''!l.'Q)A'DI~,llQ)\M!!Ul\'lf'llHrES~ ~~S!Ei\~ SfL@D,~l\f!El;J
`UNITED STATES DEPART\1ENT OF C OMMERCE
`United States Patent and Trademark Office
`
`Ma rch 17, 2022
`
`THIS IS TO CERTIFY THAT ANNEXED HERETO IS A TRUE COPY FRO:\1
`THE RECORDS OF TIDS OFFICE OF:
`
`Pre-Grant Publication Number: 2005/0113564
`Pre-Grant Publication Date: May 26, 2005
`
`By Authority of the
`Under Secretary of Commerce fo r Intellectual Pr operty
`and Director of the United States Patent and Trademark Office
`
`Miltenyi Ex. 1003 Page 1
`
`

`

`1111111111111111 IIIIII IIIII 11111 1111111111 1111111111 11111 lllll lllll 11111111111 1111 111111111
`US 20050113564Al
`
`(19) Uni1ted States
`c11> Patent Application Publication
`Cam~•ana ct al.
`
`(JU) Pub. No.: US 2005/0113564 Al
`M ay 26, 2005
`(4'.1) Pub. Date:
`
`(54) CH I M.EIUC Rl~C~PTORS WITH 4-!BU
`STIM ILJl ATORY SIGNALING DOMAIN
`
`(75)
`
`lnvcntors: Dario Campana, Oerrnanlown. TN
`(US): C hihnya lmai, Germantown, TN
`(US)
`
`Correspondence Address:
`ST. J U DE CHI L DRe N'S RESEARCH
`HOSP ITAL
`OFl•'JCE OF Tl1CHNOLOGY LICENSING
`332 N. IAUDERDALE
`Ml?.M!flHIS. TN 3t'l05 tUS)
`
`(73) Assignee: St. Jude Cbildrc n's Rcscurcb Hospit!ll
`
`(21) Appl. INu.:
`
`10/981,352
`
`(22) Filed:
`
`Nov. 4, 2004
`
`Related U.S. Application Dato
`
`(60) Provisic>nal application No. 60/517,507, filed on Nov.
`5, 2003.
`
`Publication Classification
`
`(5 L)
`
`Int. Cl.7
`
`(52) U.S. Cl .
`
`. .. ........... ........... C07K 14/74; C07K 16/44;
`C07H 21/04
`..................... 530/350; 536/235; 530/:'itl7~1;
`435169. I: 43S/320.1; 43 5/325
`
`ABSTRACT
`(57)
`Tbe pr~ol invention relate$ to a chimeric receplt1r capabl.:
`of signaling both a primary and a co-stimulatory pathway.
`1hus allowing acliv:Hfon of 1he co-stimulatory path,vay with(cid:173)
`nut binding to the natural ligand. The cytopla~mic domain of
`tbe receptor cQntains a portion of the 4-JTIB signaling
`domain. Embodiments of the invootion relate to polynuclc(cid:173)
`otides that encode the receptor, vectors and host cells
`encoding a cbimoric receptor, particularly including 'I' i.:clls
`and nuturul killer (NK) cdls and methods of use. ;\Jso
`included is u method for obtaining an enriched populatllOD of
`NK cells from a mixed population of NK .:ell~ and T cells.
`
`Miltenyi Ex. 1003 Page 2
`
`

`

`-..... '"'- '
`
`[) CD8a.signal peptide
`
`■ I ir.l,cr
`~ CD8a. hinge &transmemb. domain
`Ill C D28 intracellular domain
`~ 4189 int.racellular domain
`
`VH
`
`•1
`VH -
`
`VH ~ ~-ohain
`
`I
`
`~-chain
`
`VH ~ ~-chain
`
`I
`I
`
`•
`
`"'0
`~ ..,.
`g ..,.
`~
`....
`'O
`i:.; -
`c::
`I')
`o· =
`~
`0-
`c::
`I')
`
`~ .... o· =
`::
`
`~
`'<
`N
`:'
`N
`8 Ul
`~
`
`~
`
`~ -
`
`...,
`0
`N
`
`CD19-truncated
`
`CD19-C
`
`CD19-28-C
`
`CD19-BB-C
`
`m
`~
`m
`liJ
`rn
`t:i1
`Fl
`~
`
`VL 1·
`VL I
`VL I
`VL I
`
`+
`
`anti-CD19 scFv
`
`•
`
`signaling domain
`
`~
`
`1 kb
`
`2 kb
`
`e V,
`
`N
`0
`
`0 v, -0 --~
`
`Ul
`0--
`~
`► ,...
`
`u:------- -1
`r 1gu1it: .1
`
`Miltenyi Ex. 1003 Page 3
`
`

`

`(l)
`
`(.)
`
`120~ 11
`~ 100
`~ 80
`~ 60"
`Q) 40
`.U.
`'#-
`20:
`Q I I
`I
`,
`I
`I
`vector tr~nc.
`
`,
`
`3ao
`
`I
`
`, __ c::::,
`I
`, c:::,
`28.-<: · BB{ ·
`C,
`qhti-CD19
`
`,
`
`69t
`
`120~ T
`~ .10(:).
`:~_; 8'0
`0 u·
`~ 60~
`·~.f 40
`·b ~ · 20
`o-~~
`ve.c.tm· trunc. 1 . :za:c,. . 'tf B~(
`~anti:-CD19'
`
`I
`
`,--..,
`
`L
`
`r--
`
`120
`-~ 100~ ..--
`Q) so~ ,
`>
`0 u
`'(I) 60-
`Q) u 40,,
`~ C!
`20
`O vector tr1.Jr\¢, . ~
`2e~ .. SB{
`·anti-00.19·
`
`KOPN-5-7bi
`
`Q)
`
`120
`~ 1'00·
`> 80-
`0 u
`~ 60
`-~ AO
`'#. 20
`:o I I
`I
`I
`I
`vector trunc.
`
`' '
`
`'r,'>! ____ "
`
`r 1gu.-~ .1.
`
`""0
`
`c::
`r:,
`
`~ g -> :g
`i:.; -.... 0 =
`to:, -o· =
`
`~
`O' c::
`r:,
`
`3::
`~
`N
`:'
`N
`8 Ul
`g:
`~
`~
`N
`0 ....
`
`N
`
`-0.P-1
`
`I
`
`I
`
`I
`
`I
`I .
`I
`I
`I
`~ 2S{ BB-(,
`~nti-C'Dt9
`
`I
`
`Ul
`
`e r;r;
`N g
`0 ....
`....
`~
`.!l1.
`► ,....
`
`~
`
`Miltenyi Ex. 1003 Page 4
`
`

`

`US 2005/0113564 Al
`
`May 26, 2005
`
`CRfl\ifERIC RECEPTORS WLTH 4-lBB
`STI MIULATORY SIGNALING DOMAI N
`
`GOVERNMENT INTEREST
`[(HI01) This invention was made in pari wilh U.S. Gov(cid:173)
`eromcm supporl und1:r National Iastitutes of Health granl
`110. CA 58297. The U.S. Government may have certain
`righis in this invention.
`
`FIELD OF THE INVENTION
`[0002) This inven1ion rc::latt:s to chimeric ccU mt:mbrane
`receptors, par1icularly cbimcric T-ccll receptors.
`
`BACKGROUND
`[0003) Reg,ulalion of cell activities is frequently achieved
`by the binding of a lig,ind to a surface mcmhr.111e receptor
`comprising an extracellular and a cytoplasmic domain. The
`format ion of the complex he tween tbc ligand and the cxtrn(cid:173)
`cellular portiQn of the receptor results in a confonnalional
`change in tb,e cytoplasmic portion o( the rt!Ceptor wbicb
`resuJls in a signal traasduc.'ed within lhe cell. la some
`inslaoccs, the cbttogc io tbe cytoplasmic portion results in
`bi nding to other proteins, where olher proteins arc adivated
`and may c.~<1rry out variom; !"unctions. La some situations, the
`cytoplasmic 1portion is autophosphorylated or phospbory(cid:173)
`lated, r~ulti.rng in a drnagc in its activity. These events arc
`frequently coupled will1 secondary messengers, such as
`calcium, cycfo.: adenosine monopbospbate, inositol phos(cid:173)
`phate. diacylglyceroL ancl the like. '!be bintUng of lhe ligand
`to tbe surface membrane receptor results in a particu!Rr
`sigoal being 1!ransduced.
`[0004) For T-cclls, engagement of lhc T-ccll receptor
`(TCR) alone is not sufficient lo induce persistent activation
`of re-sting naive or memory T cells. Full. productive T cell
`activation requires a second co-slimulatmy signal from a
`competent ant igen-proscoling cell (Al'C). Co-stimulation is
`achieved natu rally by lbe interaction ol' Lhe co-st irnufolory
`cell surface receptor on tbc T cell wilb the appropriate
`counler-reccp•lor on the surface of the APC. /\n APC is
`normally a cell of host origin which displays a moiety wh ich
`will cause the s timulation of an immune response. !\PCs
`include mono-cyte/macrophngcs, de ndritic cells, U cells, and
`any number c,f virally-infcclcd or tumor cells which express
`a protein oo Lbcir surface recogo.il!1:d by T cells. To be
`immuoogcni~· APCs must also express oo tbcir surface u
`co-stimulalory molecule. Such APC~ are capable of s1imu(cid:173)
`la1ing Tcell proliferation, induci.ng cy1ok.im: production, and
`acting as targets for cytolytic T cell~ upon dirt:ct interaction
`with the T <:ell. See Linsley and Ledbellcr, Ann. Rev.
`lmmuaol. 4:191-112 (J993); Johnson and Jenkins, Lifo
`Scic-nccs 55:1767-1780 ( 1.994); June cl al., lmmunol. Today
`15:321-33 1 ( 11994); a nd Mondino and Jenkins. J. T.c uk. Biol.
`55:805-815 ( 1994).
`[0005] Eng,agemeot of
`the co-stirnulalOry molecule
`togt:tbt:r w ith the TCR is necessary tor oplimal levels of IL-2
`procluctioo, proliferation and c:looal expansioo, Rod genera(cid:173)
`tion o r effeck:>r funclions such (t!. the production of immu(cid:173)
`noregulatory cytok:iaes, induction of antibody rt:sponses
`from R cells, and induction of cytolytic activity. More
`importantly. (~ogagcme11t of the 1'CR in the abscrn.:c of the
`co-st imulatory signal results in a state of non-responsive(cid:173)
`ness, called 3taergy. Aaergic cells fail to bt:comc activated
`
`upoa subscq11co1 stimulalioo lhrougb lhe TCR, even LO the
`presence of co-stimulation, and in some cases may be
`induced to dia by a programmed self-destruct mechan ism.
`(0006] La 1.-'Crtaio situations, for example where APCs Jack
`the coua ter-reccptor molecules necessary for co-stimula tion,
`iL would be benelicial to bave lhe co--slirnulatory i;ignaJ
`induced by virtu.e of employing a Jig.ind other than the
`aalurnl ligand for the co-stimulatory receptor. This migh t be,
`l'or example, lhe snmc ligand as thal recognized hy tbe l'CR
`(i.e., lbe same moiety. such that if o ne signal i'> rec,:ived.
`both signals will be received), or another cell surface mol(cid:173)
`ecuk known to b.: preseot un the target cells (APO,).
`
`[0007] Sew,al rec1:ptors that hav.: b1:el'l r1:peirted to pro(cid:173)
`vide co-stimulation for T-cell act:ivalion, including C'D28.
`OX40, CD27, CD2, CDS, IC AM-J . LFA- 1 (CD1 la/CDL8),
`and 4-188. lnc signaling pathways utili1.cd by lhcsc co(cid:173)
`stimulatory molecu !es share the common properly of acting
`in synergy with !he primary T cell rece ptor activation signal.
`(0008) Previously tbe signaling domain of CD2S has, been
`combined wilb Lbe 'J'.ceU receptor to form a .:o-st irnullatory
`chimeric rocaptor. Sec U.S. Pat. No. 5,686,281; Geiger., T. L.
`et al., Blood 98: 2364-2371 (2001); IJombach, A e t aJ., J
`f mnmnol 167: 6123-6131 (2001); Ma her, J. et al. Nm• Bio(cid:173)
`redm.ol 20: 70-75 (2002); l laynes, N. M. et nl., ./ luQ111mol
`169: 5780-5786 (2002); Haynes, N. M. ct al., Blood 100:
`3155-3163 (2002). These co-sLi111ulat<iry receptors provide a
`signal that is synergistic with the primary effector activa1lon
`signal, i.e. the TCR sigoal or the cbimeric effector function
`receptor signal, and can complcte the requirements [or
`aclivation under conditions where stimulation of the TCR or
`chimeric uffecl0r function receptor is suboptimal a nd 1might
`otherwise be detrimental lo the fu nction o f the cell. These
`receptors can supporl lmmune responses, particularly of T
`cells, by permitting the use of ligands other than the natural
`ligand to provide the required co-stimulatory s ignal.
`[0009) Chimeric receptors tbat contain a CD19 specific
`s111gle c hain immunoglobulin exlracellular domain have
`been shown to Jysc CD 19+ target cells aod eradicate c'Dl 9+
`8 cell lymphomas eografted in mice [Coop1:r L J, <:l al..
`Oloot! 101:1637-1644 {2003) and Brentjeas R J. t:I al.,
`Nature Medicine 9:279-286 (2003)). Cooper c t al. reported
`that T-cell clones lransduced with chimeric receptors com(cid:173)
`prising anti-CDl9 scFv a nd CD3!;, produced approximately
`80% specific lysis of 8-cell Jeu.kemia ,LDt! lyrnp lmm a cell
`lines al a 1:1 effector lo target ratio in a 4-bour Cr mleasc
`assay; at Ibis ratio, percent specific lysis or o ne prtLmary
`B-linenge AL L sampfo tested was approximately 30%.
`Brcntjtms et al. reported that T-cells bearing anti-CDl91 sc.fv
`a.ad CD3!;, chimt:ric receplors could be greatJy expanded in
`the presence of exogen01J;; 11,- I 5 and arlificia I an11igca(cid:173)
`presuntiag cells lransduced with CD l 9 and CO80. The
`authors showud that these T cells significaotly improved the
`survival of immuoodef.icit:ot mice t:ngraftt:d with the~ Raji
`B-ccll lymphoma cell line. Tbeir results also coofirrui:d tbe
`importance of co-stimulation in maximi:d.ng T-cell-mediated
`aoli-lcukeruic activity. Ooly ce[L,; expres.-;ing the 87 ligands
`of CD28 elicited effective T-cell responses. Th~-;; could be a
`major obstacle in the case of B-lineage ALL because leu(cid:173)
`kemic lymphoblasts typically dn not express B7 mole,;;ules.
`(0010)
`In addition to T <.:eU immune respoose~, oatural
`killer (NK) cell responses appear to be clinically reh:nnl.
`WllilC T cells recoguit.e tumor as.~ociated peptide antigen
`
`Miltenyi Ex. 1003 Page 5
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`

`

`US 2005/0113564 Al
`
`May 26, 2005
`
`2
`
`expressed on surface l-fLA class 1 or dass II molecules.
`antigen nonspecific immune responses arc mcdi,1tod by NK
`cell~ lbat ,trc activated by Ibo failure 10 recognize cogoa1c
`"self' Hl, A class I molecules. The graf1-versus-n1mor effect
`of trnosplnots using HLA matched donors is mcdi!lted by
`antigen specific T coils, while tr!lnsplantation using HLA
`mismatched donors can aL<;0 lead to donor NK cell,-; with
`polcot anlitmnor nclivity, 111..J\ mismatched bap!o-identical
`transplants CJJ1n exert a powerful aoli-leu.kernia effect bru;ed
`oo c>.'Pansion of antigen nonspecific donor NK cclL5,
`[OOll]
`lmmuooU10rapywi1h NK cells has heen limited by
`the inability 110 obtain sullicieo1 numbers of pure NK cells
`suit.tble for manipulation and expansion. !'be cstablisbccl
`method:; for cell expaasioo (avQr T cell expaasioo aad even
`after T cdL~ are depleted, residual T cells typically be<.:0Olt!
`pmmineol aft.er stimulation. 11,us there is a need for belier
`mclhods to tlxpand NK cells from n population without
`expanding T cells.
`SUMMARY OF THE INVENTION
`((Hl l2) The pres1.1n1 invention provides a chimeric receptor
`contttiniog a co-slimulatory s~nal by iocorpurnlion oI the
`signaling do1rnain of the 4-1 rm receptor. The chimeri.:
`rnceptor comprises an ..:xtmcdJular ligand binding domain,
`a 1ransmetnhranc domain und n cyloplai,mic domain wherein
`tl1e cytoplasmic domain comprises the signaling domain of
`4-1 BB. ln orne embodiment of the invention the signaling
`domain of 4- nm used in !he chimeric rccoplor is or human
`origin. lo a preferred embodiment. buman 4-lBB consists or
`S EQ ID N0:2. In another embodiment the signaling domain
`<.:umpr:iscs amino acids 214-255 of SEQ lD N0:2.
`[0013]
`In ainother embodiment of the invention the cyto(cid:173)
`pl;1smic uomain of the chimeric receptor comprises !ht:
`signaling domain of CO31; in addition to the signaling
`domain of 4-lBJl. ln another cmbodimenl the cxtracellu lar
`domain comprises a single <.:buin variable domain of aa
`anti-CO-I 9 m,~noclonal ,intihody. In another emlwdiment the
`transmembra.11e domain comprises the hinge and traosmem(cid:173)
`bra □e domains of CD8o,. In a most preforred emhodime□ t or
`Lbe invent ion the exlracellufar domain t.'Omprises a single
`<.:hain varioble domain of an anti-CD 19 monoclonal anti(cid:173)
`body. lhe 1ransmembrane domaiJJ comprises the hinge and
`transmcmbnu1c domain of CD8a, and the cytoplasmic
`doma.ia comprises the signaling donmin of CD3~ and Lbe
`sigoaliog doooain of 4-lBB.
`[0014] O1b1:r aspects of the invcutiuu include polyoude(cid:173)
`olide sequenc:es, vectors anu bost <.:ell,; llnooding a chimeric
`receptor LhaL ,compriSt:s the signaling domain of 4-lHH. Yet
`other aspects include me I hods of enhancing T lymphocyte or
`natural killer (NK) cell aclivity in ao individual aod trcaling
`ao individu11I suffering from cancer by introducing inlo the
`individual a T lymphocyte or NK cell compr.ising a cb..imcric
`rcceplor that co,npriscs the signaling domain of 4- t BB.
`These aspects particularly include the lrca1meot of lung
`cancer, mcla aoma, breast cancer, pros talc cancer, colo n
`cancer, r<loal cell carciaoma, ovarian cancer. nuumblaste1rna,
`rhabdomyosarcoma, leukemia aad lymphc,ma. Preferred
`t;a□cer target~; [or USt: wilh the present inv1:ntion art: cancers
`of B cell orig:in, partfcularly including acute lymphoblastic
`leukemia, B-,:eJl chronic lyrnphocytic leukemia and B-cell
`non-Hodgkin's lymphoma.
`[0015] Adiffcn:01 bu1 related nsµcct of the prescm inven(cid:173)
`tion provides a method for obtaining an t:nriched NK cell
`
`population suitable for transduction witb a chimeric ret:eptor
`that comprises 1hc s ignaling domain or 4- 1 HR. This method
`c.:ompriscs 1be expansion of NK cells within a mixed 1popu(cid:173)
`lutioo of NK cells and T cells by rn-culturiog tbe m ixed
`populntioo of <.:ells with a c;:ll line Lb:u a<.:tivatcs NK c..-eUs
`aod not T lymphocytes. This NK activating cell Linc is
`composed of cells that activate NK cells, but not T lympbc1-
`cytes, :11;d which cxpruss mcmbr,u1c bound iotcrlcuJ\ in-15
`and 1l co-stimulatory Caclor ligand. In it parliculru- embodi(cid:173)
`ment tbc NK activating cell line is the 1<562 mycloid
`lcu.ke01in cell line or lbe Wilms tumor cell line IJP\VT. In
`another embodiment of the invenlioo the co-stimullatory
`factor ligand is CD137L
`
`DESCRIPTION O17 111E SEQUENC'..E LISTING
`[001 6) SEQ ID No. I is the oucleo1idesequence for buma□
`4-IBR mRNA. The coding sequence for the human 4-IHB
`protein begins at position 129 and ends at positfon 893.
`
`[0017) SEQ ID No.1 is the amino acid sequence of lw.man
`4·.lBB. The signaling domain begins :it position 214 and
`ends al position 255.
`[0018) SEQ. ill. No. 3 is tbe oudt:otide sequence for
`n111ri ne 4-18B m RNJ\. The coding sequence for tbe murine
`4-1 BB protein begins ut position 146 and ends al position
`916.
`[0019) SEQ ID. No_ 4 is the amino acid sequence of
`rnurioc 4-l BR Toe signaling domain begins at position 209
`aau eod5 at position 156.
`
`DESCRIPTION OF TIIE FIGURES
`
`[0020] FTC. 1 is a schemati(; represent~tion of the CD19-
`truncatcd, CD I 9-l;. CO 19-28-1; and CD 19-BB-t receptor
`coostrncts.
`[0021) FIG. 2 silows tbe percent of CD19-positivc leuke(cid:173)
`mia cell recovery in four dill'crcot cell lines (380, 697.
`KOPN-S7bi and OP-1) after 14 bours of cullurc witlb NK
`ce-lls witb or without n chimeric ret.-epLOr at n l :I
`ralio
`relative to cultures wit b ao NKcells. Tbe b!IIs represen1L each
`of the 4 cell lines lhat nrc co-cultured with NK cells
`containing either ''vector'' which is MSCY-IRES GFP only;
`··trnoc.'' whicb is vector cootainiag truncated anti-CD I 9;
`"l;." wbicb is vector .;ootaioiog anti-CD19-CU34; "28t'
`wbich is vector cootaini.ag aoti-CDl 9-CD28et-CD3l;; or
`"BB-!;;,. which is vector cuntai.ning anti-CD 19-4-1 BB intra(cid:173)
`cellular domaio-CO3l;, !'his figure s hows 11:iat chimeric
`receptors wafer anti-ALL activity 10 N1< celL5 which is
`improved by the addition or 1h1.: co-stimulatory molt:eLLles
`CD'.28 or 4-lBB.
`
`DETAILED DESCRIPTION OF TIIE
`INVENTION
`
`(0022] Dclioilions
`[0023) 4-1 BR: 111e term '·4-1813'' refers to a membrane
`rnceptor protein also termed CD137, wh.icb is a member of
`the tumor necrosis factor receptor (TNPR) supcrfa.mily
`expressed on tbe surface of activated T-cell~ as a typt or
`accessory molccuk [Kwon cl a l., Proc. Nu 11. Acad. Sci. USA
`86:1963 (1989); Pollok ct al .• J. lmmuool. 151:771 ( 1993)].
`4-l BB bas~ molcculur weight of 55 kDa. and is fouod as a
`bomod.inu:r. h has bee□ suggested tbaL 4-lBU mediates a
`
`Miltenyi Ex. 1003 Page 6
`
`

`

`US 2005/0113564 Al
`
`May 26, 2005
`
`3
`
`sigoal 1ransduc1ion pa1bway Erorn omside of the cell 10
`inside [Kim 1:1 al., J. lmmunol. IS i : 1255 (1993}].
`[0024] A bu1man 4-lBB gem: (SEQ 10 NO: 1) was isolated
`from a cONA.librnry m;ide from aclivnlod bumaa peripbernJ
`T-ct:11 mRNt\ (Goodwin el al., Eur. J. lmmunol. 23:2631
`(1993);]. Tbe :1111.ino add sequeoce of bumaa 4-lBB (SEQ
`m NO: 2) shows 6M'11 homology lo mouse 4-1 RB (SBQ ID
`NO:4)[Kwon et al., Proc. Nall. Acad. Sci. USA 86:J 963
`(1989); Gen l3ank No: NM_ 0 J 1612) whicb indicates tbn(
`!he sequence:s are highly conserved. As men1iom:d supra,
`4-1 BB bcloog,s 10 tbe TNFR s uperfarnily, along witb CD40.
`CD:17. TNFR-1, TNFR-U, Fas, and CD30 [AJdcrsoa et al.,
`Eur. J. lmmunol. 24:2219 (1994)), When a mouoclooal
`antibody is hound to 4-J BB expressed oa the surface of
`mouse T-cclls, aati-C03 T-ce1l activatioa is increased maay
`fold [Pollok ,it al.. .I. lmmunol. 150:77 1 (.1993)].
`[0025) 4-l B B binds lo :thigh affinity ligand (4-1 BBL, also
`tero,ccl C D137L) expressed on several antigen-presenting
`ceUs sucb as macrophages ttnd activated H cells [Pollok cl
`al., .I. lmmunol. 150:771 (1993) Scbwan ot al., Blood
`85:1043 (1995)). 4-lBUL is cl:iirned aad describt:cl in U.S.
`P:1l. No. 5,674,704. The inl~raction of 4-lDB :md its ligand
`provides a co-stimLLlatory signn l leading lo T cell uclivatio n
`and growlh l[Gooclwin et al., Eur. J. Jmmunol. 23:2631
`(1993); Alderson ct al., Eur . .I. lrnmWJol. 24:22L9 ( 1994);
`Hurtado cl al.. J. lmmuool, 155:3360 ( l 995); Pollock e1 al.,
`Eur. J. lmnmnoL 25:488 (1995); DeBcncclcttc et :11., J. fa"Jl.
`Med. 181:985 (J 995)]. These 0bScrvations suggest an
`important rohi for 4-IBB in lbe regulation ofT cell-mediated
`iromuae re~ponses [ Ig nacio ct al.. Nalurc Med. 3:682
`(1997)).
`[0026) The term TL-15 (interleukin 15) refi:rs lo a cylokine
`that st1mulah:s NK cells [fehniger T A, Caligiuri M t\.
`B lood 97(1 ): 14--32 (2001)). Tl ha~ hccomc ,1pparenl that
`l L-15 presented through cell to cell contact has a higher NK
`s1imula1ing aclivily tban soluble I L-15 [Dubois S, el al.,
`lmmuaity 17(.5):537-547 (2002); Kobayashi 11, el al., 13lood
`(2004) PMID: 15367431: Koka R, ut al., J
`lmmunoJ
`173(6):3594-.3598 (2004); Burkett P R, et al., J Exp Med
`20(~7):825-S:34 (2004)]. "fo express membrane-bound lL-15
`a tonstmct 1..-oosisting of human IL-15 matu re peptide
`(NM l 72J 74) was fused 10 tile signal peptide and transmum(cid:173)
`hrane clomai~, or human CD8o..
`[0027) To specifically expand NJ( cells means to culture a
`mixed pt1pul11tion of cells that tt,ntaias a small number of
`NK cells so tbal lhc NK cells proliferate: lo numbers grealer
`thaa other cell types in the p0pLLlation.
`[0028] To ac1ivu1e T cells and NK cells means to induce a
`change in th,::.ir biologic s tate hy w hich tbe cells express
`ac1iva1ion markers, produn' cylokincs, proliferate and/or
`become cylolox-ic to target cells. A ll these changes can be
`produced by primary stimulatory signals. C'o-stiruu lalory
`signals ampli.fy tbe magnitude- of lbe primary siguals and
`suppress cell tlentb following iai1i11l s timulation resultiag ia
`a more d umb le activation slate 1111d 1bus u big her cy1otoxic
`capacity.
`[0029) T he
`lymphocyte are inler(cid:173)
`terms T-cell and T
`c ba ngcahle a1ncl used synon ymously herein.
`
`[0030) The 1erm •·chimeric receptor" as ust:d hereia is
`defined as a c,::.11-surfocc receptor comprising an ex1rncellular
`ligand binding domain, a lraosmembrane domaia and a
`
`cytoplasmic co-s1irnula1ory signaling domain in a combina(cid:173)
`tion that is not naturally found 1ogc1lwr on a single protein.
`This particularly iac\udes receptors wherein tbe c;1.1ra,collu(cid:173)
`lar doma in and the cytoplasmic domain arc not nallurally
`found together oo a single receptor protein. Tile chimeric
`receptors of' lbe presea t invention are intende<l primarily for
`use wi1 b T cells a.ad aatu raJ killer (NK) celJs.
`
`[0031) TI1.c term ;,host cell'' rncaos any cell of any <J,rgan(cid:173)
`ism that is selected, modi fied, transformed, grown, u~:ed or
`manipulated in any way, for tbe production of a 1;ubstanicc by
`the cell, for exaraple the expression by tile cdl of a gene. a
`DNA or RNA st:quencc, a pro1ein or an enzyme, Host cells
`of the present invention include T cells and NK cells lhat
`ccmtaia 1be DNA o r RNA sequences eococliag the cbi meric
`receptor and expn:ss the chimeric receptor on lhc cell
`surface. I lost cells may be llst:d for enhaacing T lymphocy1e
`activity, NK cell activi ty, lrealmenr of cancer. and treatment
`of au toimmuae diseases.
`
`[0032] Tbe tcmJs "express" and "expression" meao ~ I low(cid:173)
`ing or ca.using lbe informalioa in a gene or DNAseq1ucnce
`to become manifest. for example producing a prote.in by
`activating the ccllulttr functioas involved in tra.oscr1i111ion
`and tran1,la1ion of a corresponding gcae or DNA scqu1encc.
`A DNA sequence is expressed ia or by a cell lo form ~n
`··cxpres1;io11 product'' such as n p rotein. T he expression
`proclucl itself, e.g. the resu lting prmein, may also be sa.icl 10
`be "expressed" by the cell. An expression product can be
`cbacacterizccl as intracellular, extra ccllulnr or Lraas1n1e01-
`brane. The term "intracellular•· means something tlla1 is
`inside a cell. The lerm "extracellular'" means something that
`is outside a cell. ·1110.: term lt'aasmcmbrane means somt,1hing
`Iha! has an exlracellul3r domain outside the cell, a portion
`embecldt:tl in the cell membrane and an intracellular clc,main
`inside lhc cell.
`
`(0033) Tbe term "transfoction" means tbe iatroduction of
`a forciga □uclcic acid iato a cell using rccombinaal DNA
`technology. 'lbe term •·traasfonnati.oo" means !be inl~ncluc(cid:173)
`tioo of a ''foroiga" (i.e. extrinsic or exlraccUuJar) gene, DNA
`or RNA seq uence to u host cell, so that the host c.-ell will
`express lbe iatroducecl gene or seq uence to produce a
`desired s ubstance, lypicnlJy a protein or enzyme cocl,~d by
`the iotroducccl gene or sequeoct:. T he inlroduccd gene or
`sequence may also he cal.led a "cloned" or "foreign•· gene or
`seq uence, may include regulatory or control seque nces,, sucb
`as stan, stop, promoter. signal. sccrc1ion, or other sequences
`used by a cell's genetic rn:tchim,ry. The gene or seq,ueace
`may include nonfuoctional sequences o r sequences wiitb oo
`known function. A hos1 cell tha t rec~ives a nd expresses
`in troduced DNA or RNA has been "lrnnsformcd·· and is a
`"transformant" or~ ·'clone." ·n1e DNA or RNA introduced
`10 a host cell can come from any source. including Ctl lls c,f
`the same genus or species :is the host cell, or cell~ c,f a
`diJferen L g.:nus or :;pccics.
`
`[0034} Tb.e term "transcluc1ion" means the in1roduc1i.on of
`a foreiga aucleic acid ioto a celJ usiag a viral vector.
`
`[0035} Tbc terms "vector", ·' cloning vector .. and "cKprc;;(cid:173)
`sion vector" mean the. vehicle by wbicb a ONA or RNA
`sequer1ce (e.g. a foreign geoc) ca n be introducud into ,i host
`ceJL so as to transform 1bc bost and promote cxpression (e.g.
`transcrip1ioo and translation) of tbc introduced scq\11cncc.
`Vectors include plasmids, phagcs. viruses, e tc.
`
`Miltenyi Ex. 1003 Page 7
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`

`US 2005/0113564 Al
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`
`4
`
`DESCRJPTJON OF THE JNVENTl ON
`
`[0036)
`In accordance with 1hc prcscnr invcmion 1hcre may
`be employed ,rnnventionaJ molecular biology, microbiology,
`and recombinant DNA techniques witbin tbc skill of tbc art.
`Sucb lecbniques are explained fuUy in tbe literature. See,
`e.g., Sambrook et al, ·'Molecular Cloning: A Luborutory
`Manual" (19H9); ''Current Protocols in Molecular 13iology"
`Volumes 1-ITT [J\usubel. R. M., ed. ( 1994)); ''Cllll Biology:
`A Laboraiory lla.adbook" Volumes 1-0I [J. E. Celis, ed.
`( 1994))); "Current Protocols in Immunology" Volumes J-]l I
`[ Coligao,J. E .. ed. (J994)]; "Oligonuclcotide Synthesis" (M.
`J. Gait ed. l984); "Nudeic /\1,;icl llyl:>ridizatiun"(IJ. D.
`I lames & S. J. lligi,,,ins eds. (J 985)): "Trunscrip1ioa And
`Translation"[l:3. 0. llami;;s & S. J. Higgins, eds. (1984)];
`·'Animal Cell Cuhure''[R. I. Freshney, ed.( ! 986)); "Immo(cid:173)
`bilized Cell,; And Enzymes"[IRL Press, ( 1986)); B. PerbaJ,
`'·A Practical Guide To Mo lecular Cloning" ( I 984); CUR(cid:173)
`RENT PROTOCOLS IN fMMUNOLOGY (J. E. Coligan,
`A. M. Kn1isbcck, 0. H. Margulies, E. M. Sbcvach and W.
`Strober, tids., 1991); ANNUAL REV!.EW O F lMMUNO.L.r
`OGY; as well as wonographs
`journals such as
`ia
`ADVANCES IN LMMUOLOGY. All pateats, patent appli(cid:173)
`c3tions, and p,ubl ications menLioned herein are hereby incor(cid:173)
`porated herei a by refereoce.
`[0037] Primary T celli, expressing chimeric receptors spe(cid:173)
`cific for niroor or viral antigens bave considcrnble therapeu(cid:173)
`tic potcnti:i.l a.s immunotbcrapy reagents. Unfortunately,
`their clinical value is limited by their rapid loss of function
`and failure to expand in vivo, presumably due 10 tbe lack or
`co-stimul ator molecules on tumor ccUs aod the inherent
`Limitations 01[ s ignaling exclusively through 1he chimeric
`rece11tor.
`
`(0038] The chimeric receptors of the prcscnl invenl ion
`ovcrcomt' thi:s limitation wherein 1hcy have the capacity to
`provide both tbc primary uffcctor ac1·ivity and 1he co-stimu(cid:173)
`latory activity upon binding of the receptor to a single
`ligand. For in1stam:c, binding of thu anti-CD 19-Bll-l; ruccp(cid:173)
`tor to lhe CO II 9 ligand provides not only the primary effector
`function, but also a proliferative and cytolytic ell.eel.
`
`[0039] TceLls transduccd with anli-CDJ9 chimeric recep(cid:173)
`tors or tbe presen1 ioventioo which contain co-s1imul01ory
`m0Jccules have rcm:1,kablc anti-ALL capacity. I l0wever, the
`usi;; or allogcnic receptor-modified T cells after bemaiopoi(cid:173)
`etic cell transplamation might carry the ri,..;k of severe
`graft-versus-host diSt:aS<: (Gvl!O). In this :;citing, the use or
`CD3-0l:gativ<, NK et:lls is attractive becaUSll 1hcy ari;; oot
`eiqiected tocmuse GvllD.
`
`[0040] Studlit:s suggest an anti-tumor effect of NK cells
`and Zeis et al., Br .1 H;1cm,11ol 96: 757-6 1 ( 1997) have shown
`in mice that NK cells contribute to a graft-vt:rsus-Jeukemia
`effect, without inducing Gvl-1O.
`
`[(N)4l] Obtaining an enriched population of NK cells for
`use in therapy has been difficult to achievti. Specific NK c:eU
`expa nsion has been problematic lo achieve wilb establishc:cl
`methods, wb,:rc CD3+ T t-ells prcforeotially oxpancl. Ewa
`al'lcr T ccU depiction, residual T cells lypicaJly become
`prominent al'iler slimu.hl.tion. I lowever, in accorda□ct with
`the teaching~. of the presc:ot invention NK cell~ may be
`exp:indcd by exposure to cells tba1 lack or poorly express
`major his1ocl11mpatibilily complex I aocl/or II molecules and
`which have bei;;o genetically modified to express membiane
`
`bound IL-15 and 4-lBB ligand (CDJ37L). Such cell l.iocs
`include, hut arc not necessarily limited to, K562 [ATCC,
`CCL 243; Louio ct al., Blood 45(3): 321-334 (1975); Klein
`ct al., Int. .l. Cancer 18: 421-43L (J976)], and the Wilms
`tumor ci:ll line lffWT. [FehoigcrT /\, Caligiuri MA. JrntRev
`immu□o1 20(3-4):503-534 (2001); 1-larada H, ct al.,, Exp
`llematol 32(7):614-621 (2004)], the uterine eadometrium
`tumor cell Linc lllIU/\, the melanonio cdl line IIMV-11, the
`hepatc;,blnstoma cell line I lul 1-6, the lung smll.ll cell ,carci.(cid:173)
`noma cell lines Lu-J30 and Lu-134-A, the ncutoblai;toroa
`cell lines NTIJ 9 and NB69, the cmbryona) carcinoma cell
`line from testis N~Ct4, the ccrvi1- crtrcinoma cclll line
`TC0-2, and 1hc bone nmrow-mctasl~led ncurohlastnmia cell
`line JN8 I [ llarada 11., et al.,Jpn. J. Cancer Res 93: 31.3-319
`(2002)]. Preferably tbe cell line used lacks or p·oorly
`expresses both MHC I and II molt:cules, such as K562 and
`the HFWr cell lines.
`[0042] Exp3nding NK celb; which can then be lransfoct.ecl
`witb cl.timeric receptors according to this mclhod rcpn:scnts
`another aspcc1 of tbe present invention.
`[0043) The chimeric receptors o r tbe present inve:nlion
`comprise an extracellular domain, a lransmerobranc dciwain
`and a cytoplasmic domain. ·111e extract:lluJUI domain nnd
`transmembrane domain can be derivtlcl from any d«isiretl
`source for such doma.im;.
`[0044) As described in U.S. Pat Nos. 5,359,046, S,686.
`281 and Ci,103.521, !be extracellular domain may bi:
`obtained frcim any of the wide variety of extracc.llular
`domains or Sl!Cretcd proteins assuciati:d with Ligand binding
`and/o r signal transduction. The cxtracellular<lomain m ;1y be
`part of a prcHein which is ownomeric, homodimeric, het(cid:173)
`erodimeric, o r associated with a l~rger number of proteins in
`a non-covaleot complex. In particular, lhe 1:xtracdlu Jar
`domain may consist of an lg heavy chain which may i1l turn
`he covalently as.5ocialed with lg light chain by virtue of the
`presence of CHl and hioge regions, or rrrny become
`covalently associated with o ther lg heavy/ light chain com(cid:173)
`plexes by virlue of tbe presence of b.ingi;;, C H2 au<l Cl-13
`tlomaias. Ia the latter case, the heavy/light chain complex
`·lbal becomes joined to lbe dlimeric construct may constitute
`an antibody with a specificity distinct from the ant ibody
`speciticily of tbe chimeric construct. Ot:pending on tbe
`fuoc1ion o[ tbc antil)ody, the desired structure aod tbe Hignal
`transduction, tbt: entire cbain may be used or a trun catetl
`cbain may be used, where alJ or a part of tbc CH L. CH2, or
`C ID domains may be removed or all or part of tbe hinge
`region may be removed,
`[0045] Wherein an anLitumor chimeric rc:ceptor is utilized,
`the: lumor may be or any kind as long as it bas a cell surfactl
`notigea which nrny be recognized by th..: chimeric rec.:ptor.
`La a specific <.:mbodimcnL. !hi;; chimeric receptor may be for
`any cancer for which a specilic monoclona l antibody ,exists
`or is capable o[ being geoerntecl .. In panicular, cancers. such
`as ocuroblas1oma, small cell lung cancer. melanoma. ovarian
`cancer, ren:1! cell c-ardnoma, colon C3J.1cer. Ilodgkin's lym(cid:173)
`pbor)Ja, and childhood acute lympboblastic leukemia have
`antigens specilic J'or lhe c himeric receptors.
`[0046] The lransmembranc domain may be conlcibuted by
`the protein contribuling the multis-pecific extrncellulnr
`inducer clustering domain, the protein comributiag the
`clfoctor function .<;igoaliog domajn. th~ protein contributing
`th~ proliferation signaling portion, ur by a totally cliDfcrent
`
`Miltenyi Ex. 1003 Page 8
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`US 2005/0113564 Al
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`
`5
`
`pro1eiu. For I be mos1 parl ii will be convcnieol lo b:wc lbc
`transmemhrane domain naturally associaled wilh one nf the
`domains. ln i;<Jmo cases it will be desirable Lo employ !be
`or
`zeta.,
`.eta.
`of
`the
`transmemhra1Je
`domain
`1-ic.epsiloo.Rl .gamma. chains wbicb contain a cysieinc resi(cid:173)
`due capable. of disulfide bonding, so lbal tbc resulting
`chimeric prmein will be able to l'orrn tlisuU.itle linked dimers
`wilb itself, or witb unmodiJied versions of lhc zeta., .cla. or
`Pc.epsilonJl) .gamma. chains or related proteins. In some
`i