PGR2025-00030
`
`Declaration of Dr. Hecht
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`––––––––––––––––––
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`––––––––––––––––––
`
`Merck Sharp & Dohme LLC,
`Petitioner,
`
`v.
`
`Halozyme Inc.,
`Patent Owner.
`
`––––––––––––––––––
`
`Case No. PGR2025-00030
`U.S. Patent No. 12,054,758
`
`––––––––––––––––––
`
`Declaration of Michael Hecht, Ph.D.
`
`Petitioner Merck
`EX1003, p. i
`
`

`

`PGR2025-00030
`
`Declaration of Dr. Hecht
`
`I.
`
`II.
`
`Table of Contents
`Introductory Matters ................................................................................... 1
`A.
`Background and Qualifications ............................................................ 1
`B.
`Compensation ....................................................................................... 3
`C.
`Person of Ordinary Skill in the Art ...................................................... 4
`D.
`Terminology Used in this Declaration; Patent Documents .................. 5
`E. Materials Considered ........................................................................... 7
`F.
`Legal Principles .................................................................................... 9
`Scientific Principles .................................................................................... 12
`A.
`Protein Structure Basics ..................................................................... 13
`B.
`Protein Engineering ............................................................................ 19
`1.
`Rational Design vs. Directed Evolution ................................... 19
`2.
`Challenges with Predicting the Effects of Multiple Mutations in
`Proteins .................................................................................... 25
`The Stability of a Protein Is Measured by Assessing the Free Energy
`Difference Between Its Folded and Unfolded States ......................... 31
`Activity Is Not Synonymous with Stability ....................................... 33
`Hyaluronidases and PH20 .................................................................. 40
`1.
`Characterization of Hyaluronidases ......................................... 41
`2.
`Chao Reports Structure of Human HYAL1 and the Hyal-EGF
`Domain ..................................................................................... 43
`Modifications at the C-Terminus of PH20 Were Poorly
`Understood in the 2011 Timeframe ......................................... 49
`III. The Common Disclosure Defines Two Mutually Exclusive Types of
`Modified PH20 Polypeptides ..................................................................... 56
`A.
`Two Types of Modified PH20 Polypeptides Are Differentiated Based
`on Possession or Absence of Hyaluronidase Activity ........................ 56
`1.
`The Common Disclosure Draws a Clear Line Between Two
`Alternative Types of Modified PH20 Polypeptides ................. 56
`Petitioner Merck
`EX1003, p. ii
`
`C.
`
`D.
`E.
`
`3.
`
`

`

`PGR2025-00030
`
`Declaration of Dr. Hecht
`
`V.
`
`B.
`
`B.
`
`C.
`
`B.
`
`2.
`
`2.
`
`3.
`
`4.
`
`5.
`
`The Experimental Results Classify Single Replacement
`Mutants of PH201-447 as Active or Inactive Mutants ................ 58
`Proposed Uses for Active Mutant Modified PH20 Polypeptides Are
`Different from Those for Inactive Mutants ........................................ 61
`IV. The Claims Capture an Immense Number of Distinct PH20
`Polypeptides ................................................................................................ 65
`A.
`The Claims Define Large Sets of Modified PH20 Polypeptides Using
`Sequence Identity Parameters ............................................................ 65
`The Claims Would Be Understood to Concern Active Mutant PH20
`Modified Polypeptides ....................................................................... 75
`All of the Claims Encompass a Single-Replacement PH201-447 Mutant
`Where L at Position 317 is Changed to Q or K .................................. 81
`Observations on the Common Disclosure ................................................ 83
`A.
`The Common Disclosure Does Not Identify the Modified PH20
`Polypeptides with Multiple Amino Acid Substitutions Encompassed
`by the Sequence Identity Parameters in the Claims ........................... 83
`1.
`The Data from Testing Single Replacement PH201-447 Mutants
`Does Not Identify a Correlation Between PH20 Polypeptides
`with 2-42 Substitutions and PH20 Proteins Having > 40%
`Hyaluronidase Activity ............................................................ 83
`The Common Disclosure Does Not Identify Any Specific PH20
`Polypeptides Having 2-42 Substitutions that Retain >40%
`Hyaluronidase Activity ............................................................ 86
`The Common Disclosure Says to Avoid Changing Certain
`Residues Involved in Glycosylation ........................................ 87
`The Common Disclosure Says to Not Include Substitutions that
`Rendered PH201-447 Inactive in Modified PH20 Polypeptides
`that Are Active Mutants ........................................................... 88
`The Prior Art and the Common Disclosure Indicate that C-
`Terminal Truncations Can Render PH20 Polypeptides Inactive
`.................................................................................................. 91
`The Sequence Identity Parameters in the Claims Encompass
`Enzymatically Active Multiply-Modified PH20 Polypeptides the
`Petitioner Merck
`EX1003, p. iii
`
`

`

`PGR2025-00030
`
`Declaration of Dr. Hecht
`
`C.
`
`2.
`
`2.
`
`Common Specification Does Not Identify, Says to Not Make and For
`Which It Provides No Meaningful Guidance ..................................... 93
`A Skilled Artisan Would Have to Engage in an Impossible Scale of
`Experimentation to Make and Identify All the Multiply-Modified
`PH20 Proteins with Hyaluronidase Activity Within the Sequence
`Identity Parameters of the Claims ...................................................... 99
`1.
`The Common Disclosure Provides Only a Research Plan for
`Discovering Multiply-Modified, Enzymatically Active PH20
`Polypeptides ........................................................................... 101
`Discovering All the Active Mutant Multiply-Modified PH20
`Polypeptides Within the Scope of the Claims Is Impossible . 108
`VI. The L317Q PH201-447 Mutant Would Have Been Obvious ................... 112
`A.
`The ’429 Patent Suggests Making Single-Amino Acid Substitutions in
`Non-Essential Regions of PH201-447 ................................................. 113
`1.
`The ’429 Patent Describes PH201-447, Its Production and Its
`Uses ........................................................................................ 113
`The ’429 Patent Says to Make Single Amino Acid Substitutions
`in Non-Essential Regions of PH20 ........................................ 122
`The Skilled Artisan Would Consider Chao for Structural Insights into
`Making Modified PH20 Polypeptides .............................................. 125
`The Skilled Artisan Would Have Identified Non-Essential Regions in
`PH20 and Suitable Amino Acid Substitutions Using a Multiple
`Sequence Alignment of Homologous Hyaluronidase Proteins ........ 126
`Both Chao and a Multi-Sequence Alignment of Proteins Homologous
`to PH20 Would Have Identified Position 317 Being in a Non-
`Essential Region and Suggested Glutamine (Q) as a Single
`Substitution at Position 317 ............................................................. 128
`Inspection of the L317Q Substitution in a PH20 Structural Model
`Confirms that the Substitution Would be Tolerated in PH201-447 .... 134
`Appendices
`
`B.
`
`C.
`
`D.
`
`E.
`
`A.
`B.
`
`Tables From U.S. Patent 12,054,758
`C.V of Michael Hecht, Ph.D.
`
`Petitioner Merck
`EX1003, p. iv
`
`

`

`PGR2025-00030
`
`Declaration of Dr. Hecht
`
`I.
`
`Introductory Matters
`A.
`Background and Qualifications
`1.
`My educational background, career history, and other relevant
`
`qualifications are summarized below. I attach to this Declaration my curriculum
`
`vitae, which provides a full and accurate description of my educational
`
`background, professional experience, and qualifications.
`
`2.
`
`I received my Ph.D. in Biology from Massachusetts Institute of
`
`Technology in 1984. My Ph.D. thesis was on the topic of “The Effect of Amino
`
`Acid Replacement on the Structure and Stability of the N-terminal Domain of λ-
`
`Repressor.” I received a Bachelor of Arts in Chemistry from Cornell University in
`
`1977. For my B.A., I wrote a Thesis regarding Studies of the α-helical
`
`Propensities of Amino Acids in Synthetic Copolymers. I completed my Post-
`
`Doctoral work at Duke University from 1986 to 1989 in the Department of
`
`Biochemistry, where I conducted research on the Design of Novel Proteins.
`
`3.
`
`I currently serve as Professor of Chemistry at Princeton University. I
`
`have been a professor at Princeton since 1990. I served as Associate Chair of the
`
`Chemistry Department from 2004-2007. From 2011-2008 I was Director of
`
`Undergraduate Studies for the Department of Chemistry. I was Master (Head) of
`
`Forbes College at Princeton from 2010-2018.
`
`Petitioner Merck
`EX1003, p. 1
`
`

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`PGR2025-00030
`
`Declaration of Dr. Hecht
`
`4.
`
`I am currently teaching courses in General Chemistry and Principles
`
`of Macromolecular Structure: Protein Folding, Structure and Design.
`
`5.
`
`I have over 4 decades of experience in the field of protein structure
`
`and design. My research interests include de novo protein design, synthetic
`
`biology, and protein folding and design. In my laboratory, we explore protein
`
`design and synthetic biology to explore novel proteins. Since 1978, I have
`
`authored over 90 peer-reviewed publications, most of which are directed to these
`
`topics.
`
`6.
`
`In 2024, one half of the Nobel Prize in chemistry was awarded to
`
`David Baker for “computational protein design,” and the other half went to Demis
`
`Hassabis and John Jumper for their work in “protein structure prediction,”
`
`specifically their AI model called AlphaFold2. The Nobel committee’s write-up of
`
`other notable contributions in the field of protein design included a description of
`
`my earlier work. See https://www.nobelprize.org/uploads/2024/10/advanced-
`
`chemistryprize2024.pdf (“Four-helix bundles thus became common targets for
`
`protein design in the early years of this field, and the concept of a ‘binary code’
`
`with hydrophobic and hydrophilic amino acid residues was further elaborated by
`
`Hecht and coworkers. These researchers conducted a large library of synthetic
`
`genes coding for the same pattern of polar and nonpolar residues and showed that
`
`most of the designed protein sequences folded into compact α-helical structures.”)
`Petitioner Merck
`EX1003, p. 2
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`
`
`

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`PGR2025-00030
`
`Declaration of Dr. Hecht
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`(citing my 1993 paper entitled “Protein design by binary pattering of polar and
`
`nonpolar amino acids” in Science, 262, 1680-1685).
`
`7.
`
`Since 2003, I have been on the Editorial Advisory Board of Protein
`
`Science and Protein Engineering, Design & Selection. In 2014, I organized a
`
`National Science Foundation Workshop on the Future of Protein Engineering &
`
`Design.
`
`8.
`
`I was a National Science Foundation Graduate Fellow from 1979-
`
`1983, and a Life Sciences Research Foundation Burroughs-Wellcome Post-
`
`doctoral Fellow from 1986-1989. I won the Protein Society’s Kaiser Award in
`
`2003.
`
`9.
`
`Since 1991, I have supervised 14 post-doctoral researchers, 32
`
`graduate students in chemistry and molecular biology, and the senior thesis of 75
`
`undergraduate students in chemistry and molecular biology.
`
`B.
`10.
`
`Compensation
`I am being compensated for my time at the rate of $700 per hour for
`
`my work in connection with this matter. I am being reimbursed for reasonable and
`
`customary expenses associated with my work in this investigation. This
`
`compensation is not dependent in any way on the contents of this Declaration, the
`
`substance of any further opinions or testimony that I may provide, or the ultimate
`
`outcome of this matter.
`
`Petitioner Merck
`EX1003, p. 3
`
`

`

`PGR2025-00030
`
`Declaration of Dr. Hecht
`
`C.
`11.
`
`Person of Ordinary Skill in the Art
`I understand that my analysis and opinions are to be provided using
`
`the perspective of a person of ordinary skill in the art in the timeframe before
`
`December 29, 2011. I will refer to this as the “2011 timeframe” in this declaration.
`
`12. The scientific field of the patent concerns proteins, and more
`
`particularly, protein structures and modification of them. I am very familiar with
`
`this field, and the individuals who work within it, including in the 2011 timeframe.
`
`13.
`
`I have been informed by counsel that a person of ordinary skill in the
`
`art is a hypothetical person who is presumed to have the typical skills and
`
`knowledge of someone working in the field of the invention. Based on my review
`
`of the patent and my experience, I believe a person of ordinary skill in the art (who
`
`I may refer to as “a skilled artisan”) would have had an undergraduate degree, a
`
`Ph.D., and post-doctoral experience in scientific fields relevant to study of protein
`
`structure and function (e.g., chemistry, biochemistry, biology, biophysics). From
`
`training and experience, the person would have been familiar with factors
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`influencing protein structure, folding and activity, production of modified proteins
`
`using recombinant DNA techniques, and use of biological assays to characterize
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`protein function, as well with techniques and tools used to analyze protein structure
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`(i.e., sequence searching and alignments, protein modeling software, etc.).
`
`
`
`Petitioner Merck
`EX1003, p. 4
`
`

`

`PGR2025-00030
`
`Declaration of Dr. Hecht
`
`14.
`
`In the 2011 timeframe, I had at least the qualifications I outline above
`
`for a person of ordinary skill in the art. The opinions I provide in this declaration
`
`are provided from the perspective of a person of ordinary skill in the art in the
`
`2011 timeframe as I have described it above.
`
`D. Terminology Used in this Declaration; Patent Documents
`15.
`I will use the following abbreviations and terminology in this
`
`declaration:
`
`(a)
`
` “PH20” refers to the human PH20 protein. In its full-length form
`
`(including its 35 amino acid signal sequence) it has 509 amino acids.
`
`Its amino acid sequence was first published in 1993.1 Its sequence
`
`also is reported as:
`
`-
`
`-
`
`-
`
`UniprotKB Accession ID P38567;
`
`SEQ ID NO:1 in U.S. Patent No. 7,767,429; and
`
`SEQ ID NO:6 in U.S. Patent No. 12,054,758.
`
`(b) When I refer to the “mature” protein sequence of PH20, I am referring
`
`to the form of the protein without the signal sequence. So, for
`
`example, the mature form of PH20 having amino acids from positions
`
`
`1 EX1029 (Gmachl), 546, Fig. 1.
`
`
`
`Petitioner Merck
`EX1003, p. 5
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`

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`PGR2025-00030
`
`Declaration of Dr. Hecht
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`36 to 509 of Uniprot ID: P38567, would have numbering that starts at
`
`position 1 and ends at position 474.
`
`(c)
`
`I will use “PH201-n” to refer to a human wild-type PH20 polypeptide
`
`sequence that starts at position 1 and terminates at position “n” of the
`
`PH20 sequence. If I do not indicate otherwise, the sequence that is
`
`being referred to is the mature form of the protein without the signal
`
`sequence. For example, PH201-447 means the polypeptide starting at
`
`position 1 and ending at position 447 of the mature human wild-type
`
`PH20 sequence (which would be 36 to 482 if numbering includes the
`
`signal sequence).
`
`(d)
`
`“AxxxB” refers to an amino acid substitution at position xxx, where
`
`the wild-type residue is A and the residue after the substitution is B.
`
`16.
`
`I was asked to assess two patent documents. One is an issued U.S.
`
`Patent, which has the number 12,054,758, which I refer to as the ’758 Patent
`
`(EX1001). The other is a U.S. patent application having the number 13/694,731,
`
`which I will refer to as the ’731 Application (EX1026).
`
`17.
`
`I understand that the disclosure of a patent consists of a narrative
`
`section called the specification, often includes drawings, and may contain sequence
`
`listings, which are nucleotide or amino acid sequences. I understand that each
`
`
`
`Petitioner Merck
`EX1003, p. 6
`
`

`

`PGR2025-00030
`
`Declaration of Dr. Hecht
`
`sequence in the patent will be assigned a number for easy reference (e.g., SEQ ID
`
`NO: 3). I understand that patents end with claims that define the invention.
`
`18.
`
`I reviewed the contents of the ’758 Patent, as well as the ’731
`
`application. I also reviewed a redline comparison of the specifications of the ’758
`
`Patent and the ’731 application (EX1045). Based on that review, the two
`
`specifications appear to be substantively the same. Because the two patent
`
`documents have the same disclosures, I will refer to the two of them together as the
`
`“common disclosure” in this declaration. For convenience, citations will be the
`
`disclosure in the ’758 Patent.
`
`E. Materials Considered
`19. My opinions are based on my years of education, research, and
`
`experience, as well as my investigation and study of relevant materials. I reviewed
`
`a number of publications in the course of my assessment, including those listed in
`
`the Exhibit List. I also relied on my extensive familiarity with the scientific
`
`literature in this field.
`
`20.
`
`I also reviewed a declaration by Dr. Sheldon Park provided to me by
`
`counsel (EX1004). Based on my review, Dr. Park’s declaration provides an
`
`accurate description of how a person of ordinary skill in the art in the 2011
`
`timeframe would have approached the task of identifying single amino acid
`
`substitutions in non-essential regions of PH20 that would have been expected to be
`Petitioner Merck
`EX1003, p. 7
`
`
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`

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`PGR2025-00030
`
`Declaration of Dr. Hecht
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`tolerated by the protein, and is the type of analysis I would rely upon from others
`
`working in my lab in evaluating mutated proteins. Dr. Park’s declaration also
`
`provides an accurate description of the techniques that were being used in the 2011
`
`timeframe to find structurally similar proteins, analyze them using sequence
`
`alignment tools, identify conserved and evolutionarily varying positions in the
`
`related proteins, and use protein structure models to assess the tolerance of the
`
`protein to individual amino acid substitutions.
`
`21.
`
`I reviewed Dr. Park’s discussion of the tools he used in his analysis,
`
`such as BLAST, Clustal Omega, SWISS-MODEL and PyMol. See EX1004 (Park
`
`Dec.), ¶¶ 20-24, 139-164. I am familiar with these tools. I also agree with Dr.
`
`Park that while the tools in their modern incarnation have different forms and
`
`additional capabilities relative to the versions of the tools that existed in 2011, the
`
`analyses Dr. Park performed using them relied on features that would have been
`
`present in the versions that existed in the 2011 timeframe. For example, sequence
`
`alignments performed by the Clustal Omega tool rely on algorithms that were
`
`largely developed in the 1990s and produce outputs that are typically evaluated
`
`manually by the user to confirm the accuracy of the alignments.
`
`22. Like Dr. Park, I also would have expected that structural models
`
`produced by the SWISS-MODEL tool today for PH20 would be very similar the
`
`models for PH20 that would have been produced by that tool in the 2011
`Petitioner Merck
`EX1003, p. 8
`
`
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`

`

`PGR2025-00030
`
`Declaration of Dr. Hecht
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`timeframe. EX1004 (Park Dec.), ¶¶ 146-159. As Dr. Park points out, the template
`
`that SWISS-MODEL used to produce the PH20 models was the HYAL1 structure
`
`published by Chao et al. in 2007 (EX1006) (structure ID: 2PE4). I also agree with
`
`Dr. Park’s observation that even if there were subtle differences in the positioning
`
`of certain side chains in the PH20 model relative to a model generated in the 2011
`
`timeframe, those differences would not have affected the overall assessment that a
`
`scientist would have made, which are based on a visual assessment of individual
`
`substitutions within the PH20 structure.
`
`F.
`23.
`
`Legal Principles
`I am not a lawyer and am not offering opinions on the law. However,
`
`I have been provided a general explanation of some of the legal requirements for
`
`obtaining a patent.
`
`24.
`
`I have been informed that a patent’s disclosure consists of the
`
`information in the specification, along with any drawings and sequence listings
`
`that accompanied the patent application. When I use the term “patent disclosure,”
`
`I am using it with this understanding.
`
`25.
`
`I have been informed that a patent claiming a set (or “genus”) of
`
`chemical compounds (e.g., polypeptides) must be supported by the patent
`
`disclosure, and that there are two distinct disclosure requirements, as summarized
`
`in the following paragraphs.
`
`
`
`Petitioner Merck
`EX1003, p. 9
`
`

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`PGR2025-00030
`
`Declaration of Dr. Hecht
`
`26.
`
`I have been informed that one of the disclosure requirements is that
`
`the patent disclosure provide a sufficient written description of the claimed set of
`
`polypeptides (“written description” requirement). I have been informed that this
`
`can be achieved by a patent disclosure that describes either (i) “a representative
`
`number of species falling within the scope of the genus” or (ii) “structural features
`
`common to the members of the genus,” with either being such that “one of skill in
`
`the art can ‘visualize or recognize’ the members of the genus.” I have been
`
`informed that an adequate written description of a genus of polypeptides “requires
`
`a precise definition, such as by structure, formula, chemical name, physical
`
`properties, or other properties, of species falling within the genus sufficient to
`
`distinguish the genus from other materials.” I also have been informed that a
`
`description that is “merely drawing a fence around the outer limits of a purported
`
`genus is not an adequate substitute for describing a variety of materials constituting
`
`the genus and showing that one has invented a genus and not just a species.”
`
`27.
`
`I have been informed a second disclosure requirement is that the
`
`patent disclosure provide a description that enables a skilled artisan to practice the
`
`claimed invention without engaging in undue experimentation (“enablement”
`
`requirement”).
`
`28.
`
`I have been informed that if a patent claims an entire class of
`
`compounds (e.g., polypeptides), the patent’s specification must enable a person
`Petitioner Merck
`EX1003, p. 10
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`

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`PGR2025-00030
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`Declaration of Dr. Hecht
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`skilled in the art to make and use the entire class, which means the specification
`
`must enable the full scope of the invention as defined by its claims. I have also
`
`been informed that a research plan that requires a skilled artisan to perform trial
`
`and error procedures in order to discover which members of a large class of
`
`polypeptides have a desired functional property is not sufficient to satisfy the
`
`enablement requirement.
`
`29.
`
`I have also been informed that factors such as the breadth of the
`
`claims, unpredictability in the field, the degree of guidance in the prior art and in
`
`the patent disclosure, and the level of skill of the skilled artisan are factors, among
`
`others, that are considered in assessing the question of enablement.
`
`30.
`
`I have been informed that a separate patentability requirement is that
`
`an invention must not have been obvious to a person of ordinary skill in the art in
`
`view of what was known in the prior art before the filling date of the patent. I also
`
`have been informed that if a patent claim encompasses a compound that would
`
`have been obvious in light of the prior art, that claim is unpatentable.
`
`31.
`
`I have been informed that for a claimed compound to be found
`
`obvious, a person of ordinary skill in the art must have found a reason in the prior
`
`art to make that compound, and must have had a reasonable expectation of success
`
`in achieving the claimed invention. I have been informed this does not require the
`
`skilled artisan to have absolute certainty about achieving a desired result, and that
`Petitioner Merck
`EX1003, p. 11
`
`
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`

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`PGR2025-00030
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`Declaration of Dr. Hecht
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`an invention can be found obvious if a result is expected but still requires some
`
`experimentation to confirm.
`
`32.
`
`I have been informed that if a particular compound exhibits
`
`unexpected properties, that evidence can support a finding that the compound is
`
`not obvious. For a claim defining a large class of compounds, the members of the
`
`class must share the unexpected property to support a finding that the class of
`
`compounds is not obvious. A claim defining a large class of compounds cannot
`
`benefit from evidence showing only one compound exhibits a particular
`
`unexpected property. Instead, the evidence must demonstrate that the unexpected
`
`property is shared by the entire class of claimed compounds.
`
`II.
`
`Scientific Principles
`33. The common disclosure discusses modified forms of a human
`
`hyaluronidase enzyme called PH20, which is one of five related hyaluronidase
`
`enzymes found to occur in humans.
`
`34. The focus of the patent claims, as I explain below, are modified PH20
`
`polypeptides that have incorporated at least one amino acid substitution at position
`
`317, and optionally may incorporate 20 to 41 additional substitutions at any other
`
`position in the wild-type sequence of 37 different PH20 polypeptides of varying
`
`length, depending on the parameters of each claim.
`
`
`
`Petitioner Merck
`EX1003, p. 12
`
`

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`PGR2025-00030
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`Declaration of Dr. Hecht
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`35. The scientific fields relevant to the subject matter of the patent are
`
`protein structure and engineering. Two textbooks that provide a useful orientation
`
`to protein structure principles are: (i) Brandon & Tooze (EX1014) (chapters 1-6
`
`and 17) and (ii) Alberts (EX1039) (chapter 3).2
`
`A.
`Protein Structure Basics
`36. Proteins are formed by sequences of amino acids, but the feature of
`
`each protein that confers its unique biological function(s) is its overall three-
`
`dimensional structure.3 That overall structure results from folding of the amino
`
`acid sequence of the protein (its “primary structure”). The sequences initially fold
`
`into regions of characteristic “secondary structures” (e.g., alpha-helices, beta-
`
`strands, loop regions). Sets of secondary structures then are positioned and
`
`arranged to form structural motifs, and those motifs and other sequences pack
`
`together to form compact globular units called domains, of which there may be one
`
`or several in the protein. That higher order structure is the “tertiary structure” of
`
`the protein. Multiple polypeptides can also form complexes, referred to as the
`
`“quaternary structure” of the protein. PH20 consists of a single polypeptide chain.
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`
`2 Brandon & Tooze (EX1014) (“Brandon”) is a textbook which I have used in
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`the graduate course that I teach for many years.
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`3 EX1014 (Brandon), 3-11.
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`Petitioner Merck
`EX1003, p. 13
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`PGR2025-00030
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`Declaration of Dr. Hecht
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`37. Amino acids are generally classified based their chemical attributes,
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`which are dictated by the side chain of the amino acid. Amino acids can be
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`classified in different ways. For example, at a high-level, amino acids can be
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`classified as being polar or non-polar, with subsets of polar residues being charged
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`(positively or negative) or uncharged.4 Amino acids also have varying sizes, which
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`can influence their ability to fit into defined protein structures.
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`4 EX1039 (Alberts), 127.
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`Petitioner Merck
`EX1003, p. 14
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`PGR2025-00030
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`Declaration of Dr. Hecht
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`38. Some amino acids have diverse chemical characteristics. For
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`example, lysine (K) and arginine (R) have terminal amine groups (which are
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`hydrophilic) and long aliphatic chains (which have a hydrophobic character).5
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`Other amino acids incorporate ring structures (e.g., tryptophan (W), phenylalanine
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`(F), tyrosine (Y), histidine (H), proline (P)), which are rigid and may be aromatic.
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`Cysteine (C) has the unique ability to form covalent disulfide bonds with other
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`cysteines, which confers significant structural stability to areas of protein structure.
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`The diversity of characteristics of amino acid side chains contributes to the
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`incredible diversity of structures that proteins have.
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`39. Folded proteins generally are arranged to have a hydrophobic interior
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`and a hydrophilic surface. The backbone or primary chain has a polar character,
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`5 EX1014 (Brandon), 6-7.
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`Petitioner Merck
`EX1003, p. 15
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`PGR2025-00030
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`Declaration of Dr. Hecht
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`which is hydrophilic and not energetically favored within a hydrophobic
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`environment.6
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`40. Secondary structures formed out of characteristic patterns of amino
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`acids, however, offset this intrinsically polar character of the backbone chain
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`within the hydrophobic interior of proteins. Secondary structures form
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`energetically favored structures within the backbone via hydrogen bonding
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`between backbone NH and C’=O groups. The secondary structures then pack
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`together to form motifs and higher order structures, both of which further stabilize
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`the interior of the protein structure.
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`41. There are two general classes of secondary structures: (i) the alpha-
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`helix (a-helix), and (ii) the beta-sheet (b-sheet) (illustrated below).7
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`6 EX1014 (Brandon), 14.
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`7 EX1039 (Alberts), 134-135.
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`Petitioner Merck
`EX1003, p. 16
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`PGR2025-00030
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`Declaration of Dr. Hecht
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`42. There are numerous variants of each class of secondary structure, each
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`associated with characteristic patterns, spacing and types of amino acids in the
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`polypeptide sequence. The structures are, to varying degrees, sensitive to the
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`positioning and patterns of residues, as well as to the types of amino acids that may
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`be in those positions.8 For example, certain amino acids tend to favor being within
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`a helical structure (e.g., leucine), while others disfavor such structures if
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`incorporated into a sequence that would ordinarily form an a-helix or b-sheet.9
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`8 See generally, EX1014 (Brandon), 16-20; EX1039 (Alberts), 131-135.
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`9 EX1014 (Brandon), 353-4.
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`Petitioner Merck
`EX1003, p. 17
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`PGR2025-00030
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`Declaration of Dr. Hecht
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`exterior of it. The interior is hydrophobic, while the exterior will generally be
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`solvent accessible. However, when the secondary structures are packed together,
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`the exterior facing side chains may interact with side chains of amino acids in other
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`secondary structures (which may be a hydrophobic environment), and the effects
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`of those interactions may be stabilizing or destabilizing.
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`44. Structural motifs form from combinations and arrangements of
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`secondary structures, and those structural motifs, in turn, pack together to form the
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`higher order structure that characterizes a protein domain. There are numerous
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`influences on how these structural motifs are formed and stabilized.
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`45. For example, sequences that form secondary structures are often
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`separated by stretches of amino acids of varying lengths. The nature and length of
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`those intervening sequences influence how secondary structures on either end of
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`the intervening sequence can interact with each other or with other secondary
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`structures in the protein. To illustrate this, consider the figures below, which
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`shows schematically how different lengths of intervening sequences between b-
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`sheet secondary structures influence the packing of pairs of beta sheets and then
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`the structural motifs created by those pairs into a higher order structure.10
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`10 EX1014 (Brandon), 77-78.
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`Petitioner Merck
`EX1003, p. 18
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`PGR2025-00030
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`Declaration of Dr. Hecht
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`46. The specific amino acids in specific positions of an amino acid
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`sequence also play a critical role in inducing the folding of the protein into its
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`destined structure, as well as in maintaining that structure.11
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`B.
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`Protein Engineering
`1.
`Rational Design vs. Directed Evolution
`47. Protein engineering aims to create novel proteins not found in nature
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`with desired properties. At a conceptual level, there are two approaches for
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`11 EX1039 (Alberts), 125-130.
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`Petitioner Merck
`EX1003, p. 19
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`Declaration of Dr. Hecht
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`creating engineered proteins, referred to as “rational design” and “directed
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`evolution.”12
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`48.
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`“Rational design” involves an in-depth study of the structural features
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`of a protein and their contribution to the stability and function(s) of the protein.
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`The insights derived from that study are used with knowledge of protein structure
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`principles to devise