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

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Serendipities of acquired immunity

Tasuku Honjo

December 7, 2018

Nobel Lecture

Kyoto University Institute for Advanced Study and Graduate School of Medicine

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My family (1955)

SLIDE 3 Saturn. Actual photo taken on June 5, 2016 https://www.nasa.gov/mission_pages/cassini/multimedia/pia17171.html

Cassini: Earth and Saturn The Day the Earth Smiled

Through the brilliance of Saturn’s rings, Cassini caught a glimpse of a faraway planet and its moon. At a distance of just under 900 million miles, Earth shines bright among the many stars in the sky, distinguished by its bluish tint. Earth

The telescopic view of Saturn fascinated me. I dreamed of becoming an astronomer.”

“

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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

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With Osamu Hayaishi

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With Jacques Lucien Monod 1966

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Diphtheria toxin inactivates protein synthesis factor by ADP-ribosylation

T. Honjo et al., J.Biol.Chem. (1968)

active inactive

NAD

+

EF-II

nicotinamide

EF-II Ribo ADP EF-II

diphtheria toxin acts as a catalyst

Ribo ADP Ribo ADP DT

+

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Donald Brown at Carnegie Institution in Baltimore 1971

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Mystery of immune response in 1950~1970 How can animals generate antibodies specific to an almost infinite number

f antigens, including artificial

chemicals?

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Why can animals generate specific antibodies to almost all unexperienced compounds?

Anti-N-benzene Ab Anti-N-phenol Ab Anti-anthracene Ab Anti-toluene-DIC Ab Anti-oxazolone Ab nitrobenzene

protein

nitrophenol

protein

anthracene

protein

toluene diisocyanate

protein
xazolone
protein

Modified from K. Landsteiner 191922

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Structure of antibody identified by 1970

H chain (heavy chain) L chain (light chain) Constant region (C)

(Antibody class determination)

Variable region (V)

(Antigenrecognition site)

C C C C

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Philip Leder at NIH 1973

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C. Brack et al., Cell (1978)

VDJ recombination generates V region repertoire during differentiation

VDJ recombination

JH Cµ

S

Cδ DH VH

S

Constant region

B cell IgD IgM

S. Tonegawa

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

SLIDE 15 10000 1000 100 10 1 7 14 21 28 35 42

Primary response Secondary response

Titer Days

Primary immunization Secondary immunization

Antibody memory generation by vaccine (antigen) administration

Increase in antigen binding capability (somatic hypermutation of variable region)

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Somatic hypermutation (SHM) mutates V region and only good antibodies are selected

Y Y Y Y Y pathogen

Y

strongest Y Y Y Y Y Y Y Y

Darwinian principle

B cell

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Increase in antigen processing ability (class switch of constant region)

10000 1000 100 10 1 7 14 21 28 35 42

Primary response Secondary response

Titer Days

IgM IgG Primary immunization Secondary immunization

Antibody memory generation by

vaccine (antigen) administration

Increase in antigen binding capability (somatic hypermutation of variable region)

?

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Class switching changes the H chain constant region and antibody function

L chain H chain

C C C C

Constant region (C) Variable region (V) IgM

C C C C

CSR

C C C C

IgG

C C C C C C C C

IgA

C C C C C C C C

IgE Y Y Y Gut bacteria Parasite Virus

C C

SLIDE 19 class switch recombination (CSR) loopedout circular DNA

Class switch recombination takes place by deletion of a large DNA segment

S S S S S S S V µ δ γ ε α γ γ γ H C C C 3 C C C 1 C 2b C 2a µ C δ C γ C 3 γ C 1 γ C 2b VH γ C 2a ε C α C S S S

T. Honjo & T. Kataoka, PNAS (1978)
T. Kataoka et al., PNAS (1980)
A. Shimizu et al., Cell (1982)
T. Kataoka
A. Shimizu

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Matthias Wabl, Göran Möller (coorganizer) Leroy Hood

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Y I Y

Discovery of AID by comparison of gene expression before and after CSR

Stimulation with CD40L, IL4, TGFβ1

M. Muramatsu et al., J.Biol. Chem. (1999)

IgM IgA Activation Induced cytidine Deaminase AID

AID B cells

Expressed in germinal center Comparison of expressed gene transcripts

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Defective IgG response to antigens (Sheep Red Blood Cell) in AID deficient mice

0.1 0.2 0.3 0.4 0.5 0.6 0.7 21 5 10 14 Days

IgM

21 5 10 14

IgG1

Antibody titer

AID +/- AID -/- AID+/- AID-/-

Days Antibody titer

M. Muramatsu et al., Cell (2000)

SLIDE 23 VH186.2 sequence (residue) 40 Q1 R98 W33 40 80

CDR1 CDR2

R S

γ1

µ µ

Mutation frequency(%)

AID+/-

AID+/- AID-/-

No mutation

AID deficient mice fail to accumulate mutations

K. Kinoshita M. Muramatsu et al., Cell (2000)

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・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.

SLIDE 25 VDJ recombination V D J µ δ γ ε α γ γ γ H H C C C 3 C C C 1 C 2b C 2a S S S S S S S

RAGs

Natural Ab Natural Ab Pathogen somatic hypermutation (SHM) mutated V gene class switch recombination (CSR) chromosomal product ＋

Antibody memory formation

loopedout circular DNA Memory Ab VDJ recombination V D J µ δ γ ε α γ γ γ H H C C C 3 C C C 1 C 2b C 2a

Repertoire formation

S S S S S S S

RAGs

Natural Ab tumors

AID engraves Ab memory in the genome for effective vaccination

AID

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Immune surveillance against cancer

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

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Cancer immunotherapy by boosting accelerators has not given convincing clinical outcomes

1. Cancer vaccine
2. In vitro activation of T lymphocytes
3. Cytokine treatment (IFNγ, IL2, IL12 etc)

This was because no immune brake molecules were known before 1995

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driving ［Attack］ accelerator ［ICOS］ brake ［PD1］〜100k/h parking ［Activation］ ignition ［CD28］ parking brake ［CTLA4］ ON/OFF

drive stop action mode

Brakes and accelerators control immune reactions like those in a car

［Drastic］［Mild］

action phase

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Y. Ishida et al., EMBO J. (1992)

Discovery of PD-1 (programmed death-1) cDNA

Structure of cytoplasmic tail suggests PD1 is a surface signaling molecule

Y. Ishida Y. Agata

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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 Nephritis and arthritis after 5M in PD1 KO x lpr/lpr background ・1998 ・1997 But overresponse to antigen stimulation Clear autoimmunity in PD1 KO by 14M

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PD-1 is a negative regulator

PD1 KO (Knock Out) Nephritis Arthritis

Y. Nishimura

et al., Immunity (1999) Dilated cardiomyopathy PD1 KO WT BALB/c NOD Diabetes NODxPD1 KO MRL Myocarditis

T. Okazaki et al., Nat. Medicine (2003)
J. Wang et al., Int. Immunol. (2010)

MRLxPD1 KO C57BL/6

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Molecular mechanism of immune inhibition by PD-1 signaling

N N

PD1

N N N

α β γ/ε δ/ε ζ ζ

Antigen receptor negative signal

P P P P

Coreceptor

T. Okazaki et al.,

PNAS（2001） ITSM Activation signal Kinase ZAP70

S-S S-S S-S S-S S-S S-S S-S S-S S-S P Y Y

Antigen

P P P P

PDL1

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Balance between immune surveillance and immune tolerance

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

SLIDE 34 Days after inoculation 5 10 15 2

Inhibition of tumorigenesis of myeloma (J558L) in PD-1-/- mice

Y. Iwai et al., PNAS (2002)

J558L BALB/c WT

Tumor volume (mm3) 5 10 15 2 N=4

J558L BALB/c PD1/

N=4 10000 7500 5000 2500

SLIDE 35 Tumor volume (mm3) Days after inoculation 5 10 15 20 500 1000 1500 2000 2500 5 10 15 20 n=10 n=10

Rat IgG P815/PDL1 DBA/2 aPDL1

Inhibition of tumorigenesis of P815/PD-L1 by anti-PD-L1

Tumor volume (mm3) 2500 2000 1500 1000 500 5 10 15 20 5 10 15 20 N=10 N=10 Days after inoculation

Tumor growth Survival rate

(%)

Rat IgG aPDL1

100 20 40 60 80 20 40 60 80 Days after inoculation

N. Minato

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Y. Iwai et al., Int. Immunol. （2005）

PD-1 blockade inhibits metastasis

f B16 melanoma (mouse model)

weight of liver (g)

2 4 6 8

WT Spleen to liver

Anti PD1 Ab

WT

Anti PD1 Ab

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PD-1 blockade by antibody against either PD-1 or PD-L1 can cure cancer

Killer T cell (CD8+) Tumor/ infected cell anti PDL1 Other immune cells PDL1 is expressed on various immune cells SHP2

PDL1 PD1 MHC TCR PDL1 PD1 MHC TCR

anti PDL1 anti PD1 anti PD1

+

+

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Human anti-PD-1 antibody

Approved as Investigation New Drug by FDA (USA; Aug 1, 2006) 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

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296 terminal stage patients recruited Nivolumab treatment for two years Complete or partial response rates 18% ( 76 patients) of non small cell lung cancer 28% ( 94 patients) of melanoma 27% ( 33 patients) of renal cell carcinoma

S. Topalian et al., NEJM (2012)

Clinical trials began in US (2006) and Japan (2008)

Summary of Phase I clinical trial

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Durable response to PD-1 blockade

“Responses were durable; 20 of 31 responses lasted 1 year or more and some even after stopping therapy” baseline tumor enlargement tumor regression

Change in target lesions from baseline(%) Patients with melanoma First occurrence

f new lesion

Patient off study Weeks since treatment initiation

S. Topalian et al., NEJM (2012)

stop treatment

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Phase II trial of anti-PD-1 antibody in patients with platinum- resistant ovarian cancer

Oct 21, 2011Dec 7, 2014 Dose total (n) CR PR SD PD NE RR DCR 1 mg/kg 10 1 4 4 1 1/10 (10%) 5/10 (50%) 3 mg/kg 10 2 2 6 2/10 (20%) 4/10 (40%) Total 20 2 1 6 10 1 3/20 (15%) 9/20 (45%)

Tumor growth stopped in 4050%

f terminal stage patients
J. Hamanishi et al., J. Clin. Oncol. (2015)

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A responder with ovarian cancer (clear cell): Nivolumab 3mg/kg

Baseline 4 months

Peritoneal dissemination ⇒ disappeared History: 60 yr. Stage Ic with progressive disease after RSO, MMC/CPT11*3, SCH+BSO, CPT/CDDP*5, TC*2

Cancer marker CA125 (U/ml)

１

2

(day) 316 16 10 200 400 50 100 150

43

J. Hamanishi et al., J. Clin. Oncol. (2015)

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Durable complete responses of

varian cancer patients to Nivolumab
J. Hamanishi, The International Federation of Gynecology and Obstetrics FIGO (2018)

50 100 150 1 2 3 4 5

CA125 (U/ml) Nivo

Case 1

CA125 (U/ml)

100 200 300 400 1 2 3 4 5

years

Nivo

Case 2

No medication > 4 years

No recurrence > 5 years

No medication > 3.5 years

No recurrence > 4.5 years

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Randomized Study on Untreated Melanoma Patients with Nivolumab and Dacarbazine (chemotherapy)

Overall survival Dacarbazine Nivolumab Patients who died median survival Hazard ratio for death, 0.42(99,7% Cl, 0.25-0.73) P<0.001 Dacarbazine Nivolumab No,/total no. mo (95% Cl) 50/210 Not reached 96/208 10.8 (9.3-12.1) Dacarbazine Nivolumab

No. at risk

210 208 185 177 150 123 105 82 45 22 8 3 Months Patients surviving (%)

C. Robert et al., NEJM (2015)

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Cancers approved for PD-1 blockade therapy

2014

melanoma

2015

lung cancer

2016

renal cancer Hodgkin’s lymphoma head and neck cancers urothelial cancer

2017

colorectal cancer gastric cancer hepatocellular carcinoma Merkel cancer all highly mutated cancers

2018

cervical cancer primary mediastinal large BCell lymphoma

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Paradigm shift of cancer therapy by anti-PD-1 treatment

1. Less adverse effects because normal cells

are unaffected

2. Effective for a wide range of tumors

(more than 1000 clinical trials)

3. Durable effects to responders after

stopping treatment

47

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Cancer cells accumulate mutations

I. Martincorena et al., Science (2015)

10000 1000 100 10 1 100 10 1 0.1

Mutations per megabase Coding mutations per tumour

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What we learned from huge cancer genome projects

1. Cancer cells accumulate a large number
f mutations to express neoantigens that can be

recognized by the immune system as nonself. This is why cancer immunotherapy is effective.

2. Too many mutations to pinpoint the dominant

mutations for targeted chemotherapy.

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Continuous mutations generate resistant tumor cells

cancer cells drug A Selection mutagenesis Resistant cells grow

Lymphocytes can recognize many more mutants & attack them

drug B Selection mutagenesis Resistant cells grow

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Current issues in PD-1 blockade therapy Biomarkers for responders

・ High mutagenesis in tumors ・ Potency of individual’s immunity

Improvement of immunotherapy

・ Accessibility of killer T cells to tumor sites ・ Potentiation of killer T cell function

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PD-1 blockade initiates killer T cell expansion in lymph nodes

Chemokines attract killer T cell

K. Chamoto et al., PNAS (2017)
1. PD1 blockade enhances

killing within tumor which secrets chemokines

2. PD1 blockade enhances

priming and induces chemokine receptor to help migration of new killer T cell towards tumor

DLN (draining lymph node) Tumor

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53

Numbers of PD1 blockade trials using combinations with : 1. AntiCTLA4 agents: 251

2. Chemotherapies: 170
3. Radiotherapies: 64
4. AntiVEGFA agents: 43
5. Chemoradiotherapy combos: 42
J. Tang et al., Ann. Oncol. (2018)

Cancer immunotherapy by PD-1-based combination studies underway in 2017

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Requirement of mitochondrial activation for killer T cell activation and proliferation

TCR stimulation Proliferation/ activation of killer T cells Mitochondria Mitochondrial biogenesis provides cell with energy

AntiPD1

PD1

+

+

? boost

tumor

SLIDE 55 AMPK PGC1α mTOR PGC1α PPARα/γ

Activation of PGC-1〈 /PPAR complex improves the efficacy of PD-1 blockade

PD1
K. Chamoto et al., PNAS (2017)

Bezafibrate

2500 2000 1500 1000 500 Days after MC38 inoculation Tumor volume(mm3) 5 10 15 20 25 Control AntiPDL1 mAb AntiPDL1 mAb + Bezafibrate Bezafibrate

Mitochondria mass + Energy boost

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TCR stimulation

+

+

Bezafibrate increases killer T cell proliferation and blocks cell death

PD1

+
Survival, proliferation &

memory generation Effector killer T cells Exhaustion & cell death

P. Chowdhury et al., Cancer Immunol. Res., (2018)

Bezafibrate + PPARα/γ

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M. Miyajima, B. Zhang et al., Nat. Immunol. (2017)

Hyperimmune activity can be read in blood biochemistry of PD-1-/- mice

Tryptophan metabolism Malateaspartate shuttle Gluconeogenesis Urea cycle Galactose metabolism Starch and sucrose metabolism Fructose and mannose degradation Aspartate metabolism Ammonia recycling Alanine metabolism Glucosealanine cycle Histidine metabolism Glutamate metabolism P value 1e01 6e01 1e+00 Fold enrichment 0 2 4 Citric acid cycle Mitochondrial electron transport chain

Mitochondrial activation & Tryptophan consumption

PC1 (24.8%) PC2 (16.6%)

PD1/

WT

Sidonia Fagarasan

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PD-1-/- mice biology is very complex

?

Metabolite shift Behavioral changes

Expansion T cells

H. Nishimura et al.

Immunity 1999

Consumption of metabolites

M. Miyajima, B. Zhang et al., Nat. Immunol. (2017)

Gut bacterial changes

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PD-1 selects IgA critical to microbiota regulation

IgAcoated bacteria in the gut

*** WT PD1/ IgA coated bacteria (%)

IgA DAPI

Less IgAcoating of bacteria in PD1/ mice Bacterial dysbiosis

Total Anaerobic Aerobic Bacteroidiaceae 103 104 105 106 107 108 109

No. bacteria/g small intestine content

*** ND Bifidobacterium Lactobacillus Streptococcus Enterobact *** *** WT PD1/

S. Kawamoto et al., Science (2012)

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GC B cells GC T cells PD1hi AIDhi

AID and PD-1 cooperate in germinal centers for high affinity IgA selection to maintain microbiome

Y Y Y AID IgA PD1 IgM

S. Kawamoto et al., Science (2012)

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AID/ WT

Critical role of AID for controlling microbiota & whole body immune homeostasis

S. Fagarasan et al., Science (2002)
K. Suzuki et al., PNAS (2004)

WT AID/

94% Lactobacillus 72% SFB 28% Clostridium

Mucosal immune activation WT AID/ Systemic immune activation, spleen

SFB

From Meyerholz et al, 2002

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Enhanced anti-tumor immunity in AID-/- mice depends on microbiota

M. Akrami, R. Menzies, M. Miyajima, Y. Nakajima. unpublished data

WT SPF AID/ SPF WT GF AID/ GF

Specificpathogen free Germ free

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Microbiome-immune system regulation

Y Y AID PD1 metabolites Y IgA System homeostasis Immune tolerance

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Changes Microbiota & Metabolites Y AID PD1 Y Y IgA blockade Anxiety, autoimmunity

Microbiome-immune system regulation

Enhanced Antitumor activity

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CLOSING IN ON CANCER

Andy Coghlan New Scientist, 5 March 2016 “We’re at the point where we’ve discovered the cancer equivalent of penicillin” says Chen. Although penicillin itself couldn’t cure all infections, it gave rise to a whole generation of antibiotics that changed medicine forever, consigning most previously fatal infections to history.

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Future prospects in cancer therapy

1. Efficacy of PD1 blockade

therapy improved.

2. Many more cancers may be treated

by immunotherapy.

3. Cancer may not completely

disappear, but be controlled by

immunotherapy. Cancer may

become one of chronic diseases. 2016 2020 2030?

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Enormous benefit by acquired immunity

20th century Penicillin Pneumonia Streptomycin Tuberculosis Eradication of infectious diseases by vaccination and antibiotics 21st century Cancer may be controlled by

immunotherapy and its improvement including microbiome manipulation

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Acquired immunity evolved in vertebrates

Molluscs Arthropods Nemertea Annelides Urochordata Platyhelminthes Echinoderms Cnidaria Protozoa Porifera Mammals Birds Amphibians Reptiles Jawed fish Jawless fish

Acquired immunity Natural immunity

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Fortunate outcomes from evolution of acquired immunity

Molluscs Arthropods Nemertea Annelides Urochordata Platyhelminthes Echinoderms Cnidaria Protozoa Porifera Mammals Birds Amphibians Reptiles Jawed fish Jawless fish

Acquired immunity Natural immunity

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Collaborators

Dept. of Immunology and Genomic Medicine,

Graduate School of Medicine, Kyoto University Sidonia Fagarasan IMS, RIKEN Fumihiko Matsuda Kyoto University

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Major outside collaborators

Antibody diversity

E. Severinson (Stockholm Univ.)
F. Alt (Harvard Univ.)
M. Nussenzweig (Rockefeller Univ.)
A. Fischer (Necker Hospital)
A. Durandy (Necker Hospital)
T. Chiba (Kyoto Univ. Hospital)

Cancer immunotherapy by PD1 blockade

G. Freeman (Dana Farber Cancer Center)
N. Minato (Kyoto Univ.)
S. Fujii (Kyoto Univ.)
I. Konishi (Kyoto Univ.)

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Acknowledgement for research funding support

ONO Pharmaceutical CO. LTD MEXT MHLW JSPS AMED NIBIOHN NIHN BristolMyers Squibb Jane Coffin Child Memorial Fund Tang Prize Foundation

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Thank you for your attention

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