Drug Discovery Michael Palazzolo and William Boyle December 11, 2014

Reminders

• Questions

– Webinar participants: chat box

– In house: microphone

• Survey questions

• Course evaluation

Overview – High Level Objectives

Learn more about this fastest growing class of new medicines

• Review of approved mabs and target classes being prosecuted

in the clinic

• How therapeutic mabs are generated

• Technologies currently in play and next generation methods

• PK/PD of monoclonal antibodies

THERAPEUTIC ANTIBODIES AND TARGET CLASSES

International non-proprietary name Trade name Type Indication first approved First EU (US) approval year

Muromonab-CD3 Orthoclone Okt3 Anti-CD3; Murine IgG2a Reversal of kidney transplant rejection 1986* (1986#)

Abciximab Reopro Anti-GPIIb/IIIa; Chimeric IgG1 Fab Prevention of blood clots in angioplasty 1995* (1994)

Rituximab MabThera, Rituxan Anti-CD20; Chimeric IgG1 Non-Hodgkin's lymphoma 1998 (1997)

Basiliximab Simulect Anti-IL2R; Chimeric IgG1 Prevention of kidney transplant rejection 1998 (1998)

Daclizumab Zenapax Anti-IL2R; Humanized IgG1 Prevention of kidney transplant rejection 1999 (1997); #

Palivizumab Synagis Anti-RSV; Humanized IgG1 Prevention of respiratory syncytial virus infection 1999 (1998)

Infliximab Remicade Anti-TNF; Chimeric IgG1 Crohn disease 1999 (1998)

Trastuzumab Herceptin Anti-HER2; Humanized IgG1 Breast cancer 2000 (1998)

Gemtuzumab ozogamicin Mylotarg Anti-CD33; Humanized IgG4 Acute myeloid leukemia NA (2000#)

Alemtuzumab MabCampath, Campath-1H Anti-CD52; Humanized IgG1 Chronic myeloid leukemia 2001 (2001)

Adalimumab Humira Anti-TNF; Human IgG1 Rheumatoid arthritis 2003 (2002)

Tositumomab-I131 Bexxar Anti-CD20; Murine IgG2a Non-Hodgkin lymphoma NA (2003)

Efalizumab Raptiva Anti-CD11a; Humanized IgG1 Psoriasis 2004 (2003); #

Cetuximab Erbitux Anti-EGFR; Chimeric IgG1 Colorectal cancer 2004 (2004)

Ibritumomab tiuxetan Zevalin Anti-CD20; Murine IgG1 Non-Hodgkin's lymphoma 2004 (2002)

Omalizumab Xolair Anti-IgE; Humanized IgG1 Asthma 2005 (2003)

Bevacizumab Avastin Anti-VEGF; Humanized IgG1 Colorectal cancer 2005 (2004)

Natalizumab Tysabri Anti-a4 integrin; Humanized IgG4 Multiple sclerosis 2006 (2004)

Ranibizumab Lucentis Anti-VEGF; Humanized IgG1 Fab Macular degeneration 2007 (2006)

Panitumumab Vectibix Anti-EGFR; Human IgG2 Colorectal cancer 2007 (2006)

Eculizumab Soliris Anti-C5; Humanized IgG2/4 Paroxysmal nocturnal hemoglobinuria 2007 (2007)

Certolizumab pegol Cimzia Anti-TNF; Humanized Fab, pegylated Crohn disease 2009 (2008)

Golimumab Simponi Anti-TNF; Human IgG1 Rheumatoid and psoriatic arthritis, ankylosing spondylitis

2009 (2009)

Canakinumab Ilaris Anti-IL1b; Human IgG1 Muckle-Wells syndrome 2009 (2009)

Catumaxomab Removab Anti-EPCAM/CD3;Rat/mouse bispecific mAb Malignant ascites 2009 (NA)

Ustekinumab Stelara Anti-IL12/23; Human IgG1 Psoriasis 2009 (2009)

Tocilizumab RoActemra, Actemra Anti-IL6R; Humanized IgG1 Rheumatoid arthritis 2009 (2010)

Ofatumumab Arzerra Anti-CD20; Human IgG1 Chronic lymphocytic leukemia 2010 (2009)

Denosumab Prolia/Xgeva Anti-RANK-L; Human IgG2 Bone Loss 2010 (2010)

Belimumab Benlysta Anti-BLyS; Human IgG1 Systemic lupus erythematosus 2011 (2011)

Ipilimumab Yervoy Anti-CTLA-4; Human IgG1 Metastatic melanoma 2011 (2011)

Brentuximab vedotin Adcetris Anti-CD30; Chimeric IgG1; immunoconjugate Hodgkin lymphoma 2012 (2011)

Pertuzumab Perjeta Anti-HER2; humanized IgG1 Breast Cancer 2013 (2012)

Raxibacumab (Pending) Anti-B. anthrasis PA; Human IgG1 Anthrax infection NA (2012)

Trastuzumab emtansine Kadcyla Anti-HER2; humanized IgG1; immunoconjugate Breast cancer In review (2013)

Vedolizumab (Pending) Anti-alpha4beta7 integrin; humanized IgG1 Ulcerative colitis, Crohn disease In review (NA)

BLyS, B lymphocyte stimulator; C5, complement 5; CD, cluster of differentiation; CTLA-4, cytotoxic T lymphocyte antigen 4; EGFR, epidermal growth factor receptor; EPCAM, epithelial cell adhesion molecule; GP, glycoprotein; IL, interleukin; NA, not approved; PA, protective antigen; RANK-L, receptor activator of NFkb ligand; RSV, respiratory syncytial virus; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor. Source: Janice M. Reichert, Editor-in-Chief, mAbs.

36 Approved Monoclonal Antibodies

Target Classes for Therapeutic Antibodies Target Class MOA Examples

Cytokines Primarily neutralization TNFa, IL-6, BlyS, RANKL, INF-g, IL-23, L-17

Cytokine Receptors Receptor antagonist, agonist, blocking and internalization

IL-1r, DR-4, DR-5, CD40, IL-6R, TPOR

Immune Modulators and CD’s Receptor antagonist, ADCC, ADC, neutralization of viral entry

CD28, CTLA-4, CD52, CD33, B7RP-1, PD-1

Growth Factors Primarily neutralization VEGF, HGF, ANG1/2, NGF, CSF-1

Growth Factor Receptors ADCC, ADC, blocking HER, HER2, IGFR-1, CSFR-1, TrkA

Cell Adhesion Molecules Blocking counter receptor binding Gp IIb/Iia, CD11 (LFA), a4b1, a4b7, ICAM

GPCR’s Neutralization, ADCC, antagonist, antagonist

Glucagon receptor, CXCR 2-5, Calcitonin R

Extracellular Matrix/Mucins/Amyloid Blocking, dissolution of aggregated plaque

Sclerostin, MUC1, b-amyloid

Compliment Pathway Blocking, protease neutralization C5a, tissue factor, factor VIII, VWF

Ig and FcR Class Neutralization and blocking IgE, FceR1, Fc receptor gIIIB

Enzymes and Serum Proteins Blocking Carbonic anhydrase, oxidized LDL, PSCK9

Gated Voltage Channels Blocking ion flux K= channel, TRPV1, PN3, SDF-1

Infectious Disease Antigens Neutralization and opsinization RSV, HIV, MRSA, HCV, Influenza

Ca 2010; non-redundant target list ~ 110 targets

MOA of Therapeutic Antibodies

• Neutralization of growth factors and cytokines

• Blocking receptors and adhesion molecules

• Cell killing and clearing via Effector functions

• Interference of Pathogen Entry and Pathology

• Interference of Enzymatic Activity

What are Therapeutic Antibodies?

IgG-Fab: Antigen Binding

Oligosaccharide

Hinge

Fc Receptors & Complement Binding

FcRn Binding

Antigen Binding

IgG-Fc: Fc Receptor Binding

• Generally Humanized Recombinant Immunoglobulins (IgG1, 2, 4)

• Heavy and Light Chain Linked via Cys Bonds

• Constant Regions Including Fc Domain

• Hypervariable Regions, CDR’s, Involved in Antigen Binding

• Ig Repitoire Capable of Generating 10E+09 Diversity

• Recombination of Germline Variable Regions

• Somatic Hypermutation to Increase Affinity and Specificity

Antibody Modalities in the Clinic

Major Industry Focus

Full length bivalent, fully human

Full-length product formats Hyphenated product formats

Variety of platforms allow for the isolation and optimization of all antibody modalities; full length and fragments to produce best-in-class therapeutics

GENERATION OF THERAPEUTIC ANTIBODIES - TECHNOLOGY

Recombination Creates Initial Antibody Diversity

How are Antibodies Formed?

Recombination Somatic Hypermutation

Antibody specificity and affinity are selected following these two critical events

Antibody Diversity Generation Recent Advances in Understanding

Somatic Hypermutation

• Natural process of mutagenesis that occurs in B cells during the selection and maturation of antibodies

• Key in vivo process for formation of antibodies with potent characteristics

• Follows an optimized design template created during Ig recombination

• Mutations are often non-obvious and hence difficult to recapitulate by in vitro mutagenesis

* * * * SHM is initiated by Activation Induced

Cytidine Deaminase (AID)

Generation of Therapeutic Antibodies

CDR grafting – most approved MAbs developed this way “Queen” IP estate - PDL, Genentech Transgenic mice – fully human antibodies from mice Abgenix/Amgen (Cell Genesys) Medarex/BMS (GenPharm) Phage display – Abbott’s Humira anti-TNF CAT, Morphosys, Dyax Yeast Display – Abbott, Adimab, Alder Alternative Routes to Humanization Morphotek Kalabios Xencor AnaptysBio BioAtla

Antibody humanization is the fundamental breakthrough that allowed antibodies to become a major therapeutic class

Select full length human mAb; save time and avoid loss due to format conversions

Ability to screen large libraries with several different types of antigens in different formats

Retain avidity and affinity from both VH and VL binding

Ability to measure simultaneously affinity and expression levels

Fast cycle times

Antibody Discovery Platform Overview: Several Advantages

Library Based Technologies

In Vivo Immunization

Cell Based Technologies

Companies

Key Limitations

Key Industry Advantages

Requirements

CAT Dyax Morphosys

Xoma Glycofi

Adimab

Abgenix Medarex Kirin

Hematech Regeneron Ablexis

Vivalis

Humabs AIMM

Theraclone Chiome 4-Antibody

Rely on partial antibody formats for discovery and format conversion

Accumulate mutations in the framework due engineering and requires germ lining/humanizing

Lack of critical read-outs (expression levels, affinity)

In vivo bias against homologous targets

Further germlining required

Limited selectivity of target epitope(s)

Search for rare events in a disease state

Further optimization is often required can lead to loss of activity

Often identify key epitopes rather than product leads

Acquired or Encumbered

Recent Start-up (2010)

Established with Partnerships

AnaptysBio Sea Lane F-Star Kymab Ablynx

Other

Morphotek

Domantis

BioAtla Silverlake

107 10-100

Mouse

immunization (e.g.,

XenoMouse®, HuMab-Mouse®

& wt mice)

Static library

approaches (e.g., antibody phage

display)

Industry adopts redundant, resource and time intensive approaches to address technical

limitations of other platforms

Industry Needs… New, powerful Ab discovery and protein optimization approaches

How industry mitigates risk

• Large pharma example (AstraZeneca): Owns/access multiple platforms incl., CAT/phage lib.,

Dyax/Phage lib., Amgen/XenoMouse®, REGN/VelocImmune, Xencor/XmAb™, MEDI/humanization, other …

• Antibody that meets design goals not always present in library

Example technical limitation

• Body does not always generate useful Abs to desired site on target

Despite emergence of phage display and transgenic mouse platforms, majority of industry continues to

utilize humanized antibodies

1 3 4 7 2 4

15 20

34 26

90 85

0

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40

60

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100

120

Approved Phase III Phase II Phase I

Humanized

TransgenicMousePhage Display

Stage of Development

Num

ber o

f Kno

wn

Antib

odie

s

Antibodies in the Clinic

• Process is well understood

• Immunization techniques are important

– Route, duration, antigen selection and formulation

• Screening techniques key for antibody selection

• From our perspective, two starting points; immunization or existing sequence

Humanization: Part I Begin with Mouse Immunization & Mab Production

Myeloma cell

Fusion and Hybridoma Formation

Immunizied spleen cells High Through-Put

Hybridoma Screening ELISA Hybridoma Selection &

Antibody Production and Bioassay

Clone and generate H&L chain sequences

• Splicing of CDR sequences obtained from a rodent (mouse) immunoglobulin onto a human immunoglobulin scaffold; i.e., CDR Grafting

• Goal is “seamless integration” of mouse and human sequences using the smallest amount of mouse sequences in the final product

100% Mouse Protein (>60%)

Ordinary Mouse

No mouse protein (~1%)

5-10% mouse protein (1-8%)

Humanized Human Chimeric

Humanization Part II: Process and Goal

~30% mouse protein (25-30%)

Antibody humanization is the fundamental breakthrough that allowed antibodies to become a major therapeutic class

• Herceptin example (~1% immunogenicity)

• Back mutations introducing mouse sequences needed in this case

• SHM technology should bypass this need and give rise to more human-like product candidates

Humanization: Herceptin Example

HEAVY 1 2 3 4 5 6 7 8 91

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5B

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2B

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C8

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3

*g4D5 E V Q L V E S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P G K G L E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q M N S L R A E D T A V Y Y C S R W G G D G F Y A M D Y W G Q G T L V T V S S

* * * * * * * * *CDR1 CDR2 CDR3

human E V Q L V E S G G G L V Q P G G S L R L S C A A S G F T F S S Y A M S W V R Q A P G K G L E W V S V I S G K T D G G S T Y Y A D S V K G R F T I S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R G R X G X S L S G X Y W G Q G T L V T V S S

Mouse residue back-mutations

KD without back-mutations: 25nM KD with back-mutations: 0.3nM

Humanized mAbs can be very close to fully human mAbs depending on the complexity of the

engineering process used

Humanization Process

Murine Antibody

Mouse Immunization

Published Mouse Ab Sequence

and immediate

grafting and affinity

maturation

CDR-Grafted Antibody CDR Grafting

(Winter method. MRC)

Random Mutagenesis and/or

PDL Technology (Queen

method)

“wet” approach

• Murine Mabs still to be the largest source of new antibody medicines in the future

• Traditional methods use both Winter and Queen patents to humanize murine monoclonal antibodies for therapeutic uses

• Winter IP is close to expiration • Queen Patent still in force and an issue for humanization • Alternative methods for affinity improvement

“Dry” approach

Immunogenicity of Therapeutic Antibody Types

EU and FDA guidelines acknowledge the immunogenic potential of monoclonal antibodies

Product Name Indication Ig Modality Reported Immunogenicity

OKT3 Acute rejection of organ transplants

Murine 80%

Rituxan B-cell lymphoma

Chimeric 1.1%

Herceptin Metastatic breast cancer

Humanized >1%

Humira

RA/Crohn’s Human Phage display

15%

Panitumumab Colorectal cancer

Human Transgenic

4%

Remicade RA/Crohn’s Chimeric 45%

Zenapax Acute rejection of organ transplants

Humanized 34%

Tysabri MS Humanized 10%

Avastin Colorectal cancer

Humanized >1%

Source: Product labels and patient information leaflets

• Ig isotype, route of administration, method of optimization

and human V gene scaffold usage are important

determinants in HAHA formation – may impact PK/PD

• No major safety issues, but efficacy can be compromised

• Fully human antibodies remain the ideal choice, but

depending on the methods used, humanized abs are as

good as fully human antibodies wrt immunogenicity

Technology Timelines: Comparison of processes

XenoMouse

Humanization **

Phage Display

Antibody Discovery Cell Line Development

Antibody Engineering/ or SHM Optimization

Manufacturing Scale-up

3 mon.

3 mon.

3 mon. XenoMouse

+ optimization

Humanization *

3 mon.

* “Paper” starting point

** “Wet Lab” starting point

Enhancing Efficacy

• Loss of patent exclusivity and the emergence of non-antibody scaffolds is driving a need for product differentiation, ideally via improved efficacy

Glycosylation Strategies – defucosylation to increase affinity to the FcgRIIIa receptor, enhancing ADCC (Antigen Dependent Cellular Cytotoxicity) GlycoFi – acquired by Merck Glycart – acquired by Roche BioWa – multiple partnerships Fc Region Engineering – ADCC enhancement, half-life extension, Genentech Xencor – multiple partnerships Macrogenics – Lilly deal Also, antibody-drug conjugates, ADC’s Seattle Genetics, Immunogen, CovX

Antibody Drug Conjugates – Adcetris and Kadcyla Pave the wave

Improvements in linker technology, payload characteristics and ability for site-directed conjugation have enabled this new class of antibody drugs

Pharmacology of Recombinant Antibodies

Relationship between Antibody Affinity and Dose

• Critical Concepts:

– Unique Features of Antibodies; • High specificity for target antigen

• Efficacy and safety generally correlated with the extent of interaction with the target antigen

– Adequate affinity is required to: • Achieve the maximum therapeutic benefit

• With the intended route of administration

• At a dose associated with an acceptable cost of goods

• Will facilitate patient compliance

Binding Affinity Design Considerations

• Target Antigen Concentration

• Fraction of Antigen bound

• Antigen Turnover Rate

• Antibody PK and Biodistribution Properties

• Recruitment of any Effector Function

• Impact of Administration Route

Impact of Target Antigen Concentration

• When antigen conc is less than antibody affinity:

i.e. Ag <<KD

• Binding is governed by the affinity of the antibody for the antigen (Region 1)

• Therefore an improvement in affinity leads to an improvement in potency

Impact of Target Antigen Concentration

• When antigen conc is greater than antibody affinity:

i.e. Ag > KD

and when affinity is reduced to approx. 1/10th of antigen conc

• A potency ceiling is reached (Region 2)

• Therefore further improvements in affinity do not lead to significant improvements in potency

Time (day)

0 7 14 21 28

Seru

m C

once

ntra

tion

(ng/

mL)

0.1

1

10

100

1000

10000

100000

1000000

Time (day)

0 7 14 21 28

Urin

ary

N-T

x/U

CR

T %

Cha

nge

from

Bas

elin

e

-100

-80

-60

-40

-20

0

20

40

60

80

100Mean (±SD) Urinary N-

Tx % Change from Baseline 1 mg/kg SC

Mean (±SD) PK Profiles 1 mg/kg SC

AMG 162 vs. AMG 0007 in Cynods

Take Away Points

• Therapeutic Mabs are the largest growing area of human therapeutics

• Diverse targets with preference to specific classes – some classes are relatively intractable

• Multiple modality opinions; naked and ADC’s

• PK/PD is critical for design goals

• Other properties TBD – manufacturability, IP and Integration of Target Biology and Drug Concept

Acknowledgements

• UCLA Pharmacology

• UCLA CTSI

• UC BRAID CAI

• NCAI (UC, Harvard, Ohio)

• NHLBI

Upcoming Webinar: UC CAI I-Corps Stephanie Marrus Friday, January 23, 2015 @ 11 AM PST Archived Webinars: http://uccai.ctsi.ucla.edu/pages/education