cd8 t cell
DESCRIPTION
P. P. Chames G. Rosas L. Rem R. Willemsen R. Bolhuis HR. Hoogenboom. Affinity-matured MHC-peptide specific phage-antibodies for targeting T-cell rejection antigens on melanoma cells. CD8 T cell. Target cell. Tumors are immunogenic…. - PowerPoint PPT PresentationTRANSCRIPT
Affinity-matured MHC-peptide specific phage-antibodies for
targeting T-cell rejection antigens on melanoma cells
CD8 T cell
Target cell
PP. ChamesG. Rosas L. RemR. WillemsenR. BolhuisHR. Hoogenboom
2m (12 KDa)
Heavy chain (35KDa)
peptide 8-9 aa
Tumors are immunogenic…
A large number of tumor antigens have been described, especially on melanomas.
A large number of them are peptides presented by MHC complexes.
Their expression often limited to tumor cells make them attractive for immunotherapy
• MAGE-A1 expressed in 40% of melanoma tumors and in some other tumors
• 25 % of the population is HLA-A1 positive => the HLA A1-MAGE-A1 complex is expressed in 10% of all melanoma tumors.
• Not expressed at all in normal tissues (apart from testis, but no HLA expression)
The complex HLA-A1/MAGE-A1
An antibody directed against this complex would be interesting to:
• Check the availability of the complex before immunotherapy (in vitro MAGE-A1 loaded DC)
• As a targeting reagent (immunotoxin/cytokine, T cell retargeting)
Adapted from Garcia KC et al. 1996 Science 274:209–19
The peptide-MHC complex is recognized by the TCR
Separate cloning of the HLA A1 and 2m in intracellular expression vectors
Production of a recombinant complex
Purification of the inclusion bodies from E. coli
Dissolution in 8M urea buffer
Dilution in a reconstitution buffer in the presence of the three partners (reduced and oxidized glutathion)
buffer
• After reconstitution and concentration, the mix is analyzed by gel filtration and SDS-page
In vitroIn vitro Refolding: Results Refolding: Results
HC 2m
1
2
3
1 2 3
Refolding
concentration
50 17 5 2 0.6
0
10
20
30
40
50
60
70
Complexes (µg/ml)
A1MA1 - CTL82/30
A2MA3 - CTL82/30
A1MA1 - CTL413/13
TN
F (
pg
/ml)
TNF CTL 82/30
OD 4
50
nm
0
0.5
1
1.5
2
PBS W6/32 HK HB28 HC-A2 TÜ114 TÜ155
Aggregates
2m
Complex
Strep/biot. Complex
The complex is correctly refoldedThe complex is correctly refolded
ss
b
DTT 50 mM
Round 4
Repertoire: 3.7x1010
Selection from a large non immunized human Fab Selection from a large non immunized human Fab fragment repertoirefragment repertoire
Strep.
Strep.+ pMHC
= Streptavidin
EADPTGHSY EVDPIGHLYPeptide MAGE-A1 Peptide MAGE-A3
0
0.5
1
1.5
2
2.5
A1 D2 G8 Tü 155 HK
OD450 nm
Out of 14 different binders:
11 were pan-reactive
2 had a differential binding (D2)
1 was fully MAGE-A1 specific (G8)
Screen for peptide Screen for peptide specificityspecificity
G8 binds the pMHC complex on cell G8 binds the pMHC complex on cell surfacesurfaceB cells (HLA-A1 positive or neg.) are in vitro loaded with MAGE-
A1 or MAGE-A3 and used in FACS experiment with the phage-antibody G8
HLA-A1- HLA-A1+ HLA-A1+
fd G8
fd H2
LG2-EBV AVL3-EBV MZ2-EBV
Loaded with MAGE-A1 Loaded with MAGE-A3
Fab-G8 can efficiently retarget primary Fab-G8 can efficiently retarget primary human lymphocyteshuman lymphocytes
Fusion of G8 with a signaling element (FcR1) and retroviral transfection of human PBL
A1/MAGE1
Melanoma cells
Fab-G8fusion
Transfectedhuman T-cells
% C
r re
leas
e
Transfected lymphocytes kill MAGE-A1 loaded cells
Transfected lymphocytes kill MAGE-A1 + melanoma cells
Fab-G8 can efficiently retarget primary Fab-G8 can efficiently retarget primary human lymphocyteshuman lymphocytes
Production of cytokines by transfected human T cells upon incubation with MAGE-A1 loaded cells
APD APD + APD +
0
20
40
60
80
100
FLU MAGE-A1 FLU MAGE-A1
APD APD + APD +
0
50
100
150
200
250
Or with MAGE-A1+ melanoma cells
MZ2-MEL3.0 BLM MEL78 SKRC17 cl4
0
10
20
30
40
50
60
MZ2-MEL 3.0 BLM MEL 78 SKRC cl4
0
20
40
60
80
100
120
MAGE-A1+ MAGE-A1+
GEL FILTRATION
Yield: 1.2 mg/l (from periplasmic fraction, after metal affinity chromatography and gel filtration)
Purification of the recombinant Fab fragmentPurification of the recombinant Fab fragment
L FT E L FT E + DTT - DTT
SDS PAGE
45
30
Surface plasmon resonance Surface plasmon resonance measurementsmeasurements
590 604 618 632 646 660
Time (s)
0
100
200
300
400
RU
MAGE-3 (625 nM)
MAGE-1 (625, 542, 459, 375, 292, 208 nM)
kon: 1.8x105 M-1S-1 koff: 45x10-3 S-1
Kd = koff / kon = 250 nM
G8 has a moderate affinity
Affinity maturationAffinity maturation
• 250 nM may be enough for diagnosis purposes (FACS and tissue staining)• but we need at least a 10 fold better affinity for in vivo targeting
The problem
• We want to increase the affinity BUT
• we don’t want to lose the specificity
The new interactions have to be made ONLY with the peptide (around 20% of the interface)
• Chain Shuffling library
Affinity maturation: The libraries Affinity maturation: The libraries
VC VHCH1G8
The G8 light chain is shuffled with a kappa + lamdba repertoire
• CDR H3 spikingEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGGFHYYYYGMDVWGQGTTVTVSS
VH G8
SPIKING: for each base of the codon, 90% WT and 10% of ACGT -> few mutations per clone, scattered over CDR H3
Diversity 3.107
Diversity 2x108
Selection of mutations with additive effect
Selection: ResultsSelection: Results
Name kon (105 M-1s-1) koff (10-3 s-1) Kd (nM)
G8 1.8 45 250 3H2 2.1 16 75hyb3 3.8 5.4 14
-50
0
50
100
150
200
250
300
350
400
640 660 680 700 720 740
Time s
Re
sp
on
se
RU
G8
3H2/ 4E3 (direct selections)
HYB3 (combination)
Fine specificity analysisFine specificity analysis
Affinity-matured clones did not lose their fine specificity
00.20.40.60.8
11.21.41.61.8
TÜ155 G8 lac7 Hyb3 H2
MAGE-A1
MAGE-A3
M3A
M3T
M3S
INF
INFA
INFT
INFS
OD
405n
m
E A D P T G H S Y
E V D P I G H L Y
E A D P I G H L Y
E V D P T G H L Y
E V D P I G H S Y
C T E L K L S D Y
C A E L K L S D Y
C T E L T L S D Y
C T E L K L S S Y
The selected sequencesThe selected sequencesVl sequences
VH sequences
Difficult to localize the crucial mutations (long range effects…)
Fab-G8 model (swissprot)Fab-G8 model (swissprot)
Val111
Gly100
Tyr107
Lys30
VH
VL
ConclusionsConclusions
Abstract
-> We have produced a recombinant version of the tumor-related peptide-MHC complex HLA-A1/MAGE-1 and used it for phage selection (works as well as HLA tetramer to stain and sort specific T cells) -> We have selected a fully human Fab fragment binding to this epitope in vitro (ELISA, BIAcore) and on cell surface (FACS)
-> We have increased the affinity of the antibody by a factor 18 without loss of specificity.
-> We have shown that the Fab fragment can be efficiently used as a surface receptor to retarget human T cells against HLA-A1 / MAGE-A1 positive cells
PerspectivesPerspectives
Crystallography
-> G8 and HLA-A1/ MAGE-A1 have been produced and purified in high amounts for crystallographic studies
->The pMHC-Fab complex can be purified by gel filtration
-> The high affinity version will also be produced