Lecture 16: Business of Science Outsourcing and Collaborative Drug Discovery Research

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Biotech and Pharma Activity Across the Discovery R&D Value Chain

• Biotech companies are funded by investors (typically VCs) who get a payout when projects are licensed or company enters stock market or is purchase

• (typically start w early stage research concept and last a few years before being bough out or closing a few have brough products all the way to the market like Gilead and Biogen)

• Pharma companies earn income from a portfolio of marketed products and invest some profits in R&D

• (R&D historically performed in house, now pharma pipeline is increasingly filled with projects in license from smaller biotech’s)

• Today both pharma and biotechs outsource much of their R&D to CROs

What is a CRO?

-encompasses a huge range of companies that provide services to biotechs and pharma companies

-their defining feature is that work performed is for immediate payment, not investment for future sales profit

-this funding models means a CRO can start very small and grow gradually as business develops

-others are launched with VC investments to fund larger labs and or expensive infrastructure from the beginning

-28 000 CROS working in medical R&D. 830 in Canada, 230 in Montreal

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CRO specializations in drug discovery and development

Some CROs offer a broad range drug discovery services across multiple departments,

• Protein expression and purification

• Biophysics and structural biology

• Biochemical and cell assay panels

• Custom biochemical and/or cell assay development

• High-throughput screening

• Electrophysiology (cells, in vivo and/or ex vivo)

• Drug Distribution, Metabolism and Pharmacokinetics (DMPK) assays

• General chemistry services (med-chem and synthesis)

• Cloning and other molecular biology

• DNA/RNA synthesis or peptide synthesis

• Engineered cell lines

• High-throughput DNA sequencing (“NGS”)

• Protein biotherapeutic production (“GMP” or “non-GMP”)

• In vivo safety and toxicology studies (“GLP”)

• Chemical manufacturing on large scale (“GMP”)

• Radiolabeled chemical synthesis

• Drug substance physical analysis and formulation

• Bioanalysis of clinical samples (Mass spec, Flow

cytometry and/or ELISA and related)

• Storage and distribution of research compounds or

clinical samples

• Clinical trial coordination and monitoring

Comments on How Pharma and Biotech’s work with CROs

• Big Pharma increasingly outsource project work, especially compound synthesis and primary

testing in routine biology and DMPK assays

• Some CROs in India and China construct whole buildings dedicated to one particular Pharma

• Pharma companies will also use specialized CROs for some services, while they maintain their own

dedicated labs for others

• There is recent trend for some Pharma to carry out some outsourcing of work with North American

CROs, coupled with an expectation of higher intellectual input into projects

• Very small Biotechs often have no labs and outsource everything.

• They may coordinate efforts of many CROs themselves, or work with a CRO that can handle further outsourcing as needed

• Many small companies have a computational/AI focus for their own employees, but outsource all “wet lab” work

• More established Biotechs work with CROs that provide additional resources for their discovery efforts

• Faster and less expensive to contract for 10 people at a CRO than to hire 10 more employees and find space for them to work

• Some Biotechs have labs for one department (e.g. biology), but not others (e.g. chemistry or DMPK)

Intro to Paraza Pharma

MTL Canada based fully integrated preclinical R&D organization committed to the discovery and delivery of drug candidates for client partners

expertise in research:

chemical sciences, biology, drug metabolism and PK

key hallmarks:

creative problem solving, intellectual input, diligent and flexible team, consultative approach, risk reduction mindset

provide stand alone or integrated services to support a clients specific needs

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Paraza Involvement Within the Discovery R&D Value Chain

Target ID and validation, Hit identification, lead identification, lead optimization, candidate selection

Resourcing models: Fulltime Equivalent

Our FTE rates are built to be inclusive

• Chemistry FTE includes most chemicals and

routine analytical support (including chiral

separations)

• For Biology FTEs, project-specific materials are

charged as a pass-through cost

• No charges for project management oversight on

projects

• Most of our projects are FTE basis

• Many projects are fully integrated across the three

departments

Resourcing models: fee for service

DMPK is based on the FFS model

• Can be the favored approach for some applications

in Chemistry and Biology

• Specific compound synthesis or reaction/route

optimization

• Pharmacology studies

• Well-defined biochemical or cell biology studies

• High-throughput screening

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How do Paraza scientists interact with colleagues at client companies

quite flexible in how we work, depending on needs and expectations of different clients

• Some clients have very minimal staff and ask us to manage all day-to-day work, setting priority

goals and giving scientists freedom to propose and organize specific activities

• Level of oversight varies, as in academic labs: sometimes the client checks in daily, sometimes once a month. Biweekly updates are typical

• Some virtual companies expand to occupy their own labs and some want to remain virtual

• In some cases the client company has their own labs, and our Scientists interact with theirs as part of one team.

• Some clients contact us for very specific services (defined study or performance of an

optimized assay), and there is minimal contact beyond providing results

• We generally have more strategic involvement when collaborations are broader (especially if across multiple department

Medicinal Chemistry Overview

complex molecule synthesis

multi-parameter optimization

non-classical modalities

computer-aided drug design

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Chemistry-Enabling Technologie

high throughput screening

parallel chemistry

chemical synthesis and development

analytical support

DMPK Capabilities Overview

In vitro:

Suite of well-qualified in vitro compound profiling assays;

customized as needed: solubility, logD, metabolic stability

(mics, heps), protein binding, permeability and efflux,

P450 inhibition & induction, efflux transporters,

blood/plasma partitioning, etc.

• Profiling and screening modes available for most assays

In vivo:

Facilities accredited by the Canadian Council on Animal

Care (CCAC)

• Rapid turnaround for routine rodent PK; dog and NHP

available

• Formulation expertise for all routes of administration

• Dose escalation, MTD and metabolic studies, as well as

collection of multiple biofluids and tissue

Bioanalysis:

Eight LC-MS/MS systems to support analysis of in vitro

ADME, PK and PD samples

• Bioanalytical method expertise for different drug

modalities

• Specialized services include Met-ID and proteomic

analyses using HRMS system

Biological sciences overview

Highly trained scientists with expertise

in custom assay development:

target validation, compound screening, mechanism of action, late-stage profiling

Broad experience handling various

drug modalities:

small molecule inhibs or activators, degraders, RNA-targeted therapies, peptides and antibodies

Value Proposition:

develop assays tailored to your program

provide reproduable accurate data

interpret complex results provide clear analysis, advise on options to maximize success, data-driven decision making, open and transparent communication

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Protein Expression and Purification

-purification of MBH-His Kinase fusion protein expressed in insect cells

-challanges low-expression of full-length protein, tendency to aggregate at higher concentration and in low salt buffers

-concentrating the cut protein below 2mg-mL avoided aggregates in void volume on SEC

-all POI eluted in P2 peak pooled into one fraction, final protein 1.2mg

Surface Plasmon Resonance to Measure Ligand-Protein Binding

Low-volume microfluidics with

sensitivity down to 100 Daltons

▪ Quantifies binding affinity

and kinetics

▪ POI and control protein

immobilized in parallel

▪ Examples, peptides from

phage display screen

1. “Typical” binding

2. Slow binding

3. Promiscuous binding

APOBEC Adenosine Deaminase Assay Development & Application of New APOBEC Assay to Screening

No small molecule inhibitors and no published assay suitable for screening

▪ Method of published assay attractive, but coupling enzyme too inefficient

▪ Alternative 3-step method adapted from gel-based assays worked out

Application of New APOBEC Assay to Screening

▪ Assay reproduced published activity for modified-

oligo inhibitor

▪ Screening several hundred computationally designed

candidate inhibitors was not successful

▪ Most active hit show

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Ternary Complex Formation Assay to Screen for Molecular Glues

TR-FRET and FP biochemical assays were developed to measure interaction between ubiquitin protein ligase and fluorescent-labelled peptides

▪ The wild type degron sequence was used as positive control (high binding affinity)

▪ Disease relevant mutant peptides with medium or weak binding affinity were used to screen compounds

acting as molecular glue to enhance binding to the ubiquitin protein ligase

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Biochemical and Cellular Assays to Quantify Compound-Protein Interaction

Both assays measure displacement of fluorophore labelled ligand

▪ Fluorescence Resonance Energy Transfer (TR-FRET) used for assay with purified proteins

▪ Bioluminescence Resonance Energy Transfer used in cells

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Viability Assays to Profile Cell-Active Inhibitors

Compound potency evaluated in expected sensitive and non-sensitive cell lines

• Based on DepMap analysis

• Viability quantified using cell-permeable CyQUANT Green nucleic acid stain

• Target inhibition tracks with genetic predictions

• Viability results have been good predictors for xenograft efficacy studies

Characterization of Splicing Modulators

mRNA splicing can be modulated by ASOs and small molecule modulators to increase or

decrease the level of active protein produced in cells

▪ Here RT-qPCR confirmed increase in copy number of the cryptic exon and decrease

of the canonical exon after small molecule treatment

▪ Decrease in protein confirmed by Western Blot

Targeted Protein Degradation (TPD) and PROTAC

heterobifunctional degraders becoming modality of choice for many targets

▪ Validation of mechanism by Western Blot and viability/apoptosis assays shown here

• Degradation measured at ~6 hrs, apoptosis and viability at 2-7 days

Higher Throughput Assays for Targeted Protein Degradation

HiBiT system uses split luciferase which recombines to give luminescent signal

proportional to POI intracellular concentration

▪ 384-well homogenous assay

▪ HiBiT tag inserted by CRISPR at endogenous location

• Contrast to NanoLuc fusion which is overexpressed from plasmid

▪ Note hook effect at higher degrader concentration

Ion Channel Screening Using the FLIPR

FLIPR enables fluorescence-based kinetic assays in 384 well plates

• Assays based on calcium-sensitive dyes are commonly used for GPCRs and ion channels

• Example here is screen for agonists of an acid-sensitive channe

Kinase Autophosphorylation by AlphaLIS

The AlphaScreen principle was initially developed in Montreal (BioSignal - Perkin Elmer - Revvity)

▪ Homogeneous assay widely applied to protein-protein interactions and enzymatic assays

▪ Used with both purified proteins and in cell lysates

▪ Data shown here is for a cellular assays for inhibitors of a receptor tyrosine kin

MSD Assay Development for Protein Phosphorylation in Mouse Tissue

-Meso Scale Diagnostics technology commonly used for sensitive, multiplexed, high-throughput detection of proteins in biological samples

-Commercial kit was not sufficiently sensitive for the mouse protein (very small tissue samples)

-Testing multiple antibody combinations and plate types gave a significant improvement in performance

-Combinations 12 and 18 were selected to measure total and phosphorylated POI respectively in lung tissues

Use of MSD Assay for Ex Vivo Tissue Phosphorylation PK/PD Studie

-Optimized MSD has replaced western blot, giving tighter and mor reproducible data in a shorter time

-Fifteen minutes after in vivo mouse stimulation, a strong phosphorylation of the POI was observed in lung tissues

-This phosphorylation was inhibited by lead compounds, confirming potency observed in cellular ass

KO Mice as Animal Model of Metabolic Enzyme Deficiency

Only one publication on this mouse model

• Developed by an academic group in Sweden

• Mice gain weight in a similar manner to W until 6 weeks of age, then begin to decline

• Relative liver, heart and kidney weights were significantly larger than in WT mice

• Breeding HET animals received from the Swedish group

• Colony maintenance and experimental animal production carried out at Paraza

Effect of GOI-mRNA-LNP Treatment on Body Weight

mRNA-LNP treatment, resulting in expression of missing enzyme, induces

a delay (6-7 days) in the typical strong BW decreases of KO mic

Effect of Treatment on Liver Biochemistry Paramete

PreRx liver markers are within normal limits

- Liver markers collected at 48 and 72 h after last dose

present the same pattern

- mRNA-LNP for deficient enzyme significantly reduces

all liver dysfunction marker

multidisciplinary example using DMPK MS capabiliti

SILAC proteomics analysis to

identify other proteins modified by

a clinical-stage covalent CDK7

inhibitor

• Primary goal: identify possible

sources of toxicity

• Secondary purpose: supporting

conclusions of

phosphoproteomics investigation

of CDK7 signalling pathways

• Test compound highly specific

for target kinase

• Data published in Genes &

Development, 202