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`PATENT NUMBER:9,987,308
`ISSUE DATE: June5, 2018
`
`By Authority of the
`UnderSecretary of CommerceforIntellectual Property
`and Director of the United States Patent and Trademark Office
`Md. Few
`
`Miguel Tarver
`Certifying Officer
`
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`Miltenyi Ex. 1001 Page 1
`
`

`

`Jun. 5, 2018
`(45) Date of Patent:
`
`(54)
`
`(75)
`
`(3)
`
`(*)
`
`(21)
`
`(22)
`
`(86)
`
`(87)
`
`(65)
`
`METHOD AND COMPOSITIONS FOR
`CELLULAR IMMUNOTHERAPY
`
`Inventors: Stanley R. Riddell, Sammamish, WA
`(US); Michael Hudecek, Leipzig (DE)
`
`Assignee: Fred Hutchinson Cancer Research
`Center, Seattle, WA (US)
`
`Notice:
`
`Subject to any disclaimer,the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 672 days.
`
`Appl. No.:
`
`14/006,641
`
`PCTFiled:
`
`Mar. 23, 2012
`
`PCT/US2012/030388
`
`PCT No::
`§ 371 (€)Q),
`(2), (4) Date: May 5, 2014
`
`PCT Pub. No.: WO2012/129514
`PCT Pub. Date: Sep. 27, 2012
`
`Prior Publication Data
`US 2014/0314795 Al
`Oct. 23, 2014
`
`Related U.S. Application Data
`
`(60)
`
`Provisional application No. 61/466,552,filed on Mar.
`23, 2011.
`
`(1)
`
`(2010.01)
`(2015.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2015.01)
`
`‘Int. Cl.
`C12N 5/0783
`AG61K 35/17
`CO7K 14/725
`CO7K 16/28
`CO7K 16/30
`A61K 39/00
`AOIK 35/12
`(62) US. CL
`CPC eeesessee AGIK 35/17 (2013.01); CO7K 14/7051
`(2013.01); CO7K 16/28 (2013.01); CO7K
`16/2803 (2013.01); CO7K 16/3061 (2013.01);
`CI2N 5/0636 (2013.01); AIK 2035/122
`(2013.01); A6IK 2035/124 (2013.01); A61K
`2039/5156 (2013.01); AGIK 2039/57
`(2013.01); A6IK 2039/572 (2013.01); CO7K
`2317/622 (2013.01); CO7K 2319/00 (2013.01);
`C12N 2501/515 (2013.01); C12N 2510/00
`(2013.01)
`
`(58)
`
`(56)
`
`Field of Classification Search
`None
`See application file for complete search history.
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,690,915 A
`9/1987 Rosenberg
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`6,040,177 A
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`6/2008 ‘Riddell etal.
`2008/0131415 Al
`20 14/0314795 Al* 10/2014 Riddell
`....00... C07K 14/7051
`424/184.1
`
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`
`WO
`WoO
`
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`rather than memory subsets possess superior traits for adoptive
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`Hudeceketal., “The B-cell tumor-associated antigen ROR! can be
`targeted with T cells modified to express a ROR |-specific chimeric
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`Kershawet al., “Gene-Engineered T Cells as a Superior Adjuvant
`Therapy for Metastatic Cancer,” The Journal of Immunology 173:
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`
`Primary Examiner — Michael Burkhart
`(74) Attorney, Agent, or Firm — Seed IP Law Group LLP
`
`ABSTRACT
`(57)
`The present invention provides methods and compositions
`to confer and/or augment immune responses mediated by
`cellular immunotherapy, such as by adoptively transferring
`genetically modified tumor specific CD8+ T cells in the
`presence of tumor-specific, subset specific genetically modi-
`fied CD4+ T cells, wherein the CD4+ T cells confer and/or
`augment a CD8+ T cells ability to sustain anti-tumor reac-
`tivity and increase and/or maximize tumor-specific prolif-
`eration of the tumor-specific CD8+ T cells of interest.
`Pharmaceutical formulations produced by the method, and
`methods of using the same, are also described.
`
`31 Claims, 18 Drawing Sheets
`
`Miltenyi Ex. 1001 Page 2
`
`Miltenyi Ex. 1001 Page 2
`
`

`

`(56)
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`
`* cited by examiner
`
`Miltenyi Ex. 1001 Page 3
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`Miltenyi Ex. 1001 Page 3
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`

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`Miltenyi Ex. 1001 Page 4
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`ROR1-CARCD8+CTL
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`Miltenyi Ex. 1001 Page 5
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`Miltenyi Ex. 1001 Page 7
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`FIG. 5A
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`Miltenyi Ex. 1001 Page 11
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`Miltenyi Ex. 1001 Page 11
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`Miltenyi Ex. 1001 Page 14
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`Miltenyi Ex. 1001 Page 14
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`FIG. 11A
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`1.0x10%9CDO8+ CM CD19-CAR
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`Miltenyi Ex. 1001 Page 15
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`Miltenyi Ex. 1001 Page 15
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`Miltenyi Ex. 1001 Page 16
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`Miltenyi Ex. 1001 Page 16
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`Miltenyi Ex. 1001 Page 17
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`Miltenyi Ex. 1001 Page 17
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`

`

`CELLULAR IMMUNOTHERAPY
`
`This applicationis being filed on 23 Mar. 2012, as a PCT
`International Patent application in the name of Fred
`Hutchinson Cancer Research Center, a U.S. national corpo-
`ration, applicant for the designationofall countries except
`the U.S., 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 claims priority to U.S.
`Patent Application Ser. No. 61/466,552 filed on 23 Mar.
`2011, the disclosure of which is incorporated herein by
`reference in its entirety.
`
`STATEMENT REGARDING FEDERALLY
`SPONSORED RESEARCH
`
`This invention was made with government support under
`CA018029 awardedby the NationalInstitutes of Health. The
`government has certain rights in the invention.
`
`FIELD OF THE INVENTION
`
`The present invention relates to the field of biomedicine
`and specifically methods useful for cancer therapy. In par-
`ticular, embodiments of the invention relate to methods and
`compositions for carrying out cellular immunotherapy.
`
`BACKGROUNDOF THE INVENTION
`
`Studiesin rodents have demonstratedthat adoptive immu-
`notherapy 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 cells of a desired antigen speci-
`ficity or to engineer T cells to express receptors that target
`infected or transformed cells, and then expand thesecells in
`culture”"!*. Thetransfer ofT 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 stemcell transplan-
`tation, the administration to recipients of T cell clones from
`the donorthat target pathogens or malignant cells can avoid
`graft-versus-host disease that occurs with infusion of unse-
`lected donor T cells***!5. However,
`it
`is apparent from
`clinical studies that the efficacy of cultured T cells, particu-
`larly cloned CD8* T cells,
`is frequently limited by their
`failure to persist after adoptive transfer'®'”.
`The pool of lymphocytes from whichT cells for adoptive
`immunotherapy can be derived contains naive and long-
`lived, antigen experienced memory T cells (T,,). T,, can be
`divided further into subsets of central memory (T¢,,) and
`effector memory (T;,,) cells that differ in phenotype, hom-
`ing properties and function’*. CD8* T.,, express CD62L
`and CCR7at the cell surface, which promote migration into
`lymph nodes, and proliferate rapidly if re-exposed to anti-
`gen. CD8* Ty, lack cell surface CD62L andpreferentially
`migrate to peripheral tissues, and exhibit immediate effector
`function’’. In response to antigen stimulation, CD8* Ter,
`and T,, both differentiate into cytolytic effector T cells (T,)
`that express a high level of granzymesand perforin, but are
`short-lived*°, Thus, the poor survival of T cells in clinical
`immunotherapy trials may simply result from their differ-
`entiation during in vitro culture to T, that are destined to
`die’’???, There is a need to identify cell populations and
`methods that provide enhanced survival of adoptively trans-
`ferred T cells in vivo.
`
`15
`
`20
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`25
`
`30
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`35
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`40
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`45
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`50
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`55
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`60
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`65
`
`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 tumor-specific, subset specific genetically modi-
`fied CD4+ T cells, wherein the CD4+ T cells confer and/or
`augment the ability of CD8+ T cells to sustain anti-tumor
`reactivity and increase and/or maximize tumor-specific pro-
`liferation.
`the present invention provides a
`In one embodiment,
`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 prepa-
`ration that provides a cellular immuneresponse, wherein the
`cytotoxic T lymphocyte cell preparation comprises CD8+ T
`cells that have a chimeric antigen receptor with an extra-
`cellular 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 produce other cytokines, elicits direct
`tumor recognition and augments the genetically modified
`cytotoxic T lymphocyte cell preparations ability to mediate
`a cellular immuneresponse, wherein the helper T lympho-
`cyte cell preparation comprises CD4+ T cells that have a
`chimeric antigen receptor comprising an extracellular anti-
`body variable domain specific for the antigen associated
`with the disease or disorder and an intracellular signaling
`domain of a 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 sameordifferent. In alternative embodi-
`ments, the T cells can be modified with a recombinantT cell
`receptor (TCR). TCR could be specific for any antigen,
`pathogen or tumor. There are TCRs for many tumorantigens
`in melanoma (MART1, gp100,
`for example),
`(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
`preparationthat elicits a cellular immune response, wherein
`the cytotoxic T lymphocyte cell preparation comprises
`CD8+ T cells that have a chimeric antigen receptor with an
`extracellular variable domain antibody specific for an anti-
`gen associated with the disease or disorder and an intracel-
`lular signaling domain of a T cell or other receptors, such as
`acostimulatory domain, and a 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 ability of genetically
`modified cytotoxic T lymphocyte cell preparations to medi-
`ate a cellular immuneresponse, wherein the helper T lym-
`phocyte 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.
`In yet another embodiment, the present invention pro-
`vides an adoptive cellular immunotherapy composition hav-
`ing a chimeric antigen receptor modified tumor-specific
`CD8+ cytotoxic T lymphocyte cell preparation that elicits a
`cellular immuneresponse, wherein the cytotoxic T lympho-
`cyte cell preparation comprises CD8+ T cells that have a
`chimeric antigen receptor comprising an extracellular single
`
`Miltenyi Ex. 1001 Page 22
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`Miltenyi Ex. 1001 Page 22
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`

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`BRIEF DESCRIPTION OF THE DRAWINGS
`
`20
`
`25
`
`30
`
`specific for an antigen associated with the diseaseor disor-
`der and anintracellular signaling module ofa T cell receptor.
`These and other embodiments of the invention are
`described further in the accompanying specification, draw-
`ings and claims.
`
`diseaseor disorder and an intracellular signaling domain of
`a T cell receptor, and an antigen-reactive chimeric antigen
`receptor modified naive CD4+ T helpercell that is 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 prepa-
`ration that elicits a cellular immune response comprising
`FIG. 1: shows the phenotype and analysis of chimeric
`CD8+ T cells derived from the patient together with an
`antigen receptor (CAR) expression in a CAR-transduced
`antigen-reactive chimeric antigen receptor modified CD4+ T
`with ROR1-CAR encoding lentivirus, and an untransduced
`CD8+ T cell line as a control. The RORI-CAR cassette
`helpercell that elicits a Th! cytokine response and augments
`contains a truncated EGFR that serves as transduction
`the CD8+ immune response to pathogens, wherein the
`_ 5
`helper T lymphocytecell preparation with CD4+Tcells that
`marker and can be detected by staining with anti-EGFR
`have a chimeric antigen receptor with an extracellular anti-
`monoclonal antibodies. Truncated Fc-RORI fusion protein
`body variable domain specific for the antigen associated
`binds directly to the antigen-binding domain of the RORI-
`with the disease or disorder and an intracellular signaling
`CARandselectively stains the ROR1-CARtransduced but
`domain ofa T cell receptor.
`not the untransduced control T cell line. Expression of the
`RORI-CARonthecell surface of CD8+ T cells is measured
`In another embodiment, the present invention provides an
`adoptive cellular immunotherapy composition with an anti-
`directly by binding to ROR1-Fcfusionprotein andindirectly
`gen-reactive chimeric antigen receptor modified CD4+ T
`by expression of a truncated EGFR that is encoded down-
`helpercell that elicits direct tumor recognition and augments
`stream of a 2A sequence in the vector.
`the CD8+ immune response to pathogens, wherein the
`FIG.2: showscytolytic activity of CD8+ T cells express-
`helper T lymphocyte cell preparation comprises CD4+ T
`ing a RORI-specific chimeric antigen receptor against a
`cells that have a chimeric antigen receptor comprising an
`panel of human ROR1-positive tumorcell lines (K562) and
`extracellular antibody variable domain specific for an anti-
`primary tumorcells (B-CLL)and autologous normalB-cells
`in a *!Cr release assay. Consistent with the uniform expres-
`gen associated with a disease or disorder andan intracellular
`signaling domain of a T cell receptor.
`sion of ROR] on malignant but not on mature normal B
`In anotheraspect, the present invention provides a method
`cells, genetically modified CD8+ RORI-CAR T cells only
`of manufacturing an adoptive immunotherapy composition
`lysed ROR1+ tumorcells but not mature normal B cells.
`by obtaining a chimeric antigen receptor modified tumor-
`CD8+ RORI-CAR T cells exert specific lytic activity
`specific CD8+ cytotoxic T lymphocyte cell preparation that
`against ROR1-positive tumorcells including primary CLL,
`elicits a cellular immune response and an antigen-reactive
`but not against normal B cells.
`chimeric antigen receptor, wherein the modified cytotoxic T
`FIG. 3: shows the phenotype and CAR expression of a
`RORI-CARtransduced and an untransduced CD4+ T cell
`lymphocyte cell preparation comprises CD8+ T cells that
`have a chimeric antigen receptor with an extracellular anti-
`line as a contro]. Expression of the RORI-CARonthe cell
`body variable domain specific for an antigen associated with
`surface of CD4+ T cells is measured by specific binding to
`the disease or disorder andanintracellular signaling module
`ROR1-Fc fusion protein. Truncated Fe ROR] fusion protein
`of a T cell receptor; and obtaining a modified naive CD4+ T
`but not Fe protein alone binds directly to the RORI-CAR
`helpercell that elicits a Thl cytokine response, wherein the
`andselectively stains the ROR1-CARtransducedbutnot the
`modified helper T lymphocyte cell preparation comprises
`untransduced control CD4+ T cell line confirming expres-
`CD4+ cells that have a chimeric antigen receptor with an
`sion of the ROR1-CAR onthe cell surface and binding to
`extracellular antibody variable domain specific for the anti-
`ROR1-protein. Expression of the ROR1-CAR on thecell
`gen associated with the disease or disorder and an intracel-
`surface of CD4+ T cells is measured by specific binding to
`lular signaling domain of a T cell receptor.
`ROR1-Fe fusion protein, but not to a control Fe fusion
`In another embodiment, the present invention provides a
`protein.
`method of manufacturing an adoptive immunotherapy com-
`FIG.4: (i.e., FIGS. 4A-4B,collectively) shows weak but
`position by obtaining a modified naive CD4+ T helpercell
`specific cytolytic activity of CD4+ ROR1-CART cells ina
`51Cr release assay. against a panel of ROR1-positive tumor
`that elicits a Thl cytokine response, wherein the modified
`helper T lymphocyte cell preparation comprises CD4+ T
`cells including primary CLL, the mantle cell lymphomaline
`cells that have a chimeric antigen receptor comprising an
`Jeko-1, K562 cells that were stably transfected with RORI
`extracellular antibody variable domain specific for the anti-
`(K562/ROR1), but not native ROR1-negative K562 cells.
`gen associated with the disease or disorder and anintracel-
`CD4+ RORI-CAR T cells exert weak but specific lytic
`lular signaling domain of a T cell receptor, and combining
`activity against ROR] -positive tumorcells.
`the modified naive CD4+ T helper cell with an antigen
`FIG.5: (i.e., FIGS. 5A-5B,collectively) show the results
`specific central memory CD8+ cytotoxic T lymphocytecell
`from an IFNy ELISA (FIG. 5A) and multiplex cytokine
`preparation that has a chimeric antigen receptor with an
`assay (FIG. 5B). Cytokine secretion of CD4+ and CD8+
`RORI-CAR T cell
`lines. CD4+ ROR1-CAR and CD8
`extracellular antibody variable domain specific for the anti-
`60
`RORI-CARTcells were co-incubated with ROR1+ tumor
`gen associated with the disease or disorder and anintracel-
`lular signaling domain of a T cell or other receptors.
`cells, and levels of interferon gamma (IFNg) was measured
`In one embodiment,
`the present
`invention provides a
`by ELISA (5A), and IFNg, TNFa, IL-2, IL-4, IL-10 and
`method of performing cellular immunotherapy in subject
`IL-17 were measured by Luminex assay (5B). CD4+ ROR1-
`having a disease or disorder by administering to the subject
`CAR modified T cells specifically recognize RORI-positive
`a genetically modified helper T lymphocyte cell preparation,
`tumorcells and tumorcell lines and produce higher amounts
`wherein the modified helper T lymphocyte cell preparation
`of Thi cytokines including IFN-y, TNF-o. and particularly
`IL-2 than CD8+ RORI-CAR modified T cells. These data
`comprises CD4+ T cells that have a chimeric antigen recep-
`
`40
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`65
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`Miltenyi Ex. 1001 Page 23
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`— 0
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`45
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`350
`
`effector functionsafter stimulation through the ROR1-CAR
`cells, demonstrating that naive
`and in addition to mediating direct anti-tumor reactivity,
`FIG. 10: shows the superior ability of CD4+ CART-cell
`lines derived from the naive subset
`to augment
`could also be utilized to augment
`the ability of CD8+
`ROR1-CAR modified T cells to mediate a cellular immune
`specific proliferation of central memory-derived CD8+ CAR
`CTL in co-culture experiments with CD8+ CD19-CAR
`response.
`CTLs and CD4+ CD19-CAR T-cell lines, stimulated with
`FIG. 6 dep