1. Affinity-matured MHC-peptide specific phage-antibodies for targeting T-cell rejection antigens on melanoma cells CD8 T cell Target cell P P. Chames G. Rosas L. Rem R. Willemsen R. Bolhuis HR. Hoogenboom

2. 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

3. 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)

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

5. 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)

6. buffer After reconstitution and concentration, the mix is analyzed by gel filtration and SDS-page In vitro Refolding: Results HC 2m 1 2 3 123 Refolding concentration

7. 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 TNF (pg/ml) TNF CTL 82/30 Aggregates 2m Complex Strep/biot. Complex The complex is correctly refolded

8. ss b DTT 50 mM Round 4 Repertoire: 3.7x10 10 Selection from a large non immunized human Fab fragment repertoire Strep. + pMHC = Streptavidin

9. EADPTGHSY EVDPIGHLY Peptide MAGE-A1 Peptide MAGE-A3 0 0.5 1 1.5 2 2.5 A1D2G8Tü 155HK OD 450 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 specificity

10. 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 HLA-A1- HLA-A1+ HLA-A1+ fd G8 fd H2 LG2-EBV AVL3-EBV MZ2-EBV Loaded with MAGE-A1 Loaded with MAGE-A3

11. Fab-G8 can efficiently retarget primary human lymphocytes Fusion of G8 with a signaling element (Fc R1 ) and retroviral transfection of human PBL A1/MAGE1 Melanoma cells Fab-G8 fusion Transfected human T-cells % Cr release Transfected lymphocytes kill MAGE-A1 loaded cells Transfected lymphocytes kill MAGE-A1 + melanoma cells

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

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

14. Surface plasmon resonance measurements 590604618632646660 Time (s) 0 100 200 300 400 RU MAGE-3 (625 nM) MAGE-1 (625, 542, 459, 375, 292, 208 nM) k on : 1.8x10 5 M -1 S -1 k off : 45x10 -3 S -1 K d = k off / k on = 250 nM

15. G8 has a moderate affinity Affinity 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 dont want to lose the specificity The new interactions have to be made ONLY with the peptide (around 20% of the interface)

16. Chain Shuffling library Affinity maturation: The libraries V C VHCH1 G8 The G8 light chain is shuffled with a kappa + lamdba repertoire CDR H3 spiking EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAP GKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTAVYYCAR GGGFHYYYYGMDV WGQGTTVTVSS VH G8 SPIKING: for each base of the codon, 90% WT and 10% of ACGT -> few mutations per clone, scattered over CDR H3 Diversity 3.10 7 Diversity 2x10 8

17. Selection of mutations with additive effect Selection: Results Namek on (10 5 M -1 s -1 ) k off (10 -3 s -1 ) K d (nM) G81.845250 3H22.11675 hyb33.85.414 G8 3H2/ 4E3 (direct selections) HYB3 (combination)

18. Fine specificity analysis Affinity-matured clones did not lose their fine specificity 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 TÜ155G8lac7Hyb3H2 MAGE-A1 MAGE-A3 M3A M3T M3S INF INFA INFT INFS OD 405nm 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

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

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