BioTech at Regeneron Leveraging

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					      Biotech & Pharma

        The Science,
The Jobs, & Skills for Success

         Neil Stahl Ph.D.
    Regeneron Pharmaceuticals

• Science at a Company vs. Academia
• Attributes for Success at a Company
• Biotech vs. Big Pharma
• Biotech : Innovation and Risk
• Drug Development 101
• Job Opportunities Outside of “Research”
• Getting Hired
                           My Experience
•   BS Zoology Duke 1978
      Duke Marine Lab 1977, 78, 79 brought me to Science
•   PhD Biochemistry Brandeis 1979-1985
      Quantitative fundamentals of equilibria, kinetics,
       & how to make a conclusion
•   Post-Doc at UCSF with Stan Prusiner - Scrapie Prions
      Learned many fields ranging from protein chemistry, cell biology,
       transgenics, human genetics
•   Regeneron Discovery, 1991 (65 employees)
      Explored mechanisms of how cytokine receptors are activated and activate
       cytoplasmic signaling pathways
      Figured out a way to make tight binding cytokine blockers based on the
       mechanism of cytokine activation, using multiple cytokine receptor
       components in a recombinant fusion protein = “Cytokine Trap”
•   Regeneron Drug Development (1999-2005; now 565 employees)
      Established preclinical development group - put 3 Traps into clinical trials
      Joined Senior Management - reorganized program management, clinical
       project teams, jointly manage all aspects of Research and Clinical
       Development, present to investors, analysts, potential partners
                  Science at a Company

• Scientific endeavor on a project can be carried out at a scale
  that is very rare in a University setting

• Teams of competent people aligned toward a common goal can
  accomplish more than any individual scientist

• Discoveries can be translated into therapeutic opportunities with
  the potential to create new drugs and technologies
     Understand molecular and cellular pathways defining a
      particular biology and how it goes wrong in disease
     Create a drug to impact those pathways
     Explore how that drug works in animals and humans
     Design Clinical Program to prove that the drug is safe &
     Register the drug with the FDA and Rest of World
    Differences Between Academia & Industry

• You will have access to far more resources, equipment, core
  facilities, and collaborative colleagues to advance your project
•    You will be required to work on projects of the company’s
• You may be asked to switch to (or add on) new projects
• Although you will report to one person, you will interact with
  many Scientists instead of a single PI
• Participate and present in cross-functional meetings where data
  is vetted and the future directions of a project are established by
  discussion and consensus
      More heads are better than 1!
• You are likely to publish and attend scientific conferences
              Some Myths of Industry
•   You have failed if you don’t pursue an academic position
      That’s what some told me, but there are many, many incredibly
       competent people doing Science & Drug Discovery in Industry
•   The working day is 9-5
      Hard, effective work is expected and rewarded!
•   Compensation is dramatically better than academia
      Entry level scientist positions (3-5 year postdoc) are compensated
       similarly to Assistant Professors, but much better than post-docs,
       and there are stock options!
      However, opportunity for advancement is more frequent and more
       rapid than Academia
•   You never get to publish
      I published more rapidly at Regeneron than anywhere else! Also,
       compensation is based on contributions beyond publishing
•   You can’t move from Industry to Academia
      More and more, Universities value Industry experience and
       perspective, making a reverse move more likely
  Attributes for Success at a Company

• Team player who can collaborate effectively with others

• Ability to become interested in a wide variety of different
  scientific areas - learning is a continuous Life-long experience!

• Superb analytical, communication, and presentation skills

• All of us have particular skills that make us good Scientists,
  although my exact skill set may not be the same as yours

• Contribute your particular talent and expertise toward the
  common goal

• Success means that your project grows so that hundreds of
  people work on it!
              Biotech                         vs          Big Pharma
Often more innovative, high-risk scientific     Typically more traditional small molecule
approaches                                      Drug Discovery, unless partnered with
More informal working environment, with a       Typically more hierarchical
“we’re all in this together” spirit.
                                                Employees can become pigeon-holed in a
A “do what it takes to get the job done”        particular function.
attitude that may provide more variety
                                                Larger organizations usually have more
More likely to participate in decision-making   rules!
                                                Much larger experience base
More resources than Academia, but often         Can bring huge resources to bear on a
partners with Pharma for expensive late         project, although there is always internal
stage clinical programs                         competition for resources
Can be acquired, have layoffs, or slowly go     Can be acquired, or have periodic layoffs
out of business

More opportunities for advancement than         Base compensation often higher than
Academia or Pharma if company grows             Biotech, but usually doesn’t have as large a
                                                stock option upside
Stock options can provide financial windfall
if company successful                           Promotion may occur more slowly
              Biotech : Innovation & Risk
•   Biotech companies have traditionally been founded to exploit cutting edge ideas
    and technology. Examples include:
      Using our own cytokines, growth factors, and enzymes as drugs
      Engineering human fusion proteins, combining functionalities to achieve
       new properties
      Creating Humanized and Human Monoclonals as drugs
      Transcriptional control
      siRNA
      Ribozymes
      Aptamers
      Gene Therapy
•   Many Biotech ventures are unsuccessful, often because there is not a realistic
    business plan of how to create an income-generating product before their ability
    to raise money runs out
•   You need to assess whether the company’s scientific and business plan makes
    sense, their history and future potential of raising capital, partnering deals they
    have closed, and how soon they will generate revenue
The Promise of the Human Genome Sequence

• The Hype:
    Drug Development will be revolutionized following the
     identification of novel genes in “druggable” classes

• The Fact:
    Identifying novel genes is the first baby step.
    Understanding their biology and creating therapeutics
     against them is the difficult step that, in many instances, can
     take decades
    Accelerating these steps is the key to creating novel
     therapeutic opportunities
      Target Validation : Velocigene Allows Rapid Creation of Mutant
              Mice, and Detailed Visualization of Expression

1 Nature Biotechnology paper described
10% of KO’s ever made!
 Huge Opportunities in Protein-Based Therapeutics

• Good Drug Targets are hard to come by
    Many companies make “Me Too” drugs against targets for
     which drugs already exist
• Many Interesting Targets are large proteins (eg Cytokines and
  Growth Factors) that drive broad biological responses
• These pathways cause disease if inappropriately stimulated
    These targets are usually not amenable to small molecule
    Current successful approaches include monoclonal
     antibodies that block cytokine action, and receptor - Fc
     fusion proteins
• As we learn more about Biology, we will uncover an ever
  growing number of Targets that will require protein-based
  interventional approaches
             Protein Therapeutics - Examples

Success Stories
•   Insulin - First administered to humans in 1922                    QuickTime™ and a
                                                            TIFF (Uncompress ed) decompress or
                                                               are needed to see this picture.
•   Interferons
•   Erythropoietin - 1989
•   Growth Hormone
•   Enbrel - a receptor-Fc fusion protein
•   Antibodies - eg Herceptin, Rituxan, Remicade, Humira,
Less Successful Stories
•   Mouse immunoglobulins - antigenicity
•   Thrombopoietin (Tpo)- efficacy, immunogenicity
                                                                       QuickTime™ and a
•   Lenercept - Receptor-Fc fusion - immunogenicity          TIFF (Un compressed) decompressor
                                                                are neede d to se e this picture.

•   Leptin - misunderstood mechanism - efficacy
     Protein Therapeutics Strengths & Weaknesses

•   high specificity compared to small molecules
•   Little off-target toxicity - less likely to fail in early trials
•   Block Targets not amenable to small molecules - eg growth factors &
•   More difficult to manufacture
•   Potential immunogenicity even from fully human proteins
      Low abundance proteins that don’t circulate
      Protein variants (aggregates, oxidation, deamidation) that break
•   More difficult to evaluate toxicology
•   injectable
             Regeneron Technology: Heteromeric Soluble
               Receptors Form Tight-Binding “Traps”
  Antibody Structure

        Heavy                         Ra    Rb

                (Drives                Fc

                                Kd = 5 nM             Kd = 10 pM

                 Ra                              Rb
                       In-Line Heteromeric Traps

Ra        Ra       Ra        Rb       Rb        Rb

•    Must Overexpress 2 cDNAs

•    Must Purify Heterodimer away from

•    Waste Cell’s Production Capacity with Unwanted

•    In-Line fusion of 2 receptors without intervening
     linkers creates simple homodimer with very high
     Making a Proof of Concept into a Drug

• Make T-shirts
• Do Preclinical Work
• File Investigational New
  Drug Application with FDA
• Clinical Trials
• File Biologics License
      PreClinical Development Checklist

•   BioMolecular Engineering
•   Cell line Development - FASTR
•   Process Development
•   Formulation
•   Assay Development
•   Pharmacology
•   Pharmacokinetics
•   Toxicology
•   Regulatory - IND = Investigational New Drug Application
               BioMolecular Engineering

• Create Trap candidates with different
                    IL1 Receptor Complex                        IL1 Single Chain Trap
  receptor order (eg: ab-Fc, ba-Fc,
  a-Fc-b, b-Fc-a), different fusion
               IL1R Type 1      IL1R-AcP
  position in receptor sequence +

  linkers to increase flexibility                    IL1R-AcP
                                   Extr ace llular
                                   Doma ins
• Evaluate for bioactivity, high                                                    Extr ace llular
                                                                                    Doma ins
                                Ce ll M e mbrane
  expression level from CHO cells,
                                                          IL1R Type 1
  clean folding               Cytoplasmic
                              Doma ins
                                                                            s s
 a-Fc with no extraneous linkers                                         s s

                                                                hIgG Fc

     Something Old, Something New
Ways to Isolate Over-Expressing Cell Lines
 How to isolate clones after transfection:

 Traditional:     Random isolation of clones
            GOI                   Pick                                           Dilution
                                 clones    ELISA                                  clone     ELISA

  FASTR :   Isolation based on expression / characteristic of secreted protein

            GOI                           FACS
                   FASTR Cell Line Selection

• Flow cytometry-based Autologous
  Secretion Trap
•   CHO Parental cell with doxycyline-
    inducible expression of FcR
                                                QuickTime™ an d a
•   Binds Trap internally and displays    Motion JPEG A decompressor
                                         are need ed to see this p icture .
    on cell surface
•   Whole population of transfected
    cells can be sorted by FACS with
    fluorescent anti-Fc
•   Allows selection of highest
    expresser from amongst millions of
    transfected cells
•   Turn off expression of FcR after
    selection to allow unhindered
    secretion for manufacturing
                        Process Development

•   Goal is to have protein secreted
    from CHO (Chinese Hamster Ovary
    cells) which have low viral burden
    and make human carbohydrate
•   Batch-Fed Bioreactor Process -
    yield is 1-3 g/L after 10-12 days of
•   Start at 2L scale, eventually to
    10,000 L, which yields 10 kg at
    expression of 1 g/L
•   3 step purification process (Protein
    A, ion exchange, hydrophobic
    interaction chromatography) with up
    to 70% yield

•   Desire high concentration with adequate stability to give > 2 year shelf-
•   Add GRAS (Generally Regarded as Safe) excipients to stabilize protein
    from aggregation, deamidation, oxidation, fragmentation
      Polysorbate, sucrose, amino acids, PEG
•   IV formulations generally <10 mg/ml
•   Subcutaneous (SC) - 25-100 mg/ml
•   IL1 Trap: liquid at 50 mg/ml or lyophilized at 80 mg/ml
     Assay Development/Pharmacokinetics

• Immunoassays to measure                             100000

  Trap and their complexes                             10000
  with target cytokines in

                                  IL-1 Trap (ng/mL)
• Assays to detect formation of
                                                          10        Group 4: 3 mg/kg IV
  antibodies against the Trap                                       Group 5: 3 mg/kg SC
• Use to measure PK - how
  the blood levels change over                                 0   50        100           150   200   250

  time, which often guides                                                         Time (hr)

  dosing frequency and active
  dose levels                                           IV & SC pharmacokinetics in Monkeys
               Pharmacology: Murine Model of
                 Collagen-Induced Arthritis
•   CIA model in dba-1 mice is the

                                         Arthritis Severity Index
    most widely accepted model of                                   4
    rheumatoid arthritis                                                                                     Vehicle

•   Injection of bovine collagen II                                 3
    induces immune response that
    results in progressive                                                                                  Trap 10 mg/kg
    autoimmune joint destruction
•   Injection of zymosan IP at day                                  1                                       Trap 31 mg/kg
    30 gives more robust and
    synchronous arthritis response                                  0
                                                                    26            30   34   38    42   46    50
•   Arthritis severity index grades                                                         Day
    inflammation, swelling, and
•   IL1 Trap blocks cartilage                                           Vehicle
    erosion, as well as joint swelling
    and deformity
•   Usually, new drugs are tested at high doses in 2 animal species to identify
    NOAEL (No Adverse Event Level) and MTD (Maximum Tolerated Dose)
•   Test drugs at >10x higher doses than expected human dose
•   Many protein therapeutics have strict species specificity, and can only be tested
    in primates, but often KO data in animals is predictive of safety issues
•   IL1RI KO shows no adverse phenotype except increased susceptibility to some
    types of bacterial infections
•   Moreover, human proteins are often immunogenic in animals
 Immunogenicity in animals not predictive of Ab response in humans
•   IL1 Trap only binds primate IL1
•   6 week toxicology study in monkeys showed no evidence of toxicity, but an
    antibody response was observed after a few weeks that resulted in clearance of
    Trap from circulation
•   No MTD observed, adequate safety to proceed to clinical trials!

•   FDA regulates testing of
    experimental drugs in people
•   Must submit IND - Investigational
    New Drug Application
•   Usually takes us ~1 year to
    complete, and may involve ~100
•   Describes everything you know
    about the manufacturing and
    structure, PK, pharmacology,
    formulation, stability, toxicology,
    proposed clinical plan for Phase I
•   FDA gets 30 days to respond,
    allowing you to go forward, or
    request more information, or to
    tweak your clinical trial design…
                 Clinical Trial Overview

Phase I
•   Safety Dose Escalation in Volunteers or Patients
Phase II
•   Dose Ranging Efficacy Studies to decide on dose and interval
Phase III
•   Proof of Efficacy
•   Treat larger number and broader range of patients to evaluate overall
    safety and look for less frequent adverse events (AEs)

 As few as 4 clinical studies (each one a single “experiment”) could
  suffice to get a drug approved for use in humans!!
       Entry Level Positions in Biotech

Research Post-Doctoral Scientist
• Analogous to Academia, except more resources and mentoring
• As in academic post-doc, a good publication record should allow
  return to Assistant Professor route
Pharmaceutical Post-Doctoral Scientist
• Contribute to Clinical Development Projects or Core
  Technologies in ways that may not result in high profile
• Would lead to a career in Biotech/Pharma
• 2-5 years post-doctoral experience
Staff Scientist
• 3 years experience following Post-Doc
    Career Opportunities Outside of “Research”
•    Preclinical Development
       Immunoassays & Sample Analysis from Human Clinical Trials
       Formulation Development
       Pharmacology - Assessing Drugs in Animal Models
•    Protein Sciences
       Cell line generation to overexpress recombinant proteins
       Protein characterization
       New technology and assay development
       Protein Manufacturing Process Development
•    Program Coordination & Management
•    Core Facilities
       Methodology Oriented (DNA, in situ, FACS, Mass Spec, Biacore)
•    Clinical
•    Regulatory - understand FDA Guidance, liason for company to FDA, EU
•    Scientific Writing
•    Quality Control
•    Business Development
Getting Hired
        Application & Hiring Process

• Typically, job descriptions are posted, applications
• Human Resource personnel (non-scientists) review
  applications, winnowing down to those that match job
  description, and pass on to Hiring Scientists
• Unsolicited applications to HR and Hiring Scientists
  can sometimes hit paydirt and find an opening before
  it’s even listed
           CV & Cover Letter Essentials
•   Must communicate to multiple audiences
      Scientists - trying to figure out if you have the raw materials that
       they can mold into a productive scientist and useful contributor
      Human Resources - non-scientists checking for a match between
       your CV and a job description
•   Usually your First & Only Chance to make a positive impression
•   Should convey your
      Intelligence & ability to communicate (Clear Writing = Clear Mind!)
      Perspective of your field beyond your own project
      Accomplishments - aimed at a non-expert and placed in context of
       the open questions in your field
      Skill set - techniques that you really know as well as those for which
       you may have a passing knowledge and vocabulary
      Enthusiasm!
•   Same CV can be used for all applications
•   Need not be 1 page - can be 3-4 or longer
•   Research summary
      explain in 1 paragraph your projects and conclusions
      aimed at someone who is not in your field
      Can also briefly describe rotation & graduate research
•   Clearly identify core skill sets
      Don’t exaggerate - you’ll get busted
      just because you have seen a mass spectrometer doesn’t mean
       you should list it as a core competency!!!
•   Presentations
•   Awards/Grants
•   Initiatives that you’ve undertaken outside your core requirements
•   Publications - including submitted / in preparation
•   Supervisory & Collaborative experiences
Summary & Technical Skills
                       Cover Letter

• Ideally should be customized for each application
• Should connect your skill set and experience to the job you are
  applying for so that it’s easy for HR to understand and pass on
  to hiring scientist
• Should describe your project and findings in the broad context of
  your field - often the best way to convey to the Hiring Scientist
  that you were not just a skilled set of hands directed by your PI
• Rarely is an applicant “perfect” for the job - often we look for
  someone that appears to be smart, communicates well, and can
  grow into a job
• Therefore, it’s usually a stretch to say that you can “make
  Regeneron a success…”
• More reasonable to emphasize your flexibility and ability to learn

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