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`
`CERTIFICATION
`
`It is hereby certified that the attached copy is a true copy of the record copy of
`International Application No. PCT/US2012/030388 filed with the United States Patent
`and Trademark Office as receiving Office on 23 March 2012 and received by the
`International Bureau on 26 May 2012.
`
`By: The International Bureau
`
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`
`Miltenyi Ex. 1005 Page 1
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`

`

`For receiving Office use only
`
`
`PCT/US12/30388
`
`International Application No.
`
`
`23 MAR 2012 (23.03.12)
`International Filing Date
`
`
`The undersigned requests that the present
`haONTERNATIONAL APPLICATION
`international application be processed
`
`according to the Patent Cooperation Treaty.
`
`
`
`Nameof receiving
`Applicant's or agent's file reference
`if desired) (12 characters maximum) 15950.11WOU]
`
`PCT
`
`REQUEST
`
`é
`
`Office and "PCT International Application"
`
`
`
`BoxNo.I
`
`TITLE OF INVENTION
`
`
`
`METHOD AND COMPOSITIONS FOR CELLULAR IMMUNOTHERAPY
`
`Nameand address:
`
`
`
`Box No.1] APPLICANT[| This person is also inventor
`
`
`Telephone No.
` (Family namefollowed by given name; for a legal entity, full official designation. The
`address mustinclude postal code and nameofcountry. The country ofthe address
`
`
`indicatedin this Boxis the applicant's State (thatis, country) ofresidence ifno State of
`
`residence is indicated below.)
` Facsimile No.
`
`
`
`
`FRED HUTCHINSON CANCER RESEARCH CENTER
`
`
`1100 Fairview Avenue North
` Applicant's Registration No. with Office
`
`Mailstop: J6-200
`
`Seattle, Washington 98109-1024
`
`United States of America
`
`
`
`
`
`
`State (that is, country) of nationality:
`State (that is, country) of residence:
`US
`US
`Thisperson isapplicant [| all designated x alldesignated States except [| the United States [| the States indicated in
`for the purposesof:
`States
`Z\|
`the United States ofAmerica
`ofAmerica only
`the Supplemental Box
`
`
`
`
`Box No. II] FURTHER APPLICAN1(S) AND/OR (FURTHER) INVENTOR(S)
`<I
`Further applicants and/or (further) inventors are indicated on a continuation sheet.
`Box No. IV AGENT OR COMMON REPRESENTATIVE; OR ADDRESS FOR CORRESPONDENCE
`
`
`
`Theperson identified below is hereby/has been appointed to act on behalf
`| agent
`commonrepresentative
`
`~Z™
`ofthe applicant(s) before the competent International Authorities as:
`
`Telephone No.
`Nameand address:
`(Family namefollowed by given name;for a legal entity, full official designation. The
`
`
`
`
`er612/ 336-4621
`address musi includepostal code andname ofcountry.)
`
`
`Facsimile No.
`
`
`(612) 336-4751
`DEMASTER,Eric E.
`
`
`Merchant & Gould P.C.
`
`
`P.O. Box 2903
` Agent's Registration No. with Office
`
`55,107
`Minneapolis, Minnesota 55402-0903
`
`
`United States of America
`
`
`Address for correspondence: Markthis check-box where no agent or common representative is/has been appointed and the
`
`
`space above is used instead to indicate a special address to which correspondence should be sent.
`Form PCT/RO/101(first sheet) (January 2007)
`See Notes to the requestform
`
`
`
`
`
`Miltenyi Ex. 1005 Page 2
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`

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`Sheet No.
`
`.......2
`
`FURTHER APPLICANTS AND/OR (FURTHER) INVENTORS
`
`State (thatis, country) of residence:
`US
`
`Applicant's registration No. with Office
`
`
`
`
`If none ofthefollowing sub-boxes is used, this sheet is not to be included in the request.
`
`Nameand address
`(Family namefollowed by given name; for a legal entity, full official designation. The address
`
`
`This person is:
`must include postal code and nameofcountry. The country ofthe address indicated in this Box
`
`is the applicant's State (thatis, couniry) ofresidenceifno State ofresidenceis indicated below.)
`
`
`[| applicant only
`
`
`RIDDELL,Stanley R.
`xX applicant and inventor
`1763 268th Place SE
`
`
`
`
`Sammamish, Washington 98075
`
`[| inventoronly (Ifthis check-box is
`
`United States of America
`
`marked, do notfill in below.)
`
`
`
`State (that is, country) of nationality:
`CA
`
`
`
`
`[] all designated all designatedStates except DX theUnited States [| the States indicated in
`
`
`This person is applicant
`
`States
`the United States of America
`LCN
`ofAmerica only
`the Supplemental Box
`for the purposesof:
`
`
`
`
`Nameand address
`(Family namefollowed by given name;for a legalentity, full official designation. The address
`
`
`
`This personis:
`must include postal code and name ofcountry. The country ofthe address indicated in this Box
`
`
`
`
`is the applicant's State (that is, country) ofresidenceifno State ofresidenceis indicated below.)
`
`[] applicant only
`
`
`
`
`HUDECEK,Michael
`NZ
`DX]
`applicant
`and
`
`
`inventor
`“J applicant and
`3010 NE 117th Street
`
`Seattle, Washington 98125
`
`[| inventoronly (Ifthis check-box is
`
`United States of America
`
`
`marked, do notfill in below.)
` Applicant's registration No. with Office
`
` State (thatis, country) ofnationality:
`State (thatis, country) of residence:
`US
`
`
`DE
`
`
`
`
`This person is applicant[| the States indicated in
`[| all designated [| all designated States except Xx
`theUnited States
`
`
`the Supplemental Box
`States
`the United States ofAmerica
`of America only
`
`
`for the purposesof:
`
`Nameand address
`(Family namefollowed bygiven name;fora legal entity, fill official designation. The address
`
`
` This person is:
`mustincludepostal code andname ofcountry. The country ofthe address indicated in this Box
`
`is the applicant's State (that is, country) ofresidenceifno State ofresidenceis indicated below.)
`
`[| applicant only
`
`[| applicant and inventor
`[| inventor only (Ifthis check-box is
`marked, do notfill in below.)
`
`
`
`Applicant's registration No. with Office
`
` State (thatis, country) ofnationality. State (thatis, country) ofresidence:
`
`
`
`
`all designated
`the United States [| the States indicated in
`all designated States except [|
`
`
`
`L| States
`the Supplemental Box
`ofAmerica only
`the United States ofAmerica
`This person is applicant
`
`
`for the purposesof:
`
`
`(Family namefollowed by given name;‘fora legalentity, full official designation. The address
`Nameand address
`
`
`
`This personis:
`must include postal cade and nameofcountry. The country ofthe address indicated in this Box
`
`
`is the applicant's State(thatis, country) ofresidenceifno Siate ofresidenceis indicated below.)
`[| applicant only
`
`
`[ applicant and inventor
`[| inventor only (ifthis check-box is
`marked, do HO
`Hh beiow
` Applicant's registration No. with Office
`
`
`
`State (that is, country) of residence:
` State (that is, country) of nationality:
`
`
`all designated States except[| the United States [| the States indicated in
`
`for the purposesof:
`LJ
`
`the United States of America
`ofAmericaonly
`the Supplemental Box
`
`Further applicants and/or(further) inventors are indicated on another continuation sheet.
`
`
`
`Form PCT/RO/101 (continuation sheet) (April 2007)
`See Notes to the requestform
`
`
`
`This person is applicant
`
`all designated
`States
`
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`Sheet No.
`
`.......3
`
`Box Ne. V
`DESIGNATIONS
`Thefiling ofthis request constitutesunder Rule 4.9(a), the designation ofall Contacting States bound by the PCT on the internationalfiling date, for the
`grant ofevery kind ofprotection available and, where applicable, for the grant ofboth regional and national patents.
`However,
`
`(J DE Germanyis not designated for any kind ofnational protection
`(0 JP Japan is not designated for any kindofnational protection
`CIKRRepublic of Koreais not designated for any kind of national protection
`DO RU Russian Federation is not designated for any kind ofnational protection
`
`national application:
`country
`or Member ofWTO
`US
`
`Where earlier application is:
`regional application:*
`regional Office
`
`international application:
`receiving Office
`
`
`
`
` (The check-boxes above may be usedto exclude (irrevocably) the designations concernedin order io avoidthe ceasingofthe effect, under the national
`law,ofan earlier national applicationfrom which thepriority is claimed. See the Notes to Box No. Vas to the consequences ofsuch national law
`
`
`provisions in these and certain ofStates.)
`
`Box Ne. VI
`PRIORITY CLAIM
`Filing date
`
`Number
`ofearlier application
`
`
`ofearlier application
`(day/month/year,
`
`
`item (1)
`
`
`23 March 2011
`61/466,552
`
`23.03.2011
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CT Furtherpriority claims are indicated in the Supplemental Box.
`
`Transmit certified copy: the receiving Office is requested to prepare and transmit to the International Bureau a certified copy ofthe earlier application(s)
`
`
`(only ifearlier application was filed with the office which for the purposes ofthis international application is the receiving Office) identified above as:
`
`
`
`all items
`(item (1)
`(11 item (2)
`(item (3)
`(1 other, see Supplemental Box
`
`
`
`jority for the earlier application(s) identified above orin the
`
`Restore the right of priority: the receiving Office is requested to restore the right ofpri
`_). (See also the Notes to Box No. VI;further information
`
`
`Supplemental Box as item(s) (
`
`
`
`must be provided to support a request to restore the right ofpriority.)
`
`
` Incorporation by reference: where an elementofthe international application referred to in Article 11(1\(iii)(d) or (e) or a part ofthe description, claims
`or drawingsreferred to in Rule 20.5(a) is not otherwise contained in this international application but is completely contained in an earlier application
`
`whosepriority is claimed on the date on which one or more elements referredto in Article 1 1(1)(iii) werefirst received by the receiving Office, that
`
`
`
`elementorpart is, subject to confirmation under Rule 20.6, incorporated by reference in this international application for the purposes ofRule 20.6.
`
`
`
`
`
`INTERNATIONAL SEARCHING AUTHORITY
` Box No. VII
`Choice of International Searching Authority (ISA)
`(iftwo ormore InternationalSearchingAuthorities are competentto carryoutthe internationalsearch, indicate the Authority chosen; the two-lettercode
`
`may be used):
`ISA / EP
`Request to use results ofearlier search; referenceto that search(ifan earlier search has been carried out by or requested from the Intemational
`Searching Authority):
`Date (day/month/year):
`Number:
`Country (or regional Office):
`
`
`
`
`
`
`
`
`
`
`
`Box No. VIET.
`~=DECLARATIONS
`
`
`
`Numberof declarations
`The following declarations are contained in Boxes Nos. VIII (i) to (v) (mark applicable check-boxes below and indicate in
`
`
`
`
`
`
`the right column the number ofeach type ofdeclaration).
`(J Box No. VI(i)
`Declaration as to identity ofthe inventor
`1 Box No.VIII (ii)
`Declaration as to the applicant's entitlement,as to the internationalfiling date, to
`
`
`apply for and be granted a patent
`
`
`(1 Box No. VII (iii)
`Declaration as to the applicant's entitlement, as to the internationalfiling date, to
`claim the priority of the earlier application.
`
`
`1 Box No. VII (iv)
`Declaration ofinventorship (only for the purposes ofthe designations ofthe United
`States ofAmerica)
`
`
` C1 Box No. VIII (v)
`Declaration as to non-prejudicial disclosures or exceptions to lack ofnovelty
`
`
`‘See Notes to the requestform
`Form PCT/RO/101 (last sheet) (April 2007)
`
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`.......4
`Sheet No.
`
`
`Box No. 1X
`CHECK LIST; LANGUAGEOF FILING
`
`
`
`
`
`This international application contains:
`Numberofitems
`This international application is accompanied by the item(s) (mark the applicable
`check-boxes below and indicate in right column the numberofeach item):
`(a) on paper, the following numberofsheets:
`
`
`request (including declaration
`1.
`[1
`fee calculation sheet
`
`:4
`and Supplemental sheets)
`2.
`(1
`original separate power ofattomey
`
`
`description (excluding
`_.
`sequencelisting and/ortablesrelated thereto) : 46
`3.
`[1 original general powerofattorney
`claims
`: 6
`4.
`[1
`copy ofgeneral powerofattorney; reference number,ifany:
`
`
`abstract
`2
`5.
`(1
`statement explaininglack of signature
`
`
`
`745
`drawings
`6.
`[priority document(s) identified in Box No VI as item(s):
`Sub-total number ofsheets
`: 72
`:
`,
`.
`oe
`7.
`(1
`translation of international application into (language):
`_.
`sequencelisting
`tables related thereto
`8.
`[1
`separate indications concerning deposited microorganism or other
`biological material
`
`
`:
`(for both, actual numberofsheets iffiled in
`wg
`paperform, whetheror notalsofiled in computer readable
`sequencelisting in electronic form
`
`form; see (¢) below)
`(indicate type and number ofcarriers)
`
`Total numberof sheets
`+ 72
`
`
`[copy submitted for the purposes of international search under Rule
`
`13ter only (and notas part ofthe international application)
`(ii) 1 (only where check-box (b)(i) or (¢)(i) is marked in left column)
`(b) [7 onlyelectronic form
`additional copies including, where applicable, the copy for the
`(Section 801(a)(i))
`
`purposesof international search under Rule 13¢er
`
`
`
`(i) O sequencelisting
`[1 together with relevantstatementas to the identity of the copy or copies
`(ii) [7 tables related thereto
`
`with the sequencelisting mentioned in left column
`(c) CJ alsoin electronic form
`
`(Section 801 (a)(ii)
`
`
`
`tablesin electronic form related to sequencelisting
`(i) (0 sequencelisting
`(indicate type and numberofcarriers)
`
`(ii) D0 tables related thereto
`
`
`Type and numberofcarriers (diskette, CD-ROM,
`(copy submitted for the purposes of international search under Section
`
`
`
`CD-Ror other) on which are contained the
`802(b-quater) only (and notas part of the international application)
`
`
`
`(J sequencelisting:
`;
`
`
`(1 (only where check-box (b)(ti) or (c)(ii) is marked in left column)
`
`;
`C tables related thereto:
`additional copies including, where applicable, the copy for the
`(additional copies to be indicated underitems 9(ii)
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`purposesof international search under Section 802(b-quater)
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`and/or 10(ii), in right column)
`
`(iii) 1] together with relevant statementas to the identity of the copy or copies
`with the tables mentioned in left column
`
`11. 1__Other (specify):
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` Figure of the drawings which
`Language offiling of the
`should accompanythe abstract:
`international application: English
`
` Box No. X
`SIGNATURE OF APPLICANT OR AGENT OR COMMON REPRESENTATIVE
`
`Next to each signature, indicate the nameoftheperson signing andthe capacity in which the person signs (ifsuch capacity is not obviousfrom reading the request).
`
`
`
`
`
`
`
`
`
`
`
`
`
`9.
`
`[J
`()
`
`
`
`(iii)
`
`10.
`
`[1]
`
`(i)
`
`(i)
`
`
`hit
`
`
`DeMaster, Eric E.
`
` 7 For receiving Office use only
`
`
`1. Date of actual receipt of
`the purported
`
`
`2. Drawings:
`
`international ap lication: me
`23 MAR 2012 (23 03.12
`
`3. Corrected date of actualreceipt dueto later but
`
`
`
`[| Teceived:
`
`timely received papers or drawings completing
`
`
`the purported international application:
`
`L] not received:
`4.
`Dateoftimely receipt ofthe required
`
`
`
`corrections under PCT Article 11(2):
`
` Transmittal of search copy delayed until search fee is
`5.
`international Searching Authority
`
`
`(if two or more are competent):
`ISA/
`
`
`
`For International Bureau use only
` Date ofreceipt of the record copy
`
`by the International Bureau:
`
`
`See Notes to the requestform
`Form PCT/RO/I01 (last sheet) (April 2007)
`
`
`
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`

`METHOD AND COMPOSITIONS FOR CELLULAR IMMUNOTHERAPY
`This application is being filed on 23 March 2012, as a PCT International
`Patent application in the name of Fred Hutchinson Cancer Research Center, a U.S.
`national corporation, applicant for the designation ofall countries except the US.,
`and, Stanley R. Riddell, a citizen of Canada, and Michael Hudecek,a citizen of
`Germany, applicants for the designation of the U.S.only, and claimspriority to U.S.
`Patent Application Serial No. 61/466,552 filed on 23 March 2011, the disclosure of
`whichis incorporated herein by referencein its entirety.
`
`Field of the Invention
`The presentinvention relates to the field of biomedicine and specifically
`methodsuseful for cancer therapy. In particular, embodimentsofthe invention relate
`to methods and compositions for carrying out cellular immunotherapy.
`
`Statement Regarding Federally Sponsored Research
`This invention was made with government support in the form of grants
`RO1CA18029 from the United States Department of Health and HumanServices,
`NationalInstitute of Health and Leukemia and Lymphoma Society SCORE grant.
`The United States government hascertain rights in the invention.
`
`Backgroundof the Invention
`Studies in rodents have demonstrated that adoptive immunotherapy with
`antigen specific T cells is effective for cancer andinfections, and there is evidence
`this modality has therapeutic activity in humans'®.Forclinical applications,it is
`necessary to isolate T cells of a desired antigen specificity or to engineer T cells to
`expressreceptors that target infected or transformed cells, and then expand these
`cells in culture’'*. The transfer of T cell clonesis appealing because it enables
`control of specificity and function, andfacilitates evaluation of in vivo persistence,
`toxicity and efficacy. Additionally, in the setting of allogeneic stem cell
`transplantation, the administration to recipients ofT cell clones from the donorthat
`target pathogensor malignant cells can avoid graft-versus-host disease that occurs
`with infusion of unselected donor T cells?*!5, However,it is apparent from clinical
`studies that the efficacy of cultured T cells, particularly cloned CD8°T cells,is
`i
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`10
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`frequently limited by their failure to persist after adoptive transfer",
`The poolof lymphocytes from which T cells for adoptive immunotherapy
`can be derived contains naive and long-lived, antigen experienced memory T cells
`(Tm). Tw can be divided further into subsets of central memory (Tc) and effector
`memory (Tew) cells that differ in phenotype, homing properties and function'®,
`CD8* Tew express CD62L and CCR7atthe cell surface, which promote migration
`into lymph nodes, andproliferate rapidly if re-exposed to antigen. CD8* Tem lack
`cell surface CD62L andpreferentially migrate to peripheraltissues, and exhibit
`immediate effector function’’. In response to antigen stimulation, CD8* Tow and
`Tzu both differentiate into cytolytic effector T cells (Tg) that express a high level of
`granzymes and perforin, but are short-lived’. Thus, the poor survival of T cells in
`clinical immunotherapytrials may simply result from their differentiation during in
`vitro culture to Tg that are destined to die'’?!, There is a needto identify cell
`populations and methodsthat provide enhanced survival of adoptively transferred T
`cells in vivo.
`
`Summary of the Invention
`In one aspect, the present invention relates to methods and compositionsto
`confer and/or augment immune responses mediated by cellular immunotherapy,
`such as by adoptively transferring tumor-specific, subset specific genetically
`modified CD4+ T cells, wherein the CD4+ T cells confer and/or augmenttheability
`of CD8+ T cells to sustain anti-tumor reactivity and increase and/or maximize
`tumor-specific proliferation.
`In one embodiment, the present invention provides a method of performing
`cellular immunotherapy in a subject having a disease or disorder by administering to
`the subject a genetically modified cytotoxic T lymphocyte cell preparation that
`provides a cellular immuneresponse, wherein the cytotoxic T lymphocytecell
`preparation comprises CD8+ T cells that have a chimeric antigen receptor with an
`extracellular antibody variable domain specific for an antigen associated with the
`disease or disorder and anintracellular signaling domain of a T cell or other
`receptors, such as co-stimulatory domains; and a genetically modified helper T
`lymphocyte cell preparation that exhibits a predominant Th1 phenotype as well as
`produceother cytokines,elicits direct tumor recognition and augments the
`
`2
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`genetically modified cytotoxic T lymphocytecell preparations ability to mediate a
`cellular immuneresponse, wherein the helper T lymphocyte cell preparation
`comprises CD4+Tcells that have a chimeric antigen receptor comprising an
`extracellular antibody variable domain specific for the antigen associated with the
`disease or disorder and an intracellular signaling domain ofa T cell receptor.
`Various modifications of the above method are possible. For example, the chimeric
`antigen receptor modifying the CD4+ T cell and the CD8+ T cell can be the same or
`different. In alternative embodiments, the T cells can be modified with a
`recombinant T cell receptor (TCR). TCR could be specific for any antigen, pathogen
`or tumor. There are TCRs for many tumorantigens in melanoma (MARTI, gp100,
`for example), leukemia (WT1, minor histocompatibility antigens, for example),
`breast cancer (her2, NY-BR1, for example).
`In another embodiment, the present invention provides an adoptive cellular
`immunotherapy composition having a genetically modified CD8+ cytotoxic T
`lymphocyte cell preparation that elicits a cellular immune response, wherein the
`cytotoxic T lymphocytecell preparation comprises CD8+ T cells that have a
`chimeric antigen receptor with an extracellular variable domain antibody specific for
`an antigen associated with the disease or disorder and an intracellular signaling
`domain of a T cell or other receptors, such as a costimulatory domain, and a
`genetically modified helper T lymphocyte cell preparation that exhibits a
`predominant Thi phenotypeas well as produce other cytokines, elicits direct tumor
`recognition and augments the ability of genetically modified cytotoxic T lymphocyte
`cell preparations to mediate a cellular immune response, wherein the helper T
`lymphocyte cell preparation has CD4+Tcells that have a chimeric antigen receptor
`with an extracellular antibody variable domain specific for the antigen associated
`with the disease or disorder and an intracellular signaling domain of a T cell
`
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`receptor.
`In yet another embodiment, the present invention provides an adoptive
`cellular immunotherapy composition having a chimeric antigen receptor modified
`tumor-specific CD8+ cytotoxic T lymphocytecell preparation that elicits a cellular
`immuneresponse, wherein the cytotoxic T lymphocytecell preparation comprises
`CD8+ T cells that have a chimeric antigen receptor comprising an extracellular
`single chain antibody specific for an antigen associated with the disease or disorder
`
`3
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`and anintracellular signaling domain ofa T cell receptor, and an antigen-reactive
`chimeric antigen receptor modified naive CD4+ T helpercellthatis derived from
`CD45RO negative, CD62L positive CD4 positive T cells, and a pharmaceutically
`acceptable carrier.
`In another embodiment, the present invention provides an adoptive cellular
`immunotherapy composition having an antigen specific CD8+ cytotoxic T
`lymphocyte cell preparation that elicits a cellular immune response comprising
`CD8+ T cells derived from the patient together with an antigen-reactive chimeric
`antigen receptor modified CD4+ T helper cell that elicits a Thl cytokine response
`and augments the CD8+ immuneresponse to pathogens, wherein the helper T
`lymphocyte cell preparation with CD4+ T cells that have a chimeric antigen receptor
`with an extracellular antibody variable domain specific for the antigen associated
`with the disease or disorder and an intracellular signaling domain of a T cell
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`receptor.
`In another embodiment,the present invention provides an adoptive cellular
`immunotherapy composition with an antigen-reactive chimeric antigen receptor
`modified CD4+ T helper cell that elicits direct tumor recognition and augmentsthe
`CD8+ immuneresponseto pathogens, wherein the helper T lymphocytecell
`preparation comprises CD4 + cells that have a chimeric antigen receptor
`comprising an extracellular antibody variable domain specific for an antigen
`associated with a disease or disorder and an intracellular signaling domain of a T
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`cell receptor.
`In anotheraspect, the present invention provides a method of manufacturing
`an adoptive immunotherapy composition by obtaining a chimeric antigen receptor
`modified tumor-specific CD8+ cytotoxic T lymphocyte cell preparation that elicits a
`cellular immune response and an antigen-reactive chimeric antigen receptor,
`wherein the modified cytotoxic T lymphocyte cell preparation comprises CD8+ T
`cells that have a chimeric antigen receptor with an extracellular antibody variable
`domainspecific for an antigen associated with the disease or disorder and an
`intracellular signaling module of a T cell receptor; and obtaining a modified naive
`CD4+ T helpercell that elicits a Thl cytokine response, wherein the modified helper
`T lymphocyte cell preparation comprises CD4+ cells that have a chimeric antigen
`receptor with an extracellular antibody variable domain specific for the antigen
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`associated with the disease or disorder and an intracellular signaling domain of a T
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`cell receptor.
`In another embodiment, the present invention provides a method of
`manufacturing an adoptive immunotherapy composition by obtaining a modified
`naive CD4+Thelpercell that elicits a Th1 cytokine response, wherein the modified
`helper T lymphocyte cell preparation comprises CD4+T cells that have a chimeric
`antigen receptor comprising an extracellular antibody variable domain specific for
`the antigen associated with the disease or disorder and an intracellular signaling
`domain ofa T cell receptor, and combining the modified naive CD4+Thelpercell
`with an antigen specific central memory CD8+ cytotoxic T lymphocyte cell
`preparation that has a chimeric antigen receptor with an extracellular antibody
`variable domain specific for the antigen associated with the disease or disorder and
`an intracellular signaling domain of a T cell or other receptors.
`In one embodiment, the present invention provides a method of performing
`cellular immunotherapyin subject having a disease or disorder by administering to
`the subject a genetically modified helper T lymphocyte cell preparation, wherein the
`modified helper T lymphocyte cell preparation comprises CD4+ T cells that have a
`chimeric antigen receptor comprising an extracellular antibody variable domain
`specific for an antigen associated with the disease or disorder and an intracellular
`signaling module of a T cell receptor.
`These and other embodiments ofthe invention are described further in the
`accompanyingspecification, drawings and claims.
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`Brief Description of the Drawings
`Fig. 1: showsthe phenotype and analysis of chimeric antigen receptor
`(CAR)expression in a CAR-transduced with ROR1-CARencodinglentivirus ,and
`an untransduced CD8+Tcell line as a control. The RORI-CARcassette contains a
`truncated EGFRthat serves as transduction marker and can be detected by staining
`with anti-EGFR monoclonal antibodies. Truncated Fe-ROR1fusion protein binds
`directly to the antigen-binding domainofthe ROR1-CARandselectively stains the
`ROR1-CARtransducedbut not the untransduced control T cell line. Expression of
`the ROR1-CARonthe cell surface of CD8+ T cells is measured directly by binding
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`to ROR1-Fcfusion protein andindirectly by expression of a truncated EGFRthatis
`encoded downstream of a 2A sequencein the vector.
`Fig. 2: shows cytolytic activity of CD8+ T cells expressing a RORI-specific
`chimeric antigen receptor against a panel ofhuman ROR1-positive tumorcell lines
`(K562) and primary tumorcells (B-CLL) and autologous normal B-cells in a Cr
`release assay. Consistent with the uniform expression ofROR1 on malignant but
`not on mature normalBcells, genetically modified CD8+ ROR1-CAR T cells only
`lysed ROR1+ tumorcells but not mature normalB cells. CD8+ ROR1-CART cells
`exert specific lytic activity against ROR1-positive tumorcells including primary
`CLL,but not against normal B cells.
`Fig. 3: shows the phenotype and CAR expression of a RORI-CAR
`transduced and an untransduced CD4+T cell line as a control. Expression ofthe
`RORI-CARonthe cell surface of CD4+T cells is measured by specific binding to
`ROR1-Fcfusion protein.Truncated Fc ROR fusion protein but not Fe protein alone
`binds directly to the ROR1-CARand selectively stains the ROR1-CAR transduced
`15
`but not the untransduced control CD4+Tcell line confirming expression ofthe
`RORI-CARonthecell surface and binding to ROR1 -protein. Expression ofthe
`RORI-CARonthecell surface of CD4+ T cellsis measured by specific binding to
`RORI-Fc fusion protein, but not to a control Fe fusion protein.
`Fig. 4: (i.e., FIGS 4A-4B, collectively) shows weak but specific cytolytic
`activity of CD4+ RORI-CAR T cells ina °'Crrelease assay. against a panel of
`RORI-positive tumorcells including primary CLL,the mantle cell lymphomaline
`Jeko-1, K562 cells that were stably transfected with ROR1 (K562/ROR1),but not
`native ROR1-negative K562 cells. CD4+ RORI-CART cells exert weak but
`specific lytic activity against RORI-positive tumorcells.
`Fig. 5: (.e., FIGS 5A-5B, collectively) show the results from an IFNy
`ELISA (Fig. 5A) and multiplex cytokine assay (Fig. 5B). Cytokine secretion of
`CD4+ and CD8+ RORI-CARTcell lines. CD4+ RORI-CAR and CD8 ROR1-CAR
`T cells were co-incubated with ROR1+ tumor cells, and levels of interferon gamma
`(IFNg) was measured by ELISA (5A), and IFNg, TNFa,IL-2, IL-4, IL-10 and IL-17
`were measured by Luminex assay (5B). CD4+ ROR1-CAR modified T cells
`specifically recognize ROR1 -postive tumorcells and tumor cell lines and produce
`higher amounts ofThl cytokines including IFN-y, TNF-o and particularly IL-2 than
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`CD8+ ROR1-CARmodified T cells. These data demonstrate that CD4+ RORI-
`CART cells exert helper effector functionsafter stimulation through the ROR1-
`CARandin addition to mediating direct anti-tumorreactivity, could also be utilized
`to augmentthe ability of CD8+ ROR1-CAR modified T cells to mediate a cellular
`
`immuneresponse.
`Fig. 6 depicts the results ofa proliferation study showing that CD4+ RORI-
`CART cells are inducedto proliferate after stimulation with ROR1-positive tumor
`cell lines and primary tumorcells (CFSE assay)and that both the percentage of
`proliferating cells and numberofcell divisions that the proliferating subset
`underwent weresignificantly higher compared to CD8+ ROR1-CARmodified T
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`cells. CD4+ ROR1-CARTcells proliferate more vigorously after stimulation with
`ROR1-positive tumorcells (K562/ROR1, primary CLL, and Jeko MCL) compared
`to CD8+ ROR1-CAR CTLs.
`Fig. 7: Polyclonal unselected CD4+ RORI CARTcells provide help to
`CD8+ ROR1-CAR CTLsby promoting their proliferation in response to tumor.
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`CD4+ RORI-CART cells (derived from bulk CD4+ T cells) significantly increased
`proliferation of polyclonal unselected CD8+ ROR1-CAR CTLs(18% in individual
`culture > 31.5% after co-culture with CD4+ CAR T cells).
`Fig. 8: (i.e., FIGS 8A-8D, collectively) shows the generation of CD4+ CAR
`T cell lines from flow sort purified CD4+ naive, central memory and effector
`memory subsets and analysis ofT-cell function. Cytokineprofile and proliferative
`capacity suggest that CD4+ RORI-CART cells derived from naive CD4+ T cells
`may bebest suited to provide help to CD8+ CTLs. Similar data were obtained in
`experiments comparing the function ofCD4+ CAR T-cell lines expressing a CD19-
`specific CAR. Figure 8A shows flow sort purification ofnaive, central and effector
`memory CD4+Tcells based on expression ofCD45RA, CD45RO, CD62L.Figure
`8B showsanalysisofproliferation of ROR1-CART celllines that were derived by
`lentiviral transduction ofsort purified naive, central and effector memory CD4+ T
`cells (CFSE assay). Figure 8C shows analysis ofcytokine secretion ofRORI-CAR
`T cell lines from sort purified naive, central and effector memory CD4+Tcells
`30
`(Luminexassay). Figure 8D shows analysis of cytokine secretion of CD19-CAR T-
`cell lines from sort purified naive, central and effector memory CD4+T cells
`(Luminex assay). The cytokine profile obtained by multiplex cytokineanalysis (FIG.
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`8B)andproliferative capacity by CFSEstaining (FIG. 8C) shows that CD4+ ROR1-
`CARmodified T cells derived from the naive subset produced the highest levels of
`Thl cy