Serendipities of acquired immunity Nobel Lecture December 7, 2018 - PowerPoint PPT Presentation

Serendipities of acquired immunity Nobel Lecture December 7, 2018 Tasuku Honjo Kyoto University Institute for Advanced Study and Graduate School of Medicine

My family (1955)

Cassini: Earth and Saturn The Day the Earth Smiled “ Earth The telescopic view of Through the brilliance of Saturn’s rings, Cassini caught a glimpse of a far­away Saturn fascinated me. planet and its moon. At a distance of just under 900 million miles, Earth I dreamed of becoming an shines bright among the many stars in the sky, distinguished by its bluish tint. astronomer.” Saturn. Actual photo taken on June 5, 2016 https://www.nasa.gov/mission_pages/cassini/multimedia/pia17171.html

Inspired by biography of Hideyo Noguchi (1876~1928) Rockefeller Univ. ・ Identified Syphilis spirochete as the cause of progressive paralysis ・ Died in Ghana during pursuit of yellow fever pathogen

With Osamu Hayaishi

With Jacques Lucien Monod 1966

Diphtheria toxin inactivates protein synthesis factor by ADP-ribosylation ADP ADP Ribo NAD Ribo ADP DT + Ribo nicotinamide EF-II EF-II + EF-II diphtheria toxin active inactive acts as a catalyst T. Honjo et al., J.Biol.Chem. (1968)

Donald Brown at Carnegie Institution in Baltimore 1971 8

Mystery of immune response in 1950~1970 How can animals generate antibodies specific to an almost infinite number of antigens, including artificial chemicals?

Why can animals generate specific antibodies to almost all unexperienced compounds? nitrobenzene Anti-N-benzene Ab -protein nitrophenol Anti-N-phenol Ab -protein anthracene Anti-anthracene Ab -protein toluene diisocyanate Anti-toluene-DIC Ab -protein oxazolone Anti-oxazolone Ab -protein Modified from K. Landsteiner 1919­22

Structure of antibody identified by 1970 Variable region (V) Constant region (C) (Antigen­recognition site) (Antibody class determination) C C C C H chain (heavy chain) L chain (light chain) 11

Philip Leder at NIH 1973

VDJ recombination generates V region repertoire during differentiation C µ C δ V H D H J H S Constant region VDJ recombination IgM IgD S B cell C. Brack et al., Cell (1978) S. Tonegawa

University of Tokyo, Dept. of Nutrition (Professor Yoshinaga Mano) 1974

Antibody memory generation by vaccine (antigen) administration Primary immunization Secondary immunization Primary response Secondary response 10000 1000 100 Titer 10 1 0 Days 0 7 14 21 28 35 42 Increase in antigen binding capability (somatic hypermutation of variable region)

Somatic hypermutation (SHM) mutates V region and only good antibodies are selected Y Y Y Y Y Y strongest Y B cell Y Y Y Y pathogen Y Y Y Darwinian principle

Antibody memory generation by vaccine (antigen) administration Primary immunization Secondary immunization Primary response Secondary response 10000 1000 IgG 100 Titer IgM 10 1 0 Days 0 7 14 21 28 35 42 Increase in antigen binding capability (somatic hypermutation of variable region) Increase in antigen processing ability ? (class switch of constant region)

Class switching changes the H chain constant region and antibody function C C C C C C Variable C C IgA Gut bacteria region (V) Constant region (C) C C C C C C C C Y C C C C IgG H chain CSR Virus IgM L chain C C C C C Y C C C C C Y IgE Parasite

Class switch recombination takes place by deletion of a large DNA segment µ δ γ γ γ γ ε α V C C C 3 C 1 C 2b C 2a C C H S S S S S S S class switch recombination (CSR) γ C 3 γ C 1 S δ C µ C γ C 2b looped­out T. Kataoka A. Shimizu circular DNA S S VH γ ε α C 2a C C T. Honjo & T. Kataoka, PNAS (1978) T. Kataoka et al., PNAS (1980) A. Shimizu et al., Cell (1982)

Matthias Wabl, Göran Möller (coorganizer) Leroy Hood

Discovery of AID by comparison of gene expression before and after CSR Y Y IgM IgA I Stimulation with CD40L, IL­4, TGF­ β 1 Comparison of AID expressed gene transcripts A ctivation I nduced cytidine D eaminase B cells AID M. Muramatsu et al., J.Biol. Chem. (1999) Expressed in germinal center

Defective IgG response to antigens (Sheep Red Blood Cell) in AID deficient mice 0.7 IgG1 IgM 0.6 AID +/- AID +/- AID -/- AID -/- 0.5 Antibody titer Antibody titer 0.4 0.3 0.2 0.1 0 0 5 10 14 21 0 5 10 14 21 Days Days M. Muramatsu et al., Cell (2000)

AID deficient mice fail to accumulate mutations CDR1 CDR2 80 γ 1 AID +/- R S Mutation frequency(%) 0 µ 40 AID +/- 0 40 µ AID -/- 0 No mutation Q1 W33 R98 VH186.2 sequence (residue) K. Kinoshita M. Muramatsu et al., Cell (2000)

・ AID deficiency in human is the cause of Hyper IgM Syndrome Type II: exactly the same phenotypes as mouse. P. Revy et al., Cell (2000) ・ Thus, AID is the enzyme that engraves antigen memory in the antibody gene, the mechanistic basis of vaccination.

AID engraves Ab memory in the genome for effective vaccination µ µ δ δ γ γ γ γ γ γ γ γ ε ε α α C C C 3 C 1 C 2b C 2a C C V D J C C C 3 C 1 C 2b C 2a C C V D J H H H H S S S S S S S S S S S S S S Natural Natural Natural VDJ VDJ Ab Ab Ab RAGs RAGs recombination recombination Repertoire formation somatic class switch recombination AID Pathogen hypermutation (CSR) chromosomal product (SHM) mutated ＋ V gene Memory Ab looped­out Antibody memory tumors circular DNA formation

Immune surveillance against cancer Proposed by Sir Frank Macfarlane Burnet (1970) However, numerous attempts to develop immunotherapy were unsuccessful.

Cancer immunotherapy by boosting accelerators has not given convincing clinical outcomes 1. Cancer vaccine 2. In vitro activation of T lymphocytes 3. Cytokine treatment (IFN γ , IL­2, IL­12 etc) This was because no immune brake molecules were known before 1995

Brakes and accelerators control immune reactions like those in a car drive stop action mode action phase ignition parking brake ON/OFF parking ［ CD28 ］ ［ CTLA4 ］ ［ Activation ］ ［ Drastic ］ 〜 100k/h accelerator brake driving ［ ICOS ］ ［ PD­1 ］ ［ Attack ］ ［ Mild ］

Discovery of PD-1 (programmed death-1) cDNA Structure of cytoplasmic tail suggests PD­1 is a surface signaling molecule Y. Ishida Y. Agata Y. Ishida et al., EMBO J. (1992)

A long journey to understanding the function of PD-1 ・ 1994 PD­1 knock out (KO) on mixed background mice, no phenotype change ・ 1996 PD­1 KO on C57BL/6, no phenotype change for 6M But over­response to antigen stimulation ・ 1997 Nephritis and arthritis after 5M in PD­1 KO x lpr/lpr background ・ 1998 Clear autoimmunity in PD­1 KO by 14M

PD-1 is a negative regulator C57BL/6 BALB/c WT PD­1 KO PD­1 KO (Knock Out) Nephritis Arthritis Dilated cardiomyopathy Y. Nishimura et al., Immunity (1999) MRL NOD NODxPD­1 KO MRLxPD­1 KO Diabetes Myocarditis T. Okazaki et al., Nat. Medicine (2003) J. Wang et al. , Int. Immunol. (2010)

Molecular mechanism of immune inhibition by PD-1 signaling Antigen receptor Antigen Coreceptor PD­1 α β N N PD­L1 S-S S-S N N N γ/ε δ/ε S-S S-S S-S S-S S-S S-S S-S ζ ζ ITSM Y P P P P Kinase Y P P P P P ZAP70 negative Activation T. Okazaki et al. , signal signal PNAS （ 2001 ）

Balance between immune surveillance and immune tolerance Immune surveillance Immune tolerance Hyper immunity Immune suppression PD­1 blockade Treatment of autoimmunity Treatment of infectious Higher risk of infectious diseases diseases and cancer and cancer Risk of autoimmunity

Inhibition of tumorigenesis of myeloma (J558L) in PD-1 -/- mice J558L J558L BALB/c BALB/c PD­1 ­/­ WT 10000 N=4 N=4 Tumor volume (mm 3 ) 7500 5000 2500 0 5 10 15 2 0 5 10 15 2 0 Days after inoculation Y. Iwai et al. , PNAS (2002)

Inhibition of tumorigenesis of P815/ PD-L1 by anti-PD-L1 P815/PD­L1 DBA/2 Tumor growth Survival rate Rat IgG a­PD­L1 N. Minato 2500 2500 (%) N=10 n=10 n=10 N=10 Tumor volume (mm3) 100 Tumor volume (mm 3 ) 2000 2000 80 1500 1500 60 a­PD­L1 1000 1000 40 500 500 20 Rat IgG 0 0 0 0 40 60 80 0 5 10 15 20 0 5 10 15 20 20 0 5 10 15 20 0 5 10 15 20 Days after inoculation Days after inoculation Days after inoculation

PD-1 blockade inhibits metastasis of B16 melanoma (mouse model) Spleen to liver 8 weight of liver (g) 6 4 2 0 WT WT Anti PD­1 Ab Anti PD­1 Ab Y. Iwai et al. , Int. Immunol. （ 2005 ）

PD-1 blockade by antibody against either PD-1 or PD-L1 can cure cancer Tumor/ Killer T cell Other immune (CD8 + ) infected cell cells anti anti anti anti PD­L1 PD­1 PD­1 PD­L1 PD­L1 PD­1 PD­1 PD­L1 - - SHP2 + + MHC TCR TCR MHC PD­L1 is expressed on various immune cells

Human anti-PD-1 antibody Synthesized in mice containing human immunoglobulin gene by Medarex Subclass: IgG4S228P mutant IgG4 (S228P) stabilizes the protein and reduces ADCC (antibody­dependent cell­mediated cytotoxicity) KD = 2.6 nmol/L Named Nivolumab Approved as Investigation New Drug by FDA (USA; Aug 1, 2006)