Biased Antibody Repertoires From Concept to Implementation Juan
Biased Antibody Repertoires: From Concept to Implementation Juan C. Almagro, Ph. D Department of Biological Sciences Florida International University Miami, Florida May - July 2005
Plan of the talk 1. Statement of the problem 2. Structure-Function relationships in antibodies 3. Design and Validation of a VH Repertoire with Tailored Diversity for Protein and Peptide Antigens 4. Design and Validation of Topography-Biased Antibody Libraries
Statement of the problem • Today: ~ 40 K sequences are available in databases, including the complete repertoire of germline genes from several species (IMGT) • Today: ~ 600 structures are available at PDB (ABG; Antibody: Structurefunction web site and IMGT) However, we cannot predict the specificity of a given antibody sequence or structure. Hence, Our understanding of the evolution of the antibody repertoire is limited antibodies cannot be designed de novo.
Plan of the talk 1. Statement of the problem 2. Structure-Function relationships in antibodies 3. Design and Validation of a VH Repertoire with Tailored Diversity for Protein and Peptide Antigens 4. Design and Validation of Topography-Biased Antibody Libraries
Canonical structures (Chothia and Lesk, J. Mol. Biol. 196: 901, ‘ 87) Taken from Andreas Plückthun’s home page, with permission
Type 3 L 1 Type 1
Predicting the Specificity of Antibody Sequences Based on the Structure of the Antigen-Binding Site ~ 4, 000 antibody sequences Complete sequences VL: VH dimmers Canonical structure in L 1, L 2, L 3, H 1 and H 2 381 VL: VH sequences
Canonical structure classes in the known sequences Expected Found L 1: 5 L 2: 1 L 3: 5 x x 25 H 1: 3 H 2: 4 x 12 = 300 10 Canonical Structure Classes make ~ 90% of the sample Only L 1 and H 2 contribute to the structural diversity Vargas-Madrazo et al. , J. Mol. Biol. 254: 497, ‘ 95
Canonical structure classes classified in gross specificities Antigen size Vargas-Madrazo et al. , J. Mol. Biol. 254: 497, ‘ 95
Topography-specificity relationship Anti-protein Anti-peptide Anti-hapten Model to correlate loop lengths (in particular L 1 and H 2) with the specificity
Predicting the Specificity of Antibody Sequences Based on the Structure of the Antigen-Binding Site ~ 300 structures 59 unique antibody structures 19 anti-protein 18 anti-peptide 22 anti-hapten Determine residues in contact
Residues in contact with proteins, peptides and haptens SDR usage Some positions in the antigen-binding site interact with the antigen very often (> 70% of the antibodies). Others do so with a frequency between 30% and 70%. L 1 L 2 L 3 A third group interacts with the antigen infrequently (<30%). SDR usage The frequency of contacts differs depending upon the type of antigen with which the antibody interacts. H 1 H 2 H 3 Almagro. J. Mol. Recognit. 17: 132, ‘ 04
Contact usage - specificity relationship Anti-protein Anti-peptide Anti-hapten Model to create diversity in the antigen-binding site as a function of the specificity
Conclusions I 1. Model to correlate the structure of the antigen binding site with its specificity. 2. Guide for tailoring the antigen-binding site diversity depending upon the type of antigen the antibody interacts with.
Plan of the talk 1. Statement of the problem 2. Structure-Function relationships in antibodies 3. Design and Validation of a VH Repertoire with Tailored Diversity for Protein and Peptide Antigens 4. Design and Validation of Topography-Biased Antibody Libraries
VH Repertoire with Tailored Diversity Dp 47 scaffold Full diversity: 20 aa + 1 amber codon in positions often found in contact with proteins and peptides R/K All the germline genes have R at this To explore all amino acid variants in position, except dp 47 that has K positions with high contact usage Limited Diversity: YDAS (Felluose et al. , PNAS 34, 12467, ‘ 04) To simplify the diversity in positions of medium usage while avoiding stop codons Theoretical diversity: 720 x 94 x 2 = 6. 7 x 1014 variants
Construction of the VH repertoire The repertoire was synthesized by overlapping PCR (Stemmer et al. , Gene. 164: 49, ‘ 95) in a single-step PCR reaction by using 10 internal oligonucleotides and two amplification primers 1 10 20 30 40 50 60 70 80 90 100 110 |. . . . |. . abc. . |. . . . |a. . . . |. . 1 3 5 7 9 Leader |||||||<<<<|||||||<<<<<<|||||||<<<<<||||||| |||||||<<< LLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTF OOOOMOWVRQAPGKGLEWVS OIOOOOGOTOYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA OOOOOYFDYWGQGTLVTVSSGGG >>>>>|||||||>>>>|||||||>>>|||||||>|||||||linker 2 4 6 8 10 D 1. 3 -VL VH repertoire linker p. IT * (His)5 Size: 3 x 108 members
Validation of the VH repertoire Selection Chimeric library Washed away Trypsin to elute non-bound Ф Characterization HEL-coated immunotubes Polyclonal ELISA In Round 2, the ELISA signal was similar to D 1. 3 displayed on the phage
Frequency of sc. Fv’s (hit rate) select clones Grow in 2 x. TY IPTG Test in ELISA
Expression and relative affinity *Relative Scale: 1 - (Max-O. D. / Max - Min) The chimeric sc. Fvs are 7 -9 times better expressed than D 1. 3 as suggested by the ED 50 The affinity for HEL may be similar to D 1. 3 as suggested by the slope of the curves HEL-D 1. 3 affinity 5 n. M ( Foote and Winter, J. Mol. Biol. 224: 487, ‘ 92)
Conclusions II 1. A VH repertoire with tailored diversity 2. to recognize proteins and peptides was designed and constructed 2. It was cloned with the VL chain of D 1. 3 to yield a chimeric library of 3 x 108 members. 3. After the second round of selection on HEL-coated Immunotubes, diverse sc. Fvs against HEL were obtained, thus validating the library as source of VH domains. 4. The sc. Fvs dominating the population are well expressed in E. coli and may have affinities in the n. M range.
Plan of the talk 1. Statement of the problem 2. Structure-Function relationships in antibodies 3. Design and Validation of a VH Repertoire with Tailored Diversity for Protein and Peptide Antigens 4. Design and Validation of Topography-Biased Antibody Libraries
Topography biased antibody libraries Dp 47 scaffold with tailored diversity for proteins and peptides (Almagro et al. , J. Mol. Biol. Submitted) Theoretical diversity: 2. 1 x 1010 Invariant VL chain with a long L 1 Invariant VL chain with a short L 1
Invariant VL chains Use Frequency (%) Short L 1 Long L 1 A 27 Human Germline Genes (IGMT) Graft L 1 of B 3 in A 27 The difference between repertoires is reduced to one insertion of 5 amino acids at the tip of L 1 and 5 mutations in L 1, positions: 28, 29, 30 a and 31
Combination with different invariant VL chains VH repertoire linker A 27/Jk 1 p. IT Anti-protein repertoire * (His)5 Size: 3. 6 x 108 members Insertion of 5 aminoacids at L 1 VH repertoire Anti-peptide repertoire linker A 27/Jk 1 mod p. IT * (His)5 Size: 6 x 107 members
Panel of Selectors V 3 loop gp 120 (V 3) 14 aa; 1. 6 KDa Hen Egg White Lysozyme (HEL) 129 aa; 14. 3 KDa Bovine Serum Albumin (BSA) 583 aa; 66. 4 KDa V 3 -BSA conjugate ND Selections conducted as described for VH-D 1. 3
Polyclonal ELISA after Round 3 V 3 selections BSA selections V 3 -BSA selections HEL selections Red: Anti-peptide library Blue: Anti-protein library
Frequency of positive clones 91/96 92/96 select clones 32/96 64/192 Grow in 2 x. TY 42/192 0/96 3/96 0/96 IPTG Test in ELISA Anti-peptide library yields more sc. Fvs for V 3 and V 3 -BSA than for proteins Anti-protein library yields more sc. Fvs for proteins than for. V 3 or V 3 -BSA
Unique sc. Fvs Determined by DNA sequencing
Specificity of unique clones V 3 and V 3 -BSA selections O. D. 450 nm BSA selections The sc. Fv selected from the anti-peptide lib. on V 3 is specific for V 3 and V 3 -BSA Sc. Fvs selected on proteins are specifics O. D. 450 nm The sc. Fv selected from the anti-protein lib. on V 3 -BSA is specific for the carrier HEL selections
Expression and Relative Affinity O. D. 450 nm HEL selections Different dynamic ranges, better slopes and higher ED 50 indicating differences in binding (different epitopes? ) Sc. Fvs from SL 1 (anti-protein library) look better than the those isolated from LL 1 (anti-peptide library)
General Conclusions 1. Anti-protein library produced diverse specific sc. Fvs against two protein models: BSA and HEL. 2. Anti-protein library did not produce sc. Fvs against the peptide model, free or conjugated. Only against the carrier. 3. Anti-peptide library produced specific sc. Fvs against the peptide. 4. Anti-peptide library produced less binders against HEL than the anti-protein library. 5. Anti-peptide library did not produce sc. Fvs against BSA and against HEL produced less binders than the anti-protein library. 6. Together, these results suggest that antibody libraries can be biased toward the recognition of different kinds of antigens based on structural principles
Acknowledgments Florida International University Alvaro Velandia Matt Osentoski Sylvia L Smith National University of Mexico Luisa Fernadez Alejandra Blancas Enesto Ortiz Baltazar Beceril Lourival Possani Alejandro Alagon This work was supported by: • Grant 1 R 03 AI 057752 -01 from NIH/NIAID • Sub-Contract DAAD 13 -03 -C-0065 from CBD/USF.
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