11 United Stateg Patent
`1411113311 set 31.
`
`0.:
`
`(111) Patent N
`(45) Data 61' Patent:
`
`US 9,869,961 112
`Jan. 16., 2918
`
`S W986
`
`
`
`/’\Ur1.. \./
`
`4"\x} 'Jl 1.-
`
`911311101118 AND {IGRIE’USITIQNS FOR
`LOCALIZEB SECRETION 0F ANTlnCTLA—s't
`ANTIBODIES
`
`Inventor's: James Allismn New York. NY (:13);
`
`WIicI'iael Cia1'1'amNew Yet1,
`-IS)
`
`Assigned The Regents (if the University 01'
`Califflrnia. Oakland, CA (US)
`
`N01105:
`
`Suhiect to any disclaimer, the term (if this
`patent is extended or adjusted under 35
`11.81:. 154(k) by 2097 days.
`
`{913111.111}:
`
`12/294,889
`
`PCT 1:111:11:
`
`‘Mar. 39, 2997
`
`Pti'i‘ No.2
`
`PCT/U 829971997983
`
`1% 371 411,,
`(2,I, (4) Date:
`PCT P1311, No.1
`
`Oct. 29, 2011}
`
`“1020071123737
`
`PCT Put-.13
`Datc:Nav.1 2997
`
`131101“ Publication Data
`
`US 201141044953 Al
`US 2017/0114364 1419
`
`Feb. 24. 21311
`Apr. 27. 21.117
`
`(51))
`
`Reiatefl US. Applicatifln Data
`”’7
`Provisional application No. (BU/70.972. filed 011N131:
`30, 2006, pmvisinnfll application N1). 60/737,973,
`tiled on Mar. 31, 2006.
`
`(2006.01)
`
`lat. Cl.
`CIA/V 115/86
`U .S .
`CI.
`
`CFC
`C121‘Vl5/86 (2013.01)
`Field (11' Ciassuficatmn Search
`Name
`
`
`See application 17116 {Uroomph.Us search history
`
`(55,;
`
`Referee E31398 Cited
`
`U.S. PATENT DOCUMENTS
`
`5 ,978,996 A
`5 .998, 792 A
`5 ,4.36 146 A
`5 643,323 A
`5,6.37 ,433 A
`5 ,686 ,279 A
`5,731 199 A
`5,753. 599 A
`5,756.986 A
`5 ,,779442 A
`5,311,997 A *
`5,846, 767 A
`5,846,7' 2 A
`5 9 '
`
`
`
`5,935,299 A
`6,933,674 A
`6,949,183 A
`6,957,155 A
`
`1/1992 Conlon, 111 et al.
`3/1992 Zimmerman et 31.
`7/1995 Shenk at 31.
`8/1996 McClell3nd at 3‘1.
`6/1997 Dranofi‘ Ct 31.
`11/1997 Finer at 31.
`3/1998 Wickh3m 0131.
`5/1998 Shank at 31.
`5/1998 McClelland at 3.1.
`6/1993 ‘Wickham et 31.
`............. 424/1441
`9/1993 Allison et’ 31.
`12/1999 Halpin. e1 3‘1.
`12/1998 Wickh3m et 31.
`5/1999 Diana-ff e1 31.
`7/1999
`Roy
`8/1999
`Chatterjee et al.
`
`11/1999 Jaf‘fee et 31.
`3/2999 .3ffee et 31.
`3/2999 Ferrari 61 31.
`5/2999 \Vicltham e131.
`
`7/2999 Ferrari at 31.
`6,993.579 A
`9/2999 MacPhee et 31.
`6.117.425 A
`19/2999 Cryzstal ct 31.
`6,127,525 A
`2/2991 Panic“ at 31.
`6 18/,396 Bi
`12/2991 Cabilly at 31.
`6,331,415
`1
`2/2992 Jalfee et 31.
`6,359,445 Bl
`19/2992. Levitsky e1 31.
`6,464.973
`‘1
`4/2993 Samulski et 31.
`6.548.286 B1
`
`4/2993 Curiel
`6,5‘
`,368 131
`8/2993 1.01111 e13}.
`6,692,593 Bl
`9/2993 Ledbctter et 31.
`6,623,949 ,‘1
`1/2994 Curiel 4&1 31.
`6,683,179 B2
`2/2994 Yu et 31.
`6.692.736 B2
`1/2996 Koi‘man et 31,
`6,984,729 B1
`
`4/2996 Cai'ren
`et 31.
`7,934,121 132
`8/2997 Elle, Horn et 31.
`7,256,937 B2
`2/2999 bang at 31.
`7,485,291 152
`9/2999 (irihben et 31.
`7,592,997 152
`4/2911 Simrrmris at a}
`7,919.979 152 ’3
`......... 424/9.) 2 1
`8,196,992 B21/29128ghom‘ne et 31.
`299219939581 [11*
`4/2992 Carmen-:3 et al.
`............ 42411173 1
`299219161342 A1
`11/2992 “fang et :11.
`.. 424/ 131. 1
`,
`299419292659 Al * 19/2994 Grih‘ €11 61.31.
`2994/9298859 A1 *
`19/2994 Flienh-fim at 3‘1 ............
`4 24/93 .2
`29939992916 Al
`1/2995 31:14:11.5 at 31.
`299819914222 .41 *
`1/2998 Simimms at 3‘:
`8/2998 913130111116 et 31.
`21998191757513 Al "‘
`170 RE 1GN PAT ENT DOCUMENTS
`
`CA
`EP
`‘Nfi
`VV®
`W@
`Wfi
`
`2648967 A3
`2992263 Bl
`“’9 97/1 299 A1
`‘WO 99/39734 A1
`W’@ 99142535 Ai
`‘Wfi 99/31/594 A2
`
`11/2997
`6/2914
`5/1997
`8/1999
`8/1999
`6/2999
`
`(Cmitirmed)
`
`0111 ER PUBLICATIONS
`
`801123 et 31. (Brazilian Journal -Z?l1‘v1<fdi-Zal and Biological Reseaxcli
`
`(2995) 38: 599— 52?).
`van Y'1535 61" 31..
`19:91 130:: Med 199: 355-3(6. 4‘
`'
`‘113 at 31.,
`Enhanceitiem of 311111311101” Ammunity against 1316
`
`301113 11111101 using gmietintally modified denditic col 5
`to
`pl'uuuxlfi uytulfin-c‘ s.”
`Gene Theiapy, V01. 7, pp. 2113-2121 (2999).
`Allisufi, “8101;112:111: of T 12611 inhibitory signals: 3 new
`paradigm in
`iumm immuxw L116} 31w?"
`Camer lmmun. V01. 5(1), p. 9 (2995').
`Altschul 6131.. “B359: 10031 alignment 5631911 1001.” Mol. Biolyol.
`215(3), pp. 493-419 (19
`as).
`Anki 6131.,
`“Expression of 1111111113 intetieukin 7 in 3 murine glinma
`
`cell
`line 13.1
`PNAS vol.
`m in reduced i‘urnm‘igenicity in Viva."
`
`5411
`89(9), pp. ..859-3.
`992)
`Armstrong et .11.,
`“Cytokine modifi ed tum-fir v3cc mes . ”
`Clin. N Am vol.
`11(3). pp 681696 (2992)-
`Attia et 31., “Autoinuminity correlates with tumor regression in
`pail-6111:; with metastatic 111-2131101113
`treated with anti—cytotoxic
`113311111106er antigen-4." Clin. 611cc}. vol. 23(25). pp. 6943-6953
`(2995}
`Barber, 61 31.,
`
` ‘.€{.1 CD81" cells during,“Restoring 1imc1im1 in ex“
`chronic viral infection."
`Nature vol. 439(7
`pp. 682-681(2996}
`(CO nti timed)
`
`Burg. 9111031.
`
`Prim ar'v Exam inc-r
`
`1113 Ouspemski
`(74) Azmrmy Agent or Firm 44444 TOdd A. 11,.01'51121
`
`ABSTRAC'E‘
`(57)
`16336th invention mvides CO. n‘lOSltlfifiS 2111C} 126431130113
`111:?
`,
`1'1
` rid
`; 11.
`\1
`I?
`10? QTTQCHEQQVAU the 1013311255131 43X Ere/551101] 0,1
`NIL-A4
`mfiibady pmximal to a target tissue 111 a gatifiut.
`
`'14 Claims, 14 Drawing Sheets
`
`

`

`tis 9,868,961 an
`Page 2
`
`(5c)
`
`'WQ
`lit/Q
`W6
`V316
`W8
`Vi/Q
`'v’v’Q
`V’s/Q
`V1165
`we
`
`References Cited
`
`FOREIGN l’A’l‘EiT'l‘ DGCUMENTS
`
`“’6 66/6757-5 Al
`\VQ 66/72686 Al
`“WE fill/54732 Al
`"WE 93/666816 A2
`we 2%Mi’65886l A2
`Wfi 2664/65’3S3l A2
`“’8 2664/1645? A2
`\VE 2665/663298 A2
`‘WQ 2665/6l7l49 Al.
`EVE 2.1995/627966 AZ
`
`11/2666
`12/2666
`3/2661
`3/2693
`7/2664
`l 1/2664
`12/2664
`1/2665
`2/2665
`3/2695
`
`0TH ER PU Bl. lCA’l‘lONS
`
`
`‘ 'tionar PER L
`liatzer et al. ”Enhanced evo
`trig oligoniicleotide:
`‘ Nucleic Acit __
`with inosine at the 3'v—terrnin t,
`
`
`Res. vol. 19(i8), p.
`568]. (I991).
`Bett et al., “Packaging capacity and stability of human adenovius
`
`type 5 vec )rs." J. Virol. vol. 67GB), pp 591L592]. @993).
`Bluestonc ct al., “C ll.
`A4lg: bridging the basr
`c immunology with
`clinical application.” Immunity vol. 24 (3), pp. 233—2323 (2666).
`Bode); et al, “Failure of canceraaccines: the significant limitations
`of this approach to imin 'notherapy.” Anticancer Res. vol. 26(4). pp.
`266"—2676 (2996).
`Bossis and Chlorini, “Cloning of an aVian adeno—associated virus
`(AAAV) and generation of recombinant AAA‘J particles.” J. Virol.
`vol. 7711.12), pp, 67996816 (2663),
`Brunner et al, “C’l‘l.A—4—mediatcd inhibition of early events of ‘l‘
`
`c "l proliferation.” l
`ltrniiunol. vol. 16200) pp. 58136326 U999).
`
`C‘.
`rnbers et al,
`A-4-rnediated inliib
`n in regulation of T
`
`
`cell
`responses: mechanisms
`and manipulation
`in
`tumor
`innntinotherapy.” Annu. Rev. lrrnnunol. vol. 19, pp. 565-5943 (2661).
`Chang et al, “lmmunogenetic therapy of human melanoma utilizing
`autologous tumor cells transduced to secrete granulocyte--macro-
`
`phage colon
`' nulating factor.” Human Gene Therapy vol. l l, pp.
`839-856 (266%),
`Chaplin et at. “Production ol'interleukin- 12 as a self—process in 2A
`polypeptu‘le.” .t. lnterferon (.‘ytokine Res. val. l9t3), pp. 235-241
`(l 999),
`Chu and Sharp, “EV/46 DNA transfection of cells in suspension:
`analysi of the efficiency of transcription and translation of Banti-
`gen." G
`ol. l3(2), pp.
`l97—262 (1981).
`
`
`(norm
`.., “Identification and loc Elation ofnnpairerl cysteine
`residues in monoclonal antibodies by fiorescence labeling and mass
`spectrometry." Anal. Chem. vol. 6i, pp. 641191-64 57 (2669).
`Darrow et al. “The role of llLA class 1 antigens in recognition of
`
`
`melanoma cells by tinnor—spec'fic cytotoxic T lymphocytes. evi-
`dence for shared tumor antigens." J.
`lrtnnunol. vol.
`l42(9), pp,
`33296335 (1989),
`
`{le
`.lipe and lzquierdo, “Trieistronic and tetracistronic retroviral
`
`vectors for gene transfer." Hum. G ' Ther. vol.
`ll(l3), pp.
`l92l—l93l (299m.
`
`
`e from foot—and-mouth
`"pc ct al, Use of the 2A scqu
`
`virus in the generation of retroviral vectors for gene
`therapy.” Gene "liner. vol. 6(2), pp.
`l98—268 (1999).
`Donnelly et at, “Analysis of the aphthovirus ZA/Z'B polyprotein
`' mechanism indicates not a proteolytic reaction. but a
`
`novel translational etfect
`: a putative rilaosomal ‘skip’.” J, Gen.
`
`Donnelly et al .9 “Ti
`
`virus 2A
`directed mutants and n
`.
`.
`
`
`J. Gen. Virol, vol, 82(5), p
`1627-1941 (26m).
`segue
`
`
`Dranofl et al, “Vaccination with irradiated tumor cells engineered
`to secrete murine grantilocyte—macrophage colony—stimulating fac—
`tor stimulates potent, specific,
`'
`g anti-tumor immu-
`
`nity.” PNAS vol. 96(8), pp. 33'
`Dull et al.. “A third-generation lentivirtm vector with a conditional
`packaging system.” .I Virol. vol, 72(l1), pp. 8463—847 1 (1995).
`Hummer, “CA/AX (Cell Genesysl." Curr 8pm lnvestig Drugs. vol.
`2(6), pp, $44-$43 {266i}.
`
`Eniens ct al. “A phase 1 vaccine safety and chemotherapy dose-
`finding trial of an allogeneic GM—CSF—secretmg breast cancer
`
`vaccine
`given
`in
`a
`specifically
`timed
`sequence wi’li
`irnrnunomodulatory doses of cyclophosphmnide and doaorubicin."
`
`Hum. Gene Tn
`vol, 15(3), pp. 313637 (2%Q4).
`liearon et al., “interleukin-2 prodt ction by tumor cells bypasses ‘l‘
`helper function in the generation of an antiturnor rt ponse.” Cell
`vol. 66(3). pp, 397-463 U996).
`
`Fields Virology, 3rd Edition et’lilied lw EN. l‘ields et al., Lippincott
`
`Raven Publislie
`ch. 58, ' Atrovidae: the viruses and their rclica~
`
`tion,” (Classification, pp.
`l/f35~l'7'7'7, including Table l (1996).
`Finer et al., “kart ahigh-elliciencyretroviral transduction system for
`primaw human l‘ l 3r'mpliocytes." Blood vol. 830), pp. 43-56 ( U394).
`
`
`Gansbacher et al., “interleukin 2 gene transfer into tunic-r ce' 0
`
`
`blue;
`tes tun
`. city and induces protective immunity ’
`Med. vol. HA4), pp. l2l‘7--l224 (1996‘).
`Gansbacher et al., “Retroviral vector—mediated ganuna-interteron
`gene transfer into tumor cells generates potent and long lasting
`antlturnor immunity.“ Cancer Res. vol. 56(24), pp. 7826-7825
`(i999).
`(jar; et al., “Adeno-associated viruses undergo substantial evolution
`in primates during natural
`infections.” PNAS vol. ”3906), pp.
`66316686 (2663).
`
`Gao et al.. “Novel arieno-associ. ed viru'es from rhesus inonlte‘ys as
`vectors for hunan gene therapy." PNAS vol. 99(18), pp. 11854-
`] ”33739 (2663).
`Liolnmbeclc et al., ”treatment of established renal cancer of tumor
`cells engineer to secrete interleukin-4e.” Science vol. 254 (5 632), pp.
`'71'3—7l6 (1991).
`Graham et at. “A new technique for the assay of infectivlty of
`human ailenovirus 5 DNA”. Virology vol. 52(2), pp, 456-457
`(1973).
`Greenwald et al, “'ihe B7 family revisiterlf’ Ann. Rev. lmmunol.
`vol. 23, pp. 515—548 (2665).
`Grit‘eln et al., “Development and applications of deface—linked
`
`single chain antibodies against ilk-cell antigens”. l . 1
`incl. Meth-
`
`ods, vol. 248 (l -2) pp. 77-96 (266i).
`llalpin et al., “Self—processing ZtX-pOlYpIOKClHSW 2* system for coor—
`dinate expression of multiple proteins in transgenic plants.” Plant J.
`vol. 17(4) pp. 453459 (1999).
`
`vii uses in
`Hartley '
`tut-ally occurring murine leukc
`wild mice: characterization of a new ‘amphotropic’ class." J Virol,
`vol. 19(1). pp. 1925 0976).
`“interleukin 7 induces CD4 + T cell-dependent tumor
`
`
`Fxp. Merl. vol l74(fi), pp 1291—1298 (1991).
`l’lOGJ et al., Biologic activity of cytotoxic l lymphocyte associated
`antigen 4 antibody blockade in previously vaccinated metastic
`melanoma and ovarian carcinoma patients.” PNAS, vol. 16%. pp.
`47l2—47l7 (2663).
`Hom et al, “Common expression of melanoma tumor-associated
`
`
`
`antigen recognized by him
`turner
`intilt 'atin,
`lymphocyt‘s:
`
`analysi by human lymphocytes antigen restriction.” J lmniunother.
`vol, 16(3), p i. 153~164 (Milli).
`‘Irv[>3
`
`
`Huang et al. “Role of bone marrow—derived eel
`
`
`MHC class i
`stricted tumor antigens." Science vol
`9614965 0994).
`Huez et al., “Two independent internal ribosome entry sites are
`involved in translation initiation of vascular endothelial growth
`factor mRNA.“ [Viol Cell Biol. vol. 18(11), pp. ol’78u6196 (1995).
`Jackson and Kaminslti, “lnternal
`initiation of translation in
`eultaryotes;
`the picornavirus paradigm and beyond.” RNA vol.
`lth). pp, 9 ‘” 1666 (1995).
`
`.. kson et al
`The novel meclianisrn of initiation of picornavh'us
`
`
`
`RNA tran
`ion.” Trends Biochern. bttl. vol, 15(12), pp. 477—483
`(1.96).
`JalIee and Pardoll, “Gene therapy: its potential applications in the
`treatment of renal-cell carcinoma." Semin @ncol. vol. 2 (l), pp.
`81-91 U995).
`
`Jatfee et al.. “Cli
` 'iical protocal: a pl]: ”e l clinical trial of lethally
` irradi ‘cd allogeneic pancreatic tumor cells transfected with the
`
`GM-
`H gene for the treatment ot‘ pancreatic atlenorcarcinoma.”
`
`
`Hutn. Gene 'l'her. vol. 9 (13), pp.
`14971 (1993),
`
`in presenting
`164(5 lot), pp.
`
`

`

`US 9,868,961, B2
`Page 3
`
`(flU1 5
`
`\./
`
`References Cited
`
`
`DTP {R PUBLlCi TlClNS
`
`Jaifee et al., “Development and characterization of a cytokirre-
`secreting pancreatic adenocarcinoma vaccine from primary tumors
`for use in clinical trials.” Cancer J. Sci. Am. vol. 4(3), pp. 154-2183
`(1993).
`Jafiee et al., “Nove1 al1ogeneic granulocyte-macrophage colony—
`stimulatifig factor—secreting tumor vaccine forpancreat'c cancer: a
`phase 1 trial of safety and immune activation.” .1. Clin. @ncol. vol.
`19(1), pp. 145-156 (2551,).
`
`
`Kawakami et a .
`“Shared human rrielanorna antigen s: Rec vgnition
`by tumoivinfiltiating lyrtipltocytes in Hi.A-A2.l—transfectcd. inela-
`rromas.” J. hnrnunol. vol. 145(2), pp. 538-643 (1992).
`
`Klein et al., “Properties oftl'
`.
`62 cell line, derived from a pa errt
`with chronic rnycloid lei),
`1a." int.
`J. Cancer vol. 18(4), pp.
`
`421—431 (197-5).
`
`Kohler and Milstein, “Derivation of specific antibody-produ-c
`
`tissue c
`
`lture and tumor lines by cell fusion.“ E -. J. linrnunol. vol.
`
`6(7), pp. 511-5 9 (1976).
`
`Korinan et al.,
`luinor immunorherapy: preclinical and clinical
`activity ot‘anti—CTLAA: antibodies.”Curr. Qpirr. lnvestig. Drugs vol.
`6(6), pp. 582-591 (29135).
`Leach, et a1. “Enhancement of anLitumor im nunity 13y CTLA—4
`
`blockade." Sci
`e vol. 2.71., pp.
`l734—1736. (1996).
`Lee et a ., “Genetic imrriurrotlterapy of established tumors with
`adenovirusanurine
`granulocyte-macrophage
`colony—stintulating
`factor.” Hum. Gene Ther. vol. 8(2), pp. 137493 0997).
`Londrigan et a1. “Frolonged local expression ot‘anti-CDL‘: antibody
`
`by adenovially transduced allografts tan promote .ong-terrn graft
`survival ” .l. Gene Medicine vol. RU}, pp. 42—52 GEMS)
`Lyerly, “Quantitating cellular immune tesponses to cancer vac~
`cines.” Semin. fincol. vol. 318(3 Suppl 8), pp. 9--l6 (213133).
`McCarty et al., “Self-co 1p1ementary recombinant adeno associated
`
`
`
`
`Virus (scAA‘V) vect
`p
`note efficien transduc in independently
`ofDNA synthesis.” Gene 'lher. vol. 806), pp. 1248-114 (23%).
`Miller, “Human genetherapycmnes ofage.”l‘ilaturevol . 357033 78).
`
`pp. 455~46E (190?“
`
`pression vector system based on the chicken
`Miya?
`L et al.,
`beta~actin promoter dire‘ts elicient production of interleukinvS.”
`Gene vol. 79(2), pp. 269—277 (1989).
`
`Mulligan and Berg, ‘ Expression of a bacterial gene in mamirn
`
`
`cel
`Science vol. 2&9(44f
`' pp. 1. 22-1427 (198%).
`Nagai et al., “irradiated tum-Jr cells adenovira1ly engineered to
`secrete granulocyte/macrophage-colony-stimulating the tor establish
`antitumor immunity and eliminate pro-existing tumors in syngeneic
`
`rnicc.” Cancer lmmunol. lmmunother. vol. 47(2), pp. 72-86 (f
`Needlernan and W'un sclt. “A general metliod applicable to the search
`for similarities in the amino acid sequence of two proteins.” J. Mol.
`
`Biol. vol. 48(3), pp. 443453 (W79).
`
`Nemnrraitis et al., ”Granulocyte macrophage. colony . mutating
`factor gene—inofified an tolegous tumor vacr. nes in non77777small~cell
`lung cancer.” J. Natl. Cancer lnst. vol. 96(4), pp. 326—331 (2994).
`
`fiett gen et al., “The history of cancer innn
`herapy.” Biologic
`Therapy of Cancer, cl}. 6, pp. 87-119 (1991).
`
`
`$htsu1<a et al., “An alternativ
`oproach to tleoxyoligonu leotiites
`as hybridization probes by insertion of deoxyinosine at ambiguous
`codon positions.” J Biol Client. vol. 268(5), pp. 26%5—4668 (1935').
`@no et al., “Production 01‘ arrtiprion scFv-EC fiision proteins by
`recombinant animal cells.” J, Eioscn Biocng. vol. 95(3), pp.
`231~238 (2&93).
`
` ckaging cell line for produc-
`wry et al., “A stable human~derived 1:
`gt
`
`tion of high
`titer
`retrovirus/x1e:
`.otnatitis
`virus
`(7.-
`
`pseudotypes.” l’NAS vol. 93(21). pp. 11499-11496 (1996).
`Palmenberg, “1‘roteolytic processing of picoinaviral po1yprotein.”
`Annu. Rev. Microbiol. vol. 44. pp. 693-623 (1.99%).
`Pearson and Liprnan, “improved tools for biological sequence
`
`
`compai
`'on.” PNAS vol. 85(8), pp. 2444-2448 (198.8).
`Peggs et al. “Principles and use of anti-. .Tl A4 ant ibody in ltutnan
`cancer
`’
`unotherapy.” Curr Epin.
`immunol. vol. 18(2), pp.
`
`21936-213
`.fififi).
`
`
`
`Perreau and lirerner. “Frequency. proliiEration. and activati-"n of
`
`human memory T cells induced by a nonhuman wienrvirusf’" J.
`
`Virol. vol. 7903), pp. 146696—1461} {23855)
`Phan ct al., "Cancer regression
`nd autoirrununity induced by
`
`cytotoxic T lymphocyte—associat:rl antigen 4 blockade in pati
`
`with metastatic melanoma." PNAS vol. 181904), pp. 8372-83/7
`(2983).
`
`Fist
`'1, et al. “Ariti~C’il.A—4 human scFv antibodies pieverrt’l‘-cell
`activation in transp1antation.“ Trans. Proceedings vol. 33(1-2), pp.
`285-287 (2961).
`
`Porgarior at al., “lmrnunotlrerapy of tumor metastasis via gene
`
`therapy” Nat. lnnnun. vol. 13(2—3), pp. 113—133 (1994).
`
`“tvenetically rriotl‘iierl dendritic cells for cancer
`liibas et
` ’ Current Gene ”llierapy, vol. 5 (6), pp. 619—628
` :.1.,
`“lmrnun-zisuppressive
`effects of
`interleukin-12
`coexprelsion in melanoma antigen gene~modilierl dendritic cell
`
`vaccines.” Cancer Gene Therapy vol. 9, pp. 3
`.33 (2313?
`
`
`
`
`Ross
`et al., “Synthesi of foot-andr-n j-uth dis
`‘e virus capsid
`proteins in insect cells using liaculovirus expression vectors.” 1.
`Gen. Virol. vol. 71 (Pt, 3), pp. 17%34711 (199%).
`
`Rossolini et al., “Use ofdeoxyino sine—containing primers vs dege
`erate printers for pol‘ inerase chain reaction based on amMguous
`sequence information.” Mol. Cell Probes. vol. 8(2), pp 91—95
`(1.994).
`Ryan and Drew, “Foot-and-mouth disease virus 2A oiigopeptide
`mediated cleavage titan artificial polyprotein.” Flt/18$ .1. vol. 13(4),
`
`pp. 928-933 (19:94).
`Ryar ‘t al., “Cleavage ol‘fooband—mouth disease virus polyprotcin
`is mediated by resi lues located. within a 19 amino acid sequence."
`
`3. Gen. Virol. vol. 72(Pt ll), pp. 2.12
`' 732 (1991).
`Ryan et al., “Specificity of enzyme—substrate interactions in footv
`and—mouth disease virus polyprotein processing.” Virology vol.
`
`173(1), pp.
`C1—45 (1989).
`Salgia et al., “ ’accination with irradiated autoiogous tumor cells
`
`eniy
`red to secrete granu1ocyte»macrophage colony-stimuhting
`factor augment.) antitunior immunity in sornepatients with meta-
`static non-small-cell lung carcinoma J. Clin. finial. vol. 21(4). pp.
`
`624
`$6 (2663).
`Sanderson et al., “Autoimmunity in a phase l trial ot‘a tiilly liuman
`anti-cytotoxic t—lymphocyte antigen-4 monoclonal antibody with
`
`multiple melanoma peptides and nroatanide isa 5 1 for patients with
`resectetl stages iii moi iv melanoma.” J. Clin. Bncol. vol. 23(4), pp.
`7:31-759 (was).
`Shi et al., "Liranulocyte--macrophage colony-stimulating factor
`(GM-CSF) secreted by cDNA-transfected tumor cells induces a
`
`
`more potent antitum or response than exogenous GM ~CSl‘ Cancer
`Gene Therapy vol. 6 (1), pp. 81—33 (1:999).
`Simmons el, al., “Local secretion of arrit—C'l‘iAé’t antibody from a
`iM—CL‘F-secretin , tumor cell vaccine enhances vaccine efficac}
`Proceedings 01‘ the American AssoCiation for Cancer Research
`Annual Meeting, vol. 4’17, p. 359 (2EG-5).
`Simons et al., “Bioactivity of autologous irradiated renal cell
`carcinoma vaccines generated by ex vivo granulocytc—rnacrcphagc
`colony~smnulating factor gene transfer.” Cancer Res. vol. 57(8). pp.
`1537—1546 (1997).
`
`Simons et al., “l.
`tuction of immunity to prostate cancer antigens
`results of a clinical trial of vaccination with irradiated autologous
`prostate tumor cells engineered to secrete granulocyte~rnacropltage
`colony-stimulating factor using ex vivo gene transfer.” Cancer Res.
`vol. 59(219), pp. 51619-5168 (1999‘).
`Smith and ‘Waterman, “Comparison of biosequerrces." Adv. Appl.
`l latlt, vol. 2(4), pp. 482-439 (198i).
`
`Smith et al., “Localized expres_'jn of an anti—TN? single—chain
`
`
`antibody prevents development ol‘cmlagerr-indnced arthritis.” verre
`Therapy vol. 16(15) pp. 12484257 (2MB).
`Soifier et al., “Vaccination with irradiated autoiogous melanoma
`cells engineered to secrete human granulocyte-macrophage colony-
`stirnulating factor generates potent antitumor immunity in patients
`With metastatic melanoma.” PNAS vol. 95(7),), pp. 13141-13146
`(1998).
`
`

`

`U“; 9,868,961 nz
`Page 4
`
`(flU\ f
`
`\./
`
`References Cited
`
`OTHER PU Bill :1 TlClNS
`
`
`Sniffer et al, “Vaccination with irradiated, autologc 3 melmirjma
`
`cells engineered to seer {e grannlo-syte-macrepliage ‘ lony~stimu-
`
`lating fact-3r by adeneviralv—niediated gene transfer
`Emerita anti--
`tumor immunity in patients with metastatic iiieianoma." J. Clin.
`flincol. vel. 21(1’7), pp. 3'1""? ’ 359 (26153).
`
`Souza (it 31., “Recombin__t
`:
`-: against viral dis--
`eases.” Brazilian J. Med. Bic}. Research vol. 33(4), pp. 599—522
`(:Zfififi).
`Sugden e: 9.1., “A vector that, replicates as a, plasmid and, can be
`efficiently selected in b-lymphoblasts transformed by epstein-barr
`virus.” Mol. Cell. Biol. vol. 5(2), pp. 4l§-4l3 (lQBS).
`Szymezak et al ., “Con‘ectinn Ufmulthgene deficiency in VlVf} u Sing
`a single ‘self—eleaving’ 2A peptide-based reti‘ovii‘al vector.” Nat.
`Biotechnol. vol. 22(5), pp. 589-594 (2M4).
`
`Teng, et al, “Long-term in ihition of tumor growth by tum-Br
`necrosis factor in the absence of cachexia or ‘l-Cell
`immimity.”
`PNAS vol. 335(9), pp. 3535-3539 (1991).
`
`van Blsas e! 31., “Combinatmn irnniunotlierapy of B] 6. melanoma
`using miti~cytotoxic T lyrnphocyte-associated antigen 4 ('CTLA—ll)
`
`and,
`"uioeyte/maernphage colonyetimulnting fa clor (GM «(33?)»
`producing vaccines induces rejection of subcutane us and meta—
`static rumors accompani :d by a utoiimnun : depigmentation.” J. Exp
`Med. vol. was), pp. 355.3% (.1999).
`
`Wu et a1. “Multimerization 01' a v: meric anti-CDZE single-chain
`
`EV—Fc
`fusion p teiii
`is mediated tlzrmign variable domain
`
`exchange.” Prot.
`:ng. vol. 14(12), pp. la} 1933 (2am).
`Zernmu et al., HlV—l genome nuclear import is mediated by a, central
`DNA flap. Cell vol. lala‘.) pp. 173-185 (Zfififi).
`Zheng et 211., “8’7-CTLA4
`[ion enhances both production ofr'
`
`
`antimmor cytotoxrc ‘l
`lymphocytes and reerstanee to [F.1if110Y chal—
`lenge”, PNAS vol. 95 (11), pp) 6284—6289 (1998).
` 511g.
`Zijtferey et al., Self~iuaetivat
`lentivirus vector for safe and
`
`/.
`efficient in viva gene delivery. J. Virol. vnl. 72(12), pp. 9873-933“
`{1993).
`
`* Cited by examiner
`
`

`

`ELS, Patent
`
`Jan.16,2918
`
`Sheet 1 0f 14
`
`US 958685361 BE
`
`“371?;m$.44“!
`
`km3
`
`”MWn w-
`‘12
`
`
`
`
`
`if}
`
`

`

`U5. Patent
`
`Jan.16,2918
`
`Sheetz @E14
`
`US 958689961 32
`
`
`
`
`
`«mmmm“mum8MmumQuaHummummum8“gmmumGUMmumHumgmHumWmam3..fig5%mg”2%WE@mefilmEmSm0mmmamgm“mashmemxxgama
`
`
`
`mmmmmmmmmwmmmmmmam“mm“mmmmmammmmmow.mm:“WEEEE“m@
`
`“ms.E.@{wmg39@{fi25mfiwumm
`
`mmmumm“mmmamummMam“mmmmm“mmmamQBmamum”Wmmgmxfimma
`”mammMSG»@«m@«m903%Kg@me0mmmmm
`
`fimw04$dig@mfi@«QmmmwwfiNewmmmmmmm39gm”mmme“ma
`
`mam.
`
`”m30%m@«OEmgSammg,
`
`
`
`“mwmgmfimmm
`
`Nmum
`
`“figEmmi“
`
`

`

`ELS, Patient
`
`Jan. 16, 2818
`
`Sheet 3 92’ 14
`
`E:
`
`S 9,8683%} 32
`
`“am3”3339mmax3“
`
`.wxfimmm"
`
`
`
`“33¢.avm
`
`Em«a
`
`mg”a.
`
`
`
`mmfimwmfi.3%53%
`
`25%am5%3..
`
`“35m2
`
`.2.an
`
`”92ngma,.
`a?a».
`
`§{\
`
`
`
`.Emmmémpgmmmmkm
`
`gm”“£3
`
`
`
`muskam“.
`
`”fig”
`
`saga”R3&2;
`
`5%.:41%mvm
`
`«am$9
`
`$3wimum
`
`“.353Km
`
`.EgamEN
`
`Q
`
`flawgm
`
`SEmvm
`
`a?E,“
`
`
`
`$8“N34a3mmmghmw.
`M\Em3353
`
`gamma.£3353”..
`
`
`E33?»w£35m
`
`Em2m,3mEm
`
`”ENE.”
`
`EEu.8%3%:xmmm
`
`3%RE9xmax
`
`2%8233E;
`
`mm“Emx@2an
`Wag”mam
`
`_,
`
`fl
`
`EmmmmmmwmLMYaw.Ema
`
`
`
`:35“gm”mgwmahm
`
`3:5
`
`Emma?
`
`3%:21
`
`”Emma
`
`
`.-man2%am;”an“am
`3,camMg;”32:5
`“NewgmawM$qu
`,.g,ma$§mim%.M..£325
`
`
`
`
`
`

`

`RS. Patent
`
`Jan.16,2fi18
`
`Sheet4 ufifi
`
`S 9368,9435} BE
`
`34«noand“3.H355ta84as“332.
`
`
`
`an???an:.3.3”SE”35..
`
`£33.“Spat“Pa..23$5$6SE“53USp8Eb8pEud3.5493Ha25as.E28US“38“US.5.
`
`\‘ill‘r.
`
`nmammaamammmumHmaomwgmwwamayvawwpwmwmmwo
`
`Eu«3can3w«8SawSo92‘E3,“26Suv.3EupupE«8u?cupta«.2mi.v.2PSEb0.3.«3any»EuwoeSn303RuUs“8m66NE.3.»u:
`
`
`
`2.3%SxflmSEE.“33w
`
`
`
`ahwmumwmhmumwwwémwkmhmmSqmwwcummosmmmqwh
`
`M
`
`
`
`".333anwmwmw.833“322%
`
`
`
`
`
`.33Em$5$2«.5.«.32b93.on:SuEu3%E8492.Su“<5Eu«8.Eu«:3E8.“.U4»isCm"rum23:.5698F32:»£3.3.“3,.»2a93as«3at
`
`admxwmumhowwkkquummwnvmwwwbuqmmwmbmmmmumhm
`
`
`
`$2me33%.“.$23...wmfiwmw
`
`
`
`
`
`
`
`0.3wflan»«E,.GuawnPuu.34g95v.5.80£5.5“.3wEuEb3.4wowv.55.3wasfiwwvuuHaw45wuwuhave5%mgSm,(«Duuup«Gobum«.5“GamumSohow
`
`
`
`
`
`Ea.v354Em”mm9%EuHum.52taE35EN8.”En.finand.wfi.SmBu9343SA.3SHSHBa26.3”v.5Ea29«BE.Su.22.5“.Ha
`
`
`
`wmhnwmwmampwmmudmxummwaxnwhmmhmPMUmM‘MUm
`
`mu>m4uaxbgbmwmuaamwwmmuwmwmumwwmmwwhkaua
`
`
`
`
`
`RigaSign“SSS$3...me
`
`.36#5(EuEL.3%Su32mg93Ehv.2an”and$2va$335hwma,uwwammHa.maawMww.wmmmma5.Mau.MMwMmMamMEu:$430SHufiShv.5quahummg.35huh«um95BaufiPSmg8%ufi.HE34own93uuh3a.
`
`
`
`“FmaaaM,.9mmmHamahma.aHaMaaamwa.mmnw.53uuw$5«muEd.8wmun.«muwow34S4.34mum.and3%0.5humu:as.wuq36.3093.03um.“
`
`
`hum.PM.“.uwhuum59"35$:
`
`wmm
`
`
`
`Hmwxemoflammxwmmflmmxwwm335m
`
`
`
`
`
`
`

`

`ELS, Fatwa
`
`Jan. 16,2033
`
`Sheet 5 M14
`
`US 9,8685%} E32
`
`"m“.
`
`an...uy-<.<~mx\\\wwvrmlyvfliNuIJ/WWMv-Wflt‘fmww' )yumxnm~.\mmmekk‘awmtulw’mm‘uxvm’
`
`Wm
`
`

`

`$8.. Patent
`
`Siam. 16,, 2018
`
`Sheet 6 (3f 14
`
`US 998689961 82
`
`3.83mmmumEmmg,¢
`
`HEW???
`
`.
`
`I
`.38
`
`l.
`
`
`
`Sufi0;!
`
`x...a
`
` 3%mamM{In-Ill!!!IIIIIIIIIIIL.....:.
`
`
`3%gm
`
`
`
` a.VE5mmmmwmmfifi
`
`
`
`
`
`5mm?gamma}mfi83mm
`
`xxnew“?EmanormwgommM0%m5,”N
`
`mmay“
`
`
`
`
`
`

`

`ELS, Fatwa
`
`Jan. 16,2033
`
`Sheet 7 M14
`
`US 9,868,961 E32
`
` E t
`
`
`
`
`S,
`
`S 21
`
`w. w
`= A:
`\
`.
`W”:,,J,Wm.mmmwmmmwwm
`
`
`
`
`
`

`

`ELS, Fatwa
`
`Jan. 16,2{918
`
`Sheet 8 M14
`
`US 9,8685%} E32
`
`3t:
`
`

`

`"1
`
`u
`
`E}
`
`Patent
`
`Jam. 16,, 2018
`
`Sheet 53 0f 14
`
`US 998689961 82
`
` ,.3§§E§E§x§§£5;.....$5$$§£S:
`
`
`
`

`

`ELS, Fatwa
`
`Jan. 35,2913
`
`Sheet 10 (2f 14
`
`US 9,8685%} E32
`
`
`
`
`wW
`P'‘34,
`km?
`
`

`

`HS. Patent
`
`Jan. 16, MRS
`
`Sheetll 0f14
`
`U
`
`S 9,868,961 E32
`
`
`

`

`US. Patent
`
`Jam 16., 2018
`
`Sheet :2 of 14
`
`US 9368,96} BE
`
`93%Emfimfih
`
`NVmi
`
`Nv“
`
`$3
`
`N
`
`an
`
`mm
`
`0 $523 +£dxafif+8€313
`
`
`

`

`US” Pat‘em
`
`Jan. 16., 2813
`
`Sheet 33 9f 14
`
`US 9986836} 82
`
`
`
`
`
`mammmefiaflmw+mwmfimfimmmm33%Ewfimmfimamwwm
`
`am,mw.,%§§mfim
`
`mmanaemia-mmflm
`
`gig-fig8%
`
`gmA:25A,gmaE4Em4%;EM.
`
`
`
`“.85A:flowA?“A“EmAug..Em
`
`Rel/EEG %
`
`mu.GE
`
`93
`
`mmwmxofififi
`
`
`
`

`

`E1186 Patent
`
`Jam 16,2618
`
`Sheet 14 af 14
`
`US 9936836}; 82
`
`B€€~€3MCSF~LW2
`
`aim
`
`$33
`
`3%{$3
`
`m
`
`2%;
`
`§§§§$
`N
`4"“? W
`
`mungzm, gamma . mum 1 awaits
`
`333%
`
`as14
`
`

`

`US 9,868,96l 82
`
`l
`S EGR
`
`NIETHODS AND CQBIPOSI'E‘IG
`LUCALIZED SECRETION OF A
`
`Ell-C Tins-4
`ANTREUDIES
`
`CROSS REE ll 3N IE TO RELATED
`AP jLlCA’l‘lONS
`
`
`lhis is the National Stage filed under 35 U .C. §37l of
`
`International App cation PCT/l lSZOG7/fifi7983 filed on
`l‘vlar'.
`'30, 2007 , which desi nated the United States of
`America, the disclosure of which is nicorporated herein by
`reference. 'lhe present application claims priority from US
`
`l’ro
`nal Patent Application No. 60/787,972 lilcd on 30
`Mar. 2066, and US. Provisional Patent Application No
`60/787,978 filed on 3i Mar 2066,
`the contents each of
`whic i are incorporated herein by reference.
`The disclosure cl
`inis benefit under 35 11.811. §l lgte) of
`US. Provisional Application Ser. No, 60/7873? . tiled l‘vlar.
`
`30, 2306, and ll S. Provisional Appli ation Ser. No 601787,
`9‘67 filed Pillar. 3 l, 2006, each of which is incorporated herein
`by reference in its entirety.
`RE El} RELNC I TO A SEQUENCE LlS’l‘li iii
`
`
`A Sequence Listing is provided in this patent document as
`
`
`a t
`. e entitled “l 87609US3__ST25.txt” and create . Sep,
`30, 2010 sise 10 Kb) The contents of this file is incorpo-
`rated herein by reference in its entirety.
`
`FIELD OF lHE INVENTION
`
`e
`'lhe disclosure relates
`the localized secretion ofanli~C'l‘Lfir—4 '
`
`use in immunotherapy.
`R A CK GR Ol INT)
`
`Cytotoxic T lymphocyte antigen-4 {CTLA-Jv) is a member
`or” the CD28-B7 irrnnunoglohu in superiamily of immune
`regulatory molecules. Greenwald et al., Ann. Rev. Immunol.
`
`
`23"“ 5—548 (2005/. Although initially mis ‘haracterixed as a
`,
`
`
`
`pot
`ive regulator based on homology to its co—s
`iulatory
`
`
`counterpart CD25, C A—Aé l: s now been recognized as one
`o l'the key negative regulators of adaptive immune responses
`in general, and T cell prolife ation and etlector functions in
`
`particular. Peg ,
`et al,, Curr. Opin.
`imnnmol,
`'18:206—213
`
`(2006). Unlike eonst’
`tively-expressed (,ZJJZS, Cl Li‘s-4
`expression is tightly reg latcd and short—lived on activated T
`
`cells, and exhibits signif “antly higher afilnities for the B7
`ligands it shares with CD28.
`A number or" clinica ellbrts are underway to therapeuti—
`cally exploit the important biological function of this mol—
`
`
`ecule. On the one hand, C'lll N415?
`sioi niolecu‘
`have
`been created and employed as immune suppressants in vivo
`based on its higher atiinity for 87 and the consequent
`inhibition oliCl)28--B7 mediated 'ostinrulation. Bluestone et
`
`al., Irmmmi. 241233-38 (2006‘). The hCTLA~lg fusion pro-
`tein Orcncra M (abatacept) recentl‘
`received FDA approval
`as a fu‘st-in-class antagonist of CD28 costimulation in rheu—
`nratoid arthritis.
`On t re other hand, (I'l‘er—él blockade is being explo red as
`a promising approach to cancer immunotherapy, employing
`monoclonal antibodies directed against CTLA-‘l to prevent
`its negative regulati n and thereby enhance t e cellular
`immune response. Peggr, supra. Ongoing clinical investi—
`
`gations utilizing, systemic administr: ion of these antibodies
`have provided dramatic successes, but have also produced
`
`2
`undesirable toxicities. in particular, adverse immune events
`as
`immune-mediated colitis.
`(All?)
`such
`hypophysitis,
`uveitis and hepatitis have been observed, and serious Alli
`often correlate Witt antitumor responses or freedom. from
`relapse. Attia at al., J, Clin. Oneal. 23:6043—53 (2005).
`Accordingly, altlio ug i the curre it systemic approach may be
`clinically acceptable
`in a
`patient
`population having
`adv anccd disease and very limited treatment options, further
`improvement is
`
`lc early warranted to dissociate the positive
`anti-tumor effect from the negative adverse events.
`C
`S ‘MMARY OF THE lNVEN'l‘lON
`
`'l‘he present invention resolves this unmet need in the art
`by etieetuating localized CTlUA—él hloekami in vivo, such
`that the distribution and ellect of the desired ITLA-u’l
`blockade.
`is substantially restricted to a particular target
`tissue. More specifically, cells, viral vectors, compositions
`and methods are provided for the localized, in vivo secretion
`of anti—JlLA—4 antihodies from in vivo modified endog—
`enous cells or e: vivo modified autologous or allogeneic
`cellular vaccines that are subsequently administered.
`ln one aspect,
`the invention provides a viral vector
`encoding an anti-CTlLA-A antibody (Ab) that binds to the
`extracellular domain of Clllrilr-A and inhibits (“TEA-4 sig-
`
`naling. ln preterred em iodiments for ex vivo tr' nsfonna—
`tion,
`the viral vector can be an integrative viral vector
`‘apahle of providing constitutive expression when trans—
`feeted into a human cell such as, e.g., adcno—associated viral
`(AA‘V) vectors and lentiviral vectors.
`In preferred embodi—
`me its for in
`o trar slomration, the viral vector can be :1
`
`
`
`
`nor.
`.egrative
`
`'iral vector capable of providing transie .
`expression when transiected into a human cell such as, e.g.,
`an adcnoviral vector or an alpha virus vector. ln certain
`preferred embodiments, the vector is an alpha virus vector
`selected irom the group consisting of an SFV and an VEE
`vector.
`
`The vector can further include a nucleic acid encoding a
`eytolcinc. ln preferred embodiments, the cytokinc is ’iM—
`ESE.
`
`the invention provides a modilied
`in another aspect,
`
`human cell expres ng an anti—C “L; —-l antibody that s e—
`cilically binds to the extracellular domain ot‘ TEA-4 and
`
`inhibits CTLA~4 signaling. l’relerably, the modified human
`cell
`is capable of expressing the anti~CTlA Al antibody
`proximal to a tumor cell ii a cancer it atient in need thereof.
`
`111 one embodiment, the cell is an endogenoi ‘ cell modified
`in vivo in a patient to effectuate localized expression of
`anti—C'l’LA—él antibody in a target tissue. ln an alternative
`embodiment, the human cell is an autologons or allogeneic
`
`cell modified ex vivo to effectuate localized expression ol
`
`aiiti-CTLA--4 antibody upon adin . istration to a target tissue.
`ln a preferred embo '
`ent, the target tissue is tumor tissue.
`in a further embodiment, the human cell is further modi-
`lied to secrete at
`least one additional
`immune ell'ector'
`molecule such as, e.g., G-CTE, GM~CSR Ila-2 and the like.
`
`
`1
`elerrcd embodiments. hu: an cells suitable for modi-
`fication in accordance with the invention include tumor
`
`cells, T cell" and antigen—presenting cells such as, e.g.,
`dendritic cells. Su h cells may be endogenous and/or autolo—
`
`gous to the pa ient for personalized cellular therapies