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Drug Discovery Michael Palazzolo and William Boyle December 12, 2014
Reminders
• Questions
– Webinar participants: chat box
– In house: microphone
• Survey questions
• Course evaluation
Overview – High Level Objectives
3
Case Study of Therapeutic Antibody Generation and Development:
• Drug concept, competitive landscape and strategy for success
• Use of technology for optimal activity
• Preclinical testing
• Generation of new intellectual property (IP)
• Manufacturing and IND enabling studies
• Business development and partnering for clinical development
CASE STUDY
Best-in-class anti CD22 antibody drug conjugate(ADC) for the treatment of
B-cell cancers
4
CD22 Superfamily of Sialoadhesions
CD22 Biology
6
CD22 B-cell specific surface receptor that plays a key role in humoral immune response
Expressed at high levels during B-cell differentiation and in a majority of lymphomas and leukemias
B-cells implicated in the molecular pathology of a variety of autoimmune diseases, e.g. RA, Lupus, MS
CD22 is an extracellular signaling receptor and is internalized, therefore making it an attractive therapeutic target for both naked antibodies and ADCs
Mab LL2 / Epratuzumab binding to CD22 and internalization negatively regulates B-cell tumor cell growth
Established pattern of CD22 expression
7
Frequency of NHL subtypes
CD22 is expressed in most non Hodgkin’s lymphomas and lymphocytic leukemias, including the major categories
WHO classification of tumours, 2001
CD22 is expressed in most non Hodgkin’s lymphomas and lymphocytic leukemias
CD22 is known to be expressed only in developing B cells and in B-cell lymphomas and leukemias. CD22 expression has not be reported in other tissues (Stein, R., Belisle, E., Hansen, H. J., and Goldenberg, D. M. Epitope specificity of the anti-B-cell lymphoma monoclonal antibody, LL2. Cancer Immunol. Immunother., 37:293-219989,3).
CD22 is a 135-kDa B-cell-restricted sialoglycoprotein expressed on the B-cell surface only at the mature stages of differentiation (Dorken B, Moldenhauer G, Pezzutto A, et al. 39 (B3), a B lineage restricted antigen whose cell surface expression is limited to resting and activated human B lymphocytes. J Immunol 1986;136:4470–9.).
CD22 is expressed in almost all lymphomas and B-cell leukemias. In B-cell NHL, CD22 expression ranges from 91% to 99% in the aggressive and indolent populations, respectively (Cesano A, Gayko U, Brannan C, et al. Differential expression of CD22 by indolent and aggressive NHLs: implications for targeted immunotherapy. Blood 2002;100:350a.).
Using immunohistochemistry staining of formalin-fixed paraffin-embedded samples, CD22 positivity has shown to be highly variable between samples and within the same samples in terms of the percentage of positive cells, intensity of staining, and localization (i.e., membrane versus cytoplasm).
Anti-CD22 Antibodies in Development
Pre-clinical Phase I Phase II Phase III IND
VivaMab Naked mAb
RA
Genentech CD22-MMAE
Onc
UCB Emab
Autoimmunity
Pfizer Ino-ozo
Onc
VivaMab CD22-ADC
Onc
Humanized IgG4 with calicheamicin ADC
Deprioritized in Ph 2
Humanized IgG1 (LL2)
Difficult indication for approval (SLE)
Onc indications with ADC in early stages
Humanized IgG1 with Aurestatin E ADC POC achieved in Ph 1
Humanized IgG1 (LL2 derivative) with superior affinity and internalization
Ready for cell line development and IND enabling studies
ADC antibody w/ chemotherapy is appropriate for leukemia and lymphoma
Early Stage programs with enhanced antibodies represent next generation
biobetter opportunities
Epratuzumab (UCB-Immunomimedics) Currently in late stage clinical trials for
Lupus (Phase III) Development of naked antibody for
lymphoma and CLL stopped due to lack of efficacy
UCB and Immunomedics, Inc. announced epratuzumab, has provided a significant reduction in disease activity with moderate to severe active systemic lupus erythematosus (SLE) in Phase IIb study
EMBLEM study in the combined index responder rates were numerically higher in all groups given epratuzumab at 600 mg per week (p = 0.0265 *) o Emblem was a 12-week, multicenter, phase
IIb, randomized, double-blind, controlled versus placebo to evaluate the efficacy and safety of epratuzumab and define a dose and diet in patients with moderate to severe SLE. The primary efficacy endpoint in the emblem was a test of combined response index, including several indices of SLE activity
Two phase III studies, EMBODY™ 1 and 2, underway for pipeline drug epratuzumab in December 2010. Results due in 1Q2015
Inotuzumab Ozagamicin (Pfizer)
Currently in clinical development for hematological malignancies
Summary of Phase I results (JCO, 2010) o Seventy-nine patients enrolled o MTD was determined to be 1.8 mg/m2 o Common AEs at MTD were thrombocytopenia
(90%), asthenia (67%), and nausea and neutropenia (51% each)
o Objective response rate at the end of treatment was 39% for the 79 enrolled patients, 68% for all patients with follicular NHL treated at the MTD, and 15% for all patients with diffuse large B-cell lymphoma treated at the MTD
o Median PFS was 317 days (approximately 10.4 months) and 49 days for patients with follicular NHL and diffuse large B-cell lymphoma, respectively
Although high response rate, hematological tox profile may be limiting
CD22-MMAE Genentech/Roche
Genentech (gRED) has developed an IgG to CD22 that is conjugated to MMAE (Seattle Genetics)
Phase I trial completed
Good effects in lymphoma patients – may have issues with potency
Review of the competition indicates that a best-in-class anti-CD22 antibody combined with the best linker-toxin ADC represents a bio-
superior drug for the oncology market
CD22 Targeted Therapeutics in Development
Emab UCB
Inotuzumab Pfizer
CD22-MMAE Genentech
VM101 VivaMab
X X
X X
X X
X Microtubulin inhibitor
Nucleic acid inhibitor
CIAOTM
VivaMab has used Comprehensive Integrated Antibody Optimization (CIAOTM) to evolve Epratuzumab (Emab) into an improved, target-specific antibody therapeutic; enabled generation of an anti-CD22 ADC for use with a tumor appropriate chemotherapy
X
X DNA Binder
X X
X X
X X
Differentiation in CD22 Targeted Therapeutics
Best-in-class antibody with tumor appropriate drug conjugation for a highly differentiated CD22- targeted bio-chemotherapy
Properties Emab UCB
Inotuzumab Pfizer
CD22-MMAE Genentech
VM101 VivaMab
Affinity + +++ + +++++
Internalization + + ++ +++++
MOA Neg growth regulator
Delivers calicheamicin
Delivers auristatin
Neg growth regulator by delivering nucleic acid inhibitor
Isotype IgG1 IgG4 IgG1 IgG1
Linker Stability
NA Unstable Stable Stable
Stage of Development
Phase III Phase II Phase I Preclinical
Anti-CD22 Target Product Profile
Profile VivaMab 101 CD22 affinity ~200 pM
Isotype Human IgG1
Projected half-life ~21 days
Route of delivery IV (initially); SQ subsequently
Dosing schedule Q 4 wk
Safety Predictable & manageable
Proof of mechanism (POM) Healthy volunteers or Psoriasis
IND ~9 months from funding
First patient In ~10-12 months from funding
Biomarkers CD22 receptor occupancy; B cell depletion
Proof of Concept (POC) Severe to moderate Psoriasis; RA; Hem-Onc
Expanded indications with partner
RA, CD, MS, SLE, ALL, NHL
CD22 Selection of VM101
VM101 Selection - Summary
Lead selection based on key design goals; high affinity, surface binding, internalization and expression levels and
good manufacturing characteristics
Twenty top humanized VM101 candidates obtained from murine LL2 monoclonal using CIAO!TM
o Selected based on FTO, affinity, surface binding and internalization (FACS)
Ten variants analyzed by confocal immunofluorescent microscopy
o Rapid surface binding and internalization
Three candidates with a clear lead selected
o Lead shows greatest internalization, superior expression levels, lack of oxidizable residues in V-gene regions (Asn, Cys, Met)
o High level expression in CHO cells
o Other two candidates retained as backups and for ADC conjugation assessment
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 humanization and affinity improvement – Framework grafting
“Dry” approach
VivaMab Antibodies for Life
Humanization of Murine Mab to Therapeutic Target
Framework grafting used to produce humanized clone Analysis of humanized clones: 33 hits with specific activity >75% of wild type 12 hits with specific activity equal to or better than wild type
Frame works fully human No back mutations needed to restore binding activity
Several Hits with improved activity by Elisa
Summary of VM101 Engineering Strategy
Anti-human CD22 mouse mAb LL2
Clone BA006-12E11-RVG
Clone VM101
Affinity maturation CDR sequence diversification
Humanization Characterization (ELISA, SPR, Internalization, Sequence, Expression) Lead Selection
KD ≈ 20 nM
KD ≈ 1 nM
KD ≈ 0.22 nM
Epratuxumab KD ≈ 5 nM
Internalization – Top Lead Analysis
Relative Mean Fluorescence
Emab Vm 101
10 208
0 min 90 min 0 min 90 min
Quantitative confocal immunofluorescent microscopy demonstrates dramatic improvements in surface binding and internalization
Internalization (minutes incubated at 37oC)
Emab Vm 101 Vm 101 Emab
VivaMab Antibodies for Life
Induction of Tyrosine Phosphorylation
Humanized anti-CD22 mAbs induce Tyrosine-Phosphorylation of CD22
Immunoprecipitation from Daudi cells
Anti-Phospho-Tyrosine blot
Anti-CD22 blot
Unt
reat
ed
Ant
i-hu
man
IgG
Fc
Ant
i-hu
man
IgM
Fab
BA00
6 (E
-mAb)
H
um18
H
um
43
H
um01
H
um02
2 % 3% 177% 100% 100% 142% 26% 20% Tyrosine Phosphorylation relative to BA006
VM101 Lead Candidate
Clone Vm 43 consistently expresses at highest levels and lacks Asn, Cys and Met residues in the variable domain – good manufacturing characteristics;
Vm 02 and 04 selected as backups
Clone KD (nM) Daudi Internalization by Confocal (RMF)
LL2 (mouse Mab) 20.00 ND
Epratuzumab 5.00 ~10
Vm 01 0.50 ~100
Vm 02 0.22 ~100
Vm 04 0.33 ~150
Vm 05 0.34 ~150
Vm 07 0.80 ~100
Vm 018 2.70 ~200
Vm 20 1.00 ~200
Vm 43 0.26 ~200
Production of recombinant VM101-ADC
PBD payload conjugate Characterization In vitro bioactivity
Payload -Pyrrolobenzodiazepines (PBDs)
Original natural products isolated from streptomyces species Bind sequence selectively (purine-guanine-purine motifs) in the
minor groove of DNA Synthetic PBD dimers crosslink DNA without distortion.
• Selective activity in tumor cells vs. normal cells • “10,000 times more active that potent microtubulin inhibitors...”
No cross resistance with cisplatins and mustards
SG2000 in Phase II clinical trials for cisplatin refractory ovarian carcinoma
HP-SEC Analysis Before Conjugation
HP-SEC Analysis of VM101 ADC
VM101-ADC Superior to Emab-ADC in vitro
CD22 Preclinical
Development
Purpose: Determine Tumor Growth Delay of the Daudi human Burkitt's lymphoma with treatment of a novel ADC
Established tumors ~120 mm
Dosed once week 3X (Day 1, 7, 14) CX1 = VM101 in PBS CX2 = VM101-ADC in PBS Rituximab = Rituximab in Saline vehicle = vehicle
Daudi-e219 experiment
Day 60 data complete; study extended to 60 days to monitor durability of complete responses in the VM101 ADC and Rituxan treatment arms
10/10 complete responses in VM101 ADC 0.5 and 2.5 mg/kg groups; 8/10 complete responses in 0.1 mg group
Body weights are within the safety range
Summary of Results
Mouse Tumor Xenograft Model – Day 60 Group Mean Summary
• 10/10 complete remission of existing tumor in VM101 ADC 0.5 and 2.5 mg/kg. 8/10 CR’s at 0.1 mg/kg – Body weight remains normal
• Some dosage effect, but MED not determined (>0.1 mg/kg)
• Approximately 2 log superiority to Rituximab
• One animal in the positive control arm (Rituxan) reached the study endpoint and was sacrificed
• No animals in any of the VM101 ADC dose groups reached an endpoint
Summary
Pilot study to test the effects of VM101 in a tumor xenograft model. VM101 ADC was not fully optimized with respect to conjugation chemistry and comparison of in vitro activity – Potent activity in vitro and in vivo
VM101 lead clone and back-up clone have been assessed for manufacturability – Test expression levels comparable to Herceptin (trastuzumab)
PK analysis in rodents – T1/2 equivalent to expectations for a human IgG1
Optimization of VM101-ADC chemistry and production underway – cell line development are the next steps
CD22 recently found to be expressed on lung cancer cell lines and primary tumors – potential new use as an important drug to lung cancer treatments
Mab Discovery Timeline and Costs
Manufacturing
36
• Expensive process - ~US$ 5.0 MM
• ~18 months from lead antibody to cGMP material needed for IND enabling studies
• Contract cell line development, process development and clinical manufacturing available from various CMO’s (e.g. BI, Lonza)
• Expression yields are in the 5g/l range
Timing is Critical to Remain on Track
37
Timing, Contd.
Mab Discovery Timeline and Costs
IND Exit
Clincal POC Exit
Preclinical Exit
Therapeutic Antibodies – Early Stage Exits
Company Target Partner Stage Year Upfront Milestones (Royalty Net Sales)
Aveo RON JNJ/Centocor Preclinical 2011 $15 MM $555 MM
Alder BioPharma
IL-6 BMS Ph II 2009 $85 MM $765 MM
Merrimack HER3 Sanofi Aventis Ph I 2009 $60 MM $410 MM
Agensys PsCA Merck Preclinical 2005 $17.5 MM $200 MM
NovImmune IFN/CD3 Serono Preclinical 2005 $15 MM $105 MM
Micromet EpcAM Serono Ph 2 2004 $147 MM ND
BioStratum Laminin-5 Novo Nordisk Preclinical 2004 $80 MM ND
Seattle Genetics 5T4 Pfizer Ph 1 2011 $8 MM $200 MM
Seattle Genetics EGFR Abbive Preclinical 2 014 $25 MM $225 MM CytomX EGFR and
Platform Pfizer Preclinical 2014 $25 MM $610 MM (tiered double digit)
Ablynx IL-6 Abbvie Ph 2a 2013 $175 MM $675 MM (tiered double digit)
Partnering
• VM101 licensed to ADC Therapeutics Sarl
• At preclinical stage prior to cell line development
• Financial terms not discussed
– Upfront cash
– Milestones
– Joint ownership
• Preclinical data indicates it is the most potent molecule in its class
– Eradicates Rituximab resistant tumors
• Ongoing development – Tox studies in non-human
primates
….
Key Areas for IP Consideration and Generation
FTO Assessment on Mabs to
Target
Antibody Claims to Target
Target IP and generic claims to antibodies
Method of Treatment
Claims
Composition on Clinical
Candidate
Monocloncal antibody development plan Time Frame (in Months)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Target selection and Mab generationSelect target based on key criteriaFTO ScrubAntigen production and mab generationScreening and in vitro characterizationIn vivo testingHumanization & Clinical candidate selection *Manufacturing of clinical material
IND enabling Preclinical Studies
IND *POC Clinical Studies
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 are manufacturability, IP and Integration of Target Biology and Drug Concept, strategy for clinical development
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
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