DocCode — SCORE
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`SCORE Placeholder Sheetfor IFW Content
`
`Application Number: 14997136
`
`Document Date: 01/15/2016
`
`The presence ofthis form in the IFW record indicates that the following documenttype wasreceived
`in electronic format on the date identified above. This content is stored in the SCORE database.
`
`Since this was an electronic submission, there is no physical artifact folder, no artifact folder is
`recorded in PALM, and no paper documents or physical media exist. The TIFF images in the IFW
`record were created from the original documents that are stored in SCORE.
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`Drawing
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`e External customers may access SCORE content via PAIR using the Supplemental Content
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`Form Revision Date: August 26, 2013
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`DocCode — SEQ. TXT
`
`SCORE Placeholder Sheetfor IFW Content
`
`Application Number: 14997136
`
`Document Date: 01/15/2016
`
`The presence ofthis form in the IFW record indicates that the following documenttype wasreceived
`in electronic format on the date identified above. This content is stored in the SCORE database.
`
`Since this was an electronic submission, there is no physical artifact folder, no artifact folder is
`recorded in PALM, and no paper documents or physical media exist. The TIFF images in the IFW
`record were created from the original documents that are stored in SCORE.
`
`SequenceListing
`
`At the time of documententry (noted above):
`e USPTO employees may access SCORE content via eDAN using the Supplemental Content
`tab, or via the SCORE webpage.
`e External customers may access SCORE content via PAIR using the Supplemental Content
`tab.
`
`Form Revision Date: August 26, 2013
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`Preliminary Amendment
`U.S. Patent Application No. To be assigned
`Attorney Docket No. 046483-6001US13(01088)
`
`Electronically Filed
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`In re Applicationof:
`Carl H. June, etal.
`
`Group Art Unit: To be assigned
`
`Application No.: To be assigned
`
`Examiner: To be assigned
`
`Filed: Herewith
`
`Attorney Docket No.
`046483-6001US13 (01088)
`
`Title: Compositions and Methods for Treatment of Cancer
`
`PRELIMINARY AMENDMENT
`
`Prior to examination on the merits, kindly amend the above-identified
`
`application without prejudice, as follows. Please charge any applicable fees to deposit
`
`account number 50-4364.
`
`AMENDMENTTO THE SPECIFICATIONbeginson page 2.
`
`AMENDMENTTO THE CLAIMSbeginson page 3.
`
`REMARKSbegin on page7.
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`Preliminary Amendment
`U.S. Patent Application No. To be assigned
`Attorney Docket No. 046483-6001US13(01088)
`
`Amendmentto the Specification
`
`On page1, line 2, please replace the title of the invention with the followingtitle:
`
`--“COMPOSITIONS AND METHODS FOR TREATMENT OF CANCER”--
`
`On page 1, line 5 of the specification; please replace the paragraph with the
`
`following:
`
`-- CROSS-REFERENCE TO RELATED APPLICATION
`
`This application is a continuation of U.S. Patent Application No. 13/992,622,
`
`filed June 7, 2013, which is a U.S. national phase application filed under 35 U.S.C. § 371
`
`claiming benefit to International Patent Application No. PCT/US2011/064191, filed on
`
`December9, 2011, whichis entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional
`
`Patent Application No. 61/421,470, filed on December9, 2010 and U.S. Provisional Patent
`
`Application No. 61/502,649, filed on June 29, 2011, each of which application is hereby
`
`incorporated herein by reference inits entirety.
`
`STATEMENT REGARDING FEDERALLY SPONSORED
`
`RESEARCH OR DEVELOPMENT
`
`This invention was made with government support under grant numbers K24
`
`CA11787901, RO1CA120409, 1RO1CA105216, RO1AI057838 and RO11113482 awarded
`
`by the National Institutes of Health. The Government therefore has certain rights in the
`
`invention. --
`
`“2 -
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`Preliminary Amendment
`U.S. Patent Application No. To be assigned
`Attorney Docket No. 046483-6001US13(01088)
`
`Amendment to the Claims
`
`The listing of the claims will replace all prior versions, andlistings, of the
`
`claims in the application.
`
`1-89. (canceled)
`
`90. (New) A pharmaceutical composition comprising an anti-tumor effective amountof a
`
`population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding
`
`a chimeric antigen receptor (CAR), wherein the CAR comprises a CD19 antigen binding
`
`domain comprising, from the amino to the carboxy terminus, a light chain variable region
`
`and a heavy chain variable region, wherein the CAR further comprises a transmembrane
`
`domain, a 4-1BB costimulatory signaling region, and a CD3 zeta signaling domain.
`
`91. (New) The composition of claim 90, wherein the anti-tumor effective amount of T cells
`is 10* to 10” cells per kg body weight of a human in needofsuch cells.
`
`92. (New) The composition of claim 90, wherein the anti-tumor effective amount of T cells
`is 10° to 10° cells per kg body weight of a human in needofsuch cells.
`
`93. (New) The composition of claim 90, wherein said antigen binding fragmentis a scFv.
`
`94. (New) The composition of claim 93, wherein the scFv comprises the amino acid
`
`sequence of SEQ ID NO:20.
`
`95. (New) The composition of claim 90, wherein the transmembrane domain is CD8a,
`
`transmembrane domain.
`
`96. (New) The composition of claim 95, wherein the CD8a transmembrane domain
`
`-3-
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`Preliminary Amendment
`U.S. Patent Application No. To be assigned
`Attorney Docket No. 046483-6001US13(01088)
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`comprises the amino acid sequence of SEQ ID NO: 22.
`
`97. (New) The composition of claim 90, wherein the CAR further comprises a hinge
`
`domain.
`
`98. (New) The composition of claim 97, wherein the hinge domain is a CD8a hinge domain.
`
`99. (New) The composition of claim 98, wherein the CD80. hinge domain comprises the
`
`aminoacid sequence of SEQ ID NO:21.
`
`100. (New) The composition of claim 90, wherein the 4-1BB costimulatory signaling region
`
`comprises the amino acid sequence of SEQ ID NO:23.
`
`101. (New) The composition of claim 90, wherein the CD3 zeta signaling domain comprises
`
`the amino acid sequence of SEQ ID NO: 24.
`
`102. (New) The composition of claim 90, wherein the CD19 antigen binding domain is
`
`encoded by a nucleic acid sequence comprising SEQ ID NO: 14.
`
`103. (New) The composition of claim 95, wherein the CD8a transmembrane domainis
`
`encodedby a nucleic acid sequence comprising SEQ ID NO: 16.
`
`104. (New) The composition of claim 99, wherein the CD8a hinge domain is encoded by a
`
`nucleic acid sequence comprising SEQ ID NO: 15.
`
`105. (New) The composition of claim 100, wherein the 4-1BB costimulatory signaling
`
`region is encoded by a nucleic acid sequence comprising SEQ ID NO: 17.
`
`106. (New) The composition of claim 101, wherein the CD3 zeta signaling domain is
`
`-4-
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`Preliminary Amendment
`U.S. Patent Application No. To be assigned
`Attorney Docket No. 046483-6001US13(01088)
`
`encoded by a nucleic acid sequence comprising SEQ ID NO: 18.
`
`107 (New) The composition of claim 90, wherein the CAR comprises the amino acid
`
`sequence of SEQ ID NO:12.
`
`108. (New) The composition of claim 107, wherein the CAR is encodedby a nucleic acid
`
`sequence comprising SEQ ID NO:8.
`
`109 (New) The composition of claim 90, wherein the CAR further comprises a CD28
`
`costimulatory signaling region.
`
`110. (New) The composition of claim 90, wherein the T cells are T cells of a human having
`
`a Cancer.
`
`111. (New) The composition of claim 110, wherein the cancer is a hematological cancer.
`
`112. (New) The composition of claim 90, wherein the T cells comprise a vectorthat
`
`comprises the nucleic acid sequence.
`
`113. (New) The composition of claim 112, wherein the vector is a lentiviral vector.
`
`114. (New) The composition of claim 112, wherein the vector further comprises a promoter.
`
`115. (New) The composition of claim 114, wherein the promoter is an EF-1a promoter.
`
`116. (New) The composition of claim 90, wherein the pharmaceutical composition further
`
`comprises a pharmaceutically acceptable carrier, diluent or excipient.
`
`117. (New) The composition of claim 90, wherein the pharmaceutical composition
`
`-5-
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`Preliminary Amendment
`U.S. Patent Application No. To be assigned
`Attorney Docket No. 046483-6001US13(01088)
`
`comprises a buffer.
`
`118. (New) The composition of claim 117, wherein the buffer is neutral buffer saline or
`
`phosphate buffered saline.
`
`119. (New) The composition of claim 90, wherein the pharmaceutical composition further
`
`comprises a carbohydrate.
`
`-6-
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`Preliminary Amendment
`U.S. Patent Application No. To be assigned
`Attorney Docket No. 046483-6001US13(01088)
`
`REMARKS
`
`The present application is a continuation of U.S. Patent Application No.
`
`13/992,622,filed June 7, 2013, a national phase application of PCT/US2011/064191, filed on
`
`December9, 2011, whichis entitled to priority to U.S. Provisional Patent Application No.
`
`61/421,470, filed on December9, 2010 and U.S. Provisional Patent Application No.
`
`61/502,649,filed on June 29, 2011.
`
`Amendmentto the Specification
`
`The specification is amended herein to properly reflect the priority
`
`information of the present application.
`
`The specification is also amendedherein to provide the statement regarding
`
`federally sponsored research or development.
`
`No new matteris introduced by way of these amendmentsto the specification.
`
`Amendmentto the Claims
`
`Claims 1-89 have been canceled herein and new claims 90-118 have been
`
`added. Referring to US 2013/0287748, the publication of parent U.S. Patent Application No.
`
`13/992,622, support for new claims 90-118 is found in the application as follows:
`
`Claim 90 - support for the recited cells comprising the recited CAR is found
`
`throughout the specification, in the Examples and in SEQ ID NO:20.
`
`Claims 91-92 - Support for “anti-tumor effective amount” is found throughout
`
`the specification and for example, in paragraph [0224].
`
`Claim 93 - Support for “CD19 antigen binding fragmentis an scFV” is found
`
`throughoutthe specification and for example, in paragraph [0135].
`
`Claims 94 and 102 — Support for “SEQ ID NO: 20” and “SEQ ID NO: 14”is
`
`found throughoutthe specification and for example, in paragraph [0135] and in Table 5.
`
`-7-
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`Preliminary Amendment
`U.S. Patent Application No. To be assigned
`Attorney Docket No. 046483-6001US13(01088)
`
`Claims 95, 96 and 103 — Support for “CD8qa transmembrane domain”and
`
`“SEQ ID NO: 22” and “SEQ ID NO: 16”is found throughout the specification and for
`
`example, in paragraph [0139] and in Table 5.
`
`Claims 97, 98, 99 and 104 — Support for “hinge domain,” “CD8«a hinge
`
`domain,” “SEQ ID NO: 21” and “SEQ ID NO: 15”is found throughoutthe specification and
`
`for example, in paragraph [0140] and in Table 5.
`
`Claims 100 and 101 - Support for “SEQ ID NO: 23” and “SEQ ID NO: 24”is
`
`found throughoutthe specification and for example, in paragraph [0151] and in Table 5.
`
`Claims 105 and 106 - Support for “SEQ ID NO: 17” and “SEQ ID NO: 18”is
`
`found throughoutthe specification and for example, in paragraph [0150] and in Table 5.
`
`Claims 107 and 108 - Support for “SEQ ID NO: 12” and “SEQ ID NO: 8”is
`
`found throughoutthe specification and for example, in paragraph [0154] and in Table 5.
`
`Claim 109 — Support for “wherein the CAR further comprises a CD28
`
`costimulatory signaling region is found in paragraphs [0055] and [0149].
`
`Claim 110 - Support for “wherein the T cells are T cells of a human having a
`
`cancer” is found throughout the Examples.
`
`Claim 111 - Support for the “hematological cancer” is found throughout the
`
`specification and for example, in paragraphs [0200] and [0201].
`
`Claims 112 and 113- Support for “wherein the T cells comprise a vector” and
`
`“the vector is a lentiviral vector” is found throughout the specification and at least in Figure
`
`1.
`
`Claims 114 and 115 — Support for a promoter and an EF-1a promoter is found
`
`throughout the specification and at least in Figure 1.
`
`Claims 116, 117, 118 and 119— Support for a pharmaceutically acceptable
`
`carrier, diluent or excipient, buffer and carbohydrate is found at least in paragraph [0222].
`
`No new matter is added by wayof the addition of these claims.
`
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`Preliminary Amendment
`U.S. Patent Application No. To be assigned
`Attorney Docket No. 046483-6001US13(01088)
`
`Summary
`
`Applicants respectfully submit that the pending claims are fully supported in
`
`the specification as filed, and that no new matter has been added by wayofthe present
`
`Preliminary Amendment.
`
`Favorable examination and allowanceof the claims is hereby requested.
`
`January
`
`wv
`15, 2016
`Date
`
`Respectfully submitted,
`
`CARL H. JUNE, ET AL.
`
`s
`“ ?
`; a ar Oi é
`
`LLGLEG
`
`By:
`fp
`foo
`f
`Kathryn Doyle. Ph.D., JD.
`Registration No. 36,317
`SAUL EWING, LLP
`Centre Square West
`1500 MarketStreet, 38th Floor
`Philadelphia, PA 19102
`Phone: 215.972.7734
`Fax: 215.972.1818
`Email: kdoyle@saul.com
`Attorneyfor Applicant
`
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`Attorney Decket Ne. 046483-6001US 13(81088)
`
`USE OF CHIMERIC ANTIGEN RECEPTOR-MODIFIED T CELLS TO TREAT
`
`TITLE OF THE INVENTION
`
`CANCER
`
`rt
`
`CROSS-REFERENCE TO RELATED APPLICATION
`
`This application claims priority to U.S. Provisional Application No.
`
`61/421,470, filed December 9, 2010, and U.S. Provisional Application No.
`
`61/502,649, filed June 29, 2011, all of which are hereby incorporated herein by
`
`reference in their entireties.
`
`BACKGROUNDOF THE INVENTION
`
`The large majority of patients having B-cell malignancies, including
`
`chronic lymphocytic leukemia (CLL), will die fromtheir disease. One approach to
`
`treating these patients is to genetically modify T cells to target antigens expressed on
`
`tumor cells through the expression of chimeric antigen receptors (CARs}. CARsare
`
`antigen receptors that are designed to recognize cell surface antigens in a human
`
`leukocyie antigen-independent manner. Attempts in using genetically modified cells
`
`expressing CARsto treat these types of patients have met with very limited success.
`
`See for example, Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668: Morgan et
`
`20
`
`al., 2010, Molecular Therapy, published online February 23, 2010, pages 1-9; and,
`
`Tull et al., 2008, Blood, 112:2261-2271.
`
`In most cancers, tumor-specific antigens are not yet well defined, but
`
`in B cell malignancies, CD19 is an attractive tumortarget. Expression of CD19 ts
`
`restricted to normal and malignant B cells (Uckun, et al. Blood, 1988, 71:13-29), so
`
`that CD19is a widely accepted target to safely test CARs. While CARs can trigger
`
`T-cell activation in a manner similar to an endogenous T-cell receptor, a major
`
`impediment to the clinical application of this technologyto date has been limited in
`
`vivo expansion of CAR+T cells, rapid disappearance of the cells after infusion, and
`
`disappointing clinical activity Gena, et al., Blood, 2010, 116:1035-1044; Uckun,et al.
`
`30
`
`Blood, 1988, 71:13-29).
`
`Thus, there is an urgent need in the art for compositions and methods
`
`for treatment of cancer using CARs that can expand in vivo. The present invention
`
`addresses this need.
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`SUMMARY OF THE INVENTION
`
`The present invention provides an isolated nucleic acid sequence
`
`encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen
`
`binding domain, a transmembrane domain, a costimulatory signaling region, anda
`
`rt
`
`CD3 zeta signaling domain, wherein the CD3 zeta signaling domain comprises the
`
`amino acid sequence of SEQ ID NO: 24.
`
`In one embodiment, the nucleic acid sequence encodes a CAR
`
`cornprising the amino acid sequence of SEQ ID NO: 12.
`
`In one embodiment, the nucleic acid sequence encoding a CAR
`
`comprises the nucleic acid sequence of SEQ ID NO: 8.
`
`In one embodiment, the antigen binding domain in the CAR is an
`
`antibodyor an antigen-binding fragment thereof. Preferably, the antigen-binding
`
`fragment is a Fab or a scFv.
`
`In one embodiment, the antigen binding domainin the CAR binds to a
`
`tumor antigen.
`
`In one embodiment, the tumor antigen is associated with a
`
`hematologic malignancy.
`
`In another embodiment, the tumor antigen is associated
`
`with a solid tumor.
`
`In yet another embodiment, the tumorantigenis selected from the
`
`group consisting of CD19, CD20, CD22, ROR1, mesothelin, CD33/EL3Ra, c-Met,
`
`PSMA, Glycolipid F77, EGFRvIL, GD-2, NY-ESO-1 TCR, MAGE A3 TCR,and any
`
`20
`
`combination thereof.
`
`In one embodiment, the costimulatory signaling regionin the CAR
`
`comprises the intracellular domain of a costimulatory molecule selected from the
`
`group consisting of CD27, CD28, 4-IBB, OX40, CD30, CD40, PB-1, ICOS,
`
`tymphocyte function-associated antigen-f (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-
`
`H3, a ligandthat specifically binds with CD83, and any combination thereof.
`
`in one embodiment, the CD3 zeta signaling domain in the CARis
`
`encoded by the nucleic acid sequence of SEQ ED NO: 18.
`
`The invention also provides an isolated CAR compnsing an antigen
`
`binding domain, a transmembrane domain, a costimulatory signaling region, and a
`
`30
`
`CD3 zeta signaling domain, wherein the CD3 zeta signaling domain comprises the
`
`amino acid sequence of SEQ ID NO: 24.
`
`The invention also provides a cell comprising a nucleic acid sequence
`
`encoding a CAR, wherein the CAR comprises an antigen binding domain, a
`
`bo
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`transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling
`
`domain comprising the amino acid sequence of SEQ ID NO: 24.
`
`In one embodiment, the cell comprising the CAR is selected from the
`
`group consisting of a Tcell, a Natural Killer (VI) cell, a cvtotoxic T lymphocyte
`
`rt
`
`(CTL), and a regulatory T cell.
`
`In one embodiment, the cell comprising the CAR exhibits an antt-
`
`turnor immunity when the antigen binding domain of the CAR bindsto its
`
`corresponding antigen.
`
`The invention also provides a vector comprising a nucleic acid
`
`10
`
`sequence encoding a CAR, wherein the CAR comprises an antigen binding domain, a
`
`costirnulatory signaling region, and a CD3 zeta signaling domain, wherein the CD3
`
`zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 24.
`
`The invention also provides a method for stimulating a T cell-mediated
`
`immune response to a target cell population or tissue ina mammal.
`
`In one
`
`embodiment, the method comprises admunistering to a mammal an effective amount
`
`of a cell genetically modified to express a CAR wherein the CAR comprises an
`
`antigen binding domain, a costimulatory signaling region, and a CD3 zetasignaling
`
`domain comprising the amino acid sequence of SEQ 1D NO: 24, wherein the antigen
`
`binding domain is selected to specifically recognize the target cell population or
`
`20
`
`tissue,
`
`The invention also provides a method of providing an anti-tumor
`
`immunity ina mammal.
`
`In one embodiment, the method comprises administering to
`
`amamnnal an effective amount of a cell genetically modified to express a CAR
`
`wherein the CAR comprises an antigen binding domain, a costimulatory signaling
`
`region, and a CD3 zeta signaling domain comprising the amino acid sequence of SEQ
`
`ID NO: 24, thereby providing an anti-tumor immunity in the mammal.
`
`The invention also includes a method of treating a mammal having a
`
`disease, disorder or condition associated with an elevated expression of a tumor
`
`antigen.
`
`In one embodiment, the method comprises admunistering to a marnmal an
`
`30
`
`effective amount of a cell genetically modified to express a CAR wherein ihe CAR
`
`comprises an antigen binding domaim, a costimulatory signaling region, and a CD3
`
`zetasignaling domain compnising the amine acid sequence of SEQ TD NO: 24,
`
`therebytreating the mammal.
`
`In one embodiment, the cell is an autologous T cell.
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`In one embodiment, the tumor antigen is selected from the group
`
`consisting of CD19, CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met, PSMA,
`
`Glycohpid F77, EGFRvHL GD-2, NY-ESO-1 TCR, MAGE A3 TCR, and any
`
`combination thereof.
`
`rt
`
`The invention also provides a method of treating a human with chronic
`
`lymphocytic leukemia.
`
`In one embodiment, the method comprises administering to a
`
`human a Tcell genetically engineered to express a CAR wherein the CAR comprises
`
`an antigen binding domain, a costimulatory signaling region, and a CD3 zeta
`
`signaling domain comprising the amino acid sequence of SEQ [DB NO: 24.
`
`In one embodiment, the human is resistant to at least one
`
`chemotherapeutic agent
`
`in one embodiment, the chronic lymphocytic leukemiais refractory
`
`CD19+leukemia and lvmphoma.,
`
`The invention also includes a methodof generating a persisting
`
`population of genetically engineered T cells in a human diagnosed with cancer.
`
`In
`
`one embodiment, the method comprises administering to a human a T cell genetically
`
`engineered to express a CAR wherem the CAR comprises an antigen binding domain,
`
`a costimulatory signaling region, and a CD3 zeta signaling domain comprising the
`
`amino acid sequence of SEQ ID NO: 24, wherein the persisting population of
`
`20
`
`genetically engineered T cells persists in the human for at least one month after
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`adroinistration.
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`In one embodiment, the persisting population of genetically engmeered
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`T cells comprises at least one cell selected frormthe group consisting of a T cell that
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`was administered to the human, a progeny of a T cell that was administered to the
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`human, and a combination thereof.
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`In one embodiment, the persisting population of genetically engineered
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`T cells comprises a memory T cell,
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`In one embodiment, the persisting population of genetically engineered
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`T cells persists in the human for at least three months after administration.
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`In another
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`30
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`embodiment, the persisting population of genetically engineered T cells persists in the
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`human for at least four months, five months, six months, seven months, eight months,
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`nine months, ten months, eleven months, twelve months, two years, or three years
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`after administration.
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`In one embodiment, the chronic lymphocytic leukermiais treated.
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`The invention also provides a method of expanding a population of
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`genetically engineered T cells in a human diagnosed with cancer.
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`In one
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`embodiment, the method comprises administering to a human a T cell genetically
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`engineered to express a CAR wherein the CAR comprises an antigen binding domain,
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`rt
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`a costimulatory signaling region, and a CD3 zeta signaling domain comprising the
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`amino acid sequence of SEQ [D NO: 24, wherein the administered genetically
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`engineered Tcell produces a population of progeny T cells in the human.
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`In one embodiment, the progeny T cells in the human comprise a
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`memory T cell.
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`10
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`In one embodiment, the T cell is an autologous T cell.
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`In another embodiment, the human 1s resistant to at least one
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`chemotherapeutic agent.
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`In one embodiment, the canceris chronic lymphocytic leukemia. fn
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`another embodiment, the chronic lymphocytic leukemia is refractory CD19+ leukemia
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`and lymphoma.
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`In one embodiment, the population of progeny T cells persists in the
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`human for at least three months after administration. In another embodiment, the
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`population of progeny Tcells persist in the human for at least four months, five
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`rnonths, six months, seven months, eight months, nine months, ten months, eleven
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`20
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`months, twelve months, two years, or three years after administration.
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`In one embodiment, the canceris treated.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`The following detailed description of preferred embodiments of the
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`mvention will be better understood when read in conjunction wrth the appended
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`drawings. For the purpose of illustrating the invention, there are shown in the
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`drawings embodiments which are presently preferred.
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`[t should be understood,
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`however, that the invention is not limited to the precise arrangements and
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`instrumentalities of the embodiments shown in the drawings.
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`30
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`Figure 1, comprising Figures 1A through IC, is a series of images of
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`the schematic representations of the sene-transfer vector and transgene, gene
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`modified T cell manufacturing and clinical protocol design. Figure LA depicts the
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`lentiviral vectors and transgene that showthe major functional elements. A vesicular
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`stomatitis virus protein G pseudotyped clinical grade lentiviral vector (designated
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`pELPs 19BBz) directing expression of anti-CD19 scFvderived from FMC63 murine
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`monoclonal antibody, human CD8a hinge and transmembrane domain, and human 4-
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`iBB and CD3zeta signaling domains was produced. Constitutive expression ofthe
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`transgene was directed by inclusion of an EF-1a (clongationfactor-la promoter);
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`rt
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`LTR, long terminal repeat, RRE, rev response element. (cPPT) and the central
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`termination sequence (CTS). Figure is not to scale. Figure 1B depicts T cell
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`manufacturing. Autologous cells were obtained via an apheresis, and T cells were
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`enriched by mononuclearcell elutriation, washed and residual leukemia cells depleted
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`by addition of anti-CD3/CD28 coated paramagnetic beads for positive selection and
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`10
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`activation of T cells. Lentiviral vector was added at the time of cell activation and
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`was washed out on day 3 post culture initiation. Cells were expanded on a rocking
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`platform device (WAVE Bioreactor System) for 8-12 days. On the final day of
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`culture the beads were removed by passage over a magnetic field and the CART19 T
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`cells harvested and cryopreserved in infusible medium. Figure 1C depicts the clinical
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`protocol design. Patients were given lymphodepleting chemotherapyas described,
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`followed by CART19 infusion #1 by iv. gravity flow drip over a period of 15-20
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`minutes. The infusion was given using a split dose approach over 3 days (10%, 30%,
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`60%) beginning | to 5 days after cornpletion of chernotherapy. Endpoint assays were
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`conducted on study week 4. At the conclusion of active monitoring, subjects were
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`20
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`transferred to a destination protocol for long term follow up as per FDA guidance.
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`Figure 2, cornprising Figures 2A through 2F, is a series of images
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`demonstrating sustained in vivo expansion and persistence in blood and marrowof
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`CARTI9 cells. DNA isolated from whole blood as depicted in Figure 2A through 2C
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`or marrowas depicted in Figure 2D through 2F, samples obtained from UPN 01 as
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`depicted in Figure 2A and 2D, UPN02 as depictedin Figure 2B and 2E and UPN 03
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`as depicted in Figure 2C and 2F was subjected in bulk to Q-PCR analysis using a
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`qualified assay to detect and quantify CARTI° sequences. Each data point represents
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`the average of triphcate measurements on 100-200 ng genomic DNA, with maximal
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`% CVless than 1.56%. Pass/fail parameters for the assay included pre-established
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`30
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`ranges for slope and efficiency of amplification, and amplification of a reference
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`sample. The lower limit of quantification for the assay established by the standard
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`curve range was 2 copies transgene/microgram genomic DNA: sample values below
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`that numberare considered estimates and presented if at least 2/3 replicates generated
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`a Ct value with %CVfor the values 15%. CART19 ceils were infused at day 0, 1, and
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`6
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`2 for UPN O01 and UPN 03, and days 0, 1, 2 and 11 for UPN 02.
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`Figure 3, comprising Figures 3A through 3D, ts a series of images
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`demonstrating serum and bone marrowcytokines before and after CAR T cell
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`mfusion; longitudinal measurements of changes in serurn cytokimes, chemokines and
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`rt
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`cytokine receptors in UPN 01 as depicted in Figure 3A, UPN 02 as depicted in Figure
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`3B and UPN 03 as depicted in Figure 3C, on the indicated day after CART19 ceil
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`infusion andserial assessments of the same analytes in the bone marrow from UPN03
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`as depicted in Figure 3D. Samples were subjected multiplex analysis using Luminex
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`bead array technology and pre-assembled and validated multiplex kits. Analytes with
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`a >=3 fold change are indicated, and plotted as relative change from baseline as
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`depicted in Figure 3A through 3C or as absolute values as depicted in Figure 3D.
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`Absolute values for each analyte at each time-point were derived from a recombinant
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`protein-based standard curve over a 3-fold 8-pomt dilation series, with upper and
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`lower limits of quantification (ULOQ, LLOQ) determined by the 80-120%
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`observed/expected cutoff values for the standard curves. Each sample was evaluated
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`in duplicate with average values calculated and %CVin most cases less than 10%.
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`To accommodate consolidated data presentation in the context of the wide range for
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`the absolute values, data are presented as fold-changeover the baseline value for each
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`analyte.
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`In cases where baseline values were not detectable, half of the lowest
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`20
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`standard curve value was used as the baseline value. Standard curve ranges for
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`analytes and baseline (day 0) values (isted in parentheses sequentially for UPNOL, 02
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`and 03), all in pg/ml: ILi-Ra: 35.5-29,318 (689, 301, 287); IL-6: 2.7-4,872 (7, 10.1,
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`8.7); IFN-y: 11.2-23,972 (2.8, ND, 4.2 ); CKCLIO: 2.1-5,319 (481, 115, 287 ); MIP-
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`1B: 3.3-7,233 (99.7, 371, 174}; MCP-L: 4.8-3.600 (403, 560, 828); CXCL9: 48.2-
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`3,700 C412, £26, 177); TL2-Ro: 13.4-34,210 (4,319, 9.477, 610); IL-8: 2.4-5.278
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`(15.3, 14.5, 14.6); IL-10: 6.7-13,874 (8.5, 5.4, 0.7); MIP-Lo: 7.1-13,778 (87.6, 57.3,
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`48.1).
`
`Figure 4, comprising Figures 4A through 4D,is a series of images
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`depicting prolonged surface CART19 expression and establishment of functional
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`30
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`memory CARs in vivo. Figure 4A depicts detection of CAR-expressing CD3+
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`lymphocytes and absence of B cells in penphery and marrow. Freshly processed
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`peripheral blood or marrow mononuclear cells obtained from UPN 03 at day 169
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`post-CART19 cell infusion were evaluated by flow-cytometry for surface expression
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`of CAR19 Gop) or presence of B ceils (bottom); as a control, PBMC obtained froma
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`healthy donor ND365 were stained. The gating strategy for the CD3~+ and B cell
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`populations is presented in Figure 9. To evaluate CARI9 expression in CD3+
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`lymphocytes, samples were co-stained with antibodies to CD14-PE-Cy7 and CD16-
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`PE-Cy7 (dump channel) and CD3-FITC, positively gated on CD3+, and evaluated for
`
`rt
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`CAR19 expression in the CD8+ and CD8-lymphocyte compartments by co-staining
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`with CD8a-PE and the anti-CAR19 icdiotvpe antibody conjugated to Alexa-647. Data
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`in plots are gated on the dump channel-negative/CD3-positive cell population. To
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`evaluate the presence of B cells. samples were co-stained with antibodies to CD14-
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`APC and CD3-FITC (dump channels) and evaluated for the presence of B cells in the
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`dump channel-negative fraction by co-staining with antibodies to CD20-PE and
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`CD19-PE-Cy-7.
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`In all cases, negative gate quadrants were established on no-stain
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`controls as depicted in Figures 4B and 4C. T cell immunophenotyping of CD4+
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`(Figure 4B) and CD8+ (Figure 4C) TF cell subsets is shown. Frozen peripheral blood
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`samples from UPN03 obtained by apheresis at dav 56 and 169 post T cell infusion
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`were rested overnight m culture mediumwith no added factors, washed, and