~A'DJr,llQ>\'J!H~!t{"~H!ES~ ~RiESE~1!l~ Smttffi,~l\I!Et:;
`UNITED STATES DEPARTMENT OF COMMERCE
`United States Patent and Trademark Office
`
`Mar ch 16, 2022
`
`THIS IS TO CERTIFY THAT ANNEXED HERETO IS A TRUE COPY FROM
`THE RECORDS OF THIS OFFICE OF:
`
`Pre-Grant Publication N umber: 2008/0131415
`Pre-Grant Publication Date: June 5, 200S
`
`By Authority of the
`Dueler Secreta ry of Commerce for Intellectual Property
`and Director of the l"nited States Patent and Trademark Office
`
`~#1-
`
`Certifying Officer
`
`Miltenyi Ex. 1011 Page 1
`
`

`

`I lllll 111111111111111111111 11111 1111111111 1111111111 11111 111111111111111 111111 1111111111111
`US 2008013 14 15Al
`
`(19) Uni1ted States
`(12) PatE!nt Application Publication
`Rid de:U et al.
`
`( IO) Pu b. No.: US 2008/0131415 Al
`Jun. 5, 2008
`(43) Pub. Date:
`
`(54) AOO P'TIVE T RANSFER OF C DS + T CELL
`C LONES DERIVED FROM CE'.1/T RAL
`MEMO RY C ELLS
`
`(76)
`
`Inventors:
`
`Stanley R. Rid dell. Sammamish.
`WA (US); C arolina Ber~cr.
`SealLle. WA (US): Michael C.
`Jensen, Sierra Madre. CA (US)
`
`Correspondence Address:
`MYRHS BIC1£L SIBU.1' & SA.JOVEC
`PO BOX37428
`RALEIGH, NC 27627
`
`(2.1) Appl. No.:
`
`11/870,776
`
`(22)
`
`flied:
`
`Oct. IJ, 2007
`
`Related U.S. Applk atioo Data
`
`(60) Provisioual applicariou No. 60/867.880. l:Jlecl on Nov.
`30, 2006.
`
`Pub lication C lassification
`
`(5 I)
`
`lnt. Cl.
`(2006.01 J
`A 61K. 351/J0
`ClZN 5//)6
`(2006.0J)
`(52) U.S. C J. .................... ,. ............. 424/93.71 :435/372.3
`ABSTRACT
`
`(57)
`
`The present invention provides u method of carrying out
`adoptive immunotberapy i.J1 a prim me subject in need rhereof
`by adminis1ering tile subject ,, cytotoxic T lymphncytes
`(CTL) p1-epamtion in a lrClltmcnt-ctll..-'Ctivc amount., The
`method comprises admlnistering as the CTL pteparul.ion a
`preparation c:011sis1i11g esseutially of au i.11 vitro expanded
`primate C"l'L population. lhe CTL population enriched prior
`to expru~io11 for cent.ml me111ory T ly111b1hocytcs. aml
`depicted prior 10 expansion of clleclor memory T lympho(cid:173)
`cytes. ln some e111hodiments, the method may further C()11J·
`prise couc11rrcntly administering lnterleukiu-15 to the subject
`in an amoum effective to increase the proliferation (1f the
`central memory T cells in the subject. Phan11aceutical fom1u(cid:173)
`la1ions produced by the method. and methods of usin,!:l the
`tiilfllC, are also described,
`
`coa-•co62L·
`
`c= __ :J
`
`PEPTIDE
`
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`
`APC
`
`CLONING
`
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`AND EXPANSION
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`t.CD19
`OR
`CD20
`
`TCELL
`CLONE
`
`1
`
`T EM DERIVED
`
`TcM DERIVED
`
`Miltenyi Ex. 1011 Page 2
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 1 of 24
`
`US 2008/0131415 Al
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`FIG. 1A
`
`Miltenyi Ex. 1011 Page 3
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 2 of 24
`
`US 2008/0131415 Al
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`
`Miltenyi Ex. 1011 Page 4
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 3 of 24
`
`US 2008/0131415 Al
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`Not I
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`
`Miltenyi Ex. 1011 Page 5
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 4 of 24
`
`US 2008/0131415 Al
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`Miltenyi Ex. 1011 Page 6
`
`

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`Patent Application Publication
`
`Jun. 5, 2008 Sheet 5 of 24
`
`US 2008/0131415 Al
`
`02269
`
`0
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`PEPTIDE (µg/ml)
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`FIG. 28
`
`Miltenyi Ex. 1011 Page 7
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 6 of 24
`
`US 2008/0131415 Al
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`
`Miltenyi Ex. 1011 Page 8
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 7 of 24
`
`US 2008/013141S At
`
`02269
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`Miltenyi Ex. 1011 Page 9
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 8 of 24
`
`US 2008/0131415 Al
`
`02269
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`Miltenyi Ex. 1011 Page 10
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 9 of 24
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`US 2008/0131415 Al
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`Miltenyi Ex. 1011 Page 11
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 10 of 24
`
`US 2008/0131415 Al
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`Miltenyi Ex. 1011 Page 12
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 11 of 24
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`US 2008/0131415 Al
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`Miltenyi Ex. 1011 Page 13
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 12 of 24
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`US 2008/0131415 Al
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`Miltenyi Ex. 1011 Page 14
`
`

`

`Patent Application Publication
`
`Jun. 5, 2008 Sheet 13 of 24
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`Miltenyi Ex. 1011 Page 19
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`Miltenyi Ex. 1011 Page 20
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`Patent Application Publication
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`Jun. 5, 2008 Sheet 19 of 24
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`

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`Patent Application Publication
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`Miltenyi Ex. 1011 Page 22
`
`

`

`Patent Application Publication
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`Jun. 5, 2008 Sheet 21 of 24
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`

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`Patent Application Publication
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`Jun. 5, 2008 Sheet 22 of 24
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`

`

`Patent Application Publication
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`Jun. 5, 2008 Sheet 23 of 24
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`US 2008/0131415 Al
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`Miltenyi Ex. 1011 Page 25
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`

`

`Patent Application Publication
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`Jun. 5, 2008 Sheet 24 of 24
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`US 2008/0131415 Al
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`Miltenyi Ex. 1011 Page 26
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`

`

`US 2008/()1314 15 A l
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`Jun.5,2008
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`ADOPirtVE TRA~SFER OF CDS + T CELL
`CLONES DERIVED FROM CENTRAL
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`REl ,ATI.lD APPt.1CA110NS
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`GOVERNMENT FUNDING
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`C!\18029, UOl!IL66947. and M01R.R00037 from
`National fnsti tutL"sOfl lcalth, The US Govemrncnl has ccrh1i11
`rights to this iinvcntion.
`
`FIBLD OF THP INVENTION
`
`[0003) The present invemion concerns methods nnd com(cid:173)
`positions for ,:urrying out adoptive iminunoth1.m1py.
`13ACKGROUND OF nm INVIlNTION
`[00041 Studlics in rodents have demonstrated lbat adoptive
`immtmotherapy with a.ntigen specific T cells is effective !or
`cfmcer and in.Jlections. and 1l1ere is evidence this modality bas
`therapeutic ac:tivi1'y i11 bumans1·'-'. For cli1rical appliCTJ1ions, it
`is necessary 1,,, isolate T cells of a desired mt.igen specificity
`or to eng:inee1· T cells lo express receptors that rnrgel infected
`or trans fonucd cells, and tJ1cn expand these cells .in cu llure9
`•
`1.1. l11e trai1slhofTcellclouei; is,,ppealingbecause ituwbles
`co111ml of spe,cilicity (md fuoc1ioo. and facil itatl'S cvaltmtion
`of in vivo persistence. toxicity nnd efficacy. Additionally, in
`the setting ol':allogencic stom cell transplantation. the admin(cid:173)
`istration to rccipiems ofT cell clones from tbe donor 1'hat
`targer pathog,ens or malignant cells can avoid grall-vcr.ms(cid:173)
`host disease that 01,curs witb infusion of unselected donor T
`cells:t.4,P, Hmvever, it is appareut from clinical studies that
`the efficacy of cultured T r.:ells. particuJarly cloned CD8 ... 1'
`cells, is frequently limited by their fai lure to persist ruler
`adoptive tr,111i;fcrt 6 • 1 ~.
`[0005] The pO<ll of lymphocytes from w hich CD8+ T cells
`for adoptive i111unu11otberapy crui be derived contruos naive
`and Jong-Jived. antigen experienced memory T cells Cl'M). T l.f
`can be divided rurther ioto subsets of ccmral 11Je111nry (T c.\,f)
`and dlector 1tnemory (f su) cells lhat differ in phenotype.
`ho111L1g properties and fi.tnction's, cos· T cM express
`C-D62L and CCR7, which promote migration into lyniph
`nodes. und pmlilc:rntc rapidly irre-exposed to antigen. cos·•
`Ti:M lack CDt62L. L'rlabling migration to peripheral tissues.
`and exhibit immediate effector l't111ction'9
`•
`[00061 In rc:sponse lo antigen stimulation. C'D8+ T cM and
`T~
`hoth diffe·rentinte into cy1'olytic e~1ector T cells (T ,;) that
`11
`express a high level of gr,mzymcs and perforin. but arc short•
`lived=0 • Thus., lbe poor surviV'dJ of'J' cells in clinical iminu(cid:173)
`noti1crapy trinls may simply result from their differentiation
`22
`during in vitro cuJlure lo 'f E lbat ure destined lo dic' 7•21 •
`.
`
`S UMM<\RY OP n-IE [NVBNTJON
`
`[0007] A firslaspect of Lite present invention is a method of
`t:arrying,olli ~tdoptive immunotherapy in a primate subject in
`111.1ed thereof by administering the subject a cytotoxic T Jym(cid:173)
`phocytus (CTL) preparation in a treatment-effective ,uuotml.
`
`The method comprises administering i,s the CTL prepairatioo
`a preparation consisting essentially of an in vitro oxp1111ded
`(e.g., grown in vitro for 1, 2. or 3 days up to 3 or 4 weeks or
`more) primateCTL population, the CTL population enriched
`prior 10 expansion for central mi!JJJory 'J' lymphor.:ytes. !lild
`depleted prior to expansion of effector memory T lympho(cid:173)
`r.:ytes. lo some cmbodimeuts. the 111ctJ10<l may further com•
`prise concurrently adm inistering lnterleukin-15 to the$,ibject
`in an amount eflective to increase the prolileration 111' the
`central memory T cells in the subject.
`(0008] A further aspect oflhe invention is a phamiuceutical
`formulation comprising, consisting essentially ofor consist(cid:173)
`mu of :lrt 1.11 vjtro expanded pri1m\lc cylt)t(Jxic T lymphocyte
`(c"'rL) populati~1l,. the CTL populatlou euriched prior to
`expansion for central memory T lymphocytes, and depleted
`prior to cxpuusion of effector memory T lymphocytes.
`10009] A further ,1spec1 ol' the invc111ion is the use, cit' a
`fornrnlatfoo as described herein for tbe preparntioo of a iniedi-
`1,amenl for c.irrying mn a method as described herein (e.g ..
`treating cancer or au infectious disc.1se in a human, m pri(cid:173)
`mate, subject).
`ln some embodiments the CTL preparatiou (or
`10010]
`population) is produced by the process of: (a ) collecting a first
`CTLpopulalion from a donor: (b)scparatinga CTLsub1popu(cid:173)
`lation enricbed for CDl;i2L • central memory T lympho cy1es
`and depleted of'CD62L • eflector memory T ly111pbocylle.o, Ill
`pmd11ce a central memory-.enriched CTL subpopulation: (c)
`expanding the central memory-eruiched CTL subpopu latioo
`in vitro in a culture medium ( e.g .. for l , 2. or 3 days up Hl 3 or
`4 wt-eks or more): lllld then (d) col lecting cells from the
`culrure medium to produce the CTL µrepara tion. fbc sepa(cid:173)
`m ting,stcp "b .. cnnin ~omccmbodimcnts bec<1rricd out by: (i)
`contacting the first CTL populution to anti-CD62L antibouy.
`wherein thc.•,m tibody is immobilized on a solid support. so thl
`ce1Jtrnl memory cells bind to tbe support: tben (ii) sepa1ratiog
`the support from the CTf population with central me,mnry
`cells bound tlicreto: (iii) and then separaling the c,entral
`memory cells from lbe solid support to produce the c,entral
`memory enriched CTL subpopulation. In some embodi(cid:173)
`ments. ·the expanding step "c" fonJ1er comprises .idminister(cid:173)
`ing Tntcrleukin-15 lo the central memory subpopuhitio11 iu
`vitro.
`In some embodiments. the central memo1y•en•
`[00 11]
`riched T cells are modified i11 vitn:, wit h at least ooe geoe that
`targets te.g., specifically binds to) c.ancer cells or other pathO•
`ge11ic cells in the sub_ject.
`
`BRlEf DliSCRl PT!ON ornrn DRAWJNGS
`(0012) FIG. 1. limlutiou and genetic modification ofCMV(cid:173)
`specific cos• 'f cell clones from CD62L- and C D621 - T
`cell subsets for adoptive transfer,
`[0013] FIG. l a. CMV TE-specific memory T cells arc
`presenl in both CD62L+ ~rnd CD62l- subsets or CD8+
`peripheral blood Jymphocyles. Pl3MC wurc sorted into
`CD62L-CD8+ and CD62L +cos· fractions a11er staining
`with anli-CD8 ru1d anti-CD62L m1ti budies (upper panels).
`111c CD62L- and CD62L+ fractious were stimuJalcd [or 6
`boors with autologous C'D40L-adivated B cells poised wit11
`medi w11 alMe or wilh C-MV-m peptide. Orelcldin 11\ was
`added for the [foal 4 hours and C'D8 .. T cells that prodluced
`lf'N-y were detected by stainiug for intracellular 1FN-y. The
`lower left panels show !FN-y production by the CD62L(cid:173)
`CD8+ subset and the lower right panels show IFN-y ptoduc-
`
`Miltenyi Ex. 1011 Page 27
`
`

`

`US 2008/()1314 15 A l
`
`Jun. 5, 2 008
`
`2
`
`tion by the CD62L +co~+ l.'Ubset. Data from macaque 02269
`is sl10w11 and jg represeuiative of CU'.IUl obtained iL1 four con(cid:173)
`secutive ruac;.1ques.
`10014] FJG. l b. Strat<!gy fonhe isol.1tiun ofCMV-$pecific
`cos+ T cell clones from CO62L-T,,M and ('()621.+ TcM
`subsets. .I\JiqLuots of P13MC were ob1;1ined from each
`macaque and sorted into CD62J .-CO8• and CD62I ·cns(cid:173)
`fractions allcir staining with anti-CDS and unli-CO62L anti(cid:173)
`bodies, The 1;orted T cells were cultured with ;n,1tologo11s
`mo nocytes pu1Jsed with U1eCMV IE peptide and utter 1 week
`of stimulation, T cells from the cultures were clo11ed by lim(cid:173)
`iting dil ution .. T cell clones were screened 10 idcnri1y lhosc
`th;it lysed amologous peptide-pulsed target cells. and then
`lransduccd wi1th a rctroviral vector that encoded a cell surfocc
`~CD! 9 or C'D20 molecule. lndividual T cell clones were
`expanded in vitro to >Sxl 09 cells by stimulation with anti(cid:173)
`CD3 and anti-('D28 antibodies in cull11ros supplemented with
`y-im1diated h uman PBMC and F.BV-LCI. prior to adoptive
`trarn;Ier.
`I 001 SJ FIG. le. Retro viral vector constructs encoding for
`macaque ceU s urface marker g.eaes. Abbreviations: Ml)SV(cid:173)
`L:T'R., myclopmlifcrative snrcoma virus rctroviral long tel11l.i(cid:173)
`nal repeal: ll'+. extended pnckaging signal: PRE, woodchuck
`ht:palitis virus posttanscriplionul
`regulutory eletuent;
`~CD 19, trunc:ated macaque CD 19 cDNA encoding. the c>.'1.m(cid:173)
`cellu lar and 1ransmembn1ae<lomains. and 4 aa of the cytoso(cid:173)
`lic tail; CDW. full-le1,g.U1 macaque CD20 cONA.
`(0016)
`(d) fmmunomagnetic bead seleclion of 6CDl 9 and
`CD20-1uoditled cos•--r cell clones. Transduced T cells were
`enriched lbr LICO 19 or CD20 expressing cells ,in day 8 after
`transduction using " two-step inunl111omagnetic scleclion
`wit h anti-CD 19 or anti-CD20 antibodies and rat anti-111ouse
`lgG coupled n11icrobeads. Aliqti.11s of unmodified (lcli panels)
`and tim 19 or CD20-modilicd T t·clls (right panels) were
`removed aJlcir selection. co-stained wil h anli-CD8 ,ind anti(cid:173)
`CDJ 9 or auti-CD20 antibodies. and analyzed by flow cytom(cid:173)
`etry to assess purity. The pcrcentugcs ofCDs·T .:ells 11osi(cid:173)
`tive for the transgene are indicatl.ld.
`100171 FIG.. 2. CMV-speciftc CD8+ T cell clones dcriv<xl
`fmrn Tc_.,andl TE,"'subsetsex.hibitan elfoctm phenotype. aud
`comparable avidity a nd pmlifcr:c1lion in vitro.
`[0018) FIG .. 2u. Individua l T CM and TE.w-derived macaque
`CMV-specific: CDs+ T cell clones ,vere exami11ed by flow
`cytoo1erry for expressio11 of C'D62L. CCR7, Cl)28, C'Ol 27,
`granzyme Band perforin (bold l ine) and stained with isotype
`control amib,xlies (doued line). Inset values represent the
`mean Jluores1:cnce iulensity (MFI). The data is shown for T
`cell clones tha11 were adoptively transferred to macaque 02269
`nod is rcprcse1ntativcofthe data fora II of the T cell clones used
`in adopt·ive trnnsl'er experiments in the three macaques.
`100191 FIG. 2b. Cytotoxic activity of ead1 pair of T,.,..f
`(filled triangl,e) and T , M (filled square) -dciived CMV-spo(cid:173)
`cifil; CD8 ... T cell clones ustxl in ndopliv~ lnrnsfor was exam(cid:173)
`ined in a 4-bour chrom.iuro release a~s.1y at ,lll effector to
`target nllio ol' 20:l using autologous targ~t .:ells pulsed witi1
`the CMV IB peptide at various concentrations. The sequences
`oJ' Lhe IE-1 peptides were KKGDIKDRV (02269) and El:il l(cid:173)
`VKf FPK (A'99l7l ). U,e sequence of the lE-2 peptide wa~
`ATil'lSLEYK (02258).
`[0020) FJG. 2c. In viiro growth or CMV-specific cos· T
`cell clones. Tbe T e,w ( filled triangle) and T CN (filled square)
`derived T cell! clones used for adoptive transter were stimu(cid:173)
`lated with a nti-CD3 ,,ud anti-ClJW in the presence ofy-im1-
`
`dimed feeder cells and IL-2 l50 LJ/ml). Cell growtb over 14
`days of culture was measured by com1ti11g viable cells using
`trypan blue excl usion.
`[00211 F IG. 2<1. "Jt'lomere length in CMV-speciflc T cell
`clones derived from CO621.• aud CD62L- subsets. The
`mediaa telomcre length M duplicate- samples was measured
`by automated flow-PlSTl in peripheral blMd T lymphocytes
`(shaded squares). u.1 the infused T cell clones (iT EM filled
`squares, iTrM open squares). and iu each of' two adcli tiooal
`randomly selected 'I eM (left hatched squares ) ond T 0 ,de(cid:173)
`rlved (right hatched sq uares ) T cell clones from eacb
`macaque.
`[0022]
`r 1G. 3. Persistence and migration c,fT.e:,,r1111d T0 ,,(cid:173)
`dcrived C l)8~ T .Ii clones in peripheral blood. bo11e m:mow.
`and lymph nodes lollowing adoptive transfer.
`[002'.\) FlGS. 3a-h In vivo persistence and migration of
`6CD 19-modi (loci T £t,rdcrived (a) and T ,,wdcrived (b) Teel I
`clones in macaque 02-269. Toe T cell clone.~ were rr.inslferred
`in sep::u·ate infusions given intravenously more than l O weeks
`apart at a cell dose of 3xl{)~/kg. Samples of PBMC were
`collcctc.'tl at intervals for 6-8 weeks a.Iler each infusion. Booe
`marrow and lymph node samples were colleoled before infu(cid:173)
`sion and fourteen days a Iler each T cell i111i.isio11. Samples
`were stained with lluorochromc-conjugalcd anti-CO3. CDS,
`and CD I 9 antibodies. re~1,cctively. The frequency of lr',ms(cid:173)
`ferred CD I 9+ T cells was detem1ined by flow cytomer.ry with
`gating oo CD3+C'D8+ cells. Lefi panels show the percen,t.ige
`ofCDS•T cells Umtexpressed CD! 9 in blood. buneimu-row
`and lymph uodes prior to ,tod at iolervals after infosiou ,ofcbe
`T,;:.,.,-derivl'<l (a) aod Tcif'dcrivcd (b) T cell clum:s. Rig.ht
`parttllS show 1he absolute numben, l\fCD 19+cu:,·cow T
`cells per ft\ of blood. This wns detcm1ined by calculating the
`ahsolll1c numberofCD3+C'D8+ T cells per pl of blood at 1he
`indicated days (% or CD3 ... CO8+ T cells in .u