modulation of immunogenicity by engineered antigen ...€¦ · david w. scott department of...
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David W. ScottDepartment of Medicine
Uniformed Services University of the Health SciencesBethesda, MD, USA
10th Open Scientific EIP SymposiumImmunogenicity of Biopharmaceuticals
Lisbon, Portugal, February 26, 2019
Modulation of immunogenicity by engineered antigen-specific regulatory T cells:
Fighting fire with fireman or police CARs
Our lab has focused on developing novel approaches to induce specific unresponsiveness (“immune tolerance”) and applying these to modulate human diseases and prevent/reverse adverse immune responses.
One such approach is regulatory T-cell therapy, which has been proposed to treat autoimmune diseases, allergy and transplant rejection, as well as to suppress undesirable antibody responses to bio-therapeutics, such as FVIII.
Dealing with immunogenicity and
adverse responses
Clinical studies with expanded human regulatory T-cell therapy are already in progress. However, these are polyclonal T cells that include a diverse repertoire of relativities.
Caveats:
The frequency of relevant regulatory T cells (Tregs) may be quite low.
Expanded polyclonal Tregs (multiple specificities) may be non-specifically immunosuppressive.
Rationale and issues
• Enrich and expand Tregs with antigen/tetramer, etc.
Possible solution based on chimeric antigen receptor (CAR) therapy for cancer:
Engineer specificity into polyclonal Tregs via retroviral transduction of specific “receptors”, e.g.:
T-cell receptor (TCR) CAR (scFv) or Antigen (B-cell antibody receptor=BAR)
Approaches
Approach to transduce
expanded polyclonal Tregs
Transduced antigen-specific polyclonal Tregs
Engineering antigen-specificity into polyclonal T cells:
Four flavors
Antigen-
specific
B cell
BARSignaling domains
Treg
*BAR Treg
BAR
Antigen-
specific
B cell
Signaling domains
CD8
*BAR CD8CAR Treg
Treg
ScFv
?
CARSignaling domains
APC
Teff
TCR Treg
TCRCD3
CD4
Treg
Signaling domains
*B-cell antibody receptor=BAR
Engineering antigen-specificity into polyclonal T cells:
TCR V-regions
APC
Teff
TCR
CD3
CD4
Treg
Signaling domains
TCR Treg effects
Aihong (Allan) Yong ChanZhang Kim
FVIII in hemophilia A
MBP in multiple sclerosis
Multiple systems:
Hemophilia inhibitors (FVIII)
Autoimmunity, e.g., MS (MBP) or Type 1 diabetes
Allergy (OVA)
Future targets (ADA’s)
Systems and targets
Hemophilia
X-linked blood clotting disorder
FVIII mutations cause Hemophilia A*
FIX mutations cause Hemophilia B
“Tenase” complex
*Deletions, inversions, missense, stop codons
Hemophilia
X-linked blood clotting disorder
FVIII mutations cause Hemophilia A*
FIX mutations cause Hemophilia B
“Tenase” complex
*Deletions, inversions, missense, stop codons
What is standard treatment for bleeds?
The unwelcome response to a human protein, FVIII
Hemophilia A patients can mount an immune response
to FVIII depending, in part, on the nature of their
mutation
Because they lack FVIII, they did not develop immune
tolerance to therapeutic FVIII
Specific antibodies against FVIII inhibit clotting by binding to domains required its bio-activity (“inhibitors”)
A1 A2
1 372 740
NH 22 B A3 C1 C2 COOH
1649 1689 2019 2173
A3
Application in Hemophilia A
Immunology 101: The immune Response to FVIII
Antigen presenting cells
FVIII protein
FVIII peptide
Cytokines
Activated FVIII-specific B cells
Activated FVIII-specific T cells
FVIII-specific plasma cells
FVIII peptide
FVIII-specific TCR
FVIII-neutralizing
inhibitor antibody
FVIII protein
FVIII-specific helper T cell
FVIII-specific regulatory T cell
aka “Police CARs”
Treg
(CD4+CD25hiCD127l)
T naive (helper)
(CD4+CD25-CD45RA+)
►FSC
►S
SC
►CD4►
CD
25
►CD127
►C
D2
5
►CD4
►C
D4
5R
A
►C
D2
5
►CD127
►CD127 ►CD4
►C
D2
5
►C
D4
5R
A
►Helios
►F
oxp
3
►Helios
►F
oxp
3
Tnaive
Treg
Isolation of naive T cells and regulatory T cells
from normal donor PBMC
Induction of Foxp3 and GARP
Mock
17195
Fo
xp
3
GARP
OVA
peptide
FVIII
peptide Anti-CD3e
GITRLAP
GF
P+
Mo
ck
GF
P+
17195
No antigen
pC2 (2191-2210)
Induction of LAP and GITR
Antigen-specific upregulation of Treg markers in 17195 Tregs
FVIII-C2-specific immunosuppression by Treg17195
Teff : Treg ratio
0.5 1 2 4 8 No Treg
Teff/Treg ratioYC Kim et al. Blood 125: 1107, 2015.
Can this approach work to prevent or reverse inhibitor responses in
hemophilia A mice ?
Protocol of FVIII-specific suppression of secondary antibody formation by
engineered FVIII-specific human Tregs
n wks
No FVIII + FVIII
7 d
ays
HLA.DR1XE16
Immunization in vivo Reactivation in vitro
rFVIII
ELIspot assay to detect anti-FVIII antibody
spleen
FVIII
Immunized mice
17195* Mock
TregsNo FVIII + FVIII
0
1/2
1/4
1/8
1/16
Tregs
SHOW FIGURE QUANTITATION
*TCR vs. C2 domain peptide
Kim YC et al. Blood 125: 1107, 2015.
Engineering antigen-specificity into polyclonal T cells:
Single chain (scFv) CARs
APC
Teff
TCR Treg
TCRCD3
CD4
Treg
Signaling domains
CAR Treg
Treg
ScFv
?
CARSignaling domains
CAR Treg
?
CARScFv vs. antigen
Treg
Signaling domains
What about CAR (chimeric single chain Fv) Tregs?
Antigen on B cell or APC or endothelial cell surface
Both TCR- or scFv engineered human Tregs suppress the
secondary anti-FVIII response in vitro
scFv TCR(A2) (C2)
Yoon et al., Blood, 129: 238-245, 2017.
Jeongheon Yoon
Anja N. Schmidt
TCR- or scFv engineered human Tregs suppress the anti-FVIII response
in vivo
MockTCR vs. C2scFv vs. A2
Scheme for bystander suppression of multiple
T-cell clones by a single Treg
17195 TCR
A2
C1
C2
T effector
Treg
APC
?
?
?
xxx
Question:
Can antigen-expressing “BAR” T-cell
therapy modulate antibody responses by
directly engaging antigen-specific B cells?
“BAR” = B-cell antibody receptor
FVIII-specific B cell
BAR (B-cell Antibody Receptor) Treg
FVIII-specific
BAR Treg/CD8
Structure of BAR
(B-cell Antibody Receptor)
Treg or CD8 cell
FVIII domain
KalpanaParvathaneni
Engineering antigen-specificity into polyclonal T cells:
Targeting the B cell
APC
Teff
TCR Treg CAR Treg
Treg
ScFvTCRCD3
CD4
?
Treg
CARSignaling domains
Signaling domainsBAR
FVIII-
specific
B cell
Signaling domains
Treg
BAR Treg
FVIII-
specific
B cell
BARSignaling domains
CD8
BAR CD8
A2/C2 BAR mCD8-mediated elimination of anti-FVIII B cells from E16-mouse spleen cells stimulated with LPS
OVA BARNo CD8No LPS
LPS (1 μg/mL) E/T ratio: 5:1
A. αFVIII IgM+ ELISPOT assay by LPS-stimulated E16 B cells B. Quantification of number of spots
no CD8s A2 /C2 BAR OVA BAR no LPS0
500
1000
1500
2000
2500
Naive B cells + LPS 1mg/mL
# o
f S
po
ts p
er
1xE
6 C
ells
BAR-CD8 mediated Elimination of a FVIII IgM+ B Cells
A2/ C2 BAR
FVIII-coated wells
A2 BARControl 2 3 4 5
Day 18
Day 25
Day 10
OVA BARControl 2 3 4 5
Survival of 2JLO-injected NSG mice with BAR CD8 T-cell therapy
CD8
FVIII-
specific
B cell
BAR CD8
Can “BAR” engineered CD4 Tregs target and suppress FVIII-
specific B cells
Treg
FVIII-
specific
B cell
BAR Treg
FVIII
domain
Prevention of anti-FVIII antibody development in naïve E16 mice by BAR human Tregs in vivo
An
ti-F
VII
I a
nti
bo
dy
(µ
g/m
l)
1 4 2 1 2 8 3 5 4 2 4 9 5 6 6 3 7 0
0
5
1 0
1 0 0
2 0 0
3 0 0 O b 2 F 3 h T re g s
F V III B A R h T re g s
Multiple systems:
Hemophilia inhibitors (FVIII)
Autoimmunity, e.g., MS (MBP) or Type 1 diabetes
Allergy (OVA)
Future targets (ADA’s)
Systems and targets
BAR (B-cell Antibody Receptor) TregCan BAR Tregs be used to modulate allergy?
Mast cell with FceROVA-specific B
cell
Allergic response
Isolated
T cells
Viral
systems
Generic donor
Suppression of specific
T and B cells
Virally
Transduced T
cells
Engineered T
cells
Future: CAR or BAR cell therapy not only for hemophilia, but
also for allergy, transplantation, autoimmunity or other
monogenic diseases (and ADA?)
• Antigen-specific TCRs, single chain Fvs and antigen domains (BARs)
have now been engineered for retroviral transduction into human T
effectors and human (mouse) Tregs.
• These Tregs specifically suppressed both proliferation and cytokine
production by antigen-specific T effectors, and antibody formation in vitro
and in vivo in multiple model systems.
• Recent data with “BAR” CD8’s and Tregs (expressing antigen domains)
may allow multiple approaches to regulate adverse immune responses.
e.g., Ovalbumin-BAR iTregs are able to suppress both active and
passive anaphylaxis.
• Expansion of these studies to Tregs in a larger species (hemophilic
dogs) is in progress, with human clinical studies on the horizon.
Summary
Acknowledgements
Yongchan Kim Jeongheon Yoon
KalpanaParvathaneni
MahaAbdeladhim
Allan Zhang
Edward Mitre Laura Kropp
USUHS USUHS
Anja Schmidt
Frankfurt
Chris KönigsFrankfurt
Ethan ShevachNIH
Kate PrattUSUHS
Kai Wucherpfennig
Harvard
Supported by NIH/NHLBI grants RO1 HL126727 and R21 HL127495and NMSS RG1606-24524
ObrigadoGracias