8329857
`
`December 28, 2022
`
`THIS IS TO CERTIFY THAT ANNEXED HERETO IS A TRUE COPY FROM
`THE RECORDS OF THE UNITED STATES PATENT AND TRADEMARK
`OFFICE OF THOSE PAPERS OF THE BELOW IDENTIFIED PATENT
`APPLICATION THAT MET THE REQUIREMENTS TO BE GRANTED A
`FILING DATE UNDER 35 USC 111.
`
`APPLICATION NUMBER: 61/466,552
`FILING DATE: March 23, 2011
`
`THE COUNTRY CODE AND NUMBER OF YOUR PRIORITY
`APPLICATION, TO BE USED FOR FILING ABROAD UNDER THE PARIS
`CONVENTION, IS US61/466,552
`
`Miltenyi Ex. 1006 Page 1
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`

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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`DOCKET NUMBER
`
`15950.11USP1
`
`REQUEST FOR PROVISIONAL APPLICATION UNDER 37 C.F.R.§1.53(c
`
`Commissioner for Patents
`P.O. Box 1450
`Alexandria, Virginia 22313-1450
`
`DearSir:
`
`This is a request for filing a Provisional application for patent under 37 CFR § 1.53(c) entitled METHOD
`AND COMPOSITIONS FOR CELLULAR IMMUNOTHERAPYbythe following inventor(s):
`
`MD 20832/USA
`
`Second Given Name
`R,
`
`Country of Citizenship
`Canada
`State & Zip Code/Country
`WA 98075/USA
`
`Second Given Name
`
`Country of Citizenship
`Germany
`State & Zip Code/Country
`WA 98125/USA
`
`Second Given Name
`
`Country of Citizenship
`USA
`State & Zip Code/Country
`
`Full Name
`Of Inventor
`
`Residence
`& Citizenship
`Post Office
`Address
`
`Full Name
`Of Inventor
`Residence
`& Citizenship
`Post Office
`Address
`
`Full Name
`Of Inventor
`
`Residence
`& Citizenship
`Post Office
`Address
`
`Family Name
`Riddell
`
`City
`Sammamish
`Post Office Address
`1763 268"Place SE
`
`Family Name
`Hudecek
`City
`Seattle
`Post Office Address
`3010 NE 117"Street
`
`Family Name
`Rader
`
`First Given Name
`Stanley
`
`State or Foreign Country
`WA
`City
`Sammamish
`
`First Given Name
`Michael
`State or Foreign Country
`WA
`City
`Seattle
`
`First Given Name
`Christoph
`
`City
`Olney
`Post Office Address
`17705 Prince Edward Drive
`
`State or Foreign Country
`MD
`City
`Olney
`
`1.
`
`2.
`
`x]
`
`x]
`
`Enclosedis the Provisional application for patent as follows: 41 pages of specification, 51 claims,
`1 page Abstract, and 11 sheets of drawings.
`
`Small entity status is claimed pursuant to 37 CFR 1.27.
`
`Miltenyi Ex. 1006 Page 2
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`Miltenyi Ex. 1006 Page 2
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`

`

`Payment of Provisional filing fee under 37 C.F.R. § 1.16(d):
`[|
`Attached is a check in the amount of $
`.
`DX]|Paymentis being madebycredit card.
`[|
`PAYMENT OF THE FILING FEE IS BEING DEFERRED.
`
`The Commissioner is hereby authorized to charge any additional fees as set forth in 37 CFR §§
`1.16 to 1.18 which may be required by this paper or credit any overpayment to Account No. 13-
`2725.
`
`Enclosed is an Assignmentof the invention to
`checkfor $
`to cover the Recordation Fee.
`
`, Recordation Form Cover Sheet and a
`
`Also Enclosed:
`
`The invention was made by the following agency of the United States Government or under a
`contract with the following agency of the United States Government:
`
`Addressall future communications to the Attention of Katherine M. Kowalchykassociated
`with the customer number below (may only be completed by attorney or agentof record).
`
`KXOOOC
`
`Respectfully submitted,
`
`MERCHANT & GOULDP.C.
`P.O. Box 2903
`Minneapolis, MN 55402-0903
`612/332-5300
`
`
`
`Date:J741t4.23,7,Ol oof
`
`
`Kath
`
`KME:kf
`
`
`
`Miltenyi Ex. 1006 Page 3
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`15950.11USP1
`
`METHOD AND COMPOSITIONS FOR CELLULAR IMMUNOTHERAPY
`
`Field of the Invention
`
`The present invention relates to the field of biomedicine and specifically methods
`
`useful for cancer therapy. In particular, embodiments of the 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
`
`P01CA18029 and RO1CA136551 from the United States Department of Health and Human
`
`Services, National Institute of Health. The United States governmenthascertain rights in
`
`the invention.
`
`10
`
`15
`
`Backgroundof the Invention
`
`Studies in rodents have demonstrated that adoptive immunotherapy with antigen
`
`specific T cells is effective for cancer and infections, and there is evidence this modality
`has therapeutic activity in humans'*. Forclinical applications,it is necessary to isolate T
`
`20
`
`cells of a desired antigen specificity or to engineer T cells to express receptorsthat target
`infected or transformedcells, and then expandthesecells in culture”*. The transfer of T
`
`cell clones is appealing because it enables control of specificity and function, and
`
`facilitates evaluation of in vivo persistence, toxicity and efficacy. Additionally, in the
`
`setting of allogeneic stem cell transplantation, the administration to recipients of T cell
`
`25
`
`clones from the donorthat target pathogens or malignant cells can avoid graft-versus-host
`3,4,15
`disease that occurs with infusion of unselected donor T cells’"”~. However, it is apparent
`from clinical studies that the efficacy of cultured T cells, particularly cloned CD8" T cells,
`is frequently limited by their failure to persist after adoptive transfer'®'”.
`
`The pool of lymphocytes from which T cells for adoptive immunotherapy can be
`
`30
`
`derived contains naive and long-lived, antigen experienced memory T cells (Tm). Tw can
`
`be divided further into subsets of central memory (Tm) and effector memory (T_m) cells
`
`Miltenyi Ex. 1006 Page 4
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`that differ in phenotype, homing properties and function'*. CD8' Tem express CD62L and
`
`CCR7, which promote migration into lymph nodes, and proliferate rapidly if re-exposed to
`antigen. CD8* Tym lack CD62L enabling migration to peripheral tissues, and exhibit
`immediate effector function’ In responseto antigen stimulation, CD8" Tey and Tpyy 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 immunotherapy
`
`trials may simply result from their differentiation during in vitro culture to TE that are
`17,21,22
`
`. Thus, there is a need to identify cell populations and methodsthat
`
`destined to die
`
`provide enhancedsurvival of adoptively transferred T cells in vivo.
`
`10
`
`Summaryof the Invention
`
`In one aspect, the present invention relates to methods and compositions to confer
`
`and/or augment immuneresponses mediated by cellular immunotherapy, such as by
`
`adoptively transferring genetically modified tumor specific CD8+ T cells in the presence
`
`15
`
`of tumor-specific, subset specific genetically modified CD4+ T cells, wherein the CD4+ T
`
`cells confer and/or augment a CD8+ T cells ability to sustain anti-tumorreactivity 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
`
`20
`
`genetically modified cytotoxic T lymphocyte cell preparation that provides a cellular
`
`immuneresponse, wherein the cytotoxic T lymphocyte cell 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 an intracellular
`
`signaling domain of a T cell or other receptors, such as co-stimulatory domains; and a
`
`25
`
`genetically modified helper T lymphocyte cell preparation that exhibits a predominant Th1
`
`phenotype as well as produce other cytokines,elicits direct tumor recognition and
`
`augments the genetically modified cytotoxic T lymphocyte cell preparationsability to
`
`mediate a cellular immune response, wherein the helper T lymphocyte cell preparation
`
`comprises CD4 + T cells that have a chimeric antigen receptor comprising an extracellular
`
`30
`
`antibody variable domain specific for the antigen associated with the disease or disorder
`
`and an intracellular signaling domain of a T cell receptor. Various modificationsof the
`
`Miltenyi Ex. 1006 Page 5
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`above method are possible. For example, the chimeric antigen receptor modifying the
`
`CD4+ T cell and the CD8+ T cell can be the sameor 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 tumor
`
`antigens in melanoma (MART1, 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 immuneresponse, wherein the cytotoxic T
`
`10
`
`lymphocyte cell 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 Th1 phenotypeas well as produce other cytokines,
`
`15
`
`elicits direct tumor recognition and augments the genetically modified cytotoxic T
`
`lymphocyte cell preparations ability to mediate a cellular immune response, wherein the
`
`helper T lymphocyte cell preparation has 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 receptor.
`
`20
`
`In yet another embodiment, the present invention provides an adoptive cellular
`
`immunotherapy composition having a chimeric antigen receptor modified tumor-specific
`
`CD8+ cytotoxic T lymphocyte cell preparation that elicits a cellular immuneresponse,
`
`wherein the cytotoxic T lymphocyte cell preparation comprises CD8+ T cells that have a
`
`chimeric antigen receptor comprising an extracellular single chain antibody specific for an
`
`25
`
`antigen associated with the disease or disorder and an intracellular signaling domain of a T
`
`cell receptor, and an antigen-reactive chimeric antigen receptor modified naive CD4+ T
`
`helper cell that is derived from CD45RO negative, CD62L positive CD4positive T cells,
`
`and a pharmaceutically acceptable carrier.
`
`In another embodiment, the present invention provides an adoptive cellular
`
`30
`
`immunotherapy composition having an antigen specific CD8+ cytotoxic T lymphocyte cell
`
`preparation thatelicits a cellular immune response comprising CD8+ T cells derived from
`
`Miltenyi Ex. 1006 Page 6
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`the patient together with an antigen-reactive chimeric antigen receptor modified naive
`
`CD4+ T helpercell that elicits a Thl cytokine response and augments the CD8+ immune
`
`response 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 receptor.
`
`In another embodiment, the present invention provides an adoptive cellular
`
`immunotherapy composition with an antigen-reactive chimeric antigen receptor modified
`
`naive CD4+T helpercell that elicits direct tumor recognition and augments the CD8+
`
`10
`
`immuneresponse to pathogens, wherein the 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 a disease or disorder and
`
`an intracellular signaling domain of a T cell receptor.
`
`In another aspect, the present invention provides a method of manufacturing an
`
`15
`
`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 domain specific for an antigen
`
`20
`
`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 associated with the disease or disorder and an intracellular
`
`25
`
`signaling domain of a T cell receptor.
`
`In another embodiment, the present invention provides a method of manufacturing
`
`an adoptive immunotherapy composition by obtaining a modified naive CD4+ T helper
`
`cell that elicits a Thl cytokine response, wherein the modified helper T lymphocyte cell
`
`preparation comprises CD4+Tcells that have a chimeric antigen receptor comprising an
`
`30
`
`extracellular antibody variable domain specific for the antigen associated with the disease
`
`or disorder and an intracellular signaling domain of a T cell receptor, and combining the
`
`Miltenyi Ex. 1006 Page 7
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`modified naive CD4+ T helper cell 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
`
`immunotherapy in 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
`
`10
`
`with the disease or disorder and an intracellular signaling module of a T cell receptor.
`
`These and other embodiments of the invention are described further in the
`accompanying specification, drawings and claims.
`
`Brief Description of the Drawings
`
`15
`
`Fig. 1: shows the phenotype and analysis of chimeric antigen receptor (CAR)
`
`expression in a CAR-transduced and an untransduced CD8-+Tcellline as a control. The
`
`RORI1-CARcassette contains a truncated EGFRthat serves as transduction marker and can
`
`be detected by staining with anti-EGFR monoclonal antibodies. Truncated Fc-ROR1
`
`fusion protein binds directly to the antigen-binding domain of the ROR1-CAR and
`
`20
`
`selectively stains the ROR1-CARtransduced but not the untransduced control T cell line.
`
`Expression of the ROR1-CARonthe cell surface of CD8+ T cells is measured directly by
`
`binding to ROR1-Fc fusion protein and indirectly by expression of a truncated EGFRthat
`
`is encoded downstream of a 2A sequencein the vector.
`
`Fig. 2: showscytolytic activity of CD8+ T cells expressing a ROR1-specific
`
`25
`
`chimeric antigen receptor against a panel of human ROR1-positive tumorcell lines and
`
`primary tumorcells and autologous normal B-cells. Consistent with the uniform
`
`expression of ROR1 on malignant but not on mature normalB cells, genetically modified
`
`CD8+ RORI-CART cells only lysed ROR1+ tumorcells but not mature normal B cells.
`
`CD8+ RORI-CART cells exert specific lytic activity against ROR1-positive tumorcells
`
`
`
`30
`
`including primary CLL, but not against normal B cells.
`
`Fig. 3: shows the phenotype and CAR expression of a ROR1-CARtransduced and
`
`Miltenyi Ex. 1006 Page 8
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`an untransduced CD4+ T cell line as a control. Truncated Fc-ROR1 fusion protein but not
`
`Fe protein alone binds directly to the ROR1-CARandselectively stains the RORI-CAR
`
`transduced but not the untransduced control CD4+ T cell line confirming expression of the
`
`RORI-CARonthecell surface and binding to ROR1-protein. Expression of the ROR1-
`
`CARonthe cell surface of CD4+ T cells is measured by specific binding to ROR1-Fe
`
`fusion protein, but not to a control Fe fusion protein.
`
`Fig. 4: (.e., FIGS 4A-4B, collectively) shows weak but specific cytolytic activity
`
`of CD4+ RORI-CART cells against a panel of ROR1-positive tumorcells including
`
`primary CLL, the mantle cell lymphomaline Jeko-1, K562 cells that were stably
`
`10
`
`transfected with ROR1 (K562/ROR1), but not native ROR1-negative K562 cells. CD4+
`
`RORI-CART cells exert weak but specific lytic activity against ROR1-positive tumor
`
`cells.
`
`Fig. 5: (.e., FIGS 5A-5B, collectively) show the results from an IFNy ELISA (Fig.
`
`5A) and multiplex cytokine assay (Fig. 5B). CD4+ ROR1-CAR modified T cells
`
`15
`
`specifically recognize ROR1-postive tumorcells and tumorcell lines and produce higher
`
`amounts of Th1 cytokines including IFN-y, TNF-a and particularly IL-2 than CD8+
`
`ROR1-CAR modified T cells. These data demonstrate that CD4+ ROR1-CART cells
`
`exert helper effector functions after stimulation through the ROR1-CARandin addition to
`
`mediating direct anti-tumorreactivity, could also be utilized to augmentthe ability of
`
`20
`
`CD8+ ROR1-CAR modified T cells to mediate a cellular immuneresponse. For these
`
`studies, CD4+ RORI-CAR and CD8 ROR1-CART cells were co-incubated with ROR1+
`
`tumorcells and levels of interferon gamma (IFNy) was measured by ELISA (SA), and
`
`IFNy, TNFa, IL-2, IL-4, IL-10 and IL-17 were measured by Luminexassay (5B).
`
`Fig. 6: depicts the results of a proliferation study showing that CD4+ ROR1-CAR
`
`25
`
`T cells are inducedto proliferate after stimulation with ROR1-positive tumorcell lines and
`
`primary tumorcells and that both the percentage ofproliferating cells and numberofcell
`
`divisionsthat the proliferating subset underwent weresignificantly higher compared to
`
`CD8+ ROR1-CAR modified T cells. CD4+ ROR1I-CART cells proliferate more
`
`vigorously after stimulation with ROR1-positive tumor cells (K562/ROR1, primary CLL,
`
`30
`
`and Jeko MCL) compared to CD8+ ROR1I-CAR CTLs.
`
`Fig. 7: Polyclonal unselected CD4+ ROR1 CART cells provide help to CD8+
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`Miltenyi Ex. 1006 Page 9
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`ROR1-CAR CTLsby promotingtheir proliferation in response to tumor. CD4+ ROR1-
`
`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+ CART cells).
`
`Fig. 8: (.e., FIGS 8A-8C, collectively) shows the generation of CD4+ CART cell
`
`lines from flow sort purified CD4+ naive, central memory and effector memory subsets
`
`(FIG 8A). The cytokine profile obtained by multiplex cytokine analysis (FIG. 8B) and
`
`proliferative capacity by CFSEstaining (FIG. 8C) shows that CD4+ ROR1-CAR modified
`
`T cells derived from the naive subset produced the highest levels of Thl cytokines and
`
`10
`
`proliferated most vigorously after stimulation with ROR1-positive tumorcells, suggesting
`
`they may bebest suited to augment CD8+ ROR1I-CAR CTLs. Generation of ROR1-CAR
`
`T cell lines from sort purified naive, central and effector memory CD4+ T cells and
`
`analysis of effector function. Cytokine profile and proliferative capacity suggest that CD4+
`
`RORI-CART cells derived from naive CD4+ T cells may be best suited to provide help to
`
`15
`
`CD8+ CTLs. Fig. 8A: Flow sort purification of naive, central and effector memory CD4+
`
`T cells based on expression of CD45RA, CD45RO, CD62L. Fig. 8B: Analysis of
`
`proliferation of RORI-CART cell lines that were derived by lentiviral transduction of sort
`
`purified naive, central and effector memory CD4+Tcells (CFSEassay). Fig. 8C: Analysis
`
`of cytokine secretion of ROR1-CART cell lines from sort purified naive, central and
`
`20
`
`effector memory CD4+ T cells (Luminex assay).
`
`Fig. 9: showsthat co-culture of CD8+ ROR1-CAR modified T cells with CD4+
`
`RORI1-CAR modified T cells (but not untransduced control CD4+ T cells) leads to an
`
`increase in tumor-specific proliferation of the CD8+ subset, and that maximum
`
`proliferation of the CD8+ subset is observed after co-culture with CD4+ ROR1-CAR T
`
`25
`
`cells derived from naive CD4+ T cells, demonstrating that naive CD4+ RORI-CART cells
`
`are best able to provide help to CD8+ ROR1-CART cells. Co-culture of CD8+ ROR1-
`
`CAR CTLs and CD4+ ROR1-CART cell lines derived from naive, central and effector
`
`memory subsets to define the optimal combination of CD8+ and CD4+Tcells that would
`
`allow maximum proliferation of CD8+ RORI-CAR CTLs. CD4+ naive ROR1-CAR T
`
`30
`
`cells provide the greatest proliferation of CD8+ central memory ROR1-CAR CTLs.
`
`Fig. 10: shows the augmentation and synergistic effect CD4+ ROR1-CAR modified
`
`Miltenyi Ex. 1006 Page 10
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`T cells on the anti-tumorefficacy of CD8+ROR1-CAR CTLsin a mouse tumor model of
`
`systemic mantle cell lymphoma (NSG/Jeko-1-ffLuc). Anti-tumor efficacy of ROR1-CAR
`
`modified CD8+ and CD4+ T cells in a mouse tumor model of systemic aggressive mantle
`
`cell lymphoma (NSG/Jeko-1). Analysis of tumor burden using bioluminescence imaging
`
`after adoptive transfer of CD8+ RORI-CAR CTLs, CD4+ ROR1I-CART cells or a
`
`combination of CD8+ and CD4+ ROR1-CART cells T cells.
`
`Detailed Description of the Preferred Embodiments
`
`"T cells" or "T lymphocytes" as used herein may be from any mammalian,
`
`10
`
`preferably primate, species, including monkeys, dogs, and humans. In some embodiments
`
`the T cells are allogeneic (from the same species but different donor) as the recipient
`
`subject; in some embodiments the T cells are autologous (the donor and the recipient are
`
`the same); in some embodimentsthe T cells arc syngeneic (the donor andthe recipients are
`
`different but are identical twins).
`
`15
`
`Cytotoxic T lymphocyte (CTL) as used herein refers to a T lymphocyte that
`expresses CD8on the surfacethereof(i.e., a CD8" T cell). In some embodimentssuchcells
`
`are preferably "memory"T cells (Tw cells) that are antigen-experienced.
`
`"Central memory" T cell (or "Tcy") as used herein refers to a CTL that expresses
`CD62L onthesurface thereof(i.c., CD62L*” CD8" cells).
`
`20
`
`"Effector memory” T cell (or "Tzm") as used herein refers to a CTL that does not
`express CD62Lonthesurface thereof(i.e., CD62L,CD8" cells).
`
`"Enriched" and "depleted" as used herein to describe amounts ofcell types in a
`
`mixture refers to the subjecting of the mixture of the cells to a process or step whichresults
`
`in an increase in the numberof the "enriched" type and a decrease in the numberofthe
`
`25
`
`"depleted" cells. Thus, depending upon the source of the original population ofcells
`
`subjected to the enriching process, a mixture or composition may contain 60, 70, 80, 90,95,
`
`or 99 percent or more (in numberor count) of the "enriched"cells and 40, 30, 20, 10, 5 or
`
`1 percent or less (in numberor count) of the "depleted"cells.
`
`Interleukin-15 is a known and described in, for example, US Patent No.
`
`30
`
`6,344,192.
`
`Miltenyi Ex. 1006 Page 11
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`

`Modesof the Disclosure
`
`CD4+ T lymphocytes during in vitro culture significantly increase proliferation,
`
`persistence and anti-tumorreactivity of tumor-specific CD8+ T cells in vitro and in vivo.
`
`In some embodiments, naive CD4+ T cells possesses an intrinsic programmingthat leads
`
`to superior helper activity compared to CD4+ T cells derived from central and effector
`
`memory, or bulk CD4+ T cells.
`
`In embodiments, tumor-reactive CD4+ T cells are modified with a single-chain
`
`antibody-derived chimeric antigen receptor (CAR) specific for the orphan tyrosine kinase
`
`receptor ROR1. ROR] is uniformly expressed on chronic lymphocytic leukemia (CLL)
`
`10
`
`and mantle cell lymphoma (MCL) and ROR1-specific CAR from an anti-ROR1
`
`monoclonal antibody (mAb) confers specific recognition of malignant, but not mature
`
`normal B-cells when expressed in CD8+ cytotoxic T cells (CTLs). ROR1-CART cells
`
`from bulk and flow sort purified naive, central and effector memory CD4+Tcells are
`
`obtained from the peripheral blood of both healthy donors and CLL patients. CD4+ CAR T
`
`15
`
`cells had specific but weak cytolytic activity against ROR1+ tumors including primary
`
`CLL, the MCLline Jeko-1, and K562 cells transfected with ROR1. Multiplex cytokine
`
`analysis detects high-level production of Thl cytokines with significantly higher levels of
`
`IFNy, TNFa, and particularly IL-2 compared to CD8+ CAR CTLs. CFSEstaining shows
`
`dramatically higher proliferation after stimulation with ROR1-positive tumorcells, with
`
`20
`
`both the percentage of cells that were inducedto proliferate and the numberofcell
`
`divisionsthat the proliferating subset underwentbeing significantly higher compared to
`
`CD8+ CAR CTL. CD4+ T cells obtained from both healthy donors and CLL patients
`
`acquire anti-tumorreactivity after genetic modification with a ROR1-specific CAR.
`
`Moreover, the ability to proliferate in the absence of exogenous cytokines and to produce
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`high levels of Thl cytokines demonstrates that CD4+ CARTcells exert typical helper
`25
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`functions after stimulation through the CAR and suggests that in addition to conferring
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`direct anti-tumoreffects, could be utilized to augment tumor-specific CD8+ CTL.
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`The cytokine profile and proliferative capacity of ROR1-CART cells derived from
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`flow sort purified CD4+ naive, central and effector memory subsets is obtained. The CD4+
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`CART cells, derived from the naive CD45RA+ CD45RO- CD62L+subset, producesthe
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`highest levels of Thl cytokines, especially IL-2, and proliferates in response to RORI+
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`tumorcells. Indeed, in co-culture experiments, the addition of CAR-transduced, but not
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`untransduced CD4+Tcells leads to a significant increase in tumor-specific proliferation of
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`CD8+ CAR CTLs. In some embodiments, CAR-modified CD4+ T cells derived from
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`naive rather than central and effector memory subsets or bulk CD4+Tcells results in
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`enhanced proliferation of CD8+ CAR CTL.
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`CD8+ central memory T cells have an intrinsic programming that allows them to
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`persist for extended periods after administration which makes them the preferred subset of
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`CD8+ T cells for immunotherapy. In embodiments, ROR1-CAR modified CTLs from sort
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`purified CD8+ central memory T cells and CD4+ naive CAR-modified T cells provide
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`10
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`enhancedproliferation of the CD8+ T cell subset. In embodiments, tumor-specific CD4+ T
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`cells exert anti-tumorreactivity and provide help to tumor-specific CD8+ T cells in vitro
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`and in vivo. In a specific embodiment, tumor-specific CD4+ T cells from the naive subset
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`are utilized.
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`In another embodiment, the CD8+ and CD4+ T cells can be modified with a T cell
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`15
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`receptor (TCR). The TCR could be specific for any antigen, pathogen or tumor(there are
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`TCRsfor many tumorantigens in melanoma (MART1, gp100 for example), leukemia
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`(WT1, minor histocompatibility antigens for example), breast cancer (her2, NY-BR1 for
`
`example).
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`20
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`Detailed Description
`
`Compositions
`
`The disclosure provides for an adoptive cellular immunotherapy composition
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`comprising a genetically modified CD8+ cytotoxic T lymphocyte cell preparation that
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`elicits a cellular immune response, wherein the cytotoxic T lymphocyte cell preparation
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`25
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`comprises CD8+ T cells that have a chimeric antigen receptor comprising an extracellular
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`single chain antibody derived domain specific for an antigen associated with the disease or
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`disorder and an intracellular signaling domain of a T cell receptor or other receptors, and a
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`genetically modified helper T lymphocyte cell preparation that augments the genetically
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`modified cytotoxic T lymphocyte cell preparations ability to mediate a cellular immune
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`30
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`response, wherein the helper T lymphocyte cell preparation comprises CD4 + T cells that
`
`have a chimeric antigen receptor comprising an extracellular antibody variable domain
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`10
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`specific for the antigen associated with the disease or disorder and an intracellular
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`signaling domain of a T cell receptor or other receptors.
`
`In some embodiments, an adoptive cellular immunotherapy composition comprises
`
`a chimeric antigen receptor modified tumor-specific CD8+ cytotoxic T lymphocyte cell
`
`preparation that elicits a cellular immuneresponse, wherein the cytotoxic T lymphocyte
`
`cell 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 and an intracellular signaling domain of a T cell receptor, in combination with an
`
`antigen-reactive chimeric antigen receptor modified naive CD4+ T helper cell derived
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`10
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`from CD45ROnegative, CD62L positive CD4 positive T cells, and a pharmaceutically
`
`acceptable carrier.
`
`In other embodiments, an adoptive cellular immunotherapy composition comprises
`
`an antigen specific CD8+ cytotoxic T lymphocyte cell preparation that elicits a cellular
`
`immune responsederived from the patient combined with an antigen-reactive chimeric
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`15
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`antigen receptor modified naive CD4+ T helpercell that augments the CD8+ immune
`
`response, wherein the 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 of a T cell receptor.
`
`20
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`In a further embodiment, an adoptive cellular immunotherapy composition
`
`comprises an antigen-reactive chimeric antigen receptor modified naive CD4+ T helper
`
`cell that augments the CD8+ immuneresponse, wherein the 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 a disease or
`
`25
`
`disorder and an intracellular signaling domain ofa T cell receptor.
`
`In alternative embodiments, the T cells can be modified with a recombinantT cell
`
`receptor. TCR could be specific for any antigen, pathogen or tumor. There are TCRsfor
`
`many tumorantigens in melanoma (MARTI, gp100, for example), leukemia (WT1, minor
`
`histocompatibility antigens, for example), breast cancer (her2, NY-BR1, for example).
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`30
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`1]
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`Selection and Sorting of T lymphocyte populations
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`The compositions described herein provide for antigen reactive CD4+ and CD8+ T
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`lymphocytes.
`
`T lymphocytes can be collected in accordance with known techniques and enriched
`
`or depleted by known techniquessuchas affinity binding to antibodies such as flow
`
`cytometry and/or immunomagnetic selection. After enrichment and/or depletion steps, in
`
`vitro expansion of the desired T lymphocytes can be carried out in accordance with known
`
`techniques (including but not limited to those described in US Patent No. 6,040,177 to
`
`Riddell et al.), or variations thereof that will be apparent to those skilled in theart.
`
`10
`
`For example, the desired T cell population or subpopulation may be expanded by
`
`adding an initial T lymphocyte population to a culture medium in vitro, and then adding to
`
`the culture medium feeder cells, such as non-dividing peripheral blood mononuclearcells
`
`(PBMC),(e.g., such that the resulting population of cells contains at least about 5, 10, 20,
`
`or 40 or more PBMCfeedercells for each T lymphocyte in the initial population to be
`
`15
`
`expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T
`
`cells). The non-dividing feeder cells can comprise gamma-irradiated PBMCfeedercells. In
`
`some embodiments, the PBMC are irradiated with gammaraysin the range of about 3000
`
`to 3600 rads. The order of addition of the T cells and feeder cells to the culture media can
`
`be reversed if desired. The culture can typically be incubated under conditions of
`
`20
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`temperature and the like that are suitable for the growth of T lymphocytes. For the growth
`
`of human T lymphocytes, for example, the temperature will generally be at least about 25
`
`degrees Celsius, preferably at least about 30 degrees, more preferably about 37 degrees.
`
`The T lymphocytes expanded include cytotoxic T lymphocytes (CTL) and helper T
`
`lymphocytes that are specific for an antigen present on a human tumoror a pathogen.
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`25
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`Optionally, the expansion method may further comprise the step of adding non-
`
`dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be
`
`irradiated with gammaraysin the range of about 6000 to 10,000 rads. The LCL
`
`feeder cells may be provided in any suitable amount, such as a ratio of LCL feedercells to
`
`initial T lymphocytesof at least about 10:1.
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`30
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`Optionally, the expansion method may further comprise the step of adding a