IMMUNOGEN 2109, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`lint}
`
`\tl;\l.lii?,\']]]3 runs scrences.
`1.
`
`La mobilité cle oe derive triméthyle diflere (le cellos des de’rixés 1.3.4 et
`3 . .1.(i. mais est identique 51 (:elie du trimetliyl—i 4.6 n-fructose.
`
`L‘) Séi‘tnce thl 3 i'nal's i955.
`.-\. \\ inrts'i'm‘m ct P. Li: intixr, Cunt/ares mutiny. 2E2,
`("j
`,\. AllCll-UIBAL‘L'I‘.
`.l. E.
`l,.‘.ou;rrors,
`29.36. p. 281—7) rt
`[fix/l. S'r'm. Claim. 8501.. 33. 19515, {1. “2:4 Ilit et 1). ”3341'“.
`
`I Lahoruroirc ([3 ("I'M-mic Brnfogr'r U18. Mien/M r/c [’frru'mucie. Pur'r'xf
`..
`/
`
`.t‘lIiIJECIAE EXPERIMENTALE. — E/fff .rrrr [a [erred/ink lZZlU (Ir: [a .S'oui'zh' d’rme
`cornbr’nairon par diasomzion a"[lsrmfl/ioptérz'rze (’1:
`(1’6 “ii—globuiz'ner dc hamsters
`por'lcurr d8 CEIIK? leucémie par ficifcirogi‘efle. Note de MM. GEORGES Mum's,
`rrRAN “A Lee et. JEAN BERNARD. pre’sentée par M. Léon Binet.
`
`ll est montrt? qnc ls comlJinnison par (liazore'netion d’un nnlimétabolite [el que
`l’A-métlioptérine aver.
`les y—gloiinlines (le hamsters porteurs rle leucémie 1210 par
`lit-iterogrelie a one activilé sur- la leueemie 12H] nettement supe'rienre 51
`l’:\—Inétl101k
`térine on aux Teg'loliulines seules on it l’adtlition dos dutix (non combinés).
`
`Nous evens observe qu‘il est possible (le grefl'er la leuee’mie 1210 de la Sonris
`dens la porrlie juggele de hamsters (U et (M) Le but (in present travail est rle
`reehercher si les y—globulines rile hamsters portenrs de cette leueémie par liétéw
`rogrelic sont eapablcs do transporter un arrtime‘taljolite electivement snr lcs
`rellules de la laurénlie l'Qlll isogreliée CltCZ la Sonris. L‘Hntimetabolite choisi
`est l’A—methopterine, compose portenr de denx fonctions amines aromatiques
`grt’iee auxquelles on pent le combiner par diazoreaetion aux T—gloliiulines.
`
`£1 son 487‘: passage
`111356,”?! cl utédsodcr. m La leucémie l2lll utilisée est
`sursonris de lelignée Dl3_£\._.. Trente hamsters out recu clans one poeltejuggale
`une injection do 20 millions rle eellnles lencémiques, puis sept‘jours apres, one
`injection identiqne dons l'autre poclte. Le sang it été preleve (lens le sinus
`caverneux scpt jours epres la seconde grelie. On a verilié que les tumeurs
`éltticnt en pnrl‘aite condition. Les “.r—glolmlines (in serum ont ete isolées par la
`métliode de l'nlcool 51 froirl (1). Les anticorps non speciliques out eté adsorbés
`antnnt qne possible en mett'rtnt ces y—globulines en contact d’un mélange de
`moelle, rle foie et de rate de souris a in temperature do 4“ pendant 2.411. La
`eombinaison cle y—glol‘mlines et d‘Arméthoptérine a éte faite par diazoréae—
`tion (9). 4'5 sonris 1?.(CH Bl ><DB_X2) de 30 g ont reen nne injection de
`I million de cellules leueémiques et, le iendemain, elles out recu un des traile—
`Inents suivants :
`
`1° Groupe A : Rien.
`2" Groups B : Tugiobnlines de hamstersimmunisés, a la dose de 1:25 [mg/kg.
`3” Groupe C :
`.L't—métlioptérine 51 la dose de 20 mg/kg.
`
`IMMUNOGEN 2109, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`r‘L—méthoplérinc (12.0 mg’kg) immédiatement aprés (traitement associé).
`C1" (Iroupe E : y-gluhuhnes de hamsters non immunisés combinés £1 l’,\7métho—
`ptérine par diazoréaclion (méme quantité de T—globulines et d’A—méthnptérine
`([th dans les autres groupes).
`l”:\—métho—
`(3” Groups 17 : y—glohuhnes dc hamsters immunisés comhimi-s £1
`ptérine par diazoréaction (méme quantité de Tugiobulines et d’A7méth0ptér-ine
`cine dams les autrcs groupes}
`Les tests utilisés sent 13 mononuch’eose sanguine, la survie des animuux} les
`LiilHCTISiOHS des tumeurs 01. de In rats.
`
`Ifés‘ultats. — Les I‘éSlllthS soul cxposés clans Ies tableaux I at II.
`
`(H'uuin'.
`\ .......
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`I) ......
`
`IV
`11 .......
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`TABLHAU ll.
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`
`’i‘jiuur'.
`
`1:" .100
`if; mm
`20:01.!
`In 30::
`11 film
`j‘
`ll'll]
`
`T‘fiinur.
`
`I'm Il'm
`
`Li“; ".IHJ
`38 int.»
`[3 5m:
`IU 500
`
`'.i"linur.
`
`Ii‘flinur.
`
`if’fiimna
`
`-
`
`r1” IOU
`39 now
`:14 '10'.)
`[0100
`
`7
`—
`
`7
`—
`1713!)”
`
`7
`—
`7
`
`Hi [rm
`
`Dimensions day Hummus ea: dc la rate at; moment de la man. 77 Toutes les rates
`
`des groupcs A, B, C, D at E, Observées is. l’autopsie, sont fortcment hypertro—
`phiées, 061135 (111 groups F 11" sont trés pen ou I‘estent (chez H‘ois souris)
`sensihlement égales a la rate normale.
`Les tumours des souris des groupes A, B, C, D at E sont égales 0L1 supé—
`I'ieures 21 2,5 cm at 001165 du groupe F sent
`toutes inféricures {'1 0,5 cm.
`La combinaison par diazotation d’A—Inéthoptérine et de y—glohuhnes de
`hamsters pm‘teurs par hétérogrefie delaleuce’mie 1210(1e1a Srmris est done extl‘é-
`men'lent efficace sur la lcucémie 12H) isogreflée a des souris 1*‘1((_IHBI><DBA9).
`Elle détruit complétement lcs cellules luucémiques dams deux Gas favorables
`ou retards considérahlement
`l’apparition des
`tumeuI-s. Elle a permis 1a
`guérison
`compléte
`d’une
`soul‘is. Les
`expériences Lémoins
`faitns
`aver:
`l’A—méthoptérine
`settle,
`has y—glohuhnes
`immunes
`seules,
`l’addition de
`l’A—méthoptérine et des T—glohulines immunes, 1a combinaison de l’A-méthm
`
`— ___
`
`IMMUNOGEN 2109, pg. 3
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2109, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`I628
`
`,\¢I,\Dl:‘,_\llFI
`
`[HES SITII'INLIES.
`
`pLérinc eL des 1{-glol'ntliHES «’lc hamsters non immunisés, suggéreul que les
`y—globulinesimn’luncs transpm'lent élecliwmem; gn‘lcr
`'21
`la dielzoréaclion,
`l’anlimélabolile sur les cellulrs lencémiques.
`semble ouverte,
`Une nouvelle méthodr gém‘rale ('[e chimiothéraph)
`transporLer «électiremcnl. par des T-giubulirles de sérum immun,
`:‘les agents
`uhimiques artif‘s Ijusqu’a l’ngenl palhugéne Ull aux cellules maladés, an
`11103611 d’unr t'onution amine diaminlahle rm d’unr antre uornbinaison chin’lique
`(Innt la recherche est en cmlrs.
`
`0) ll. DIir'rstlll, flirt/lads in fifedical Research, 5. 1952, p. 28:1.
`H)
`|\'. LANDSTEINL‘R cl J. VAN DER Scum-Lu. J. ISL-[min ,‘UecL, 3:5, £932, 1). 78: 3 li. LANDSI‘EINEII,
`’I’Jtrr spear/form of Seralogz}.-rrl rmrxtt'mrs, Cambridge.
`I935, Harvard l_'niw+r~;it_y Press Ed.
`(3) G. Myrur-z ct J. REHNAIH). (I R. Soc. flint, i550, 1956, p. 1708.
`("'1 G. Myrnr-r J. CIIOMr-z. e!
`.f.
`)rlanJlllL ('orrf'. [Em-op. Hémntoh. ('10peuljag‘uc, 19:37.
`
`r f'rnrr'r rl'r:
`
`l‘ff‘C/‘ref‘C/ifl r/r: f'mzlmoru'an'ou ('lcmr'lr Her/MHZ,
`Hfipia‘ai Hfirolr]. Prrr‘r'x.
`\
`
`\l IJLI'JICCINE l-TXPIJZRH-I IENT \ LE. 7511146 (hfl‘c’r’upprmmzi(/6thrombowr(£17106;'z'rm'njaler
`Me [Warm C/IE'I- [a .S'eic/m. Note ([e MM. ANTOINE Juumx, Juan lermxmm er
`Jun Cannon préseniée par M. Léon Binet.
`
`le plus grns drs vaissraux artériels qui,
`ll s’agit dr l’aorte créphaliqur,
`HP. (16:17"?th du VHTLricule,
`111011 be vars la tfibte, sons ]:1 coquillr dorsalc;
`CHE est situér :5. pet] pl-és dams 16 “plan gaging]; son lrajet PSL 1mraH€1lr
`(.‘T
`dorsal
`:1
`relui dc l’:‘1esophngr;
`e119. CSL mgagéu, mm,-
`rr.
`(1P]‘]]](‘]‘_._
`(Inns
`J’espuur conjm'wLif qui sépare 105 (leux Inoitiés du fair.
`3:) H'lll‘UCLler hismlngiqua qur nous uwms élimliét- par divurs promidés
`mammal.
`([e 'la (:Iasser (lans has arlzirrrs dr typo. muscuhirr. Elle (:nmprand
`une muique inter-Hr. avcu rndmhélimn 0t
`limiLante éhsliqnc interne,
`unr media qui s’rf'lcnd sur la majourtr parlie (,le l’épaissmr (Ir Ia paroi 6|
`qui est mJl'lstituée dc plnsieurs assism L19
`fibres kisses a‘r'nJlflaires rl. une
`l..I.u:J.iqu externr. La Tunique lnnycnnr rontienti aussi d’assez nmnbreuses
`Forumtions éiastiqurs que nous awms Ulises an évitlrm-r par In méthorh‘.
`(In Gomori
`i1 la fuchsinc—pnraldélwrhr, su’ivmll’ Gabe.
`Les ras (Ir,
`thromlmh'r. gut: 11mm avnns observr'm out [0118 étr" (h’wlrnrhrs
`[a suite de lésions expérhnentalcs (Jr
`la purni do I’am‘tr. provoquées
`{1
`par Lle pc‘tits tampons d’ouatr,
`.I'rrrbibés rle formal dilué el
`insérés it son
`niveau. Nous avons‘
`tiléierminé aver: prtfluision la mode opérn'mirc (21;
`Iixé
`le lien (l’intlfoflrlction de l’agent irritant. Nous aroma [facar'té le pmcédé
`qu'i consistait 51 enlever un valet dans la coquillr, au-desmls the I’m-tern;
`uelIe-ri é'tait mime {‘1
`111.1, wait;
`in. Lraninatisrne,
`trop brutal,
`sc révélail:
`uxtrfnnemenst donunagrahla pour In surviu uliériuurn (Iv I’nninm].
`
`IMMUNOGEN 2109, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
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`

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`[initials]
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`
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`FIRST HALF
`OF
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`1958
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`
`
`
`WEEKLY
`
`
`REPORTS
`
`OF THE ACADÉMIE DES SCIENCES
`[ACADEMY OF SCIENCES]
`
`MEETINGS
`
`
`PUBLISHED
`
`WITH THE ASSISTANCE OF THE CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE [NATIONAL CENTER FOR
`SCIENTIFIC RESEARCH]
`
`FOR THE PERMANENT SECRETARIES
`
`VOLUME 246
`
`No. 10 (March 10, 1958)
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`[stamp:]
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`RECEIVED
`93985
`UNIV. OF WISCONSIN
`LIBRARIES
`
`PARIS,
`GAUTHIER-VILLARS, PUBLISHER-PRINTER-BOOKSELLER
`Quai des Grands-Augustins, 55
`
`
`
`1958
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`IMMUNOGEN 2109, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

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`ACADÉMIE DES SCIENCES.
`
`
`
`
`
`1626
`
`The mobility of this trimethylated derivative differs from that of derivatives 1.3.4
`
`and 3.4.6 but is identical to that of trimethyl 1.4.6 D-fructose.
`
`(*) Meeting of March 3, 1958.
`(1) A. ARCHAMBAULT, J. E. COURTOIS, A. WICKSTRÖM and P. LE DIZET, Comptes rendus [Reports], 242,
`1956, p. 2875 and Bull. Soc. Chim. Biol. [Bulletin of the French Biological Chemistry Society], 38, 1956, p.
`1121-1131 and p. 1133-1141.
`
`
`
`(Laboratoire de Chimie Biologique [Biological Chemistry Laboratory],
`Faculté de Pharmacie [Faculty of Pharmacy], Paris)
`
`
`EXPERIMENTAL MEDICINE - Effect on mouse leukemia 1210 of a combination by means of
`diazotization of Amethopterin and of -globulins from hamsters with this leukemia by
`means of heterograft. Report by Mr. GEORGES MATHÉ, Mr. TRAN BA LOC and Mr.
`JEAN BERNARD, presented by Mr. Léon Binet.
`
`
`It was shown that the combination of a diazo reaction of an antimetabolite such as amethopterin
`
`with the -globulins from hamsters with leukemia 1210 by means of heterograft has an activity on leukemia
`1210 that is significantly higher than amethopterin or -globulins alone or with the addition of the two (not
`combined).
`
`We have observed that it is possible to graft mouse leukemia 1210 into the cheek
`pouch of hamsters (3) and (4). The goal of this work is to research whether the -globulins
`from hamsters with this leukemia via heterograft are capable of transporting an
`antimetabolite electively
`to
`the
`isografted 1210
`leukemia cells
`in mice. The
`antimetabolite selected was amethopterin, a compound with two aromatic amine
`functions. As a result of these two functions, it was possible to combine it by means of
`diazo reaction with -globulins.
`Material and methods. - The leukemia 1210 used was on its 487th passage in mice
`on the DBA2 line. Thirty hamsters received an injection of 20 million leukemia cells in
`one cheek pouch, and then seven days later received an identical injection in the other
`pouch. A blood sample was taken from the cavernous sinus seven days after the second
`graft. Whether the tumors were in perfect condition was verified. The serum -globulins
`were isolated using the cold ethanol method (1). The non-specific antibodies were
`adsorbed as much as possible by putting the -globulins in contact with a mixture of
`murine marrow, liver and spleen at a temperature of 4º for 24 hours. The combination of
`-globulins and amethopterin was made by means of diazo reaction (2). 45 mice F1(C57
`B1 x DBA2) weighing 30 g received an injection of 1 million leukemia cells and the next
`day they received one of the following treatments:
`1st Group A: Nothing.
`2nd Group B: -globulins from immunized hamsters at a dose of 125 mg/kg.
`3rd Group C: Amethopterin at a dose of 20 mg/kg.
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`IMMUNOGEN 2109, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
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`

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`MEETING OF MARCH 10, 1958.
`
`
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`
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`1627
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`4th Group D: -globulins (125 mg/kg) from immunized hamsters, then
`
`amethopterin (20 mg/kg) immediately thereafter (combination therapy).
`5th Group E: -globulins from non-immunized hamsters combined with
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`amethopterin by means of diazo reaction (the same quantity of -globulins and
`amethopterin as for the other groups).
`6th Group F: -globulins from immunized hamsters combined with amethopterin
`
`by means of diazo reaction (the same quantity of -globulins and amethopterin as for the
`other groups).
`
`The tests used were blood mononucleosis, the survival of animals, and the size of
`the tumors and of the spleen.
`Results - The results are shown in tables I and II.
`
`
`
`Group. Day
`5
`10
`5
`10
`5
`5
`10
`
`A…...
`B…...
`C…...
`D…...
`E…...
`F…...
`
`
`Group.
`A…...
`B…...
`C…...
`D…...
`E…...
`F…...
`
`
`Day
`6
`10
`5
`10
`5
`5
`10
`
`Day
`7
`6
`1
`10
`5
`4
`8
`
`Day
`8
`0
`0
`10
`5
`4
`8
`
`TABLE I – Survival.
`Day
`Day
`Day
`9
`10
`11
`-
`-
`-
`-
`-
`-
`8
`5
`0
`3
`3
`0
`4
`2
`0
`8
`8
`7
`
`Day
`12
`-
`-
`-
`-
`-
`6
`
`Day
`13
`-
`-
`-
`-
`-
`5
`
`Day
`14
`-
`-
`-
`-
`-
`4
`
`Day
`15
`-
`-
`-
`-
`-
`1
`
`Day
`40
`-
`-
`-
`-
`-
`1
`
`Day 4
`17,400
`14,000
`20,700
`10,300
`11,400
`7,100
`
`TABLE II – Mononucleosis.
`Day 7
`Day 9
`60,100
`-
`-
`-
`37,200
`46,100
`38,400
`39,000
`13,500
`24,200
`10,500
`10,400
`
`Day 11
`-
`-
`-
`-
`-
`15,300
`
`Day 13
`-
`-
`-
`-
`-
`16,100
`
`Size of tumors and the spleen at the time of death. - All the spleens for groups A,
`B, C, D and E, observed during the autopsy, were severely hypertrophied. There were
`very few for group F or they were (in three mice) largely the same as a normal spleen.
`The tumors for the mice in groups A, B, C, D and E were equal to or larger than
`2.5 cm and those for group F were all smaller than 0.5 cm.
`The combination by means of diazotation of amethopterin and -globulins from
`carrier hamsters via heterograft of mouse leukemia 1210 is thus extremely effective in
`terms of isografted murine leukemia 1210 F1(C57 B1 x DBA2). It completely destroyed
`the leukemia cells in two favorable cases or considerably delayed the appearance of
`tumors. It allowed for the complete recovery of one mouse. Control experiments with
`amethopterin on its own, -immunoglobulins on their own, the addition of amethopterin
`and -immunoglobulins, the combination of amethopterin
`
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`IMMUNOGEN 2109, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676
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`

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`ACADÉMIE DES SCIENCES.
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`1628
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`and -globulins from non-immunized hamsters, suggest that the -immunoglobulins
`electively transport, as a result of the diazo reaction, the antimetabolite to the leukemia
`cells.
`A new general chemotherapy method seems to have opened: electively
`
`transporting, via serum -immunoglobulins, active chemical agents to the pathogenic
`agent or diseased cells, by means of a diazo-based amine function or other chemical
`combination for which the research is ongoing.
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`(Centre de Recherche de l’Association Claude Bernard [Claude
`Bernard Association Research Center], Hôpital Hérold [Hérold
`Hospital], Paris).
`
`(1) H. DEUTSCH, Methods in Medical Research, 5, 1952, p. 284.
`(2) K. LANDSTEINER and J. VAN DER SCHEER, J. Exper. Med., 55, 1932, p. 781; K. LANDSTEINER, The
`specificity of Serological reactions, Cambridge, 1945, Harvard University Press Ed.
`(3) G. MATHÉ and J. BERNARD, C. R. Soc. Biol. [Reports from the meetings of the Biology Society], 150,
`1956, p. 1768.
`(4) G. MATHÉ, J. CHOMÉ and J. BERNARD, Conf. Europ. Hématol. [European Hematology Conference],
`Copenhagen, 1957.
`
`
`
`
`
`EXPERIMENTAL MEDICINE - On the development of experimental aortic thromboses in
`Cuttlefish. Report by Mr. ANTOINE JULLIEN, Mr. JEAN RIPPLINGER and Mr. JEAN
`CARDOT, presented by Mr. Léon Binet.
`This concerns the cephalic aorta, the largest of the arterial vessels which, coming
`
`off from the ventricle rises up towards the head, under the dorsal shell. It is located
`slightly in the sagittal plane. Its course is parallel and dorsal to that of the esophagus. It is
`held, together with the latter, in the connective space that separates the two halves of the
`liver.
`Its histological structure, which we have studied by means of various procedures,
`
`allows for its classification into a muscular-type artery. It includes tunica intima with
`endothelium and internal elastic lamina, a media that extends over a major part of the
`thickness of the wall and which is composed of several layers of smooth annular fibers
`and a tunica externa. The tunica media also contains a good number of elastic formations
`that we have highlighted using Gomori’s paraldehyde fuchsin stain, in accordance with
`the Gabe method.
`
`The cases of thrombosis that we observed were all triggered as a result of
`experimental lesions to the wall of the aorta, caused by small cotton swabs soaked in
`diluted formalin and inserted in the area. We have precisely determined the surgical
`method and have set the location for the introduction of the irritant. We have ruled out the
`process whereby a section is removed from the shell above the artery. While the artery
`was exposed, the trauma was too brutal and was extremely damaging to the subsequent
`survival of the animal.
`
`
`
`
`
`
`
`IMMUNOGEN 2109, pg. 8
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`fa
`
`TRANSPERFECT
`1~l3l'IJ‘Eil‘d
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`E f. A,
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`
`AFFIDAVIT OF ACCURACY
`
`I, Brett Whelan, hereby certify that the following is, to the best of my knowledge
`and belief, a true and accurate translation of the accompanying document from
`French to English:
`
`"Mathe 1958”
`
`Brett Whelan
`
`700 Sixth St, NW
`
`Washington, DC 20001
`
`Sworn to before me this
`22nd day of January 2015
`
`
`
`LAUREN LUBERGER
`
`
`NOTARY PUBLIC
`.,
`
`District of Columbia
`l
`
`
`as A
`.31r 2018
`'ivly :Igmmlsslon E:
`
`
`
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`IMMUNOGEN 2109, pg. 9
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2109, pg. 9
`Phigenix v. Immunogen
`IPR2014-00676
`
`