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