Expansion of regulatory and specific reactive T cells from cord blood

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Expansion of regulatory and specific reactive T
cells from cord blood

• J Garcia, DV Breier and S. Querol

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CB as a source of HP Transplants

• Proliferative potential
• Immunological permissively
• Safe product
• Immediate availability
• Stability of inventory on registry
• Possibility of ex vivo modifications
  (Engraftment, Immunotherapy)
• Regenerative medicine?

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CB as a source of HP Transplants

• Lack of available cells for DLI after CB
  transplants.
• UCB T cell immunity is immature with respect to
  adults T cell immunity
• Reduced amount of effector cells

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UCB T cell immunity is immature with respect to
adults

• Decreased numbers of CTLs
• Standard activation neonatal T cells produce
  predominant Th2 response. (soluble anti CD3 and
  standard APC)
• Enhanced conditions (co stimulation with Th1
  promoting adjuvant) produce adult level Th1
  and CTL responses (protective immunity)
• The capacity to develop allogeneic cytotoxic T
  cells is intact at birth despite overall dimished
  magnitude of responses.

Neonatal T Cells
Enhanced
default
Co stimulation
Strong Th1 adjuvant
Th1
Th2
Th2
CTL
CTL
Th1
Non response tolerance
Protective immunity
Th1 Pro inflammatory (IFNg) Th2 anti
inflammatory (IL4,IL13,IL5)
Immunology Today 99 Clinical and Experimental
Immunology 2002, Blood 2005 vol105 n4
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REDUCED GVHD IN CORD BLOOD
5 to 10 times lower numbers of
lymphocytes transplanted
Reduced alloreactivity of UCB lymphocytes

• Innate and stimulated immunity to EBV
  demonstrated in UCB lymphocytes. . There is no
  increased risk of EBV PTLD.
• T reg from cord blood may be a contributing
  factor to the low rate GVHD experienced in CBT
• Wadlow R, Porter D. Biol Blood Marrow Transplant.
  2002 8
• Ability of neonatal T cells to regulate lympho
  proliferation similar to UBM (and compares
  favourable with T cell depleted UBM).
• Barker et al. Biology of Blood and Marrow
  Transplantation 2001
• Speculate that pre existing HA-1 specific T cells
  may contribute to GvL reactivity upon CBT in HLA
  A2pos/HA-1 pos recipients
• .Mommaas et al. Blood 2005

• Differences in Lymphocyte recovery between CBT
  and BMT
• Similar immune recovery to matched related or
  unrelated BM
• (Moretta et al. Exp Haematology 2001)
• Lymphocyte reconstitution (median time to normal
  value) was
• 3 months for NK
• 6 months for B cells
• 8 months for CD8 cells
• 11.7 months for CD3 and CD4 cells
• Niehues et al. Br. Journal of Haematology 2001.

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GVHD vs. GVL

• GVL effect T cell and NK mediated
• Clinically used as Donor lymphocytes infusion to
  control
• Minimal residual disease
• Re induction of remission in relapsed leukemia
  after BMT or Peripheral SC transplantation.
• Control EBV or CMV infection
• Post transplant lympho-proliferative disease

Depletion
DLI
T cells
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Donor Lymphocytes Infusion (DLI)

• Limitations
• Not universally effective (AML,ALL)
• Treatment related mortality up to 30
• GVHD (38)
• Infections due to marrow aplasia.
• Relapse (45)

• Benefits
• Used to induced GVT
• aprox. 50 of pts.
• Acceptable compared with other options (2nd. Tx)

Porter et al. Blood 2000
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NK therapy / LAK cells

• Lymphokine activated killer
• Tumor recognition MHC I y MHC I like molecules.
• IL2 activated NK
• Facilitates bone marrow engraftment
• Enhance B cell immune reconstitution
• Attenuate T cell mediated GVHD

• But
• Relatively low anti tumor cytotoxic activity
• Difficulty on obtaining clinically relevant cell
  numbers
• Poor ex vivo expansion
• Require in vivo high dose IL2 (considerable
  toxicity)
• Leembuis T et al. Biol Blood Marrow Transplant.
  2005 Mar11(3)181-7.

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CTL Therapy

• Subsets of Lymphocyte that retain GVT effect
• but Reduce GVH
• Tumor recognition MHC class I and II.
• Potent cytolysis in vivo
• Less GVH in vivo
• (Johnson BD, et al. Biol Blood Marrow
  Transplant. 19995(3)123-32)

• But
• Difficulty on identifying tumor/viral specific
  peptides
• Highly polymorphic nature of human MHC
• Need custom-designed therapies for each patients
  donor derived T cells

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Human CIK cells

• Phase I Trial Autologous CIK cells for tx of
  relapsed HD and NH Lymphoma.
• Minimal toxicity after infusion, preliminary
  evidence of disease response.
• May be useful to reduce risk of relapse in high
  risk pt. after autologous BMT.
• Capable of graft versus leukemia effects with
  attenuated GVHD
• Large variation in the level of expansion
  observed.
• Biology of Blood and Marrow Transplantation
  11181-7(2005)

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Adoptive immunotherapy

• Rational
• Donor T cells infused with the graft, active in
  fighting against tumor cells
• Lower leukemia relapse than syngeneic graft,
• Increased risk of disease relapse in pt.
  receiving T cell depleted grafts,
• DLI effective for pt. who have relapsed.

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CTLs

• Policlonal donor T cells also contain T cells
  that are not reactive with target including T
  cells reactive to minor histocompatibility
  antigens.
• CONCERN GVHD

• Specific CTL use of different immunoeffector
  cell populations to improve control of virus
  infection or increase GVL effect
• CONCERN donor derived T cells from donor
  lacking specific immunity

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Alternatives
12.1
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Specific T cells

• CMV
• EBV
• HLA subtypes
• Minor H ags.
• Her2/neu
• CD19
• BCR ABL
• REGULATORY CD4CD25

Riddel et al. Immunotherapy with Genetically
Modified T cell Clones
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Why cord blood as a source for effector cells?

• Because
• Widespread availability
• Absence of donor risk
• Absence of donor attrition
• Low risk of transmissible infectious diseases in
  the cells
• Increased proliferative potential of HS
  progenitor and immune effector cells,
• Decreased graft versus host disease without
  increase incidence of relapse in antigen
  mismatched situations.
• BMT 14187-96,1994 Blood 904665-78,1997 Clin
  Can Res 64351,2000 Breast Cancer Research and
  Treatment 8315, 2004

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CTL from cord blood. Is that feasible?

• Generation of EBV specific CD4 cytotoxic T cells
  from virus naïve individuals
• Using EBV transformed lymphoblastoid B cell lines
  (LCL)
• Activation with autologous LCL
• (sorting of CD4CD25 after activation
  expansion of selected population might be
  sufficient to generate EBV specific CTLs. The
  Journal of Immunology 2002,168909)

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CTL from cord blood

• Policlonal Expansion of CB
• Lymphocytes achieved
• Serum containing media.
• Azuma et al. 100 fold expansion. Anti CD3 moAb
  coated flask and IL2
• Serum deprived medium
• Sanchez et al. SCFIL2IL7 7 fold expansion of
  total nucleated cells
• Our group
• Anti CD3 anti CD28 BeadsIL2
• 102 fold expansion of total nucleated cells with
  fresh CB,
• 22 fold expansion with frozen/thawed CB.

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CTL from cord blood DC generation for priming

• Salio et al. efficient priming of CB DC,
  generated on serum containing media, generating
  melan A specific T cells, which acquired markers
  of activated cells and capable of cytolytic
  activity towards peptide pulsed targets and tumor
  cells.
• Our experience DC generation from CB mononuclear
  cells is feasible under serum free media and
  standard cytokines (DV. Breier et al. ISCT 2004)

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Specific CTL from CB

• Naïve CB T cells were competent enough to
  generate CTL against a subtype of HLA A2.
• Yasaki et al. BMT(2000)26,451-53
• Her2/neu antigen specific cytotoxic T lymphocytes
  can be generated from umbilical cord blood
  mononuclear cells and demonstrated the antitumor
  cytotoxicity of them against Her2/neu positive
  breast cancer cells.
• HA-1 specific T cells can be generated ex vivo
  from HLA A2pos/HA-1neg CB samples , with
  cytotoxicity activity ( lysis of HA-1pos leukemic
  cells.)
• Mommaas et al. Blood 2005105(4)1823.

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Specific regulatory T cells.

• Two groups (three classes) of regulatory T cells
• Group 1 anergic. Required cell cell contact for
  their function
• CD4CD25 ( regulatory T cells (Tregs)) 5-10 of
  CD4 population
• Group 2 functioning by the secretion of
  inhibitory cytokines
• IL10 producing Tr1 cells
• TGF b producing Th3 cells
• Other T reg Subsets
• CD8 T reg
• TCRgdT cells
• NKT cells

• CD4/CD25 Timus derived supresor cells
• Down regulation of antigen specific effector T
  helper cells (Th)
• Control of the size of the PB T pool
• Control of self tolerance
• Modulate tolerance on organ transplantation.

• CD25
• IL2 Ra, one of the three subunits of IL2 R,
  together with IL2Rb (CD122) and common
  g-chain(CD132)

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Expansion of human CD4CD25 regulatory T cells

• CD25CD4 T cells could be expanded in vitro in
  the presence of IL-2 and allogeneic feeder cells
  and maintained their suppressive capacities.
• J Exp Med. 2001 Jun 4193(11)1295-302
• Induction of a human CD4CD25 regulatory T-cell
  line specific for a defined peptide alloantigen
  (human leukocyte antigen A2 HLA-A2 by priming
  purified CD4CD25 cells ex vivo with in vitro
  differentiated DC, together with IL2 and IL7 gt
  Limited expansion (X20)

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Expansion of human CD4CD25 regulatory T cells

• Policlonal expansion (up to 40000) from adult
  highly purified CD4CD25 T cells with T cell
  expander and IL2
• 100 fold expansion from Cord blood culture
  CD4CD25 with T cell expander and IL2 with non
  clinical grade reagent

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Future approaches

• Specific CTL production ( ex. CMV) from cord
  blood under clinical grade (GMP) conditions
• CD4CD25 Cord blood T cell expansion with
  clinical grade reagents to make it possible its
  application for the treatment of autoimmune
  diseases as well as pathogenic allo-responses
  after solid organ or stem cell transplantation.

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Specific CTL from donor with specific Immunity

• Whole virus infected antigen presenting cells
  succesfully used to generate virus specific T
  cell lines.

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Specific CTL from donor who lacks specific
Immunity

• Ex vivo production of virus specific CTLs from
  seronegative pts.
• Selective expansion of CD25-expressing T cells,
  9-11 days after activation with LCL alone, proved
  to be a simple and reliable method for generating
  EBV-CTL from all sero negative children. The
  majority of these CTL were CD4() (71 /- 26)
  and demonstrated HLA class II-restricted,
  EBV-specific killing
• J. Immunol 168(2002) 909
• Vaccination with virus specific peptides
• A canarypox CMV pp65 recombinant was studied in
  sero negative adult volunteers for its ability to
  induce CMV pp65 specific CTL, helper T
  lymphocytes, and antibodies in a phase I clinical
  trial. Canarypox CMV pp65 is the first
  recombinant vaccine to elicit CMV-specific CTL
  responses, which suggests the potential
  usefulness of this approach in preventing disease
  caused by CMV.
• J Infect Dis. 2001 Apr 15183(8)1171-9.

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Specific CTL Independent of donor immunity

• Krueger, Schneck and Oelke, new strategies to
  improve immunotherapy
• Advantages of aAPC no need leukaphereses,
  stimulation of t cells is not dependent on the
  state and availability of the pt DC, peptid
  loading of aAPC controlled, produce under GMP
  standars
• Krueger , Schneck , Oelke Trends in molecular
  medicine vol 10 no 5 may 2004.

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CTL from cord blood. Is that feasible?
Optimization of the use of the CB mini bag (5
ml) a therapeutic platform
NK/NKT/T Cell amplification gt 100X
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Remarks (CB cells)

• Production of peptide specific CTLs with
  reproducible and clinical grade applicable method
  ,such as aAPC, demonstrated in vitro similar
  functionality to their DC induced counterparts,
  but still needs to be confirmed in vivo
• Whether CB CTL may play a GVL effect in CBSCT
  needs to be elucidated.
• Expansion of human CD4CD25 Regulatory cells is
  feasible,and potential therapeutic tool in
  manipulating direct and indirect allo responses
  in vivo. (GVHD)

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Barcelona CBB Allostem project

• S. Querol
• D.V. Breier
• J. García

• C. Torrico
• C. Azqueta
• M. Santos
• L. Rodríguez
• E. Cortés