Matthew Kaufman
               Manlio Ferrarini
Chronic Lymphocytic Leukemia


Most prevalent adult leukemia of the Western world
    15,340 new cases and 4,500 deaths in 2007

Disease of aging individuals (>50) with incidence
   increasing with each subsequent decade

Usually affects men > women; Caucasian > African > Asian
Clinical dilemma

Clinical courses of patients are heterogeneous and that
      of an individual patient is unpredictable
    Some patients follow very benign courses living
      decades and dying with the disease, not from it
    Other patients follow more malignant courses
      living only a few years after diagnosis, despite
      therapy
CLL remains an incurable disease
Therefore, “wait and watch” / “wait and worry”
      approaches are taken by clinician and patient
Chronic Lymphocytic Leukemia

Lymphocytosis seen in blood, but most leukemic cells are
  in non-vascular areas including bone marrow, lymph
  nodes, and spleen

Usually detected upon routine blood workup as an
  elevated white cell or lymphocyte count (~3x1010 total
  in blood)

Clonal expansion of a CD5+ B lymphocyte with low surface
   immunoglobulin (Ig)

Most clones express predominantly IgM isotype surface
  Ig, but ~7% are predominantly IgG or IgA, though in
  those cases IgM clonal relatives can be found
Clonal disease of B lymphocytes
                                         CD19
                                         CD5
                                         CD23
               Smudge cell               smIg (BCR)




                             CLL cells




 Granulocyte
Take home messages

1. CLL results from the non-random selection and
   transformation of B lymphocytes expressing B-cell
   antigen receptors (BCRs) of restricted amino acid
   structure

2. These BCRs can be poly- and auto-reactive, binding
   natural as well as novel autoantigens generated by
   apoptosis and other catabolic processes

3. The clinically-distinct subgroups differ in the retention
   or loss of poly- and auto-reactivity, with the retention of
   polyreactivity being associated with worse clinical
   disease
Take home messages

1. CLL results from the non-random selection and
   transformation of B lymphocytes expressing B-cell
   antigen receptors (BCRs) of restricted amino acid
   structure
Ig molecules and their genes

            NH2
                      VK
               CK
                                 VH
  Fab                                       CDR3       CDR2 CDR1
                           CH1
  Fc                                  FR4        FR3    FR2        FR1     VH    D        JH   CH
            -S - S-

Hinge                      CH2
Region                                  JH D                  VH
                                                                         Chromosome 14


                                                                           VK        JH        CK
                           CH3

                                                                         Chromosome 2
           COOH                             JK             VK



         Ig Molecule                               V Region                     Ig Genes
CLL cells differ from normal CD5+ B cells
by the overuse of certain autoreactive genes




                                Fais et al. J Clin Invest 98: 1659, 1998
CLL clones differ in the degree of somatic
mutations, especially in particular IgV genes

   VH                Specific                       % Cases with
  Family             VH Gene                         Mutations

   All cases               -                              50.7
   1                      -                               33.3
                        1-69                              10.0
   3                     -                                66.7
                        3-07                              90.0

   4                     -                                41.2
                        4-34                              55.0

               Fais et al. J Clin Invest 98: 1659, 1998
Ig VH gene mutation status of CLL cells is an
 important prognostic indicator of outcome

            ≥ 2% mutation       ≥ 2% mutation




            < 2% mutation       < 2% mutation




        Damle et al.           Hamblin et al.
    Blood 94: 1840, 1999    Blood 94: 1848, 1999
Ig molecules and their genes

            NH2
                      VK
               CK
                                 VH
  Fab                                       CDR3       CDR2 CDR1
                           CH1
  Fc                                  FR4        FR3    FR2        FR1     VH    D        JH   CH
            -S - S-

Hinge                      CH2
Region                                  JH D                  VH
                                                                         Chromosome 14


                                                                           VK        JH        CK
                           CH3

                                                                         Chromosome 2
           COOH                             JK             VK



         Ig Molecule                               V Region                     Ig Genes
CLL clones are culled from the normal B-cell
  repertoire based on structural constraints of
  the B-cell antigen receptor
IgV gene segment recombination
         Heavy Chain                       Light Chain (k/λ)
    VH (44)   D (27)   JH (6)            VL (46/36)      JL (5/7)




                                           = RAG mediated
                                             recombination




HC = VH x D x JH = 44 x 27 x 6 = 7,128      VHDJH     rearrangement: ~1 : 7,000
~1% of CLL patients express a BCR with a VH 1-69
  gene exhibiting very similar HCDR3s often
  comprised of the same VH-D-JH segments

  Widhopf et al. Blood 104: 2499-2504, 2004
Ig V region gene segment recombination
       Heavy Chain                       Light Chain (k/λ)
  VH (44)   D (27)   JH (6)          VL (46/36)       JL (5/7)




                                         = RAG mediated
                                           recombination


HC = VH x D x JH = 44 x 27 x 6 = 7,128
k = Vk x Jk = 46 x 5 = 230
                                  VHDJH / VLJL rearrangement: ~1 : 3 x 106
l = Vl x Jl = 36 x 7 = 252
CLL cases with remarkably similar B-cell receptors

          V Gene   Germline         Percent              D             J
 CLL      Family    V Gene         Difference           Gene          Gene

CLL 039    VH4       4-39                0.3             6-13           5b
           VkI       O12                 0.0               -            k2

CLL 057    VH4       4-39                0.7             6-13           5b
           VkI       O12                 0.0               -            k1

CLL 114    VH4       4-39                0.3             6-13           5b
           VkI       O12                 0.0               -            k1

CLL 202    VH4       4-39                0.7             6-13           5b
           VkI       O12                 0.0               -            k1

CLL 209    VH4       4-39                0.3             6-13           5b
            ?         ?                   ?                -             ?
                              Ghiotto et al. J Clin Invest 113: 1008, 2004
IgV gene segment recombination
     Heavy Chain              Light Chain (k/λ)
VH (44)   D (27)   JH (6)   VL (46/36)     JL (5/7)




                              = RAG mediated
                                recombination
Because of the differences that occur at the
  junctions when gene segments combine, the
  likelihood that the same VHDJH- VLJL
  rearrangement with the same junctional
  characteristic would occur in two different B
  cells is even much more remote

             ≈ 1 / 1x108 – 1 : 1x1012

Therefore, if the gene structure of the Ig variable
  region found in B-CLL cells from different
  patients is very similar or identical, then this
  must indicate a selective process of
  leukemogenesis that targets B cells with a given
  type of Ig V region.
Heavy chain sequence alignment

         VH1-69               D3-16                          JH 3
            CAR          YYDYVWGSYRY                    DAFDIWG
CLL068: CAR GG DYDYVWGSYRS N DAFDIWG
CLL258: CAR GG IYDYVWGSYRP N DAFDIWG
CLL022: CAR GG DYDYVWGSYRP N DAFDIWG
CLLSMI: CAR GG NYDYIWGSYRS N DAFDIWG
aCLA*:       CAR GG NYDYIWGSYRS N DAFDIWG
 Natural autoantibody
*aCLA = anti-cardiolipin ab   Messmer et al. J Exp Med 2004; 200: 519-525
Almost 30% of patients with chronic lymphocytic leukemia
 carry stereotyped receptors

   Stamatopoulos et al. Blood 109:259-270, 2007

   Murray et al. Blood 111:1524-1533, 2008



>35% chance of fitting into a stereotypic set if U-CLL
     or if express a specific VH gene (1-69, 3-21, 4-39)
     associated with poor outcome
Take home messages

2. These BCRs can be poly- and auto-reactive, binding
   natural as well as novel autoantigens generated by
   apoptosis and other catabolic processes
Expression of recombinant CLL mAbs
                      Transfection of 293T HEK cells using Lipofectamine 2000
 1. Plasmid DNA       2.Lipofectamine 3. Lipofectamine 2000   4. Liposomes are added in
carrying heavy and     2000 Reagent    Reagent and DNA are      293T HEK cell culture
light chain Ig gene                    mixed and incubated


                                                                                          Immuno assay for
                                                                                           quantification of
                                                                                              CLL mAb



                                                                  4-5 days of culture



       293T HEK cell line
                                                    Antibody purification using
                                                         Protein G beads




                                            Wardemann et al. Science 301:1374, 2003
Herve et al. J Clin Invest 115:1636-1643, 2005
Reactivity of recombinant mAbs with viable HEp-2
 cells that were permeabilized to allow Ab entry
              [“Anti-cell antibodies”]

 ~20% of mAbs from CD5+ B cells from normal subjects

 ~75% of mAbs from CLL cells

     ~90% from unmutated CLL
     ~60% from mutated CLL

 Reactivity mainly with cytoplasmic structures and
   occasionally with nucleoli. Only one mAb from
   M-CLL patient reacted with nucleus in a
   homogeneous pattern
                              Herve et al. J Clin Invest 115:1636-1643, 2005
Autoreactivities of individual CLL BCRs/mAbs


1. VH1-69/D3-16/JH3 + VK3-20:
   Non-muscle myosin heavy chain IIA
    (Chu et al. Blood 112: 5122-5129, 2008)

2. VH4-39/D6-13/JH5 + VK1D-39/JK1
   Vimentin
    (Chu et al. Blood 112: 5122-5129, 2008)

3. 60% of U-CLL and 10% of M-CLL:
   Apoptosis-associated autoantigens
   (Catera et al. Mol Med 2008; 14: 665-674 )
Herve et al. J Clin Invest 115:1636-1643, 2005
Heavy chain sequence alignment

         VH1-69               D3-16                          JH 3
            CAR          YYDYVWGSYRY                    DAFDIWG
CLL068: CAR GG DYDYVWGSYRS N DAFDIWG
CLL258: CAR GG IYDYVWGSYRP N DAFDIWG
CLL022: CAR GG DYDYVWGSYRP N DAFDIWG
CLLSMI: CAR GG NYDYIWGSYRS N DAFDIWG
aCLA*:       CAR GG NYDYIWGSYRS N DAFDIWG
 Natural autoantibody
*aCLA = anti-cardiolipin ab   Messmer et al. J Exp Med 2004; 200: 519-525
mAb 068 binds 225KDa molecule




    C. Chu et al. Blood 112: 5122-5129, 2008
LC MS/MS
identifies 225KDa
band as
non-muscle
myosin heavy
chain IIA
(MYHIIA)




                    C. Chu et al. Blood 112: 5122-5129, 2008
CLL mAb 068 co-localizes with pAbs to MYHIIA




          C. Chu et al. Blood 112: 5122-5129, 2008
Autoreactivities of individual CLL BCRs/mAbs


    1. VH1-69/D3-16/JH3 + VK3-20:
       Non-muscle myosin heavy chain IIA
        (Chu et al. Blood 112: 5122-5129, 2008)

    2. VH4-39/D6-13/JH5 + VK1D-39/JK1
       Vimentin
        (Chu et al. Blood 112: 5122-5129, 2008)

    3. 60% of U-CLL and 10% of M-CLL:
       Apoptosis-associated autoantigens
       (Catera et al. Mol Med 2008; 14: 665-674 )
CLL mAbs react with apoptotic (not healthy) cells

                                           A                      B
                          3.70             28.17   16.77          13.86




              Annexin V
RAMOS

                          67.27             0.92   69.14           0.23


                                  CLL014                   DO13


                                           C                      D
                          52.86            18.12   55.35          16.96
              Annexin V




 Jurkat

                          28.47             0.55   27.37           0.32


                                  CLL014                   DO13



          R. Catera et al. Mol Med 14: 665-674, 2008
Apoptotic B and T cells are a source of
         autoantigens for CLL mAbs


SUMMARY of the results:

- More than 60% (18/28) of the mAbs tested reacted with
   these two cell types

- 15 of the 18 reactive mAbs used an unmutated VH gene,
    only 3 used a mutated VH gene




            R. Catera et al. Mol Med 14: 665-674, 2008
Antigens bound at the surface of apoptotic cells
have translocated from intracellular compartments
                  Cytox Orange       Annexin V        CLL 114   Merge



 Membrane blebs




 Apoptotic body
 without DNA




 Apoptotic body
 with DNA



                  R. Catera et al. Mol Med 14: 665-674, 2008
MYHIIA is one of the intracellular antigens
   that translocates to the surface and
         is bound by CLL mAbs
MEAC: MYHIIA exposed apoptotic cell
            Late apoptotic




Live




            Early apoptotic
                         Chu et al. Blood 2010 in press
CLL 068 mAb binds to MEACs


  Negative   Apoptotic          MEACs




                    Chu et al. Blood 2010 in press
Many CLL mAbs bind MEACs
   MEAC binding




 Subset
Mutation

                           Chu et al. Blood 2010 in press
Take home messages

3. The clinically-distinct subgroups differ in the retention
   or loss of poly- and auto-reactivity, with the retention of
   polyreactivity being associated with worse clinical
   disease
Polyreactivity is a feature primarily of unmutated CLL cells




               Herve et al. J Clin Invest 115:1636-1643, 2005
Ig VH gene mutation status of CLL cells is an
 important prognostic indicator of outcome

            ≥ 2% mutation       ≥ 2% mutation




            < 2% mutation       < 2% mutation




        Damle et al.           Hamblin et al.
    Blood 1999; 94: 1840    Blood 1999; 94: 1848
Binding well to MEACs correlates with poor
              patient survival

                         Lo binding
                         ?? Months (n = 9)



         Hi binding
         99 months
         (n = 15)




                         Chu et al. Blood 2010 in press
In this limited series, MEAC binding correlates better
    with patient survival than IGHV mutation status

                                 Mutated
                                 ?? Months (n = 6)



              Unmutated
              118 months
              (n = 18)




                                Chu et al. Blood 2010 in press
Many CLL mAbs bind MEACs
   MEAC binding




 Subset
Mutation

                           Chu et al. Blood 2010 in press
Inferences
1. MEACs may be a source of autoantigens
   driving CLL disease
2. The origin of many CLL clones may be cells
   that produce natural antibodies to MEACs
   or other natural products of catabolism
      B1-like cells, MZ B cells?

3. If MEAC binding is a “better” predictor
    of patient survival than IGHV mutations
    status, is it because the former implies
    antigen-binding activity whereas the latter
    directly measures it?
B-CLL           Initiation

                               M-CLL
 evolution                     U-CLL
hypothesis       Progression



MEACs
MYHIIA+
Vimentin
Filamin B
Oxidation
Chemical
  Modification

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Se1.2 chiorazzi pc aecc_retreat_5-5-10_for_website

  • 1. Immunobiology of chronic lymphocytic leukemia Nicholas Chiorazzi Departments of Cell Biology and of Medicine Albert Einstein College of Medicine and The Feinstein Institute for Medical Research North Shore – LIJ Health System
  • 2. Ig V gene sequencing Autoantigen reactivity Rajendra Damle Charles Chu Joy Yan Rosa Catera Bradley T. Messmer Manuela Woelfle Emilia Albesiano Katerina Hatzi Angelo Valetto Zev Sthoeger Fabio Ghiotto Eric Meffre Franco Fais Maxime Herve Gregg Silverman Collaborators Kanti R. Rai Steven L. Allen Jonathan E. Kolitz Matthew Kaufman Manlio Ferrarini
  • 3. Chronic Lymphocytic Leukemia Most prevalent adult leukemia of the Western world  15,340 new cases and 4,500 deaths in 2007 Disease of aging individuals (>50) with incidence increasing with each subsequent decade Usually affects men > women; Caucasian > African > Asian
  • 4. Clinical dilemma Clinical courses of patients are heterogeneous and that of an individual patient is unpredictable  Some patients follow very benign courses living decades and dying with the disease, not from it  Other patients follow more malignant courses living only a few years after diagnosis, despite therapy CLL remains an incurable disease Therefore, “wait and watch” / “wait and worry” approaches are taken by clinician and patient
  • 5. Chronic Lymphocytic Leukemia Lymphocytosis seen in blood, but most leukemic cells are in non-vascular areas including bone marrow, lymph nodes, and spleen Usually detected upon routine blood workup as an elevated white cell or lymphocyte count (~3x1010 total in blood) Clonal expansion of a CD5+ B lymphocyte with low surface immunoglobulin (Ig) Most clones express predominantly IgM isotype surface Ig, but ~7% are predominantly IgG or IgA, though in those cases IgM clonal relatives can be found
  • 6. Clonal disease of B lymphocytes CD19 CD5 CD23 Smudge cell smIg (BCR) CLL cells Granulocyte
  • 7. Take home messages 1. CLL results from the non-random selection and transformation of B lymphocytes expressing B-cell antigen receptors (BCRs) of restricted amino acid structure 2. These BCRs can be poly- and auto-reactive, binding natural as well as novel autoantigens generated by apoptosis and other catabolic processes 3. The clinically-distinct subgroups differ in the retention or loss of poly- and auto-reactivity, with the retention of polyreactivity being associated with worse clinical disease
  • 8. Take home messages 1. CLL results from the non-random selection and transformation of B lymphocytes expressing B-cell antigen receptors (BCRs) of restricted amino acid structure
  • 9. Ig molecules and their genes NH2 VK CK VH Fab CDR3 CDR2 CDR1 CH1 Fc FR4 FR3 FR2 FR1 VH D JH CH -S - S- Hinge CH2 Region JH D VH Chromosome 14 VK JH CK CH3 Chromosome 2 COOH JK VK Ig Molecule V Region Ig Genes
  • 10. CLL cells differ from normal CD5+ B cells by the overuse of certain autoreactive genes Fais et al. J Clin Invest 98: 1659, 1998
  • 11. CLL clones differ in the degree of somatic mutations, especially in particular IgV genes VH Specific % Cases with Family VH Gene Mutations All cases - 50.7 1 - 33.3 1-69 10.0 3 - 66.7 3-07 90.0 4 - 41.2 4-34 55.0 Fais et al. J Clin Invest 98: 1659, 1998
  • 12. Ig VH gene mutation status of CLL cells is an important prognostic indicator of outcome ≥ 2% mutation ≥ 2% mutation < 2% mutation < 2% mutation Damle et al. Hamblin et al. Blood 94: 1840, 1999 Blood 94: 1848, 1999
  • 13. Ig molecules and their genes NH2 VK CK VH Fab CDR3 CDR2 CDR1 CH1 Fc FR4 FR3 FR2 FR1 VH D JH CH -S - S- Hinge CH2 Region JH D VH Chromosome 14 VK JH CK CH3 Chromosome 2 COOH JK VK Ig Molecule V Region Ig Genes
  • 14. CLL clones are culled from the normal B-cell repertoire based on structural constraints of the B-cell antigen receptor
  • 15. IgV gene segment recombination Heavy Chain Light Chain (k/λ) VH (44) D (27) JH (6) VL (46/36) JL (5/7) = RAG mediated recombination HC = VH x D x JH = 44 x 27 x 6 = 7,128 VHDJH rearrangement: ~1 : 7,000
  • 16. ~1% of CLL patients express a BCR with a VH 1-69 gene exhibiting very similar HCDR3s often comprised of the same VH-D-JH segments Widhopf et al. Blood 104: 2499-2504, 2004
  • 17. Ig V region gene segment recombination Heavy Chain Light Chain (k/λ) VH (44) D (27) JH (6) VL (46/36) JL (5/7) = RAG mediated recombination HC = VH x D x JH = 44 x 27 x 6 = 7,128 k = Vk x Jk = 46 x 5 = 230 VHDJH / VLJL rearrangement: ~1 : 3 x 106 l = Vl x Jl = 36 x 7 = 252
  • 18. CLL cases with remarkably similar B-cell receptors V Gene Germline Percent D J CLL Family V Gene Difference Gene Gene CLL 039 VH4 4-39 0.3 6-13 5b VkI O12 0.0 - k2 CLL 057 VH4 4-39 0.7 6-13 5b VkI O12 0.0 - k1 CLL 114 VH4 4-39 0.3 6-13 5b VkI O12 0.0 - k1 CLL 202 VH4 4-39 0.7 6-13 5b VkI O12 0.0 - k1 CLL 209 VH4 4-39 0.3 6-13 5b ? ? ? - ? Ghiotto et al. J Clin Invest 113: 1008, 2004
  • 19. IgV gene segment recombination Heavy Chain Light Chain (k/λ) VH (44) D (27) JH (6) VL (46/36) JL (5/7) = RAG mediated recombination
  • 20. Because of the differences that occur at the junctions when gene segments combine, the likelihood that the same VHDJH- VLJL rearrangement with the same junctional characteristic would occur in two different B cells is even much more remote ≈ 1 / 1x108 – 1 : 1x1012 Therefore, if the gene structure of the Ig variable region found in B-CLL cells from different patients is very similar or identical, then this must indicate a selective process of leukemogenesis that targets B cells with a given type of Ig V region.
  • 21. Heavy chain sequence alignment VH1-69 D3-16 JH 3 CAR YYDYVWGSYRY DAFDIWG CLL068: CAR GG DYDYVWGSYRS N DAFDIWG CLL258: CAR GG IYDYVWGSYRP N DAFDIWG CLL022: CAR GG DYDYVWGSYRP N DAFDIWG CLLSMI: CAR GG NYDYIWGSYRS N DAFDIWG aCLA*: CAR GG NYDYIWGSYRS N DAFDIWG  Natural autoantibody *aCLA = anti-cardiolipin ab Messmer et al. J Exp Med 2004; 200: 519-525
  • 22. Almost 30% of patients with chronic lymphocytic leukemia carry stereotyped receptors Stamatopoulos et al. Blood 109:259-270, 2007 Murray et al. Blood 111:1524-1533, 2008 >35% chance of fitting into a stereotypic set if U-CLL or if express a specific VH gene (1-69, 3-21, 4-39) associated with poor outcome
  • 23. Take home messages 2. These BCRs can be poly- and auto-reactive, binding natural as well as novel autoantigens generated by apoptosis and other catabolic processes
  • 24. Expression of recombinant CLL mAbs Transfection of 293T HEK cells using Lipofectamine 2000 1. Plasmid DNA 2.Lipofectamine 3. Lipofectamine 2000 4. Liposomes are added in carrying heavy and 2000 Reagent Reagent and DNA are 293T HEK cell culture light chain Ig gene mixed and incubated Immuno assay for quantification of CLL mAb 4-5 days of culture 293T HEK cell line Antibody purification using Protein G beads Wardemann et al. Science 301:1374, 2003
  • 25. Herve et al. J Clin Invest 115:1636-1643, 2005
  • 26. Reactivity of recombinant mAbs with viable HEp-2 cells that were permeabilized to allow Ab entry [“Anti-cell antibodies”] ~20% of mAbs from CD5+ B cells from normal subjects ~75% of mAbs from CLL cells  ~90% from unmutated CLL  ~60% from mutated CLL Reactivity mainly with cytoplasmic structures and occasionally with nucleoli. Only one mAb from M-CLL patient reacted with nucleus in a homogeneous pattern Herve et al. J Clin Invest 115:1636-1643, 2005
  • 27. Autoreactivities of individual CLL BCRs/mAbs 1. VH1-69/D3-16/JH3 + VK3-20: Non-muscle myosin heavy chain IIA (Chu et al. Blood 112: 5122-5129, 2008) 2. VH4-39/D6-13/JH5 + VK1D-39/JK1 Vimentin (Chu et al. Blood 112: 5122-5129, 2008) 3. 60% of U-CLL and 10% of M-CLL: Apoptosis-associated autoantigens (Catera et al. Mol Med 2008; 14: 665-674 )
  • 28. Herve et al. J Clin Invest 115:1636-1643, 2005
  • 29. Heavy chain sequence alignment VH1-69 D3-16 JH 3 CAR YYDYVWGSYRY DAFDIWG CLL068: CAR GG DYDYVWGSYRS N DAFDIWG CLL258: CAR GG IYDYVWGSYRP N DAFDIWG CLL022: CAR GG DYDYVWGSYRP N DAFDIWG CLLSMI: CAR GG NYDYIWGSYRS N DAFDIWG aCLA*: CAR GG NYDYIWGSYRS N DAFDIWG  Natural autoantibody *aCLA = anti-cardiolipin ab Messmer et al. J Exp Med 2004; 200: 519-525
  • 30. mAb 068 binds 225KDa molecule C. Chu et al. Blood 112: 5122-5129, 2008
  • 31. LC MS/MS identifies 225KDa band as non-muscle myosin heavy chain IIA (MYHIIA) C. Chu et al. Blood 112: 5122-5129, 2008
  • 32. CLL mAb 068 co-localizes with pAbs to MYHIIA C. Chu et al. Blood 112: 5122-5129, 2008
  • 33. Autoreactivities of individual CLL BCRs/mAbs 1. VH1-69/D3-16/JH3 + VK3-20: Non-muscle myosin heavy chain IIA (Chu et al. Blood 112: 5122-5129, 2008) 2. VH4-39/D6-13/JH5 + VK1D-39/JK1 Vimentin (Chu et al. Blood 112: 5122-5129, 2008) 3. 60% of U-CLL and 10% of M-CLL: Apoptosis-associated autoantigens (Catera et al. Mol Med 2008; 14: 665-674 )
  • 34. CLL mAbs react with apoptotic (not healthy) cells A B 3.70 28.17 16.77 13.86 Annexin V RAMOS 67.27 0.92 69.14 0.23 CLL014 DO13 C D 52.86 18.12 55.35 16.96 Annexin V Jurkat 28.47 0.55 27.37 0.32 CLL014 DO13 R. Catera et al. Mol Med 14: 665-674, 2008
  • 35. Apoptotic B and T cells are a source of autoantigens for CLL mAbs SUMMARY of the results: - More than 60% (18/28) of the mAbs tested reacted with these two cell types - 15 of the 18 reactive mAbs used an unmutated VH gene, only 3 used a mutated VH gene R. Catera et al. Mol Med 14: 665-674, 2008
  • 36. Antigens bound at the surface of apoptotic cells have translocated from intracellular compartments Cytox Orange Annexin V CLL 114 Merge Membrane blebs Apoptotic body without DNA Apoptotic body with DNA R. Catera et al. Mol Med 14: 665-674, 2008
  • 37. MYHIIA is one of the intracellular antigens that translocates to the surface and is bound by CLL mAbs
  • 38. MEAC: MYHIIA exposed apoptotic cell Late apoptotic Live Early apoptotic Chu et al. Blood 2010 in press
  • 39. CLL 068 mAb binds to MEACs Negative Apoptotic MEACs Chu et al. Blood 2010 in press
  • 40. Many CLL mAbs bind MEACs MEAC binding Subset Mutation Chu et al. Blood 2010 in press
  • 41. Take home messages 3. The clinically-distinct subgroups differ in the retention or loss of poly- and auto-reactivity, with the retention of polyreactivity being associated with worse clinical disease
  • 42. Polyreactivity is a feature primarily of unmutated CLL cells Herve et al. J Clin Invest 115:1636-1643, 2005
  • 43. Ig VH gene mutation status of CLL cells is an important prognostic indicator of outcome ≥ 2% mutation ≥ 2% mutation < 2% mutation < 2% mutation Damle et al. Hamblin et al. Blood 1999; 94: 1840 Blood 1999; 94: 1848
  • 44. Binding well to MEACs correlates with poor patient survival Lo binding ?? Months (n = 9) Hi binding 99 months (n = 15) Chu et al. Blood 2010 in press
  • 45. In this limited series, MEAC binding correlates better with patient survival than IGHV mutation status Mutated ?? Months (n = 6) Unmutated 118 months (n = 18) Chu et al. Blood 2010 in press
  • 46. Many CLL mAbs bind MEACs MEAC binding Subset Mutation Chu et al. Blood 2010 in press
  • 47. Inferences 1. MEACs may be a source of autoantigens driving CLL disease 2. The origin of many CLL clones may be cells that produce natural antibodies to MEACs or other natural products of catabolism  B1-like cells, MZ B cells? 3. If MEAC binding is a “better” predictor of patient survival than IGHV mutations status, is it because the former implies antigen-binding activity whereas the latter directly measures it?
  • 48. B-CLL Initiation M-CLL evolution U-CLL hypothesis Progression MEACs MYHIIA+ Vimentin Filamin B Oxidation Chemical Modification

Editor's Notes

  1. I would like to thank the organizers for giving me the opportunity to discuss our research results with you. This represents the work of not only myself but of many others including… Lu, Katerina, Rosa Steve and Kanti for clinical collaboration Nick for support, guidance and inspiration and others that I will acknowledge throughout the talk
  2. This is a typical example of flow cytometry data showing that CLL 068 mAb reacts to MEACs. This is a negative control. This shows that CLL 068 binds to a subset of dead cells (AV-PE positive) and not live cells. This shows that CLL 068 binds only to MEACs (MYHIIA positive). And not other cells.
  3. This is a graph that shows the binding of 26 CLL mAbs listed on the x-axis to MEACs… … with the cutoff of 1.5 for MEAC binding. 16 mAbs bind well/. This is a grouping of the CLL mAbs based on having a shared common “stereotyped” sequence, which I do not have time to describe, … but would just like to point out that the stereotyped groups bind in the same manner. This shows which CLL mAbs are mutated or unmutated, … U = is less than 2% mutation in the IGHV, which correlates with bad patient survival … M = is greater than 2% mutation in the IGHV, which correlates with good patient outcome. All the MEAC binding mAbs are UM, except for 1 (15/16), which binds like the same stereotype subgroup “1” and is a borderline “Mut”. Those mAbs that do not bind MEACs well are a mixture of Mut (6) and four UM (4) mAbs. Because survival correlates with IGHV mutation, perhaps MEAC binding correlates with survival and help distinguish the UM that may survive better.. .
  4. Indeed binding well to MEACs correlates to poor patient survival (24 patients have survival data)… Hi binding has a median survival of 99 months (n=15), Whereas Lo binding has not reached median survival (n=9). This is statistically significant P&lt;0.0087. This significance is better than that for UM versus Mut (P&lt;0.06) in this patient cohort. … this is because one mutated (CLL 154) and two unmutated (CLL 376 and 412) IGHV CLL patients having survival outcomes contrary to that expected for their IGHV mutation status.
  5. IGHV mutation status versus patient survival (24 patients have survival data)… UM IGHV has a median survival of 118 months (n=18), Whereas Mut IGHV has not reached median survival (n=6). This is not quite statistically significant P&lt;0.06. This significance is not as good as MEAC binding. … this is because one mutated (CLL 154) and two unmutated (CLL 376 and 412) IGHV CLL patients having survival outcomes contrary to that expected for their IGHV mutation status.
  6. This is a graph that shows the binding of 26 CLL mAbs listed on the x-axis to MEACs… … with the cutoff of 1.5 for MEAC binding. 16 mAbs bind well/. This is a grouping of the CLL mAbs based on having a shared common “stereotyped” sequence, which I do not have time to describe, … but would just like to point out that the stereotyped groups bind in the same manner. This shows which CLL mAbs are mutated or unmutated, … U = is less than 2% mutation in the IGHV, which correlates with bad patient survival … M = is greater than 2% mutation in the IGHV, which correlates with good patient outcome. All the MEAC binding mAbs are UM, except for 1 (15/16), which binds like the same stereotype subgroup “1” and is a borderline “Mut”. Those mAbs that do not bind MEACs well are a mixture of Mut (6) and four UM (4) mAbs. Because survival correlates with IGHV mutation, perhaps MEAC binding correlates with survival and help distinguish the UM that may survive better.. .
  7. These results lead to the following inferences…
  8. Just to end with a MODEL cell death (CLL or other) leading to MEAC formation and exposure of MYHIIA and other epitopes observed by Rosa This could be important for the initiation of CLL or the ongoing stimulation that may be required for CLL growth and development.