immune checkpoints sot2016

© 2016 Envigo envigo.com

Chair: Greg Bannish

Presenters: Lee A. Coney and Mandy Horn

Key considerations in the safety

evaluation of drugs targeting

immune checkpoints

Company overview

We are a global contract research products and

services company dedicated to helping our

customers achieve the potential of their products

which improve human life, advance animal

welfare, and protect the environment and global

food security.

Who We Are

Our Brand Promise

Together, we make the world a

safer and healthier place to live.

Our company places the customer at the center

of everything we do. We focus on what matters

to you, our customer, and work closely with you

to secure the potential of your research and

products. We are dedicated to research that

makes a difference in the lives of people all over

the world.

52 locations Global availability of research

models

and CRO services

5 continents Extensive reach across the

Americas, Europe, Asia, and the

Middle East

3,800 employees Serving over 65 countries

150 years Combined industry experience

$500 million Approximately $500m in annual

revenues

Capacity and Reach

Contract Research Services (CRS) locations

United States of America

+ Princeton, New Jersey

Envigo laboratories are

located in The United

States of America,

Europe and the Middle

East

Europe

Corporate HQ: Huntingdon, England

+ Huntingdon, England

+ Eye, England

+ Ely, England

+ Shardlow, England

+ Rossdorf, Germany

+ Barcelona, Spain

+ Valencia, Spain

Middle East

+ Rehovot, Israel

Research Models and Services (RMS) locations

Envigo production

sites in North

America, Europe, the

Middle East and Asia

North America

+ Dublin, VA

+ Frederick, MD

+ Haslett, MI

+ Houston, TX

+ Indianapolis, IN

+ Livermore, CA

+ Madison, WI

+ Mexico City, Mexico

Europe

+ Blackthorn, UK

+ Bresso, Italy

+ Gannat, France

+ Horst, Netherlands

+ Loughborough, UK

+ Udine, Italy

+ Wyton, UK

Middle East

+ Jerusalem, Israel

+ Rehovot, Israel

+ Yokneam, Israel

India

+ Hyderabad, India

Corporate HQ:

Indianapolis, IN

European Office:

Venray, Netherlands

Key considerations in the safety evaluation of drugs

targeting immune checkpoints

+ What is cancer immunotherapy?

+ What are the potential challenges in the safety assessment

of antibodies targeting immune system checkpoints?

+ Things to consider when designing and running your non-

clinical safety programmes for immune checkpoint targets?

+ Measuring immunotoxicity / immunopharmacology

+ Case study – what if your chosen therapeutic has no

pharmacologically relevant non-clinical safety species?

+ Summary

Presentation overview

Cancer immunotherapy

The utility of the immune system

Heidi L. Nature 2014 508:24

A history of cancer immunotherapy

Kirkwood JM et al. CA Cancer J Clin 2012 62:309

+ There are multiple different platforms of cancer

immunotherapy and many very different target pathways

+ Adoptive cell transfer + TILs, TCR–transfected T-cell, CAR therapy

+ Non-specific immunotherapies + Cytokines (IL-2, IFNa) CD40 agonist mAb, TLR agonist

+ Vaccination strategy + Sipuleucel-T, MAGE-3 ASCL, OncoVEX

+ Immune checkpoint blockade + CTLA-4 blocking mAb, PD-1 blocking mAb

+ The remainder of this talk with focus upon this latter

approach so called immune checkpoint targeting

antibodies

Current state of cancer immunotherapy

+ T-cell killing of tumour cells can be switched on and off + Regulated by signals from other cells of the immune system such as antigen

presenting cells and tumours themselves

+ T-cells are effectively “switched off” by due to signalling

via immunoinhibitory (checkpoint) receptors on activated T

cells

+ T cells can be switched back on in lymph nodes or the

tumour microenvironment by drugs that target checkpoint

receptors

+ There are many such drugs in development and on the

market today with some exciting biology behind them + PD1, CTLA-4, PDL, KIR, IDO1, 4-1BB, OX40, LAG3, B7-H3, CD27, CD70, CD28,

CD30 etc.

Checkpoint inhibitors: Background

Checkpoint inhibitors: Background

Shin DS & Ribas A. Curr Opin Immunol 2015 33:23

+ CD28

+ OX40

+ GITR

+ CD137

+ CD27

+ HVEM

Potential checkpoint targets (not exhaustive)

Activating receptors

+ CTLA-4

+ PD-1

+ TIM-3

+ BTLA

+ VISTA

+ LAG-3

Inhibitory receptors

Inspired by Mellman I et al. Nature 2011 480:480

Agonistic antibodies Blocking antibodies

+ Immunoinhibitory receptors are expressed after normal

MHC class I/II adaptive T cell activation

+ Ligation of CTLA-4 by B7-1/B7-2 or PD1 by PD-L1 or PD-

L2 attenuates normal T cell activation

+ CTLA-4 and PD1 upregulation on chronically activated T

cells makes them less responsive to antigenic stimulation

+ Inhibition of these targets can restore T cell function and

enhance T-cell tumour killing

+ Checkpoint inhibitors play critical roles in maintenance of

peripheral T cell tolerance

+ Drugs that target immune system checkpoints have the

potential to promote inflammation/autoimmunity in humans

Checkpoint inhibitors: Mechanism of action

Characteristic CTLA‐4 pathway PD‐1 pathway

Expression pattern Exclusively T cells

(constitutively on Tregs)

T, B, and NK cells

(activated T cells)

Natural ligands CD80 (B7-1) and CD86

(B7-2)

PD‐L1 and PD‐L2

Expression profile of

ligands

Mainly on antigen

presenting cells (APCs)

APCs and tumour cells

Phenotype of KO mice Early fatal autoimmune

syndrome

(lymphoproliferative

disease)

Strain‐specific

autoimmunity presenting

later in life in some strains

Effect of target blockade Enhanced proliferation of

CD4+ and CD8+ T cells

with increase in ratio to

Treg cells

Enhanced proliferation of

CD8+ T cells greater than

CD4+ T cells and

enhanced cytotoxicity of

CD8+ T cells

CTLA-4 v PD-1 pathways

Greenwald RJ, et al. Ann Rev Immunol. 2005;23:515-548. Chambers CA, et al. Ann Rev Immunol. 2001;19:565-594. Dong H, et al.

Nat Med. 2002;8:793-800. Curran MA, et al. Proc Natl Acad Sci U S A. 2010;107:4275-4280. Pilon-Thomas S, et al. J Immunol.

2010;184:3442-3449.

+ CPIs control central and peripheral immune tolerance to

“self-antigens” + CPIs restrict positive selection in the thymus, inhibit the activation and function of

self-reactive T cells in the peripheral circulation, effect Treg cell development and

their function

+ Therefore inhibition of some CPI pathways may contribute

to the development of immune-mediated disease + Findings consistent with autoimmunity observed in both CTLA-4 and PD-1 KO mice

on certain backgrounds

+ Similar findings seen in humans with genetic anomalies in

certain CPI genes + SLE, RA, MS, Type 1 diabetes

+ Immune-mediated events observed in clinical studies with

CPIs and monitored in post-marketing PV

Predicted risks based on mechanism of action

Checkpoint inhibitors: Mechanism of action

Intlekofer AM et at. J Leukoc Biol 2013 94:25

+ The intended pharmacological action of each therapeutic is

immune system activation – immediate alarm bells!

+ As with most biopharmaceuticals the balance between

therapeutically desired pharmacology and clinical dose-

limiting “exaggerated pharmacology” is a delicate balance

+ Acute effects such as systemic cytokine release may be

manageable in some clinical situations

+ More chronic effects such as induction of autoimmune

disease may be more difficult to model in healthy standard

non-clinical models

+ SPF animals may also lack or have limited target

expression of checkpoint molecules

Challenges in safety assessment

+ Binds human CTLA-4 in vitro with high affinity

+ Inhibits in vitro binding of B7.1 (CD80) and B7.2 (CD86) to

human CTLA-4

+ CDC and ADCC activity investigated in vitro and in vivo

+ Colon carcinoma studies in human CTLA-4 transgenic

mouse

+ Extensive TDAR investigation in cynomolgus monkey + Hepatitis B surface antigen (HBsAg) vaccine, a melanoma cell-based vaccine (Sk-

mel), DNP (2,4-Dinitrophenyl)-Ficoll, keyhole limpet hemocyanin (KLH) and simian

immunodeficiency virus (SIV), DNA vaccines (purified plasmid DNA) expressing the

proteins for the gag (pSIVgag), env (pSIVenv), and pol (pSIVpol) portions of SIV. In

one study, the SKmel tumour line was transfected to express GM-CSF.

+ Extensive immunophenotypic analysis of T cells and subsets in these studies

+ Extensive assessment of humoral immune responses to TDAR antigens

Yervoy (Ipilimumab) anti-CTLA-4 antibody

EMA Assessment Report For Yervoy (ipilimumab)

+ Tissue cross reactivity studies

+ Safety pharmacology included as part of repeat dose

toxicity programme

+ Combination studies with anti-CD137 and anti-PD-1

antibodies conducted in cynomolgus monkeys

+ Single species toxicology programme conduced in

cynomolgus monkey as only pharmacologically relevant

species

+ Studies up to 6 months in duration at a variety of dose

levels conducted

+ No reproductive, developmental or juvenile toxicology

studies were submitted for registration purposes



+ Most findings considered adverse seen in combination

toxicity or exploratory (TDAR) pharmacology studies not

anti-CTLA-4 alone GLP studies

+ Low incidence of immune-meditated toxicities + Colitis, dermatitis, or infusion reactions

+ Responses consistent with proposed MOA of CTLA-4 in maintaining self-tolerance

+ Findings in cynomolgus monkeys correlated with findings

in humans, although with less frequently – under predict?

+ CTLA-4 blocking did result in an over stimulation of the T-

cell compatment + Few meaningful changes in immunophenotype or autoimmune organ pathology

(with exception of colitis and dermatitis)

+ Increased TDAR was observed demonstrating expected pharmacodynamics



+ Agonist antibodies + Systemic cytokine release

+ Hepatotoxicity

+ Antagonist (blocking) antibodies + Immune system activation and autoimmunity

+ Dermatitis, colitis, hepatitis, thyroiditis, hypophysitis, uveitis, pneumonitis

+ Adverse events observed in clinic include: pneumonitis,

colitis, skin effects (rash, pruritis, vitiligo), conjunctivitis,

uveitis, hepatotoxicity, thyroid toxicity, nephritis

+ Normal cynomolgus monkeys not predictive of all clinical

findings – how do we increase the potential to translate

understanding from non-clinical to clinical?

Clinical experience

+ Can studies in normal animals (were lower expression of

checkpoint receptors since lower numbers of activated T

cells) predict adverse events in cancer patients?

+ Do the immune changes in normal animals adequately

reflect the risk of immune-mediated pathology in humans?

+ Should greater focus be on safety studies in models of

autoimmunity, cancer, vaccination, where more activated T

cells & checkpoint expression / activity is higher?

+ Could these models then become over predictive?

+ Is it useful to understand relative risk of combos of

checkpoint inhibitors to monotherapy; benchmark new

therapies to anti-CTLA-4 & anti-PD-1 marketed products?

Checkpoint inhibitors: Key considerations for safety

+ “Classical” NOAEL approach less relevant for molecules

targeting check point inhibitors

+ First in human dosing likely to be based on extensive non-

clinical investigations + In vitro potency assessment including relative potency between human and non-

clinical species

+ Receptor occupancy

+ FIH dose likely to be based on PAD and / or MABEL. Generally clinical dose setting

will be conservative with slow dose escalation in the clinic

+ Extensive PK/PD modelling to inform clinical dosing and dose escalation.

Challenging given huge diversity in clinical subjects T-cell populations

Increasing the translation of non-clinical to clinical

+ Non-standard approaches to safety assessment will

continue to be evolved to increase the predictiveness of

non-clinical models + Vaccination / antigen challenge in non-clinical studies

+ Host defense approach adapted for safety evaluation

+ Comprehensive analysis of T-cell subsets by flow cytometry (Treg, Tem, Teff, Tcm,

Th1, Th2 in addition to normal panels)

+ Receptor occupancy in blood and other lymphoid tissue

+ In vitro and in vivo cytokine analysis (large multiplex panels)

+ RO, PAD/MABEL, in vitro / in vivo (EC50s), integrate exposure/PD/efficacy/safety;

close patient monitoring

+ The sample burden on animals is likely to be extensive,

increases the requirement for microsampling /

microanalysis even in non-rodent species

Increasing the translation of non-clinical to clinical

Measuring immunotoxicity / immunopharmacology

Immunotoxicology synopsis

Normal

animals

Immunosuppression Immunostimulation

Infection / cancer risk Hypersensitivity

& Autoimmunity

Morbidity

potential

Morbidity

potential

Immunotoxicology focus for immune checkpoints

Normal

animals

Immunosuppression Immunostimulation

Infection / cancer risk Hypersensitivity

& Autoimmunity

Morbidity

potential

Morbidity

potential

+ There is no single assay or suitable biomarker to measure

all aspects of immunotoxicology

+ A weight of evidence approach is undertaken + Existing understanding of drug pathway engagement / pharmacological properties

+ Immune system effects observed in animal toxicity and clinical safety studies

+ Drugs with a high predicted risk will require immune

function testing to characterise that risk and understand

dosing limitations

Measuring immunotoxicology

+ Inclusion of immunotoxicology / pharmacology endpoints in

toxicity studies aids us in… + …demonstrating the relevance of the tox species

+ …preclinical toxicity study dose selection

+ …TK / PD demonstrates durable pharmacology and exposure in studies

+ …understanding applicability of immunogenicity

+ …identifies translatable biomarkers for clinic

+ …interpretation of dose-response relationship with toxicity and identification of

clinical doses

Immunotoxicology v pharmacology

+ Can biomarkers of immune activation improve our

nonclinical models? + Understand relevance of the non-clinical model

+ Demonstrate PD and maintenance of PD in presence of immunogenicity

+ Potentially increase target expression when it requires immune activation for

expression

+ Establish pharmacodynamic range

+ Help set doses for toxicity studies

+ Help define FIH starting dose

+ Immunotoxicity – extension of the pharmacology

+ Help identify and/or understand adverse findings

+ Cytokines, complement activation, immune complexes, auto-antibodies

Immune activation biomarkers?

+ Immune system over stimulation can cause pathological

changes + Immune complex deposition

+ Hypersensitivity

+ Activation of pre-existing disorders

+ Autoimmune cross-reactivity

+ Inflammatory responses

+ How do we predict autoimmunity in animal models?

+ Molecular mimicry + Pathogen (or vaccine antigen) contains a similar linear T cell epitope or

conformational B cell epitope to host leading to a cross reactive immune response

to healthy tissue

Autoimmune cross-reactivity

Disease Implicated agent Mechanism?

Rheumatic heart disease Group A Streptococcus T cell mimicry (GAS Prt-M

with vascular heart protein)

Guillain-Barre Syndrome Campylobacter jejuni B cell mimicry (LPS

oligosaccharides cross react

with gangliosides)

Chronic Lyme arthritis Borrelia burgdorferi T cell mimicry (OspA cross

reactivity with human LFA-1

Idiopathic thrombocytopenia VZV, EBV, Rubella B cell mimicry (viral epitopes

cross reactivity with platelet

glycoprotein IIb/IIIa

Reactive arthritis Chlamydia trachomatis T cell mimicry (epitope cross

reactivity with HLA-B27)

Type I Diabetes Rotavirus/ Enterovirus Epidemiological evidence

(trigger effect?)

Infection and autoimmune disease

+ Haematology: total & absolute differential leukocyte counts

(incl. macrophages)

+ Clinical chemistry - globulin levels and A/G ratios

+ Gross pathology of lymphoid organs and tissues

+ Organ weights - thymus, spleen, lymph nodes etc.

+ Histopathology of lymphoid organs (bone marrow, spleen,

lymph node, thymus etc.)

+ Flow cytometry of T, B & NK cells

+ All included within most general toxicology studies study

General immune status

+ General immunophenotyping of cellular component of

immune system by flow cytometric methods

+ T-cell-dependent antibody response (TDAR) to model

antigens e.g. KLH, SRBC, TT, OVA, flu' immunised during

dosing & after recovery + Measure primary (IgM/IgG) & secondary (IgG) antibody response

+ Measure cellular response to vaccination with viral / DNA / tumour vaccines

+ T cell responses + DTH responses, LLNA

+ NK activity

+ Macrophage function

Measuring immunomodulation

+ Increases in in vitro cytotoxicity, receptor expression, T cell

proliferative / cytokine responses to mitogens, NK activity

+ Histopathology of target tissues for autoimmunity

+ Serum cytokine levels

+ Show immune system returns to / towards normal on

cessation of dosing / removal of drug

+ Activation quantified by increase in in vitro cytotoxicity,

cytokine secretion, receptor expression (flow cytometry)

+ General immune status- flow cytometry of T and B cells,

monocytes, histopathology of lymphoid organs,

haematology

Immune system activation

TDAR principle

1st Vaccination 2nd Vaccination

IgM

IgG

General immunophenotyping

SSC

FS

C

CD14

SS

C

CD3

CD

20

CD4

CD

8

CD3

CD

16

Lymphocytes

Monocytes

NK

T helper

T cell

B cell

T cytotoxic

Unstimulated PHA Unstained

(Isotype controls)

CD25

Fo

xP

3

Treg Treg

+ Intracellular staining: Increased CD4+CD25+foxP3+ Tregs

following stimulation of whole blood from cynomolgus

macaque

Treg cell staining by flow cytometry

CD8 T

CD4 T

CD3

CD

4

SSC

FS

C

+ A gating strategy for IL4, IFNg, IL17, IL13

Intracellular T cell activation I

+ Expression of IFNg in CD4T and CD8 T cells

Intracellular T cell activation II

CD4 T CD8 T

Unstim

Stim.

IL4

IFN

g

+ Expression of IL17 in CD4 T cells

Intracellular T cell activation III

Unstim

Stim.

IL17

IL13

CD4 T CD8 T

Control Dosed

IL-4

IFN

-g

Draining

LN

Non-

Draining LN

PBMC

Intracellular expression of IL-4 and IFNg in total CD3 T

lymphocytes

Intracellular expression of IL-4 and IFNg in CD4 T

lymphocytes

Control Dosed

IL-4

IFN

-g

Draining

LN

Non-

Draining LN

PBMC

Intracellular expression of IL-4 and IFNg in CD8 T

lymphocytes

Control Dosed

IL-4

IFN

-g

Draining

LN

Non-

Draining LN

PBMC

TEM

TCM

TNaive

TEM

TCM

TNaive

Cyno DTH - increased TEM in CD8+

Case study – What if your chosen therapeutic has no

pharmacologically relevant non-clinical safety

species?

+ The novel antibody needs a safety package but does not

bind to the checkpoint inhibitor (CPI) in any standard

toxicity species

+ An academic group has a transgenic mouse model

expressing the CPI (KO/KI) that may be suitable for safety

assessment of the therapeutic

+ The transgenic model is a good well characterised one,

the transgene expression and distribution of the CPI is

consistent with the human expression profile

+ Initiated a project to breed the transgenic mouse for use in

toxicity studies

Challenges with a checkpoint blocking antibody

+ The transgenic mouse model is on a C57BL/6N

background + One mutation is x-linked

+ One mutation is somatic

+ Currently maintained in academic institution with

unacceptable health status and poor availability due to

small colony size

Transgenic mouse model background

+ Rederive model to acceptable health status

+ Develop breeding colony + Genetic testing required to confirm genotype in offspring and future breeders

+ Model maintained mating heterozygous / hemizygous males x heterozygous /

homozygous females

+ Expand colony for provision of acceptable numbers of

animals at requested frequency

Next steps

Breeder genotype

FEMALE BREEDER

n/+

XmXm

MALE BREEDER

n/+

XmY

n

Xm

n

Xm

+

Xm

+

Xm

n

Y

n/n

XmY

n/n

XmY

n/+

XmY

n/+

XmY

n

Xm

n/n

XmXm

n/n

XmXm

n/+

XmXm

n/+

XmXm

+

Y

n/+

XmY

n/+

XmY

+/+

XmY

+/+

XmY

+

Xm

n/+

XmXm

n/+

XmXm

+/+

XmXm

+/+

XmXm

Preferred mating scheme

Female (n/+ XmXm)

Male

(n/+

Xm

Y)

+ 50% to be selected as

future breeders

+ 25% proper genotype for

research studies

+ 25% discarded due to

incorrect genotype

+ Colony mice positive for pinworms and Helicobacter spp

+ 5 x 10 breeding pairs provided for rederivation via embryo transfer

+ Females superovulated; embryos collected, pooled, washed, then surgically transferred into recipient pseudopregnant females of an approved health status

+ Pups born and genotyped; health monitoring performed to confirm approved health status

+ 4 females and 6 males provided

Rederivation

+ Breeding pairs received into flexible

film isolator to maintain approved

health status

+ Based on feedback from original

institution, mice maintained on 18%

protein, 6% fat diet and wood pulp

bedding

+ Mice maintained for six weeks with

only three females pregnant and one

cannibalized litter

Develop breeding colony

+ Housing and husbandry reviewed; mice switched to higher

fat diet (19% protein, 9% fat) and crinkle paper enrichment

added to cages

+ The addition of enrichment and diet change improved

breeding; however, two females died unexpectedly, leaving

6 breeding males and 2 breeding females in the

Foundation Colony

Root cause analysis #1

+ Concept of backcrossing discussed with geneticist

+ C57BL/6N females at an approved health status were

available

+ Females were introduced into male breeding cages (1x2)

+ Breeding efficiency improved, but now more extensive

record keeping and genetic testing needed to confirm

which mice are acceptable for future breeder selection

Root cause analysis #2

+

X+

n

Xm

n/+

XmX+

n

Y

n/+

X+Y

+

Xm

+/+

XmX+

+

Y

+/+

X+Y

Backcross generation 1

Female (+ X+)

Male

(n/+

Xm

Y)


future breeders


incorrect genotype

Intercross generation 2

Female (n/+ XmX+)

Male

(n/+

X+Y

)


future breeders


incorrect genotype

n

Xm

n

X+

+

Xm

+

X+

n

Y

n/n

XmY

n/n

X+Y

n/+

XmY

n/+

X+Y

n

X+

n/n

XmX+

n/n

X+X+

n/+

XmX+

n/+

X+X+

+

Y

n/+

XmY

n/+

X+Y

+/+

XmY

+/+

X+Y

+

X+

n/+

XmX+

n/+

X+X+

+/+

XmX+

+/+

X+X+

Intercross generation 3

Female (n/+ XmX+)

Male

(n/+

Xm

Y)


future breeders

+ 12.5% proper genotype for

research studies

+ 62.5% discarded due to

incorrect genotype

n

X+

n

Xm

+

X+

+

Xm

n

Y

n/n

X+Y

n/n

XmY

n/+

X+Y

n/+

XmY

n

Xm

n/n

XmX+

n/n

XmXm

n/+

XmX+

n/+

XmXm

+

Y

n/+

X+Y

n/+

XmY

+/+

X+Y

+/+

XmY

+

Xm

n/+

XmX+

n/+

XmXm

+/+

XmX+

+/+

XmXm

+ Backcross complete!

+ Adequate number of future breeders available to expand

colony rapidly

+ Genetic testing still necessary to confirm pups carrying

transgenes

+ Health monitoring performed quarterly to ensure

appropriate health status

Colony expansion

+ CPI expressed on activated T lymphocytes

+ Harvest spleens from naïve and transgenic mice

+ Stimulate with PMA + Ionomycin for 24 hours

+ Flow cytometry analysis of activated T lymphocytes to

evaluate endogenous and human CPI

Verification of CPI expression in T cells of Tg mouse

FSC-A

SS

C-A

0 65536 196608-2523

63644

129810

195977

262144

LM

FSC-W

FS

C-A

0 65536131072 2621440

65536

131072

196608

262144

Single_Cells

CD3AF488 FITC-A

Count

100

101

102

103

104

105

0

298

596

893

1191

T Cells

Single Cells Lymphocytes T Lymphocytes

Verification of CPI expression in T cells of Tg mouse:

Gating Strategy

PD-1 PerCP-Cy5-5-A

Count

100

101

102

103

104

105

0

9

19

28

37

CPI,

30.26

PD-1 PerCP-Cy5-5-A

Count

100

101

102

103

104

105

0

60

119

179

238

CPI,

1.75

PD-1 PerCP-Cy5-5-A

Count

100

101

102

103

104

105

0

8

16

24

32

CPI,

44.59

Isotype PerCP-Cy5-5-A

Count

100

101

102

103

104

105

0

10

19

29

38

CPI,

0.09

Naïve Mouse Transgenic

Mouse

Mouse CPI

Human CPI

no

endogenous

expression

Transgene

expressed

Verification of CPI expression in T cells of Tg mouse

CPI CPI

CPI

CD4 PE-A

CD

8 V

500-A

-103

-102

103

104

105

-102

102

103

104

105

CD4 T

CD8 T

CD28 PE-Cy7-A

CD

95

V4

50

-A

-10210

210

310

410

5

-102

102

103

104

105

46.72%0.38%

42.80%10.10%

FoxP3 PE-A

CD

25

Pa

cific

Blu

e-A

-10210

210

310

410

5

-102

102

103

104

105 CD4TregFoxP3

CD3+CD4+

T helper

CD3+CD4+

T helper

(total T)

CD

25

CD

62L

CD

8

CD4 FoxP3 CD44

T naive Tcm Tem CD8 T CD4 T T reg

Other T cell subsets evaluated for CPI expression

Rederivation

•~ 3 months

Breeding Colony Development

•~ 3 months

Backcrossing Project

•~ 6 months

Colony Expansion

•~ 5 months

Receipt of Animals

Conclusions

0 3 6 9 15 18

+ Process is time consuming and labor intensive

+ Genetic testing and proper record keeping paramount to success of

colony development and expansion

+ Provision of high quality mice approximately 17 months after initiation

of project

Summary

+ Immunomodulatory biologics are increasing in complexity

+ A translational approach to safety assessment with focus

upon the biologics mechanism of action is essential in

designing fit for purpose safety packages

+ Although standard species offer enormous value in

determining the safety profiles of drugs that target CPIs,

new approaches are being considered all the time to

produce clinically translatable data

+ Biology…biology…biology…

Summary

Thank You

immune checkpoints sot2016

Health & Medicine