`Declaration of John Fiddes Ph.D
`
`Filed on behalf of Patent Owners Genentech Inc and City of Hope by
`
`David
`Cavanaugh
`Reg No 36476
`HeatherM Petruzzi
`Reg No 71270
`Robert Gunther Jr
`Pro Hac Vice Application
`Pending
`Wilmer Cutler Pickering
`Hale and Don LLP
`1875 Pennsylvania Ave NW
`Washington DC 20006
`
`Adam
`Brausa
`Reg No 60287
`Dune
`Daralyn
`Pro Hac Vice Application
`Pending
`Dune Tangri LLP
`217 Leidesdorff Street
`San Francisco CA 94111
`
`Kushan
`Jeffrey
`Reg No 43401
`Peter Choi
`Reg No 54033
`Sidley Austin LLP
`Street N.W
`1501
`Washington D.C
`20005
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`SANOFI-AVENTIS U.S LLC AND
`REGENERON PHARMACEUTICALS INC
`
`Petitioners
`
`GENENTECH INC AND CITY OF HOPE
`Patent Owners
`
`Case IPR2O15-01624
`Patent 6331415
`
`EXPERT DECLARATION OF JOHN FIDDES PH.D
`
`EXH
`DATE
`YiIT
`PATRICIA
`
`HUBBARD
`
`5ANOFI
`
`GENENTECH
`1PR2015-01624
`EXHIBIT 2019
`
`Mylan/Merck v. Genentech
`IPR2016-00710
`Genentech Exhibit 2139 cont.
`
`Mylan v. Genentech
`IPR2016-00710
`Merck Ex. 1145, Pg. 1
`
`
`
`CaseNo 1PR2015-01624
`Declaration of John Fiddes Ph.D
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`TABLE OF CONTENTS
`
`Page
`
`INTRODUCTION AND BACKGROUND
`
`Qualifications And Experience
`
`Compensation
`
`Prior Expert Testimony
`
`II
`
`III
`
`LEGAL PRINCIPLES ON OBVIOUSNESS
`
`BACKGROUND OF THE TECHNOLOGY
`
`Genes Proteins And Antibodies
`
`Antibody Production Techniques As Of April 1983
`
`Use of Recombinant Gene Expression To Produce Proteins
`
`Basic principles of recombinant gene expression
`
`As of April 1983 Only Small Number of Monomeric
`Eukaryotic Proteins Had Been Produced Recombinantly
`
`As of April 1983 Insulin Was The Only Multimeric Eukaryotic
`Protein Which Had Been Produced Recombinantly
`
`In May 1981 When Bujard Was Filed The Speculative
`Possibility Of Using Recombinant Techniques To Produce
`Antibodies Was Highly Uncertain And Unpredictable
`
`Research Involving Antibodies And Recombinant Gene
`Expression Between May 1981 And April 1983 Confirmed The
`Uncertainty And Unpredictability Of Whether Antibodies
`Could Be Produced Recombinantly
`
`As Of April 1983 Highly Acclaimed Scientists Were Still
`Uncertain Whether It Was Even Possible To Make Antibodies
`Using Recombinant Techniques
`
`16
`
`18
`
`18
`
`20
`
`29
`
`37
`
`41
`
`46
`
`Merck Ex. 1145, Pg. 2
`
`
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`Case No 1PR2015-01624
`Declaration of John Fiddes Ph.D
`
`As Of April 1983 Nobody HadProduced An Antibody
`Recombinantly
`
`As Of April 1983 Nobody Had Produced Multimeric
`Single Host Cell
`Eukaryotic Protein Recombinantly In
`
`IV THE CLAIMS UNDER CONSIDERATION AND THEIR
`INTERPRETATION
`
`The Cabilly 415 Patent
`
`Summary Of Contested Claims
`
`The Person Of Ordinary Skill In The Art
`
`OP1NIONS REGARDING THE ASSERTED PRIOR ART
`
`Bujard
`
`What is the focus of the Buj ard reference
`
`49
`
`49
`
`53
`
`53
`
`54
`
`56
`
`57
`
`58
`
`58
`
`Do the references to multimers and one or more structural
`genes in Bujard suggest the use of the co-expression of
`single host
`multiple distinct eukaryotic genes of interest
`cell
`
`in
`
`63
`
`Do the references to multiple stop codons in Bujard suggest
`the use of the strong promoter/terminator system for the
`expression of multiple distinct genes in
`single host cell
`
`72
`
`Does Bujard at least suggest the coexpression of the heavy and
`light chains of an immunoglobulin in
`single host cell as
`the Board found
`
`75
`
`Does Bujard teach away from the production of light chains in
`one culture and heavy chains in another to be combined
`stage after their harvest and isolation as Dr
`chemically at
`Foote argues
`
`79
`
`Does the statement in Bujard that the proteins may be prepared
`single unit or as individual subunits and then joined
`as
`
`11
`
`Merck Ex. 1145, Pg. 3
`
`
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`Case No 1PR2015-Ol 624
`Declaration of John Fiddes Ph.D
`
`in appropriate ways suggest in vivo assembly of
`together
`single host cell as Dr Foote argues 84
`multimeric protein in
`
`Do you agree with the Boards finding that Bujard is more
`specific and robust than the Axel reference
`
`Riggs
`
`Itakura
`
`What is the focus of the Riggs
`
`Itakura reference
`
`86
`
`88
`
`88
`
`Itakura reference address the same problem
`Does the Riggs
`as Bujard and would the skilled artisan have had good reason
`to combine these two references in April 1983
`89
`
`Would inferences gleaned from Riggs
`Itakura have provided
`the person of ordinary skill with the motivation to selectively
`apply the teachings of Bujard to the specific production of
`immunoglobulins by means of co-expressing both the heavy
`and light chain in
`single host cell
`
`91
`
`Do you agree with Dr Footes opinion and the Boards
`preliminary fmding that Bujard in combination with Riggs
`Itakura renders the claimed invention of the Cabffly 415 patent
`obvious
`98
`
`Southern
`
`What
`
`is the focus of the Southem reference
`
`100
`
`100
`
`Do you agree with Dr Footes opinion that
`the skilled artisan
`would have been motivated to combine Bujard and Southern
`because both have as
`goal the expression of genes of interest
`single transformed host cell whether by using one Bujard
`or two Southern vectors
`102
`
`in
`
`Would inferences gleaned from Southern have provided the
`person of ordinary skifi with the motivation to selectively
`apply the teachings of Bujard to the specific production of
`immunoglobulins by means of co-expressing both the heavy
`104
`and light chain in
`single host cell
`
`ifi
`
`Merck Ex. 1145, Pg. 4
`
`
`
`Case No IPR2O1S-O1 624
`Declaration of John Fiddes Ph.D
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`Do you agree with Dr Footes opinion and the Boards
`preliminary finding that Bujard in combination with Southern
`renders the claimed invention of the Cabilly 415 patent
`obvious
`
`111
`
`iv
`
`Merck Ex. 1145, Pg. 5
`
`
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`Case No IPR2O1 5-01624
`Declaration of John Fiddes Ph.D
`
`INTRODUCTION AND BACKGROUN1T
`
`John Fiddes Ph.D have been retained by counsel for
`
`Genentech Inc and City of Hope collectively Patent Owners as an expert
`
`in
`
`this proceeding
`
`understand that in February
`
`2016 decision the Patent
`
`Trial and Appeal Board the Board instituted inter partes review as to claims 1-
`
`11 12 14 18-20 and 33 ofU.S PatentNo 6331415 the Cabilly 415
`
`patent
`
`further understand that the references relied upon by the Board in
`
`instituting inter partes review include the Bujard patent Ex 1002 the Riggs
`
`ItakurapaperEx 1003 and the Southern paperEx 1004
`
`have been asked to review the challenged claims of the
`
`Cabffly 41 patent and the references identified in the petition requesting inter
`
`partes review and evaluate whether the cited references alone or in combination
`
`render the challenged claims unpatentable As part of my review have been
`
`asked to evaluate the prior art and scientific accuracy of the observations that the
`
`Board made in the decision instituting inter partes review also was asked to
`
`evaluate certain statements that Dr Jefferson Foote made in his declaration Ex
`
`1006 sulmitted with the petition requesting inter partes review
`
`Merck Ex. 1145, Pg. 6
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`
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`Case No IPR2O1S-O1 624
`Declaration of John Fiddes Ph.D
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`list of materials have reviewed in preparation of this
`
`Declaration is attached as Exhibit
`
`have also relied upon my scientific
`
`knowledge as of April 1983 when the Cabffly 415 patent was filed
`
`Qualifications And Experience
`
`My background is summarized below and in my curriculum
`
`vitae which includes
`
`list of my publications and patents and is attached as
`
`Exhibit
`
`received
`
`Bachelor of Science degree in Biological Sciences
`
`Molecular Biology with First Class Honors froth the University of Edinburgh in
`
`1973 In 1977
`
`received my Ph.D in Molecular Biology from Kings College
`
`Cambridge University My thesis advisor was Dr Fred Sanger The title of my
`
`thesis was The Determination of Nucleotide Sequences in Bacteriophage aX174
`DNA
`
`From 1977 to 19801 was Postdoctoral Research Fellow at the
`University of California San Francisco UCSF in the laboratory of Dr Howard
`
`Goodman where worked on the human growth hormone human chorionic
`
`somatomammotropin and human glycoprotein hormone genes
`
`After my post-doe at UCSF became Senior Staff
`Investigator at Cold Spring Harbor Laboratory CSHL in Cold Spring Harbor
`
`New York position
`
`held until January 1983
`
`Merck Ex. 1145, Pg. 7
`
`
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`Case No IPR2015-01 624
`Declaration of John Fiddes Ph.D
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`My research at CSHL focused on the structure evolution and
`
`expression of the human glycoprotein hormone genes specifically human
`
`chorionic gonadotropin and human luteinizing hormone and on methods of
`
`making cDNA libraries suitable for immunological screening of expression
`
`products
`
`was also an instructor at the CSHL Advanced Cloning Course in the
`
`summers of 1982-1983
`
`10
`
`Following my academic career
`
`entered industry and spent
`
`over twenty years in drug discovery and development
`
`In January 1983 just
`
`shortly before the filing date of the Cabilly 415 patent
`
`took
`
`position at
`
`California Biotechnology Inc later renamed Scios Inc in Mountain View
`
`California The primary interest of this company was in applying recombinant
`
`DNA technologies to the production of therapeutically useful proteins
`
`11
`
`Among other things was involved in the development of
`
`systems for the production of recombinant forms of basic fibrob last growth factor
`
`and the isolation of cDNA and genomic clones for atrial natriuretic peptide
`
`vascular endothelial growth factor variant and heparin-binding EGF-like growth
`
`factor
`
`12 My last industry position was at Genencor
`
`International
`
`Inc in
`
`Palo Alto California where served as Vice President Research Health Care from
`
`2003 to 2005 Since 20051 have been an independent consultant on
`
`Merck Ex. 1145, Pg. 8
`
`
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`CaseNo IPR2O1S-01624
`Declaration of John Fiddes Ph.D
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`biopharmaceutical matters for
`
`variety of organizations including the California
`
`Antiviral Foundation and the Institute for One World Health
`
`13
`
`Based on my academic and early industry experience was
`
`well aware of the birth of recombinant DNA technology and followed the
`
`developments that eventually led to the production of recombinant forms of
`
`medically important proteins This in my view is the art to which the Cabffly
`
`415 patent pertains and
`
`believe
`
`am well-positioned to understand and address
`
`the skills and mindset of person of ordinary skill
`
`in this field circa 1982-1983
`
`Compensation
`
`14
`
`am being compensated at my normal consulting rate for my
`
`work which is $650 per hour My compensation is not dependent on and in no
`
`way affects the substance of my statements in this Declaration
`
`Prior Expert Testimony
`
`15
`
`provided expert reports and deposition testimony in Bristol-
`
`Myers Squibb Co
`
`Genentech Inc
`
`CityofHope 213-cv-05400-MRP-JEM
`
`C.D Cal and Eli Lilly
`
`Co
`
`Genentech Inc 21 3-cv-07248-MRP-JEMC.D
`
`Cal.
`
`II
`
`LEGAL PRINCIPLES ON OBVIOUSNESS
`
`16
`
`have been informed and understand that in order to invalidate
`
`patent claim as obvious in the context of an inter partes review it must be shown
`
`Merck Ex. 1145, Pg. 9
`
`
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`CaseNo 1PR2015-01624
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`by
`
`preponderance of the evidence that the claim would have been obvious to
`
`person of ordinary skill at the time the invention was made The prior art does not
`
`need to render obvious every possible embodiment within the scope of the claim
`
`the prior art renders the claim obvious if
`
`the combined teachings disclose an
`
`embodiment that is within the scope of the claim
`
`17
`
`have been informed and understand that factors relevant
`
`to the
`
`determination of obviousness include the scope and content of the prior art the
`
`level of ordinary skill
`
`in the art at the time of the invention differences between
`
`the claimed invention and the prior art and secondary considerations or objective
`
`evidence of non-obviousness
`
`18
`
`have been informed and understand that obviousness can be
`
`established by combining or modifying the teachings of the prior art to produce the
`
`claimed invention where there is some teaching suggestion or motivation to do so
`
`and that
`
`reasonable expectation of success in achieving the subject matter of the
`
`claim at issue must also be shown Further have been informed and understand
`
`that the teaching suggestion or motivation test is flexible and that an explicit
`
`suggestion to combine the prior art is not necessary
`
`the motivation to combine
`
`may be implicit and may be found in the knowledge of one of ordinary skifi
`
`in the
`
`art from the nature of the problem to be solved market demand or common sense
`
`Merck Ex. 1145, Pg. 10
`
`
`
`Case No 1PR2015-0l 624
`Declaration of John Fiddes Ph.D
`
`19
`
`have been informed and understand that
`
`patent claim
`
`composed of several
`
`limitations is not obvious merely because each limitation was
`
`independently known in the prior art Hindsight
`
`reasoning is not an appropriate
`
`basis for combining references to form an obviousness combination
`
`also have
`
`been informed and understand that it can be important
`
`to identify
`
`reason that
`
`would have prompted
`
`person of ordinary skifi
`
`in the relevant field to combine the
`
`limitations in the way the claimed new invention does
`
`20
`
`In undertaking an obviousness analysis
`
`have been informed
`
`and understand that may take into account
`
`the inferences and creative steps that
`
`person of ordinary skill would have employed in reviewing the prior art at the time
`
`of the invention If the claimed invention combines elements known in the prior
`
`art and the combination yields results that would have been predictable to
`
`person
`
`of ordinary skifi at the time of the invention then this evidence would make it
`
`more likely that the claim was obvious
`
`21
`
`have also been informed and understand that obviousness may
`
`be established if the combination of prior art elements was obvious to try even if
`
`no one attempted the combination For
`
`combination to be obvious to try
`
`however
`
`solution must be among fmite number of identified predictable
`
`solutions Where the art is uncertain or unpredictable
`
`person of ordinary skill
`
`in
`
`the art will not have
`
`reasonable expectation of success
`
`Merck Ex. 1145, Pg. 11
`
`
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`CaseNo 1PR2015-01624
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`22
`
`have been informed and understand that an obviousness
`
`analysis must take into account any secondary considerations or as they are
`
`sometimes called objective indicia of non-obviousness These secondary
`
`considerations can include the inventions commercial success long-felt but
`
`unresolved needs licenses showing industry respect the failure of others
`
`skepticism by experts praise by others teaching away by others recognition of
`
`problem and copying of the invention by competitors Such secondary
`
`considerations when present offer objective information as to the state of the art at
`
`the time of the invention and provide
`
`check to hindsight analysis
`
`III
`
`BACKGROUND OF THE TECHNOLOGY
`
`23
`
`To place the importance and innovation of the Cabffly 415
`
`patent
`
`into context
`
`have been asked to provide some background on the relevant
`
`technology
`
`Genes Proteins And Antibodies
`deoxyribonucleic acid DNA molecule encodes the genetic
`
`24
`
`instructions that
`
`living organism uses for wide variety of critical
`
`functions
`
`Sequences ofDNA nucleotides are organized into discrete structures called genes
`
`which
`
`cells machinery reads to make proteins Proteins comprised of
`
`string
`
`of units called amino acids are biomolecules that perform many of the functions of
`
`cells and organisms
`
`Merck Ex. 1145, Pg. 12
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`
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`25
`
`The basic process of making
`
`protein from gene is called
`
`gene expression First the cell copies the gene of interest DNA sequence
`
`into messenger ribonucleic acid mRNA via
`
`process called transcription
`
`Second the mRNA is converted into the corresponding sequence of amino acids
`
`called
`
`polypeptide via
`
`process called translation Finally the translated
`
`polypeptide undergoes folding and possibly post-translational modifications to
`
`assemble as the active protein structure
`
`26
`
`This step-wise process is reflected in the following illustration
`
`DNA synthesis
`replication
`
`DNA
`
`inst
`XSUNADRaWS
`
`nucleotides
`
`RNA synthesis
`transcription
`
`protein synthesis
`translation
`
`PROTEIN
`
`rH
`
`amino adds
`
`Rpur %.3 ssathlCdl ot.qy3MtG.La.dkIwa2O1C
`
`Ex 2081 Bruce Alberts et al Essential Cell Biology Chapter 3rded 2009
`
`Merck Ex. 1145, Pg. 13
`
`
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`Case No 1PR2015-01624
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`27
`
`Transcription In transcription an enzyme in cells called RNA
`
`polymerase synthesizes single-stranded mRNA in
`
`linear fashion from the
`
`doubled-stranded genomic DNA template
`
`28
`
`Translation In translation complex structures in cells called
`
`ribosomes bind to specific sites on the mRNA transcript and translate the mRNA
`
`sequence into
`
`polypeptide chain of amino acids The mRNA sequence is read
`
`three nucleotides at
`
`time with each triplet of nucleotides
`
`called
`
`codon
`
`specifying one amino acid
`
`start codon initiates translation and stop codons
`
`signal
`
`termination of translation Codons therefore provide the information that
`
`dictates the order and arrangement of amino acids in
`
`polypeptide chain and
`
`when the formation of the chain begins and ends
`
`29
`
`An exemplary coding region from protein
`
`of bacteriophage
`
`G4 is shown in the table below see Ex 2082
`
`Godson et al Nucleotide
`
`Sequence ofBacteriophage 64 DNA Nature 276236-47 1978
`
`Codon
`Amino Acid
`
`TAC
`TYR
`
`GGA
`GLY
`
`TAT
`TYR
`
`TTC
`PHE
`
`TGA
`STOP
`
`TGA
`STOP
`
`30
`
`As seen in this table above multiple stop codons written for
`
`ease of reference as DNA as opposed to mRNA can signal
`
`the end of translation
`
`of
`
`single polypeptide
`
`coding region may end with multiple stop eodons to
`
`Merck Ex. 1145, Pg. 14
`
`
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`Declaration of John Fiddes Ph.D
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`ensure termination of translation
`
`start codon would be found upstream of the
`
`region shown above
`
`31
`
`Depending on where translation begins each mRNA sequence
`
`can be translated in any one of several reading frames i.e the various ways in
`
`which the sequence may be divided into sets of nucleotide triplets
`
`32
`
`Folding As part of the process of translation the polypeptide
`
`chain folds to take on its fmal structure and to become an assembled active
`
`protein Folding allows the polypeptide to form its three dimensional structure and
`
`occurs as amino acids within
`
`polypeptide chain interact with one another
`
`Disulfide bonds or bridges which are covalent bonds formed between cysteine
`
`amino acids form scaffolding that helps maintain
`
`proteins proper three
`
`dimensional structure Disuffide bonds may also be referred to as S-S bonds
`
`Such scaffolding may be present in both simple and more complex proteins and
`
`where present is essential
`
`to the activity of protein
`
`33
`
`Post-translational modjfication This generally refers to
`
`processes by which cells may modify
`
`polypeptide after it has been produced to
`
`achieve mature product and can include any number of processes
`
`34 Monomeric andMultimeric Proteins The most simple
`
`proteins are monomeric meaning they are formed by only
`
`single polypeptide
`
`chain Other proteins exist as
`
`complex of multiple polypeptide chains called
`
`10
`
`Merck Ex. 1145, Pg. 15
`
`
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`Case No 1PR2015-01 624
`Declaration of John Fiddes Ph.D
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`multimeric proteins Multimeric proteins can be made up of multiple identical
`
`polypeptide chains or
`
`combination of different polypeptide chains For
`
`multimeric proteins amino acid interactions between individual chains and
`
`disulfide bonding between individual chains are critical
`
`to correct folding
`
`35
`
`Insulin There are many different
`
`types of multimeric proteins
`
`One example is insulin which is
`
`relatively simple and small i.e 5800
`
`Daltons multimeric protein
`
`Dalton is
`
`standard unit of measurement used to
`
`characterize the mass of protein The insulin molecule contains two different
`
`polypeptide chains
`
`an
`
`chain consisting of 21 amino acids and
`
`chain
`
`consisting of 30 amino acids
`
`that are chemically attached via two intra-chain
`
`disulfide S-S bonds
`
`36
`
`The relatively simple configuration of the insulin protein
`
`including its disulfide bonding scheme is shown in the illustration below
`
`AChiiln ic tCys CAJa ec
`
`d\
`
`chain
`
`JJY\
`
`GIuj
`
`11
`
`Merck Ex. 1145, Pg. 16
`
`
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`Case No 1PR2015-01 624
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`Ex 2083 Protein Structure Boundless
`
`https //www boundless .com/biology/textbooks/boundless-biology
`
`textbooklbiological-macromolecules-3/proteins-5
`
`6/protein-structure-3 04-11437/
`
`last visited May 12 2016
`
`37
`
`As can be seen above the two chains that comprise insulin are
`
`joined together by two inter-chain disuffide bonds there is also one intra-chain
`
`disulfide bond
`
`38
`
`Antibodies Antibodies also known as immunoglobulins are
`
`large tetrameric proteins which may form even larger complexes that are
`
`expressed and secreted by
`
`cells
`
`type of white blood cell made in the bone
`
`marrow There are five classes of antibodies IgG IgD IgE IgA and 1gM each
`
`of which is further divided into multiple different types called isotypes
`
`39
`
`naturally occurring tetrameric antibody is composed of four
`
`polypeptide chains
`
`two identical heavy chains or
`
`chains and two
`
`identical light chains or
`
`chains The heavy and light chains differ in their
`
`size and thus their respective molecular weights By way of example in
`
`antibodies of the immunoglobulin
`
`IgGl class the longer
`
`chains are
`
`naturally comprised of about 450 amino acids and each have molecular weight of
`
`about 50000 Daltons whereas the shorter
`
`chains are naturally comprised of
`
`about 212 amino acids and each have molecular weight of about 25000 Daltons
`
`12
`
`Merck Ex. 1145, Pg. 17
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`
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`Case No 1PR2015-0l 624
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`40
`
`The heavy and light chains of an antibody form what is often
`
`depicted schematically as Y-shaped molecule via multiple intra- and inter-chain
`
`disulfide bonds For example in the case of an antibody of the IgG class there
`
`are 12 intra-chain disulfide bonds and
`
`inter-chain disulfide bonds that together
`
`provide inter- and intra-chain scaffolding within the structure of an antibody as
`
`discussed above
`
`41
`
`The below figure taken from FIG of the Cabilly
`
`15 patent
`
`provides
`
`representation of an antibodys Y-shape and how the various heavy and
`
`light chains assemble via disulfide bonds to form functional antibody
`
`Fc fragment
`
`Fig
`
`42
`
`As shown above both the heavy and light chains have
`
`constant
`
`region and variable region The constant
`
`regions may be the
`
`same between different
`
`types of antibodies By contrast
`
`the variable regions
`
`13
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`Merck Ex. 1145, Pg. 18
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`differ from one individual antibody to the next these variable regions are
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`responsible for identiQying and binding to
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`particular antigen
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`substance that
`
`causes the immune system of an organism to generate antibodies that bind the
`
`antigen in order to provoke an immune response
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`43
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`In sum an antibody is much larger and more complex protein
`
`than insulin and most other proteins The molecular weight of
`
`typical antibody
`
`is approximately 150000 Daltons or more than 25 times the size of insulin
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`Further the three-dimensional shape of an antibody is more complex and requires
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`much more post-translational processing
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`on next page
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`I/I
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`I/I
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`I/I
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`I/I
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`I/I
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`I/I
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`III
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`I/I
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`I/I
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`I/I
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`14
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`Merck Ex. 1145, Pg. 19
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`44
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`The table below provides
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`to-scale comparison that illustrates
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`the relative size of insulin compared to an antibody
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`Structure
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`Insulin
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`dimer ab
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`Immunoglobulin
`tetramer a2b2
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`Size
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`51 amino acids
`5800 Daltons
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`Disulfide
`Bonds
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`1324 amino acids
`150000 Daltons
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`16
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`45
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`At my request
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`the space-filling models in the above figure
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`were generated by scientists at Genentech using PyMOL software available for
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`download at https//www.pymol.org/
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`Each sphere represents an atom with gray
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`representing carbon red representing oxygen blue representing nitrogen yellow
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`representing sulfur and green representing zinc The size difference between
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`insulin and an antibody represented above is consistent with my general
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`15
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`Merck Ex. 1145, Pg. 20
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`understanding regarding the relative overall shape and size of each See also Ex
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`2084 RCSB Protein Databank Molecular Machinery
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`Tour of the Protein Data
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`Bank http//cdn.rcsb.org/pdb
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`10 1/learnlresources/20 14-mol-mach-postetpdf
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`last
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`visited May 10 2016 illustrating the diversity of proteins with an antibody
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`shown as protein 13 and insulin as protein 16
`
`Antibody Production Techniques As Of April 1983
`
`46
`
`PolyclonalAntibodies AsofApril 1983 it was well known
`
`that antibodies could be produced by immunizing an animal with
`
`foreign antigen
`
`and then recovering the antibodies that had been produced by the animaL Under
`
`this approach
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`polyclonal serum is generated
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`mixture of antibodies with
`
`varying specificities
`
`47
`
`As of April 1983 polyclonal antibodies were being widely used
`
`to study the structure and function of antibodies Due to their varying specificities
`
`however polyclonal antibodies had limited usefulness for therapeutic and non-
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`therapeutic applications EL 1001 at 151-63 240-43
`
`48 Hybridomas In the 1970s Drs Georges Kohler and César
`
`Milstein pioneered
`
`technique for producing monoclonal antibodies i.e
`
`antibodies that have the same amino acid sequence and bind to the same location
`
`on the antigen called an epitope in the same way Their technique involved the
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`use of fused cells known as hybridomas See Ex 2013
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`Kohler
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`16
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`Merck Ex. 1145, Pg. 21
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`Milstein Continuous Cultures ofFused Cells SecretingAnti body ofPredefined
`
`Specificity Nature 256495 -497 Aug
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`1975
`
`49
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`Like the production ofpolyclonal antibodies the production of
`
`hybridoma starts with the immunization of an animal with
`
`particular antigen of
`
`interest
`
`cells from the animal are then isolated and fused to an immortalized
`
`cell called myeloma which are blood cell-derived cancer cells that have the
`
`ability to grow indefinitely in
`
`cell culture The fused cells
`
`hybridomas
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`grow continuously in culture and produce the desired antibody
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`50
`
`In the early l980s the hybridoma technique was being widely
`
`used to produce monoclonal antibodies and researchers were focusing on
`
`expanding their use even further Ex 1039 Milstein MonoclonalAnti bodies
`
`from HybridMyelomas Proceedings ofthe Royal Society ofLondon 211393-412
`
`407 1981 use of hybrid myelomas to define the complete repertoire of
`
`antibodies to single antigens
`
`is now expanding very rapidly and many
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`commercial companies are beginning to market them Ex 2020 Foote Dep 37
`
`48 describing hybridoma technique as very big deal
`
`in the early 1980s due to
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`significant achievements Ex 1001 at 164-211
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`17
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`Merck Ex. 1145, Pg. 22
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`Use of Recombinant Gene Expression To Produce Proteins
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`Basic principles of recombinant gene expression
`
`51
`
`Recombinant gene expression is method that allows for the
`
`production and isolation of protein of interest in
`
`foreign i.e heterologous
`
`host organism usually
`
`cell or host cell
`
`52
`
`As explained in the Cabilly 415 patent
`
`this process involves
`
`four fundamental steps
`
`identification and isolation of particular gene of
`
`interest EL 1001 at 410-12
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`insertion of that DNA sequence into
`
`vector or
`
`plasmid Id at417-21
`
`insertion of that vector or plasmid into
`
`suitable host
`
`cell Id at 420 and
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`expression of the sequence i.e transcribing the gene of
`
`interest into mRNA and then translating the mRNA into
`
`polypeptide by the host
`
`cell Id at 423-29 Host cells transformed with the vector are grown in culture
`
`to express the gene of interest in high quantities and the resulting protein is then
`
`isolated Id at 421-24
`
`53
`
`Vectors
`
`vector comprised ofDNA includes
`
`number of
`
`components to assist with the insertion and expression of the DNA sequence of
`
`interest For example vector will generally include
`
`number of restriction
`
`enzyme sites which are locations in DNA that can be cut by certain proteins
`
`known as restriction enzymes Restriction enzyme sites allow for the insertion of
`
`the DNA sequence of interest into the vector E.g Ex 2085 Vectors
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`survey
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`18
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`Merck Ex. 1145, Pg. 23
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`
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`Case No 1PR2015-01624
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`of molecular cloning vectors and their uses Chapter
`
`The PlasmidpBR322
`
`Rodriguez R.L andDenhardtD.T eds 1988
`
`54
`
`Prom oters/Terminators
`
`vector also includes regulatory
`
`sequences such as promoters and terminators to signal
`
`the initiation and
`
`termination of transcription Ex 1001 at 857-915 956-103
`
`promoteris
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`specific DNA sequence that signals where KNA polymerase should bind the
`
`template DNA and where mRNA synthesis should begin
`
`transcription
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`terminator is
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`specific DNA sequence that signals where mRNA synthesis should
`
`end
`
`55 Markers
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`vector or plasmid wifi also often include
`
`marker also called marker gene that allows for the identification and
`
`isolation of host cells that have taken up the vector
`
`selectable marker usually
`
`based on antibiotic resistance is used to select only the bacterial cells that have
`
`taken up the vector The antibiotic will kill all of the other cells that do not have
`
`the vector
`
`reporter marker does not provide selection but provides the ability
`
`to distinguish between cells that do or do not contain the marker For example
`
`particular marker might turn cells containing the reporter particular color in the
`
`presence of an indicator dye Id at 811-15
`
`56
`
`marker is different from gene of interest i.e the gene
`
`encoding for the protein sought to be expressed as the marker is not intended to be
`
`19
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`Merck Ex. 1145, Pg. 24
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`produced in large amounts isolated or studied Rather the only function of
`
`marker is to select or identify which host cells have been transformed by the
`
`vector
`
`understand that Dr Foote agrees Ex 1006 Foote Rep
`
`39 n.4
`
`As ofApril 1983 Only Small Number of Monomeric
`Eukaryo tic Proteins Had Been Produced Recombinantly
`
`57
`
`As of April of 1983 recombinant gene expression technology
`
`was seen as promising way to produce proteins ofinterest But many of the
`
`biological mechanisms controlling the expression of foreign DNA and the
`
`assembly of resulting proteins were stifi poorly understood at the time As
`
`result
`
`recombinant gene expression was unpredictable and raised many uncertainties
`
`understand that Dr Foote agrees with this general description of the state of the art
`
`in the early 1980s Ex 2020 Foote Dep 134-35 agreeing as set forth in the
`
`Harris publication Harris GeneticEngineering4127-84 1983Ex 1027 that
`
`it was clear that not all
`
`the rules governing the expression of cloned genes have
`
`been elaborated and those rules that do exist are still
`
`largely empirical
`
`58
`
`As of April 1983E coil aprokaryotic bacterial organism was
`
`the best characterized and most widely used host cell for recombinantly expressing
`
`protein E.g Ex 1027 providing
`
`survey of eukaryotic genes that had been
`
`expressed in
`
`co/i Yet at this time and as described in the review article
`
`published by Dr Timothy Harris in April 1983 which provided
`
`survey of
`
`20
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`Merck Ex. 1145, Pg. 25
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`Case No IPR2O 15-01624
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`eukaryotic genes that had been expressed in
`
`coli see above there were
`
`challenges in trying to recombinantly express
`
`eukaryotic protein in
`
`prokaryotic
`
`host cell Those issues included the following
`
`59
`
`First for example Harris stated
`
`Probably the most important difference between eukaryotic genes at least
`
`for higher organisms and prokaryotic genes is the presence of intervening
`
`sequences introns which interrupt
`the coding sequences Normally these
`sequences are spliced out of the initial RNA transcript producing
`cytoplasmic mRNA suitable for translation There are no introns in
`
`prokaryotic genes and consequently no splicing enzymes present soin
`general genomic DNA cannot be used as
`
`source of genes for expression in
`
`bacterial cells
`
`EL 1027at 131
`
`60
`
`In this passage Harris explained that eukaryotic gene
`
`expression in bacteria was complicated by the fact
`
`that eukaryotic genes include
`
`non-coding regions known as introns that are usually spliced out
`
`in eukaryotic
`
`cells Prokaryotic cells such as bacterial cells are unable to edit out these intron
`
`sequences
`
`Intron editing was
`
`possible option to address this issue but would
`
`require significant work because site-specific mutagenesis techniques were not
`
`well developed The preferred approach to the expression of eukaryotic genes in
`
`bacterial host cells was therefore the use of complementary DNA i.e DNA
`
`21
`
`Merck Ex. 1145, Pg. 26
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`
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`Case No 1PR2015-O1 624
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`derived from mRNA which does not include introns But just isolating cDNA
`
`was not enough This is because the regulatory elements found in eukaryotic genes
`
`were known to be different than those found in prokaryotic genes This resulted in
`
`additi