UNITED STATES PATENT AND TRADEMARK OFFICE
`
`______________________
`
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`______________________
`
`
`
`MILTENYI BIOMEDICINE GmbH and MILTENYI BIOTEC INC.
`Petitioner
`v.
`
`FRED HUTCHINSON CANCER CENTER
`Patent Owner
`
`______________________
`
`
`IPR Trial No. IPR2023-
`U.S. Patent No. 9,987,308
`Issue Date: June 5, 2018
`Title: Method and Compositions for Cellular Immunotherapy
`
`______________________
`
`
`
`DECLARATION OF DR. JONATHAN BRAMSON
`
`IN SUPPORT OF PETITION FOR INTER PARTES REVIEW OF
`U.S. PATENT NO. 9,987,308
`
`
`
`Miltenyi Ex. 1002 Page 1
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`

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`Page
`INTRODUCTION .......................................................................................... 1
`I.
`EDUCATION AND EXPERIENCE .............................................................. 1
`II.
`III. MATERIALS CONSIDERED ....................................................................... 4
`IV. TECHNOLOGY BACKGROUND ................................................................ 5
`A.
`T Cells .................................................................................................. 5
`B.
`Subpopulations of T Cells .................................................................... 6
`C. Markers of T-Cell Subpopulations ....................................................... 8
`D.
`Flow Cytometry and Cell Sorting ...................................................... 10
`E.
`Adoptive Immunotherapy .................................................................. 17
`F.
`Chimeric Antigen Receptors .............................................................. 19
`PERSON OF ORDINARY SKILL IN THE ART ....................................... 22
`V.
`VI. THE ’308 PATENT ...................................................................................... 22
`VII. CLAIM CONSTRUCTION ......................................................................... 28
`A.
`“adoptive cellular immunotherapy composition” .............................. 28
`B.
`“and/or” .............................................................................................. 31
`VIII. PRIOR ART .................................................................................................. 32
`A. Adoptive Cellular Immunotherapy Compositions With
`CD45RA+, CD45RO+, and/or CD62L+ Immunophenotypes
`Were Known ....................................................................................... 32
`1.
`Singh ........................................................................................ 32
`2. Mitsuyasu ................................................................................. 47
`The Modularity of Chimeric Antigen Receptors Was Known .......... 52
`1.
`Cooper ...................................................................................... 52
`CARs Targeting CD19, CD20, CD22, ROR1, CEA, Her2, L1-
`CAM, and Mesothelin Were Known ................................................. 54
`1.
`Hudecek I ................................................................................. 54
`2.
`Hudecek II ................................................................................ 56
`3.
`Abken ....................................................................................... 57
`
`C.
`
`B.
`
`TABLE OF CONTENTS
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`i
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`

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`E.
`
`Page
`Reckamp ................................................................................... 59
`4.
`Carpenito .................................................................................. 60
`5.
`D. Using Equivalent Numbers of CD4+ and CD8+ T Cells in
`Adoptive Cellular Immunotherapy Compositions Was Known ........ 62
`1. Moeller ..................................................................................... 62
`Benefits of TCM, which are CD62L+ T Cells, Was Well-Known ....... 63
`1. Wang ........................................................................................ 63
`2.
`Yang I ....................................................................................... 65
`3.
`Yang II ..................................................................................... 67
`4.
`Sallusto ..................................................................................... 68
`5.
`Sun ............................................................................................ 69
`Formulations for Adoptive Cellular Immunotherapy
`Compositions Were Known ............................................................... 70
`1.
`Jensen ....................................................................................... 70
`IX. LEGAL STANDARDS ................................................................................ 71
`X. GROUND 1: INDEPENDENT CLAIM 1 AND DEPENDENT
`CLAIMS 2-7, 14, 16, 21, AND 26-28 ARE ANTICIPATED BY
`SINGH .......................................................................................................... 73
`A.
`Independent Claim 1 .......................................................................... 74
`1.
`Claim Limitations Directed to Components of the
`Claimed Composition .............................................................. 75
`a.
`[a] “[a]n adoptive cellular immunotherapy
`composition” .................................................................. 76
`[b] “containing chimeric antigen receptor-modified
`CD4+ T lymphocytes and chimeric antigen
`receptor-modified CD8+ T lymphocytes,” ..................... 78
`[c] & [e] “wherein: (a) the chimeric antigen
`receptor-modified CD4+ T lymphocytes [and (b)
`the chimeric antigen receptor-modified CD8+ T
`lymphocytes] contain a chimeric antigen receptor
`that specifically binds to an antigen” ............................. 81
`
`TABLE OF CONTENTS
`(continued)
`
`ii
`
`F.
`
`b.
`
`c.
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`Miltenyi Ex. 1002 Page 3
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`

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`TABLE OF CONTENTS
`(continued)
`
`Page
`
`2.
`
`Claim Limitations Directed to Phenotypic Marker
`Surface Positivity ..................................................................... 82
`a.
`[d] & [f] “at least 50% of the chimeric antigen
`receptor-modified CD4+ helper T lymphocytes in
`the composition are surface positive for CD62L
`and/or CD45RA,” and “at least 50% of CD8+
`cytotoxic T lymphocytes in the composition are
`surface positive for CD62L and/or CD45RO” .............. 83
`B. Dependent Claims Directed to Antigens ............................................ 89
`1.
`Claims 2 and 26-28: “wherein the antigen is associated
`with a disease or disorder” ....................................................... 89
`Claims 3 and 21: “wherein the antigen is selected from
`ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22,
`mesothelin, and CEA” ............................................................. 91
`C. Dependent Claims Directed to CAR Components ............................. 92
`1.
`Claims 4 and 5: “extracellular antibody variable domain
`or single-chain antibody fragment and an intracellular
`signaling module” .................................................................... 92
`Claims 7 and 16: Claims Related to Whether the CAR is
`the Same in the CD4+ and CD8+ T Cells ................................. 94
`D. Dependent Claims Directed to Percentage Surface Positivity of
`T-Cell Markers ................................................................................... 95
`Dependent Claim Directed to Ratios .................................................. 96
`1.
`Claim 14: Composition Ratios ................................................. 96
`XI. GROUND 2: INDEPENDENT CLAIM 1 AND DEPENDENT
`CLAIMS 2-7, 14, 16, 21, AND 26-28 ARE RENDERED OBVIOUS
`BY SINGH IN VIEW OF JENSEN ............................................................. 98
`A.
`Independent Claim 1 .......................................................................... 98
`B.
`Claims 2-7, 14, 16, 21, and 26-28 .................................................... 101
`
`2.
`
`2.
`
`E.
`
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`

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`TABLE OF CONTENTS
`(continued)
`
`Page
`
`C.
`
`E.
`
`XII. GROUND 3: ALL CHALLENGED CLAIMS ARE RENDERED
`OBVIOUS BY SINGH IN VIEW OF JENSEN, HUDECEK I,
`WANG, MITSUYASU, MOELLER, ABKEN, HUDECEK II,
`RECKAMP, CARPENITO, YANG I, YANG II, SALLUSTO, AND
`SUN ............................................................................................................ 101
`A.
`Independent Claim 1 ........................................................................ 103
`B. Dependent Claims Directed to Antigens .......................................... 103
`1.
`Claims 3 and 18-25 ................................................................ 103
`Claims Directed to Percentage Surface Positivity of T-Cell
`Markers ............................................................................................. 109
`1.
`Claims 1, 6 and 9-13 .............................................................. 109
`D. Dependent Claims Directed to Ratios .............................................. 120
`1.
`Claim 14-15 ............................................................................ 120
`Dependent Claims Directed to Comparisons to Other
`Populations ....................................................................................... 122
`1.
`Claim 8 ................................................................................... 122
`2.
`Claim 29 ................................................................................. 127
`3.
`Claim 30 ................................................................................. 129
`4.
`Claim 31 ................................................................................. 131
`Remaining Challenged Claims ......................................................... 134
`F.
`XIII. GROUND 4: INDEPENDENT CLAIM 1 AND DEPENDENT
`CLAIMS 6 AND 9-16 ARE RENDERED OBVIOUS BY
`MITSUYASU IN VIEW OF SINGH AND COOPER .............................. 134
`A.
`Independent Claim 1 and Dependent Claims 6 and 9-13 ................. 134
`1.
`Claim Limitations Directed to Components of the
`Claimed Composition ............................................................ 135
`a.
`[a] “[a]n adoptive cellular immunotherapy
`composition” ................................................................ 135
`[b] “containing chimeric antigen receptor-modified
`CD4+ T lymphocytes and chimeric antigen
`receptor-modified CD8+ T lymphocytes,” ................... 136
`
`b.
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`TABLE OF CONTENTS
`(continued)
`
`Page
`
`c.
`
`2.
`
`[c] & [e] “wherein: (a) the chimeric antigen
`receptor-modified CD4+ T lymphocytes [and (b)
`the chimeric antigen receptor-modified CD8+ T
`lymphocytes] contain a chimeric antigen receptor
`that specifically binds to an antigen” ........................... 140
`Claim Limitations Directed to Phenotypic Marker
`Surface Positivity ................................................................... 140
`a.
`[d] & [f] “at least 50% of the chimeric antigen
`receptor-modified CD4+ helper T lymphocytes in
`the composition are surface positive for CD62L
`and/or CD45RA,” and “at least 50% of CD8+
`cytotoxic T lymphocytes in the composition are
`surface positive for CD62L and/or CD45RO” ............ 141
`Dependent Claims 6 and 9-13 ..................................... 156
`b.
`B. Dependent Claims Directed to Ratios .............................................. 158
`C. Dependent Claim Directed to CAR Components ............................ 159
`XIV. CONCLUSION ........................................................................................... 160
`
`
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`Miltenyi Ex. 1002 Page 6
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`
`
`I.
`
`INTRODUCTION
`1.
`I, Jonathan Bramson, Ph.D., have been retained as an expert witness on
`
`behalf of Miltenyi Biomedicine GmbH and Miltenyi Biotec Inc. (collectively,
`
`“Petitioner”) in connection with its petition (“Petition”) for inter partes review of
`
`U.S. Patent 9,987,308 (“the ’308 patent”) (Ex. 1001). I understand that the ’308
`
`patent is owned by the Fred Hutchinson Cancer Center (“Patent Owner”).
`
`2.
`
`I understand that this declaration is being submitted in support of
`
`Petitioner’s Petition of claims 1-16 and 18-31 (“Challenged Claims”) of the ’308
`
`patent, which issued from U.S. Application No. 14/006,641 on June 5, 2018. The
`
`’308 patent names Stanley R. Riddell and Michael Hudecek as inventors.
`
`3.
`
`I have reviewed the ’308 patent and in my review, considered the
`
`teachings of the scientific literature before March 23, 2011.
`
`4.
`
`I am being compensated at my usual and customary rate of $425 per
`
`hour. My compensation is in no way dependent on the outcome of this case.
`
`5.
`
`In the last 4 years, I served as an expert in, and was deposed and/or
`
`testified in connection with, the cases listed in Appendix B.
`
`II. EDUCATION AND EXPERIENCE
`6.
`A copy of my curriculum vitae, which describes my qualifications in
`
`greater detail and includes a list of publications that I authored in the past 30 years,
`
`is attached as Appendix A.
`
`
`
`1
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`Miltenyi Ex. 1002 Page 7
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`7.
`
`I am an expert in the field of engineered T-cell therapy, including
`
`chimeric antigen receptor (“CAR”)-T cells. I have significant experience designing
`
`and constructing CAR-T cells and other T-cell immunotherapies. I also have
`
`significant experience developing novel immunotherapies, including CAR T-cell
`
`therapies.
`
`8.
`
`I received my B.Sc. in Biochemistry from McGill University in 1991.
`
`I received my Ph.D. in Experimental Medicine from McGill University in 1994.
`
`9.
`
`I am currently Vice Dean of Research at the Faculty of Health Sciences,
`
`McMaster University. Prior to that, I served as Assistant Dean, Research
`
`Infrastructure, at the Faculty of Health Sciences, as well as Director at the
`
`Fraunhofer-McMaster Project Center. I am, and have been since 2021, a professor
`
`in the Department of Medicine at McMaster University. Before joining Medicine, I
`
`was a professor in Pathology and Molecular Medicine at McMaster from 2009 –
`
`2020, having been a member of that department since joining McMaster as an
`
`Assistant Professor in 1999. For a period of over 5 years (2009 – 2014), I also served
`
`as Director at the McMaster Immunology Research Centre. Before entering
`
`academia, I was a research scientist and project leader at Inex Pharmaceuticals for
`
`two years (1997 – 1999).
`
`10.
`
`I am currently a chair officer for three committees, councils, or panels,
`
`those being Triumvira Immunologics Scientific Advisory Board, Chronic Pain
`
`
`
`2
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`Miltenyi Ex. 1002 Page 8
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`
`
`Centre of Excellence Board of Directors, and CIHR Operating Grant Review Panel
`
`Cancer Biology-Therapeutics 2. I am an active member of five others, those being
`
`NIH Immuno-oncology Research Grant Review Panel, McMaster Innovation Park
`
`Board of Directors, Council of Ontario Faculties of Medicine Research Committee,
`
`Association of Faculties of Medicine of Canada Research Council, and the Canadian
`
`Cancer Immunotherapy Consortium. In the past, I have been a member, organizer,
`
`or consultant for numerous others, for which details can be found in my curriculum
`
`vitae.
`
`11. My most recent speaking experience was at the 13th Annual Myeloma
`
`Canada Scientific Roundtable, which took place in Montreal in November 2022. I
`
`presented on the status of engineered cell therapy research and clinical trials,
`
`including academic and commercial CAR-T cells, at McMaster University and our
`
`partner hospital, Hamilton Health Sciences Corporation. Before that, for the year of
`
`2022, I spoke in six other conferences, including three in Canada and one in
`
`Pennsylvania. Due to the COVID-19 pandemic, I only spoke in two conferences in
`
`2021, but before that I made an effort to speak in at least 4-5 conferences a year,
`
`preferably more. All of my topics circled around gene therapy, and later in my career
`
`I focused exclusively on engineered T cell cancer immunotherapy. I have been
`
`speaking and giving oral presentations on these topics since 2001.
`
`
`
`3
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`
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`12. Since 2012, I have served as an expert consultant to various
`
`biotechnology companies. In these roles, I have provided expert advice on program
`
`development.
`
`13.
`
`I have co-authored various publications on CAR-T cells, including
`
`those targeting CD19, HER2, and ROR1. I have also consulted about the impact of
`
`immunophenotypes on T-cell adoptive transfer.
`
`14.
`
`I have supported clinical trials of engineered T cells (NCT03880279,
`
`NCT04727151).
`
`III. MATERIALS CONSIDERED
`15.
`In forming the opinions in this declaration, I considered the materials
`
`cited in this declaration from the perspective of the general knowledge in the art
`
`before March 23, 2011.
`
`16.
`
`I also relied on my education and knowledge, as well as my experience
`
`in academic and clinical research projects, including those discussed in Section II.
`
`17.
`
`I reserve the right to amend and supplement any of the opinions in this
`
`declaration as needed in response to new information and evidence made available
`
`to me, additional analysis that leads me to conclude that supplementation is
`
`necessary, new issues that may arise, any discovery, arguments, evidence, or
`
`testimony presented in this inter partes review, and any Patent Trial and Appeal
`
`Board (“Board”) decisions or orders.
`
`
`
`4
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`Miltenyi Ex. 1002 Page 10
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`
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`IV. TECHNOLOGY BACKGROUND
`18. Below I provide a brief background of the state of the art before March
`
`23, 2011 to support my opinions on anticipation and obviousness discussed in
`
`Sections X-XIII below. A person of ordinary skill in the art (“POSA”) also would
`
`have been aware of additional references related to T cells and CARs.
`
`A. T Cells
`19. Lymphocytes are a type of white blood cell, and T cells are a type of
`
`lymphocyte. T cells work as part of the immune system by detecting foreign invaders
`
`and producing helpful cytokines in response. Cytokines are protein-based molecules
`
`that effect an immune response. See, e.g., Ex. 1018, [0005], [0016], [0009]
`
`(“transferred T cells … directly recognize and lyse tumor targets, produce cytokines
`
`that recruit and activate antigen non-specific anti-tumor effector cells …”);
`
`Ex. 1001, 2:1-31.
`
`20. T cells are commonly distinguished based on the presence or absence
`
`of surface proteins, CD4 and CD8. T cells that express the CD4 membrane
`
`glycoprotein (“CD4”) are typically referred to as CD4+ T cells. Based on their
`
`functional attributes, CD4+ cells have also been historically described as “helper T
`
`cells”. T cells that express the CD8 membrane glycoprotein (“CD8”), are typically
`
`referred to as CD8+ T cells. Based on their functional attributes, CD8+ cells have
`
`also been historically described as “cytotoxic T cells.” See, e.g., Ex. 1001, 7:7-11,
`
`
`
`5
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`
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`7:66-8:3, 9:4-13, 9:57-10:5, 10:66-11:17, 11:43-51, 26:45-48. A glycoprotein is a
`
`protein with an attached carbohydrate group. In immunology, cells that express a
`
`high level of a membrane protein can be described with the name of the protein
`
`followed by a “+” sign (e.g., CD4+ or CD8+ T cells). Immunologists generally use a
`
`“-” sign if a cell expresses a low level of a protein or complete absence thereof (e.g.,
`
`CD4- or CD8- T cells). See, e.g., Ex. 1018, [0018], [0037]; Ex. 1007, 227-28; Ex.
`
`1001, 25:35-44.
`
`B.
`Subpopulations of T Cells
`21. Both CD4+ and CD8+ T cells include several categories of T-cell
`
`subpopulations that differ in phenotype, function, and homing to their respective in
`
`vivo targets. Although there are many subpopulations of T cells, the subpopulations
`
`most relevant to the ’308 patent are “naïve” T cells, “central-memory” T cells,
`
`“effector-memory” T cells, and “effector” T cells. See, e.g., Ex. 1011, 525; Ex. 1007,
`
`227-28; Ex. 1020, 64, Ex. 1027, 12; Ex. 1001, 1:49-67.
`
`22. Naïve T cells (“TN”) are mature T cells that have not yet encountered
`
`antigenic material to which they can bind. An antigen is a substance capable of
`
`stimulating an immune response. Naïve T cells are activated by stimulation from
`
`antigenic material, most commonly short peptides known as epitopes. Once
`
`stimulated, TN cells activate, proliferate (i.e., multiply), and produce cytokines. See,
`
`e.g., Ex. 1007, 227-28; Ex. 1018, [0070]; Ex. 1001, 7:30-37, 11:52-67.
`
`
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`23. Upon activation, TN cells may differentiate into antigen-specific
`
`central-memory T cells (“TCM”), effector-memory T cells (“TEM”), or effector T cells
`
`(“TE”). TCM and TEM also have the capacity to differentiate into TE cells upon
`
`secondary exposure to epitopes from their target antigen. See, e.g., Ex. 1011, 525;
`
`Ex. 1001, 1:59-62; 7:12-42.
`
`24. Generally speaking, TE cells are the T-cell subpopulation primarily
`
`responsible for killing or eliminating infected cells from the body. However, they
`
`are short-lived. Although TE cells typically die after their target antigen clears from
`
`the body, TCM and TEM cells linger, and are capable of responding in the event of re-
`
`exposure to the antigen. After re-exposure to an antigen, TCM can differentiate into
`
`TCM-derived TE cells. See, e.g., Ex. 1011, 525 (“TCM and TEM cells . . . respond to
`
`antigen re-exposure by differentiating into a new wave of TE cells.”); id. (“some of
`
`the progeny of clonally derived TE cells retain intrinsic programming of the parental
`
`cell of origin”); see also Ex. 1001, 1:59-62 (“CD8+ TCM and TEM both differentiate
`
`into cytolytic effector T cells (TE) that express a high level of granzymes and
`
`perforin, but are short-lived.”). TEM cells differentiate into TEM-derived TE cells. See,
`
`e.g., id. There are intrinsic differences between TCM-derived, and TEM-derived, TE
`
`cells, including greater persistence in TCM-derived TE cells. See Ex. 1011, 525
`
`(“T-cell clones derived from TCM, but not those derived from TEM cells were capable
`
`of persisting long-term”). TEM-derived TE cells die rapidly after transfer. See
`
`
`
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`Ex. 1007, 228 (“T cells derived from TEM precursors die rapidly after cell transfer
`
`and do not establish persistent T cell memory.”).
`
`25.
`
` A T cell’s persistence refers to its ability to survive and continue to
`
`perform immune system functions and proliferate continually. TCM cells live, and
`
`thus persist, longer than TE cells. See, e.g., Ex. 1015, 3360, 3363-64; Ex. 1026,
`
`445-47, Ex. 1016, 2995, 2999-3002; Ex. 1007, 226-30; Ex. 1011, 525; Ex. 1017,
`
`926-27; Ex. 1001, 1:31-48, 8:49-52, 27:4-7.
`
`C. Markers of T-Cell Subpopulations
`26. T-cell subpopulations such as TN, TCM, TEM, and TE may be defined by
`
`their expression of various proteins expressed on the cell surface. These proteins
`
`may be referred to as “T-cell markers,” and can be used by researchers to understand
`
`what kinds of T cells make up a given composition. The collection of a T-cell’s
`
`markers is referred to as the T cell’s immunophenotype. See, e.g., Ex. 1003, 2966-67;
`
`Ex. 1027, 2-3, 7; Ex. 1028, S184; ; Ex. 1001, 7:12-42, 10:66-11:21.
`
`27. There are many T-cell markers, but the ’308 patent claims recite only a
`
`subset of them, including CD45RA, CD45RO, and CD62L. All were well-known
`
`T-cell markers prior to March 23, 2011. See, e.g., Ex. 1007, 227-28; Ex. 1004, 788;
`
`Ex. 1001, 7:12-42.
`
`28.
`
`In fact, as of March 23, 2011, researchers understood that those markers
`
`could be used to identify T-cell populations. According to Berger, C. et al.,
`
`
`
`8
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`
`
`“Adoptive Transfer of Virus-Specific and Tumor-Specific T Cell Immunity,” CURR.
`
`OPIN. IN IMMUNOL., 21: 224-32 (2009) (Ex. 1007) (“Berger”):
`
`lymphocyte pool from which T cells for adoptive
`The T
`immunotherapy could potentially be isolated contains CD45RA+
`CD62L+ naïve (TN), CD45RO+ CD62L+ central memory (TCM), and
`CD62L- effector memory (TEM) subsets that differ in phenotype,
`function, and homing.
`Ex. 1007, 227.
`
`29. A POSA would have understood that a TN cell was CD45RA+ CD62L+,
`
`a TCM cell was CD45RO+ CD62L+, and TEM or TE cell were both CD62L-, as depicted
`
`below:
`
`
`
`Id., 228.
`
`
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`30. However, these were not the only markers associated with TN, TCM, and
`
`TEM cells. In fact, multiple markers are used to define T-cell subpopulations,
`
`meaning that each subpopulation could be associated with more than one marker.
`
`See, e.g., Ex. 1007, 227-28; Ex. 1003, 2967; Ex. 1001, 10:66-11:17. This is
`
`consistent with how the ’308 patent defines immunophenotypes for TN, TCM, TEM,
`
`and TE cells. In the ’308 patent, each of these T-cell subpopulations is associated
`
`with immunophenotypes based on expression of CD45RA, CD45RO, CD62L, and
`
`other T-cell markers. See, e.g., Ex. 1001, 7:7-53, 10:66-11:17, 15:25-50.
`
`D.
`Flow Cytometry and Cell Sorting
`31. Generally speaking, cells are transparent and microscopic, meaning that
`
`to visualize them, researchers need to use specialized techniques and machinery to
`
`detect them. See, e.g., Ex. 1016, 2996-2997; Ex. 1025, 4099-4101; Ex. 1018, [0104];
`
`Ex. 1001, 10:16-24.
`
`32. One method of detecting T-cell markers, and characterizing the T cells
`
`by their markers to define immunophenotypes, is through the use of a flow cytometry
`
`where cells are labeled with fluorescent tags that bind to specific markers on cell
`
`surfaces allowing the presence of specific markers on specific cells to be analyzed
`
`using an instrument known as a flow cytometer. See, e.g., Ex. 1003, 2963, 2966;
`
`Ex. 1027, 3-5, 12-15; Ex. 1018, [0104], [0109]; Ex. 1015, 3361, 3363; Ex. 1014,
`
`1099; Ex. 1020, 61-62; Ex. 1004, 788-89; Ex. 1001, 10:16-24.
`
`
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`33. As an example of using flow cytometry to characterize a T cell
`
`population, to determine how many T cells express CD62L within a population of T
`
`cells, a scientist would combine the T cell population with a fluorescent reagent that
`
`is capable of binding specifically to CD62L. After the reagent binds to
`
`CD62L-expressing T cells and fails to bind to any non-CD62L-expressing cells that
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`are present, the resulting mixture would then be placed within a machine called a
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`flow cytometer. A flow cytometer analyzes each cell one-by-one by casting laser
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`beams on each cell as they pass through a component of the cytometer, known as the
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`flow cell. The fluorescent reagent(s) attached to T cells will emit photons following
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`excitation by the laser beams and those photons are captured by the flow cytometer
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`to provide a direct measure of the amount of fluorescent reagent(s) bound to each
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`T cell. Using the FACS method, T cells with specific fluorescent properties (e.g.,
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`those that bound fluorescent antibodies directed against CD62L) can be directed to
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`a collection tube, which separates them from the T cells that do not express CD62L.
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`See, e.g., id.
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`34. Scientists routinely use flow cytometry to quantify T cells expressing
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`on their surface any T-cell marker-of-interest, provided that an appropriate
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`fluorescent, specifically binding reagent is available. See, e.g., id.
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`35. Before March 23, 2011, fluorescent reagents that could specifically
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`bind to CD4, CD8, CD45RA, CD45RO, and CD62L were readily available to
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`researchers. See, e.g., id.
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`36. A typical plot of hypothetical flow cytometry data is shown below. It is
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`a two-dimensional dot plot with the x- and y-axes each representing expression of a
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`distinct, hypothetical cell surface molecule—Surface Molecule #1 and Surface
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`Molecule #2:
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`
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`See, e.g., id.
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`37.
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`In a flow cytometry dot plot, each dot represents a cell, and the position
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`of each dot on the plot shows how much of each tested cell surface marker is
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`expressed by the cell. Here, cells that express more Surface Molecule #1 will be
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`represented by a dot that is further to the right on the plot than a dot corresponding
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`to a cell that expresses less Surface Molecule #1. Similarly, cells that express more
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`
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`Surface Molecule #2 will be represented by a dot that is higher on the plot than a dot
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`corresponding to a cell that expresses less Surface Molecule #2. See, e.g., id.
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`38.
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`In the figure below, Cell #1 has high expression of both Surface
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`Molecules #1 and #2 because its corresponding dot is high on the plot, and far to the
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`right. Cell #2, on the other hand, expresses those molecules only at a low level
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`because its dot is lower on the plot, and far to the left:
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`
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`See, e.g., id.
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`39. A typical flow cytometry plot of a population of cells may have
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`thousands to hundreds of thousands of dots. This provides a visualization of the
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`population’s expression of the two surface molecules in question. See, e.g., id.
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`40. The following hypothetical plot shows a population in which most of
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`the cells either express both Surface Molecules #1 and #2 at a high level (the group
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`
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`of dots at the upper-right of the plot), or express only Surface Molecule #1 (the group
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`of dots at the lower-right of the plot):
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`
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`See, e.g., id.
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`41. Flow cytometers allow a user to visualize a cell population’s plot and
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`draw a boundary, using specific software, around a subpopulation of cells, as shown
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`below:
`
`See, e.g., id.
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`42. The user can instruct the software to isolate the cells within the drawn
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`boundary for further analysis, a process called “gating.” Further analysis can include
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`additional analysis of other surface molecules (here, Surface Molecule #3 and
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`Surface Molecule #4), as shown below:
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`
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`See, e.g., id.
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`43.
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`In this example, the large arrow from left to right indicates that the plot
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`on the right is a further analysis of the selected (boxed) group of cells on the left. A
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`majority of cells that express high levels of both Surface Molecules #1 and #2 (the
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`group of dots on the upper-right of the left-hand plot) also express a high level of
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`Surface Molecule #3, but only a low level of Surface Molecule #4 (the group of dots
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`on the lower-right of the right-hand plot). See, e.g., id.
`
`44. Overall, a flow cytometer allows for visualization and measurement of
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`expression of multiple surface molecules-of-interest in a population of cells on one
`
`or more dot plots. Those cells can be sorted into subpopulations as needed. See, e.g.,
`15
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`id. A specific application of a flow cytometer is fluorescence-activated cell sorting
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`(FACS), which allows the operator to physically isolate cells, a process known as
`
`“sorting,” using fluorescent tags that bind to specific markers on cell surfaces. See,
`
`e.g., Ex. 1003, 2963, 2966; Ex. 1027, 3-5, 12-15; Ex. 1018, [0104], [0109];
`
`Ex. 1015, 3361, 3363; Ex. 1014, 1099; Ex. 1020, 61-62; Ex. 1004, 788-89; Ex.
`
`1001, 10:16-24. As an example of using FACS to isolate a specific T cell population,
`
`a scientist would combine the T cell population with a fluorescent reagent that is
`
`capable of binding specifically to CD62L, as in the example for flow cytometry.
`
`Processing and gating is identical. However, in the case of FACS, T cells with
`
`specific fluorescent properties (e.g., those that bound fluorescent antibodies directed
`
`against CD62L) can be directed to a collection tube, which separates them from the
`
`T cells that do not express CD62L. See, e.g., id. An alternate strategy to isolate cells
`
`based on marker expression is magnetic-activated cell sorting (MACS). MACS also
`
`allows the operator to physically isolate cells, but instead of using fluorescent
`
`reagents to identify T cell markers, the operator employs magnetic tags that bind to
`
`specific markers on cell surfaces. Applying a magnetic field causes magnetically
`
`tagged cells to be separated from the overall population. As an example of using
`
`MACS to isolate a specific T cell population, a scientist would combine the T cell
`
`population with a magnetic reagent that is capable of binding specifically to CD62L.
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`Rather than selecting cells through gating using software, as in the case of
`
`
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`flowcytometry, the operator places the T cell mixture in a magnetic field which will
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`retain all the cells that bound the magnetic reagent (ie. CD62L-expressing cells). By
`
`retaining the cells in the magnetic field, non-magnetic impurities (ie. cells that do
`
`not express CD62L) can be washed away. The remaining T cells can be removed
`
`from the magnetic field to permit recovery of the desired population.
`
`E. Adoptive Immunotherapy
`45. By the 2000s, researchers were developing therapeutic compositions
`
`containing T cells genetically m