Website home page picture here The Effects of Induced Aggregation of IgG and Insulin Preparations on In Vitro T Cell Stimulation 2 nd EIP Symposium 17 th -19 th November 29
Immune Responses to Protein Therapeutics T-Dependent T-Independent receptor-mediated internalisation phagocytosis high affinty IgG 5-1nm a) Plasma Cell Immature APC repeating B cell epitopes Antigen PRR activation MHC class II +peptide antigen processing B Cell B Cell d) B Cell b) cytokines Activated CD4+ cytokines Activated CD4+ c) Mature APC costimulatory molecules CD4 TCR Resting CD4+ 2 IgM secretion cytokines PRR activation
T Cell Epitope Identification using Ex Vivo T Cell Assays 1. Human Blood Sample 2. T Cell Subset Depletion 3. Donor Characterization KLH CD4 CD4 5. Donor Selection CD8 4. Optimized Storage in Liquid Nitrogen CD8 PKYVKQNTLKLAT + MHC Class II allotyping HA 37-319 Donor Bank 6. Detection of CD4+ T Cell Responses 3 Proteins Antibodies Peptides % Donor Response 25 2 15 1 5 1 2 3 4 5 6 7 8 9 1 Peptide 3
In Vitro Time Course T Cell Assay Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 Proliferation Proliferation Assay Day 5 Day 6 Day 7 Day 8 Neg. control Sample 1 Sample 2 Pos. control Stimulation index (SI) counts test counts baseline Positive response = SI 2, p<.5 ELISpot 4
Effects of Induced Aggregation on In Vitro T Cell Stimulation Humanized IgG1 and rh Insulin Induced aggregation (Jiskoot lab); IgG -5 x freeze thawing IgG Shaking 5rpm 24 hours Insulin - Gluteraldehyde Insulin -Heating 75 o C 1 hour Test samples and controls for in vitro T cell stimulation 5 donors T cell proliferation/il-2 secretion Analysis of monocyte derived DC phenotype 5
Overview IgG1 Aggregates Lane Formulation Stress conditions 1 1 mg/ml IgG 1 mm phosphate ph 7.2 5 freezethawing cycles 2 Unstressed control 3 1 mg/ml 1 mm citrate 5% (w/v) Sucrose ph 6 Shaking 5 rpm, 24h 4 Unstressed control Structural properties non-covalent aggr. intact secondary & tertiary structure native IgG mainly non-covalent aggr. loss of secondary and tertiary structure hydrophobic aggr. native IgG Quantity /Type of aggregates <.1% particles larger 1µm.2% larger soluble aggr. 1.8% dimers 97.3% monomer 1.5% dimers 98.% monomer 14% large insoluble aggr..5% soluble aggr. 1.3% dimer/trimer 84% monomer 1.5% dimers 98.% monomer 6
Overview Insulin Aggregates Lane Formulation 5 1 mg/ml insulin 5 mm phosphate ph 7.4 Stress conditions Glutaraldehyde crosslinking 6 Unstressed insulin control 7 1 mg/ml insulin 69 mm Na 2 HPO 4 45 mm NaCl 57 µm ZnCl 2 29 µm CaCl 2, ph 7.4 Heating : 1 h 75 C 8 Unstressed insulin control Characteristic of the samples covalent soluble aggregates intact epitopes loss of secondary and tertiary structure native insulin covalent insoluble aggregates epitopes not intact loss of secondary and tertiary structure native insulin Quantity /Type of aggregates 59% sol. aggregates 17% dimer 24% monomer 54% insol. aggregates 14% sol. aggregates 1% dimer 22% monomer 7
8 7 6 5 4 3 2 1 Selection of Donor Haplotypes Frequency of Allotype (%) DRB1*1 DRB1*3 DRB1*4 DRB1*7 DRB1*8 DRB1*9 DRB1*1 DRB1*11 DRB1*12 DRB1*13 DRB1*14 DRB1*15 DRB1*16 DRB3 DRB4 DRB5 8 Leiden Donors World Population
Buffers and Humanized IgG do not Directly Modulate In Vitro T Cell Activation Normalized SI (Sample + KLH/KLH Alone) Normalized SI (to KLH-Specific T Cell Response) 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1..9.8.7.6.5.4.3.2 Buffer alone no protein Non-aggregated IgG1 Max. Min..1. Con Con Glu Con 1 3 5 7 1+IgG 3+IgG buffer alone 75 o C Con IgG1 IgG1 1 donors selected known to respond against KLH Data shown mean SI normalised data for 1 donors (sample plus KLH vs KLH alone) 9
In Vitro T Cell Stimulation with Aggregates SI 2, p<.5 (cpm or spw test vs baseline) + IL-2 ELISpot Proliferation 1
Analysis of all Donor Responses *** <.1 11
In Vitro Matured Mo Derived DC medium CD1a CD11c CCR7 CD4 MHCII CD29 R1 R3 R6 R3 R6 R1 shake R3 R6 R1 glu R1 R3 R6 12 Mature (IL-4/GM-CSNFα) DC Phenotypic Analysis
medium shake glu 13 In Vitro Matured Mo Derived DC CD8 CD83 CD86 1 8 6 4 2 CD8 Mature (IL-4/GM-CSNFα) DC Phenotypic Analysis CD83 5 4 3 2 1 1 8 6 4 2 CD86 CD11c 1 8 6 4 2 5 4 3 2 1 CD1c CD4 1 8 6 4 2 1 8 6 4 2 MHC class II CD29 1 8 6 4 2
Summary Protein aggregates can enhance in vitro T cell stimulation. Enhanced stimulation may be restricted to aggregates with specific properties e.g. size, quantity and/or physiochemical properties. Aggregates induced by shaking IgG1 induced more frequent and higher magnitude T cell responses than aggregates induced by. Large insoluble aggregates and/or loss of structure? T cell tolerance to insulin may influence in vitro responses against induced insulin aggregates an effect that is not necessarily observed with humanized IgG1 (containing engineered V regions). Aggregates do not alter Mo derived DC phenotype after maturation in vitro. Possible effects due to aggregate induced DC licensing, increased protein uptake = more effective presentation to T cells (Ho et al 28, Immunity)? 14
Acknowledgements Leiden Dr Wim Jiskoot Dr Andrea Hawe Dr Ruedeeporn (Sun) Tantipolphan Vasco Filipe Cambridge Dr Christine Bryson Ed Cloake Dr Tim Jones Leiden/Amsterdam Center for Drug Research (LACDR) Division of Drug Delivery Technology Gorlaeus Laboratories Antitope Ltd Babraham Institute Cambridge, UK 15