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Serendipities ofacquired immunity
Tasuku Honjo
December 7, 2018Nobel Lecture
Kyoto University Institute for Advanced Studyand Graduate School of Medicine
My family (1955)
Saturn.Actual photo taken on June 5, 2016https://www.nasa.gov/mission_pages/cassini/multimedia/pia17171.html
Cassini: Earth and SaturnThe Day the Earth Smiled
Through the brilliance of Saturn’s rings, Cassini caught a glimpse of a far-away 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.
EarthThe telescopic view of Saturn fascinated me. I dreamed of becoming an astronomer.” “
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
T. Honjo et al., J.Biol.Chem. (1968)
active inactive
NAD
+EF-II
nicotinamideEF-II
Ribo
ADP
EF-II
diphtheria toxin acts as a catalyst
Ribo
ADPRibo
ADP
DT
+
8
Donald Brown at Carnegie Institution in Baltimore 1971
Mystery of immune responsein 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?
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
oxazolone-protein
Modified from K. Landsteiner 1919-22
11
Structure of antibody identified by 1970
H chain (heavy chain)L chain (light chain)
Constant region (C)(Antibody class determination)
Variable region (V)(Antigen-recognition site)
CCCC
Philip Leder at NIH 1973
C. Brack et al., Cell (1978)
VDJ recombination generates V region repertoire during differentiation
VDJ recombination
JH CµS
CδDHVH
S
Constant region
B cellIgDIgM
S. Tonegawa
University of Tokyo, Dept. of Nutrition(Professor Yoshinaga Mano) 1974
10000
1000
100
10
0
1
0 7 14 21 28 35 42
Primary response Secondary response
Tite
r
Days
Primary immunization Secondary immunization
Antibody memory generation byvaccine (antigen) administration
Increase in antigen binding capability (somatic hypermutation of variable region)
Somatic hypermutation (SHM) mutates V region and only
good antibodies are selected
YY
Y Y
Ypathogen
Y strongest
Y
Y
Y
YY
Y YY
Darwinian principle
B cell
Increase in antigen processing ability(class switch of constant region)
10000
1000
100
10
0
1
0 7 14 21 28 35 42
Primary response Secondary response
Tite
r
Days
IgMIgG
Primary immunization Secondary immunization
Antibody memory generation byvaccine (antigen) administration
Increase in antigen binding capability (somatic hypermutation of variable region)
?
Class switching changes the H chain constant region and antibody function
L chain
H chain
CCCC
Constant region (C)
Variable region (V)
IgM
CCCC
CSRCCCC
IgG
CCCC CCCC
IgA
CCCC CC
CC
IgE
Y
Y
Y
Gut bacteria
Parasite
Virus
CC
class switch recombination(CSR)
looped-outcircular 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 αγ γ γH C C C 3 C CC 1 C 2b C 2a
µCδC
γC 3γC 1
γC 2b
VH γC 2a εC αCS S
S
T. Honjo & T. Kataoka, PNAS (1978)T. Kataoka et al., PNAS (1980)A. Shimizu et al., Cell (1982)
T. Kataoka A. Shimizu
Matthias Wabl, Göran Möller (coorganizer) Leroy Hood
YIY
Discovery of AID by comparison of gene expression before and after CSR
Stimulation with CD40L, IL-4, TGF-β1
M. Muramatsu et al., J.Biol. Chem. (1999)
IgM IgA
Activation Induced cytidine Deaminase
AID
AIDB cells
Expressed in germinal center
Comparison of expressed gene transcripts
Defective IgG response to antigens (Sheep Red Blood Cell) in AID deficient mice
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 215 10 14
Days
IgM
0 215 10 14
IgG1
Ant
ibod
y ti
ter
AID +/-AID -/-
AID+/-
AID-/-
Days
Ant
ibod
y ti
ter
M. Muramatsu et al., Cell (2000)
VH186.2 sequence (residue)
0
40
Q1 R98W33
0
40
0
80 CDR1 CDR2RSγ1
µ
µMut
atio
n fr
eque
ncy(
%)
AID+/-
AID+/-
AID-/-
No mutation
AID deficient mice fail to accumulate mutations
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.
VDJ recombination
V D J µ δ γ ε αγ γ γH H C C C 3 C CC 1 C 2b C 2aS S S S S S S
RAGsNatural AbNatural Ab
Pathogensomatic hypermutation(SHM)
mutated V gene
class switch recombination(CSR)
chromosomal product
+
Antibody memoryformation
looped-outcircular DNA
MemoryAb
VDJ recombination
V D J µ δ γ ε αγ γ γH H C C C 3 C CC 1 C 2b C 2a
Repertoire formation
S S S S S S S
RAGsNatural Ab
tumors
AID engraves Ab memory in the genome for effective vaccination
AID
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 (IFNγ, IL-2, IL-12 etc)
This was because no immune brake molecules were known before 1995
driving[Attack]
accelerator[ICOS]
brake[PD-1]
〜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
Y. Ishida et al., EMBO J. (1992)
Discovery of PD-1 (programmed death-1) cDNA
Structure of cytoplasmic tail suggests PD-1 is a surface signaling molecule
Y. Ishida Y. Agata
A long journey to understanding the function of PD-1
・1994 PD-1 knock out (KO) on mixed backgroundmice, no phenotype change
・1996 PD-1 KO on C57BL/6, no phenotype change for 6M
Nephritis and arthritis after 5M in PD-1KO x lpr/lpr background
・1998
・1997
But over-response to antigen stimulation
Clear autoimmunity in PD-1 KO by 14M
PD-1 is a negative regulator
PD-1 KO (Knock Out)
Nephritis ArthritisY. Nishimura et al., Immunity (1999)
Dilated cardiomyopathy
PD-1 KOWTBALB/c
NOD
Diabetes
NODxPD-1 KO MRL
MyocarditisT. Okazaki et al., Nat. Medicine (2003)J. Wang et al., Int. Immunol. (2010)
MRLxPD-1 KO
C57BL/6
Molecular mechanism of immune inhibition by PD-1 signaling
N N
PD-1
N
NNα β
γ/ε δ/ε
ζ ζ
Antigen receptor
negative signal
PPP
P
Coreceptor
T. Okazaki et al., PNAS(2001)
ITSM
Activationsignal
Kinase
ZAP70
S-S
S-S
S-S
S-S S-S
S-S
S-SS-S
S-S
P YY
Antigen
PP
P
P
PD-L1
Balance between immune surveillance and immune tolerance
Immune toleranceImmune suppression
Treatment of autoimmunityHigher risk of infectious diseases and cancer
Treatment of infectious diseases and cancerRisk of autoimmunity
Immune surveillanceHyper immunity
PD-1blockade
Days after inoculation5 10 15 20
Inhibition of tumorigenesis of myeloma (J558L) in PD-1-/- mice
Y. Iwai et al., PNAS (2002)
J558L
BALB/cWT
Tum
or v
olum
e (m
m3 )
5 10 15 20
N=4
J558L
BALB/cPD-1-/-
N=410000
7500
5000
2500
0
Tum
or v
olum
e (m
m3 )
Days after inoculation
0 5 10 15 200
500
1000
1500
2000
2500
0 5 10 15 20
n=10n=10
Rat IgG
P815/PD-L1 DBA/2
a-PD-L1
Inhibition of tumorigenesis of P815/PD-L1 by anti-PD-L1
Tum
or v
olum
e (m
m3 )
2500
2000
1500
1000
500
00 5 10 15 200 5 10 15 20
N=10 N=10
Days after inoculation
Tumor growth Survival rate
(%)
Rat IgG
a-PD-L1
100
0
20
40
60
80
020 40 60 80
Days after inoculation
N. Minato
Y. Iwai et al., Int. Immunol. (2005)
PD-1 blockade inhibits metastasis of B16 melanoma (mouse model)
weig
htof
live
r (g
)
0
2
4
6
8
WT
Spleen to liver
Anti PD-1 AbWT Anti PD-1 Ab
PD-1 blockade by antibody against either PD-1 or PD-L1 can cure cancer
Killer T cell(CD8+)
Tumor/infected cell
antiPD-L1
Other immune cells
PD-L1 is expressed on various immune cells
SHP2PD-L1 PD-1
MHC TCR
PD-L1PD-1
MHCTCR
antiPD-L1
antiPD-1
antiPD-1
+-
+
-
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: IgG4S228Pmutant IgG4 (S228P) stabilizes the protein and reduces ADCC(antibody-dependent cell-mediated cytotoxicity)KD = 2.6 nmol/L
Named Nivolumab
296 terminal stage patients recruited Nivolumab treatment for two years
Complete or partial response rates
18% ( 76 patients) of non small cell lung cancer28% ( 94 patients) of melanoma27% ( 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
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
tumorenlargement
tumorregression
Chan
ge in
tar
get
lesi
ons
from
bas
elin
e(%
)
Patients with melanomaFirst occurrence of new lesionPatient off study
Weeks since treatment initiation
S. Topalian et al., NEJM (2012)
stop treatment
Phase II trial of anti-PD-1 antibody in patients with platinum-
resistant ovarian cancer
Oct 21, 2011-Dec 7, 2014
Dose total(n) CR PR SD PD NE RR DCR
1 mg/kg 10 0 1 4 4 1 1/10(10%)
5/10(50%)
3 mg/kg 10 2 0 2 6 0 2/10(20%)
4/10(40%)
Total 20 2 1 6 10 1 3/20(15%)
9/20(45%)
Tumor growth stopped in 40-50% of terminal stage patients
J. Hamanishi et al., J. Clin. Oncol. (2015)
A responder with ovarian cancer(clear cell): Nivolumab 3mg/kg
Baseline 4 months
Peritoneal dissemination ⇒ disappeared
History: 60 yr. Stage Ic with progressive disease afterRSO, MMC/CPT11*3, SCH+BSO, CPT/CDDP*5, TC*2
Cancer marker CA125 (U/ml)
1 2
(day)
316
16 100
200
400
0 50 100 150
43J. Hamanishi et al., J. Clin. Oncol. (2015)
Durable complete responses of ovarian cancer patients to Nivolumab
J. Hamanishi, The International Federation of Gynecology and Obstetrics FIGO (2018)
0
50
100
150
0 1 2 3 4 5
CA12
5(U
/ml)
NivoCase 1
CA12
5(U
/ml)
0
100
200
300
400
0 1 2 3 4 5years
NivoCase 2
No medication
> 4 years
No recurrence > 5 years
No medication
> 3.5 years
No recurrence > 4.5 years
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
DacarbazineNivolumab
No,/total no. mo (95% Cl)50/210 Not reached96/208 10.8 (9.3-12.1)
DacarbazineNivolumabNo. at risk
210208
185177
150123
10582
4522
83
00
Months
Patien
ts s
urviving
(%)
C. Robert et al., NEJM (2015)
Cancers approved for PD-1 blockade therapy
• 2014 melanoma• 2015 lung cancer• 2016 renal cancer
Hodgkin’s lymphomahead and neck cancersurothelial cancer
• 2017 colorectal cancergastric cancerhepatocellular carcinomaMerkel cancerall highly mutated cancers
• 2018 cervical cancerprimary mediastinal large B-Cell lymphoma
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
Cancer cells accumulate mutations
I. Martincorena et al., Science (2015)
10000
1000
100
10
1
100
10
1
0.1
Mut
atio
nspe
r m
egab
ase
Codi
ng m
utat
ions
per
tum
our
What we learned from hugecancer genome projects
1. Cancer cells accumulate a large number of mutations to express neo-antigens that can berecognized by the immune system as non-self.This is why cancer immunotherapy is effective.
2. Too many mutations to pinpoint the dominant mutations for targeted chemotherapy.
Continuous mutations generate resistant tumor cells
cancercells
drug A
Selectionmutagenesis
Resistant cells grow
Lymphocytes can recognize many more mutants& attack them
drug B
Selectionmutagenesis
Resistant cells grow
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
PD-1 blockade initiates killer T cell expansion in lymph nodes
Chemokines attract killer T cell
K. Chamoto et al., PNAS (2017)
1. PD-1 blockade enhances killing within tumor which secrets chemokines
2. PD-1 blockade enhances priming and induces chemokine receptor to helpmigration of new killer T cell towards tumor
DLN(draining lymph node)
Tumor
53
Numbers of PD-1 blockade trials using combinations with :
1. Anti-CTLA-4 agents: 2512. Chemotherapies: 1703. Radiotherapies: 644. Anti-VEGFA agents: 435. Chemoradiotherapy combos: 42
J. Tang et al., Ann. Oncol. (2018)
Cancer immunotherapy by PD-1-based combination studies underway in 2017
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
-
Anti-PD-1PD-1
- + +
? boost
tumor
AMPK
PGC-1α
mTOR
PGC-1αPPARα/γ
Activation of PGC-1⟨ /PPAR complex improves the efficacy of PD-1 blockade
-PD-1
K. Chamoto et al., PNAS (2017)
Bezafibrate
2500
2000
1500
1000
500
Days after MC38 inoculation
Tum
or v
olum
e(m
m3 )
0 5 10 15 20 25
ControlAnti-PD-L1 mAbAnti-PD-L1 mAb + BezafibrateBezafibrate
Mitochondria mass + Energy boost
TCR stimulation
-+ +
Bezafibrate increases killer T cell proliferation and blocks cell death
PD-1 - +-
-
Survival, proliferation &memory generation
Effector killer T cells
Exhaustion & cell death
P. Chowdhury et al., Cancer Immunol. Res., (2018)
Bezafibrate+
PPARα/γ
M. Miyajima, B. Zhang et al., Nat. Immunol. (2017)
Hyperimmune activity can be read inblood biochemistry of PD-1-/- mice
Tryptophan metabolism
Malate-aspartate shuttleGluconeogenesis
Urea cycleGalactose metabolism
Starch and sucrose metabolismFructose and mannose degradation
Aspartate metabolismAmmonia recycling
Alanine metabolismGlucose-alanine cycleHistidine metabolism
Glutamate metabolism
P value
1e-01
6e-01
1e+00
Fold enrichment0 2 4
Citric acid cycleMitochondrial electron transport chain
Mitochondrial activation &Tryptophan consumption
PC1 (24.8%)
PC2
(16.
6%)
PD-1-/-WT
Sidonia Fagarasan
PD-1-/- mice biology is very complex
?
Metabolite shift
Behavioralchanges
Expansion T cells
H. Nishimura et al. Immunity 1999
Consumption ofmetabolites
M. Miyajima, B. Zhang et al., Nat. Immunol. (2017)
Gut bacterial changes
PD-1 selects IgA critical to microbiota regulation
IgA-coated bacteria in the gut ***
WT PD-1-/-
IgA
coa
ted
bact
eria
(%)
IgA DAPI
Less IgA-coating of bacteria in PD-1-/- mice
Bacterial dysbiosis
TotalAnaerobic
AerobicBacteroidiaceae
103 104 105 106 107 108 109
No. bacteria/g small intestine content
***
NDBifidobacteriumLactobacillus
StreptococcusEnterobact ***
***
WT
PD-1-/-
S. Kawamoto et al., Science (2012)
GC B cells GC T cellsPD1hiAIDhi
AID and PD-1 cooperate in germinal centersfor high affinity IgA selection
to maintain microbiome
YY Y
AID
IgAPD1
IgM
S. Kawamoto et al., Science (2012)
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
Enhanced anti-tumor immunity in AID-/- mice depends on microbiota
M. Akrami, R. Menzies, M. Miyajima, Y. Nakajima. unpublished data
WT SPFAID-/- SPF
WT GFAID-/- GF
Specific-pathogen free Germ free
Microbiome-immune system regulation
YYAID
PD1
metabolites
Y
IgA
System homeostasis Immune tolerance
Changes Microbiota & Metabolites
YAID
PD1 YY
IgA
blockade
Anxiety, autoimmunity
Microbiome-immune system regulation
Enhanced Anti-tumoractivity
CLOSING IN ON CANCERAndy 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.
Future prospects in cancer therapy
1. Efficacy of PD-1 blockade therapy improved.
2. Many more cancers may be treated by immunotherapy.
3. Cancer may not completely disappear, but be controlled by immunotherapy. Cancer maybecome one of chronic diseases.
2016
2020
2030?
Enormous benefit by acquired immunity
20th century
Penicillin
Pneumonia
Streptomycin
Tuberculosis
Eradication of infectious diseasesby vaccination and antibiotics
21st century Cancer may be controlled by immunotherapy and its improvement including microbiome manipulation
Acquired immunity evolved in vertebrates
Molluscs
Arthropods
Nemertea
Annelides
Urochordata
Platyhelminthes
Echinoderms
Cnidaria
ProtozoaPorifera
Mammals
BirdsAmphibians
Reptiles
Jawed fishJawless fish
Acquired immunity
Naturalimmunity
Fortunate outcomes from evolution of acquired immunityMolluscs
Arthropods
Nemertea
Annelides
Urochordata
Platyhelminthes
Echinoderms
Cnidaria
ProtozoaPorifera
Mammals
BirdsAmphibians
Reptiles
Jawed fishJawless fish
Acquired immunity
Naturalimmunity
Collaborators
Dept. of Immunology and Genomic Medicine,Graduate School of Medicine, Kyoto University
Sidonia FagarasanIMS, RIKEN
Fumihiko MatsudaKyoto University
Major outside collaboratorsAntibody diversityE. 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 PD-1 blockadeG. Freeman (Dana Farber Cancer Center)N. Minato (Kyoto Univ.)S. Fujii (Kyoto Univ.)I. Konishi (Kyoto Univ.)
Acknowledgement for research funding support
ONO Pharmaceutical CO. LTD
MEXT MHLW
JSPS AMED NIBIOHN
NIHN
Bristol-Myers Squibb
Jane Coffin Child Memorial FundTang Prize Foundation
Thank you for your attention