CD8 T cell

1. 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

2. 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 peptide 8-9 aa Heavy chain(35KDa) b2m (12 KDa)

3. 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) The complex HLA-A1/MAGE-A1 • 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)

4. The peptide-MHC complex is recognized by the TCR Adapted from Garcia KC et al. 1996 Science 274:209–19

5. Separate cloning of the HLA A1 and b2m in intracellular expression vectors 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) Production of a recombinant complex

6. In vitro Refolding: Results • After reconstitution and concentration, the mix is analyzed by gel filtration and SDS-page HC b2m 1 2 3 3 Refolding 1 concentration 2 buffer

7. TNF 70 60 50 CTL 82/30 40 TNF (pg/ml) 30 20 A1MA1 - CTL413/13 10 A2MA3 - CTL82/30 0 A1MA1 - CTL82/30 50 17 Aggregates b2m Complex Strep/biot. Complex 5 2 0.6 Complexes (µg/ml) The complex is correctly refolded

8. Selection from a large non immunized human Fab fragment repertoire Repertoire: 3.7x1010 Strep. + pMHC DTT 50 mM ss Strep. b Round 4 = Streptavidin

9. 2.5 OD 450 nm 2 1.5 1 0.5 0 A1 D2 G8 Tü 155 HK Screen for peptide specificity Peptide MAGE-A3 Peptide MAGE-A1 EADPTGHSYEVDPIGHLY Out of 14 different binders: 11 were pan-reactive 2 had a differential binding (D2) 1 was fully MAGE-A1 specific (G8)

10. LG2-EBV AVL3-EBV MZ2-EBV HLA-A1- HLA-A1+ HLA-A1+ fd H2 fd G8 G8 binds the pMHC complex on cell surface B 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 Loaded with MAGE-A3 Loaded with MAGE-A1

11. A1/MAGE1 Fab-G8 fusion Melanoma cells Transfected human T-cells Fab-G8 can efficiently retarget primary human lymphocytes Fusion of G8 with a signaling element (FceR1g) and retroviral transfection of human PBL % Cr release Transfected lymphocytes kill MAGE-A1 loaded cells Transfected lymphocytes kill MAGE-A1 + melanoma cells

12. Or with MAGE-A1+ melanoma cells MAGE-A1+ MAGE-A1+ Fab-G8 can efficiently retarget primary human lymphocytes Production of cytokines by transfected human T cells upon incubation with MAGE-A1 loaded cells

13. + DTT - DTT L FT E L FT E 45 30 SDS PAGE Purification of the recombinant Fab fragment GEL FILTRATION Yield: 1.2 mg/l (from periplasmic fraction, after metal affinity chromatography and gel filtration)

14. RU MAGE-1 (625, 542, 459, 375, 292, 208 nM) 400 300 200 100 MAGE-3 (625 nM) 0 590 604 618 632 646 660 Time (s) Surface plasmon resonance measurements kon: 1.8x105 M-1S-1 koff: 45x10-3 S-1 Kd = koff / kon = 250 nM

15. Affinity maturation G8 has a moderate affinity • 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)

16. Affinity maturation: The libraries • Chain Shuffling library The G8 light chain is shuffled with a kappa + lamdba repertoire VlCl VHCH1 G8 Diversity 2x108 • CDR H3 spiking EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGGFHYYYYGMDVWGQGTTVTVSS 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

17. HYB3 (combination) 3H2/ 4E3 (direct selections) G8 Name kon(105 M-1s-1) koff(10-3 s-1) Kd (nM) G8 1.8 45 250 3H2 2.1 16 75 hyb3 3.8 5.4 14 Selection: Results Selection of mutations with additive effect

18. Fine specificity analysis E A D P T G H S Y E V D P I G H L Y EAD P I G H L Y E V D P T G H L Y E V D P I G HSY C T E L K L S D Y CAE L K L S D Y C T E LTL S D Y C T E L K L SSY MAGE-A1 1.8 1.6 MAGE-A3 OD405nm 1.4 M3A 1.2 M3T 1 0.8 M3S 0.6 INF 0.4 INFA 0.2 0 INFT TÜ155 G8 lac7 Hyb3 H2 INFS Affinity-matured clones did not lose their fine specificity

19. The selected sequences Vl sequences VH sequences Difficult to localize the crucial mutations (long range effects…)

20. Fab-G8 model (swissprot) Lys30 Tyr107 VH Gly100 Val111 VL

21. Conclusions 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

22. Perspectives 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