Emerging Antibody Approaches

Transcription

1 Emerging Antibody Approaches Sudhir Paul University of Texas Houston Medical School With collaborations from Stephanie Planque, Yukie Mitsuda, Sari Sonoda, Sarah Murphy, Eric Brown, Yasuhiro Nishiyama (U Texas), Einar Sigurdsson (New York U), Ken Fukuchi (U Illinois), Carl Hanson (California PHS), Richard Massey (CoBio) and investigators named in our research publications Funding: NIH, Society for Engineered Negligible Senescence, Covalent Bioscience Inc Conflicts: SP has a financial interest in patents and in Covalent Bioscience Inc

2 Antibody Phylogeny, Classes and Synthesis Immunogen B cell clonal selection

3 Monoclonal Antibodies (MAbs) Specific neutralizing activity due to binding of antigenic epitope at light and heavy chain variable domains Side effects: inflammation (MAbs to amyloid β), excessive self-antigen inhibition (MAbs to VEGF, α4-integrin), cross-reaction with other antigens Generally IgG class, developed by immunogeninduced adaptive immunity processes Produced by a single B cell clone (unlike polyclonal antibodies) chemically homogeneous Magic bullets (required 25 years of development) now a ~$ 25 billion industry Large amounts needed - expensive Ab + Ag Ab Ag Fv: antigen binding Fc: accessory immune pathways Murine Fc is replaced by human Fc to reduce HAMA

4 Polyclonal Antibodies Mixtures of Abs produced by diverse B cells (generally IgG class from blood) Specificity of anti-microbial Abs is generated by immunogen-induced adaptive immunity - antisera to toxins, rabies and tetanus Intravenous immune globulin (IVIG) therapy for autoimmune diseases and infections (large amounts needed for therapy) Whole may be greater than sum of the parts Empirical findings of innate beneficial antibodies to non-self and self antigens produced without adaptive immunity. Discovery of innate catalytic antibodies evolved by Darwinian principles over millions of years

5 2014: EFFICACIOUS, SAFE PERMABODIES AND CATABODIES V domain: Large, diverse repertoire FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Innate Catabodies Electrophilic vaccines Acquired Immunity, days Permabodies Catabodies Missing link Innate Immunity, 460 million years Jawed Fish Mammals Humans Antigen Antibody (Reversible binding) Permabody (Permanent binding) Catabody (Catalytic) Covalent Bioscience Inc 2/28/2014 slide 5

6 1994: Pathogenic Catabodies to a Self-Antigen VIP: H-S-D-A-V-F-T-D-N-Y-T-R-L-R-K-Q-M-A-V-K-K-Y-L-N-S-I-L-N-NH I VIP hydrolysis, % [C], nm Wild Type S27a:A H93:A D1:A 300 CATALYTIC ANTIBODIES Antigen Fragments Does the immune system make beneficial catabodies to toxic self antigens? Antibody PERMANENT MULTI-CYCLE

7 Phosphonate CD19 Merged Merged -μ -κ/λ Catalytic rate, M E-A-R-AMC/ g Ig IgM IgG Time, h IgM IgG switch Loss of catalytic activity

8 Age Associated Protein Misfolding Diseases 27 proteins form misfolded amyloids 40 amyloid associated diseases, e.g., Alzheimer disease (amyloid β and tau ), SSA (transthyretin, atrial natriuretic peptide), Parkinson s disease (αsynuclein), Huntington s disease (huntingtin) No physiological function for age associated amyloid formation Tissues affected: brain, heart, lungs, GI tract, kidney, joints. Amyloids deposit within and outside cells. Particulate/soluble amyloid disrupts anatomy integrity, homeostasis No effective therapy or cure Target: Amyloid β Aβ peptides produced by APP processing Form insoluble amyloid plaques in brain Soluble state Toxic oligomers/fibrils with neoepitopes Soluble Aβ species kill neurons

9 Amyloid β Cleaving Catabodies Increased catabodies in old age A 40 cleaved, nm/h/ M Ab Young, < 35 y Old, > 72 y * ** IgGs IgMs Increased catabodies in Alzheimer fmol A /h/mg IgM P< IgG IgM AD Nondemented

10 Beneficial Catabodies to Amyloid β Aβ40 aggregation Neurotoxicity Catalytic IgM Non catalytic IgM 100 Cell viability, % 50 1µm 1µm Antibody Number/100 µm 2 oligomers protofibrils short fibrils Catalytic IgM 22.7 ± 3.5* 7.8 ± 2.52** 2.7 ± 0.58 Non catalytic IgM 81.3 ± ± ± Diluent IgM alone A 40 alone IgM + A 40 D A E F R H D S G Y E V H H Q K L V F F A E D V G S N K G A I I G L M V G G V V Aβ40 scissile bonds determined by HPLC/mass spectroscopy

11 Physiological IgM Catabodies Selective for Transthyretin Amyloid 14 kd blood protein protein that binds and transports retinol binding protein and thyroid hormones Physiological state is a soluble homotetramer (phyttr, ~55kD) Misfolded TTR (misttr) forms pathogenic amyloid fibrils in ~20% of humans >80 y (senile systemic amyloidosis, SSA), a disease involving frequent polyneuropathy, cardiomyopathy and systemic organ failure Associated with carpal tunnel syndrome at younger age Mutant TTRs with increased aggregation rates cause familial amyloidosis 1mer TTR hydrolyzed, nm Pre-aggregatedTTR mer 13kD 10kD 7kD Non-aggregated TTR // IgM IgG IgA IgM IgG IgA Residual turbidity, % Pre-aggregated TTR + diluent Pre-aggregated TTR + IgM 1802 Pre-aggregated TTR + IgM 1819 Non-aggregated TTR + IgM Incubation time, h

12 Brain Aβ Clearance by Catabodies Without Counteracting Adverse Effects Intrabrain IgV 2E6 injection * Non injected Intravenous IgV injection (10 days) * A plaque burden relative to left neocortex hemisphere (%) A Plaque burden, % nigv 2E6 injected aigv MMF6 aigv 2E6 Gene therapy IgV5D3 PBS nigv 2E6 nigv MMF6 aigv 2E6 aigv MMF6 No microglial activation No microbleeds Bleeds per section Microgliosis score aigv 2E6 aigv MMF6 aigv 2E6 aigv MMF6

13 Catalytic Immunotherapy Aβ clearance FcRγ Aβ fragments inflammatory Microglia mediators Inflammation Catalytic clearance of brain Aβ aggregates and peripheral of Aβ monomers X No harmful microglial activation X No microvascular dysfunction Inflammation, microbleeds Brain Antibody influx FcRn Aß deposition, Microbleeds Aß Influx RAGE LRP-1 Aß Efflux? Endothelium Blood Aβ fragments Aβ fragments Aβ clearance Aβ clearance Intravenous catalytic Ig infusion

14 Natural Catabody Defense System (CADSys) Recombinational innate V domain repertoire is ~10 billion); 30,000 genes for all other proteins Diverse innate catabody specificities developed over 500 million years

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