Transient Removal of CD46 Is Safe and Increases B-cell Depletion by Rituximab in CD46 Transgenic Mice and Macaques

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1 The Amerin Soiety of Gene & Cell Therpy originl rtile Trnsient Removl of CD46 Is Sfe nd Inreses B-ell Depletion by Rituximb in CD46 Trnsgeni Mie nd Mques Ines Beyer 1,6, Hu Co 1, Jons Persson 1, Hongjie Wng 1, Ying Liu 1, Rom Yumul 1, Zongyi Li 1, Dougls Woodle 3, Ronld Mnger 3, Mihel Gough 4, Dine Roh 4, Jlyn Bogue 4, Audrey Bldessri 4, Ronld Berenson 5, Drrik Crter 5 nd André Lieber 1,2,5 1 University of Wshington, Deprtment of Mediine, Division of Medil Genetis, Settle, Wshington, USA; 2 University of Wshington, Deprtment of Pthology, Settle, Wshington, USA; 3 Fred Huthinson Cner Reserh Center, Settle, Wshington, USA; 4 Wshington Ntionl Primte Reserh Center, Settle, Wshington, USA; 5 Compliment Corp., Settle, Wshington, USA; 6 Current ddress: Deprtment of OB/GYN, University of Düsseldorf, Düsseldorf, Germny We hve developed tehnology tht depletes the omplement regultory protein (CRP) CD46 from the ell surfe, nd thereby sensitizes tumor ells to omplement-dependent ytotoxiity triggered by therpeuti monolonl ntibodies (mabs). This tehnology is bsed on smll reombinnt protein,, whih indues the internliztion nd subsequent degrdtion of CD46. In preliminry studies, we hd demonstrted the utility of the ombintion of nd severl ommerilly vilble mabs suh s rituximb, lemtuzumb, nd trstuzumb in enhning ell killing in vitro s well s in vivo in murine xenogrft nd syngenei tumor models. We hve ompleted sled mnufturing of protein in Esherihi oli for studies in nonhumn primtes (NHPs). In mques, we first defined dose of the CD2-trgeting mab rituximb tht did not deplete CD2-positive peripherl blood ells. Using this dose of rituximb, we then demonstrted tht pretretment with reonstituted ner omplete elimintion of B ells. Further studies demonstrted tht the tretment ws well tolerted nd sfe. These findings in relevnt lrge niml model provide the rtionle for moving this therpy forwrd into linil trils in ptients with CD2-positive B-ell mlignnies. Reeived 6 August 212; epted 9 September 212; dvne online publition 23 Otober 212. doi:1.138/mt Introdution Monolonl ntibodies (mabs) hve emerged s rpidly growing lss of onology therpeutis. Despite their suess in ertin linil pplitions, the therpeuti effiy of mabs is limited, with only minority of ptients responding to these gents s monotherpies. One of the lrgest mabs in terms of sles is rituximb, sold under the brnd Rituxn. It is n nti-cd2 ntibody used to tret vriety of blood mlignnies inluding non-hodgkin lymphom (NHL). There re over 3, ptients with NHL per yer in the USA. The vst mjority reeives tretment with rituximb either lone or ombined with hemotherpy. Even when rituximb is ombined with hemotherpy, the mjority of ptients with NHL develop reurrent, tretment-refrtory disese, nd the 5-yer survivl rte for ptients is 67%. 1 3 Rituximb binds to CD2-positive lymphom ells resulting in the killing of lymphom ells. Severl studies hve doumented tht binding of rituximb indues omplement-dependent ytotoxiity (CDC) tht leds to diret ell lysis. 4 8 Complement tivtion n lso trigger other rms of the immune system (ntibody-dependent ell-medited ytotoxiity or dptive T-ell responses) 9 tht ontribute to the therpeuti effet. 1 However, lymphom ells tively blok the ntitumor tivity of rituximb by upregulting CD46, whih is key ell surfe protein tht inhibits the tivtion of omplement. 5,11 Inresed levels of CD46 re found on the membrnes of lymphom ells nd my ontribute to the ineffetiveness of rituximb in treting NHL. 12 We hve shown previously tht CD46 expression on leukemi ells ws uniform nd t lest one order of mgnitude higher thn on norml peripherl blood mononuler ells (PBMCs). 13,14 Our reserh hs shown tht severl humn denoviruses, inluding serotype 35 (Ad35), use CD46 s reeptor. 15 We generted reombinnt protein derived from the fiber knob domin of Ad35 tht binds to CD46 with piomolr ffinity. This protein,, is produed in Esherihi oli nd tightly rosslinks severl CD46 reeptors. 16 Binding results in trnsient removl of CD46 from the surfe of lymphom ells nd tumor ells from other ners, inluding brest nd olon ners for bout 72 hours fter tretment. 13 During this time, tumor ells re sensitized to CDC triggered by mabs. In previous study, we hd demonstrted tht inresed the effiy of lymphom ell killing by rituximb both in vitro in primry nd estblished humn CD2-positive lymphom/leukemi ells, nd in vivo in tumor xenogrft models. 13 This study provides dditionl in vitro nd in vivo dt to support the linil pplition of otherpy with rituximb to tret NHL. We demonstrte in nonhumn primtes The first three uthors ontributed eqully to this work. Correspondene: André Lieber, University of Wshington, Box 35772, Settle, Wshington 98195, USA. E-mil: lieber@u.wshington.edu Moleulr Therpy vol. 21 no. 2, feb

2 Enhnement of Rituximb Therpy The Amerin Soiety of Gene & Cell Therpy (NHPs) tht intrvenous injetion of is sfe nd effetive s refleted by n inrese in rituximb-medited killing of peripherl blood CD2-positive ells. Results Studies with humn ell lines viously, we hd shown tht inreses rituximbtriggered CDC in severl lymphom nd leukemi ell lines. 13 Here, we extend these in vitro studies to other tumor types nd mabs: inubtion of CD52-positive Rji lymphom ells or Her2/ neu-positive BT474-M1 brest ner ells with signifintly inresed CDC triggered by mabs tht trget these moleules, i.e., lemtuzumb nd trstuzumb, respetively (Figure 1 ). No -ssoited ytotoxiity ws observed in tumor ells lines tht did not express the trget moleule for the orresponding mab (Figure 1b,, left pnels). Consistent with these results, lthough lso removed CD46 from norml humn ells inluding PBMCs, 13 it did not use ytotoxiity on CD2-negtive primry humn ells by itself or in ombintion with rituximb. 13 The therpeuti effets of were lso demonstrted in tumor xenogrft models. For exmple, in n orthotopi xenogrft model with Her2/neu-positive brest ner ells, two yles of /trstuzumb tretment prevented tumor relpse, wheres tumors reppered fter 8 dys in ll mie treted with trstuzumb lone (Figure 1d,e). Studies in humn CD46/CD2 double trnsgeni mie In ontrst to humns, where CD46 is expressed on ll nuleted ells, CD46 is expressed only in the testis of mie. For further sfety nd effiy studies, we therefore used humn CD46 (hcd46) trnsgeni mie (strin MCP-8B) tht express CD46 in humnlike pttern. 9,17 hcd46 trnsgeni mie were rossed with hcd2 trnsgeni mie rrying 168 kb DNA frgment of the humn CD2 gene lous. 18 Suh humn CD2 trnsgeni mie with syngenei hcd2-positive lymphom ell lines hve been used before for studies with rituximb. 4,19,2 For effiy studies with nd rituximb in double trnsgeni mie, we generted syngenei mouse lymphom ell line tht etopilly expressed humn CD2 nd CD46 (38C13-hCD46/CD2) (Figure 2). When 38C13- hcd46/cd2 ells were intrvenously injeted into hcd46/cd2 mie, tumors developed in the bone mrrow nd liver within 1 week (Figure 2b). By dy 16 fter trnsplnttion, mie developed hind leg prlysis, symptom tht ws used s the end point for therpy studies. When given lone on dy 14, rituximb inresed verge survivl to 21 dys. The ombintion of nd rituximb therpy further prolonged survivl by 1 week. A seond Tumor ells 8 hours 3 minutes 3 hours mab Norml humn serum Cell vibility d Tumor ells 27 dys 1 hours 7 dys 1 hours b % Vible ells Rji-CD52 (CD52-positive) 1 5 Jurkt (CD52-negtive) e 1, 9 8 plus trstuzumb % Vible ells Mok BT474-M1 (Her2/neu-positive) 1 5 Mok MDA-MB231 (Her2/neu-negtive) Tumor size (mm 3 ) Mok Mok Time fter tumor implnt (dys) Figure 1 Studies with humn ell lines. ( ) enhnes omplement-dependent ytotoxiity triggered by (b) lemtuzumb nd () trstuzumb in vitro. Tumor ells expressing the orresponding monolonl ntibodies (mab) trgets were treted with mab (15 µg/ml) nd/ or (25 µg/ml). In previous studies, -medited derese of CD46 from the ell surfe ws highest t 8 1 hours fter ddition of. 13 On the bsis of this, the intervl between nd mab ddition ws seleted. Norml humn serum () ws dded s soure of omplement in the indited groups. Cell vibility of phosphte-buffered sline-treted ells ws djusted to 1%. The right most brs show the effet of the ombintion therpy nd n be ompred with the third br from the left s the mab lone (+). N = 3. The differene between mab/ vs. plus mab/ is signifint (P <.1). (d,e) improves trstuzumb therpy in vivo. Her2/neu-positive brest ner ells BT474-M1 were injeted into the mmmry ft pd. Mie with estblished tumors were then injeted intrvenously with (2 mg/kg) followed by trstuzumb (2 mg/kg) 1 hours lter. Tretment ws repeted 7 dys lter. N = 1. The differene between the two groups ws signifint (P <.1) from dy 1 on vol. 21 no. 2 feb. 213

3 The Amerin Soiety of Gene & Cell Therpy Enhnement of Rituximb Therpy Counts Counts % M hucd2 82% M hucd46 b Bone mrrow hucd46 Liver hucd2 Perent survivl d OD Dys fter tumor ell trnsplnttion Dy 1 Dy 14 Dy 16 Dy 18 Dy 21 Dy 28 Dy 35 rituximb rituximb 6 Dys fter tumor ell trnsplnttion e Number of spots/1 6 PMBC Control Rituximb + rituximb ( + rituximb) 2x Ad.HBeAg Ad.HBeAg HBeAg HBsAg Figure 2 Studies with hcd46/hcd2 trnsgeni mie. () Flow ytometry nlysis of humn CD46 nd CD2-expressing mouse lymphom ell line 38C13-hCD2/CD46. (b) Representtive imges of tumor loliztion in trnsgeni mie. hcd46/cd2 trnsgeni mie were intrvenously injeted with syngenei 38C13-hCD2/CD46C lymphom ells. Tissues were nlyzed 1 week lter; the upper imge shows the presene of humn CD46 positive tumor ells in brown in the bone mrrow. The lower imge shows humn CD2-positive liver metstses (green). The sle brs re 2 µm. () Survivl of hcd46/cd2 trnsgeni mie rrying 38C13-hCD46/CD2 lymphom ells. (2 mg/kg) or phosphte-buffered sline () ws injeted t dy 14, followed 1 hours lter by rituximb (2 mg/kg). In one group of mie, seond round of tretment ws given 1 week fter the first tretment ( plus rituximb) 2x. Onset of hind leg prlysis ws used s the end point in Kpln Meier survivl studies. N = 5. Control vs. rituximb: P =.8; rituximb vs. plus rituximb: P =.2. Note tht from dy 28 on, ntibodies ginst were detetble by ELISA in serum of injeted mie (see rrow). (d) Anti- serum ntibodies in the group tht reeived two rounds of /rituximb injetion. The dys of /rituximb injetion re indited on the horizontl xis by rrows. Serum ws diluted 1:1 nd nti- ntibodies were mesured by ELISA. Shown re optil density 45 bsorption vlues. N = 5. (e) Immune responses ginst the Heptitis B virus E ntigen (HBeAg) in or -treted nimls. The double trnsgeni mie were intrvenously injeted with nd (2 mg/ kg). Three dys lter, intrmusulr immuniztions with n denovirl HBe vine were performed. Twelve dys lter, spleens were hrvested nd splenoytes were stimulted either with soluble HBeAg or ontrol ntigen (HBsAg). Twenty-four hours fter stimultion, interferon-γ enzyme linked immunosorbent spots were performed. The number of spots ws expressed s the verge +/ the SD. N = 5 nimls per group. PBMC, peripherl blood mononuler ells. round of /rituximb tretment, given 1 week fter the first yle, resulted in n verge survivl spn of 54 dys (Figure 2). Of note, t the time of the seond-tretment yle, mie lredy hd lerly detetble serum ntibodies ginst (mesured by enzyme linked immunosorbent ssy (ELISA)) (Figure 2d). The inresed ntitumor effets of seond yle of in the fe of nti- serum ntibodies is in greement with our previous in vitro studies demonstrting tht remins tive in the presene of nti- ntibodies. 13 For phrmokinetis studies, hcd46/cd2 trnsgeni mie were intrvenously injeted with (2 mg/kg), nd lerne from blood ws mesured. A biologil hlflife of 12.4 hours ws lulted from phrmokineti modeling of the elimintion urve (Supplementry Figure S1). Fluoresent lbel nlysis of orgn setions reveled mrked umultion in Kupffer ells nd spleni mrophges t 3 minutes fter intrvenous injetion, inditing tht is tken up by these ells (Supplementry Figure S1b). -treted nimls showed no overt signs of distress or disomfort fter injetion of the therpeuti. Lbortory studies t 3 minutes nd 6 hours post- injetion reveled no ritil hnges in blood ell ounts nd serum hemistry vlues. To test whether CD46 ligtion might use immunosuppression, possible outome of CD46 downregultion, 9,21,22 treted hcd46/cd2 trnsgeni mie were vinted with test ntigen nd immune responses were mesured. did not mesurbly suppress dptive immune responses ginst the test ntigen (Figure 2e). Mnufturing of ws produed under regultory omplint onditions (Supplementry Mterils nd Methods). The finl produt hd purity of 96% nd ontined <2 EU/mg of endotoxin. ws formulted in phosphte-buffered sline, sterile filtered, nd stored t 8 C. No deline in poteny ws deteted fter storge for 9 months (study ongoing). Seletion of NHP model Of ll mmmls, only NHPs express CD46 in pttern similr to humns. 23 The only notble differene is tht humn erythroytes lk CD46 while it is expressed on erythroytes from NHPs. vious studies demonstrted tht CD46 lignds, suh s mesles virus, hemgglutinte erythroytes from bboons (Ppio nubis). Here, we showed tht lso triggers hemgglutintion of bboon erythroytes (Figure 3). No -medited hemgglutintion ws observed with erythroytes from two mque speies: M nemestrin (pigtil mque) nd M fsiulris (ynomolgus monkey). We then performed funtionl tests to demonstrte tht Moleulr Therpy vol. 21 no. 2 feb

4 Enhnement of Rituximb Therpy The Amerin Soiety of Gene & Cell Therpy 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/124 1/248 1/496 1/8192 1/16384 M fsiulris Ppio nubis M nemestrin Humn b Counts Neg. M1 M. fsiulris PBMCs % Of CD2 + PBMC vibility CD2+ ells Rituximb plus rituximb Autologous serum Cell vibility CD Autol. serum Autol. serum Autol. serum rituximb Autol. serum rituximb Figure 3 In vitro studies with monkey ells. () hemgglutintion ssy. Seril dilutions of were inubted with 1% erythroytes nd hemgglutintion ws ssessed 1 hour lter. (b) Downregultion of CD46 on M fsiulris peripherl blood mononuler ells (PBMC). PBMC ells were inubted with 25 µg/ml for 12 hours. CD46 levels were nlyzed using phyoerythrin (PE)-onjugted ntibodies. Shown is representtive smple. The CD46 men fluoresene intensity (MFI) in phosphte-buffered sline ()-treted ells ws 28(+/ 4) (N = 3). The MFI in treted ells ws 175(+/ 22). P <.1. () B-ell depletion in vitro. PBMCs were purified from M. fsiulris, nd ultured for 1 dy. CD2-positive ells were sorted vi flow ytometry using (rossreting) nti-humn CD2-PE ntibody. For omplement-dependent ytotoxiity ssys, CD2- positive ells were inubted with 25 µg/ml or for 1 hours. Rituximb (15 µg/ml) or ws dded, followed by utologous serum (2% finl onentrtion, s soure of omplement) 3 minutes lter. Cell vibility ws mesured fter 3 hours of inubtion bsed on trypn blue exlusion. N = 8. Rituximb vs. plus rituximb: P <.1. mque CD46 is reognized by. Inubtion of mque PBMC with resulted in signifint downregultion of CD46 on the ell surfe 12 hours fter tretment (Figure 3b). Rituximb reognizes mque CD2, whih mkes this speies idel for studying the effets of the mab in depleting NHP B ells. 24 Addition of rituximb nd utologous serum (s soure of omplement) results in CDC of flow ytometri-sorted mque CD2-positive ells in vitro (Figure 3). This effet ws signifintly enhned by. The M genus therefore represents n dequte model to study the sfety nd effiy of /rituximb ombintion therpy. We seleted the M. fsiulris speies for our studies s they re redily vilble t reltively low ost. A summry of ll nimls studied is shown in Tble 1. serum lerne nd CD46 levels on PBMCs in NHPs To test the sfety of, M. fsiulris ws intrvenously injeted with high dose (4 mg /kg). serum onentrtions were mesured by ELISA t severl time points fter injetion. Figure 4 shows the dt for three nimls. After rpid deline during the first 48 hours fter injetion, levels remined in the rnge of 2 5 µg/ml until dy 9, fter whih beme undetetble. The ltter is notble sine in our in vitro studies, showed effiy t onentrtions s low s 25 ng/ml. Modeling of the phrmokinetis using two-omprtment distribution predited biologil hlf-life Tble 1 Overview of study nimls Animl ID Gender Weight (kg) Rituximb (mg/kg) Observtion time A1278 M dys A1266 M dys A1274 M dys M5221 F dys M726 F dys M72 F dys M7183 M dys A1119 M dys A1124 M dys A11311 M dys A112 M D2 9 dys A11319 M D2 9 dys A11294 F D3 28 dys A11314 M D3 28 dys The intervl between nd rituximb injetion ws either 2 dys (D2) or 3 dys (D3). Full neropsy. of 11.2 hours (Supplementry Figure S2). Beuse the mehnism of tion for is thought to be through the trnsient removl of CD46 from the surfe of ells, we mesured CD46 on PBMC by flow ytometry s red out for funtionl tivity vol. 21 no. 2 feb. 213

5 The Amerin Soiety of Gene & Cell Therpy Enhnement of Rituximb Therpy 35 b 12 CD46 + ells 3 1 % Chnge ng/ml 35 MCP hour 3 hours 6 hours 12 hours d1 d2 d7 d21d28 A1266 A1274 A1278 A1266 A1278 A hours 12 hours d1d2 d3 d4d5 d6 d7d9 d21d28 % Chnge % Chnge IL hour 3 hours 6 hours 12 hours d1 d2 d7 d21d28 d2 d3 d4 d7 A1266 A1274 A1278 % Chnge 2,5 2, 1,5 1, 5 d21 d28 A1266 A1278 A1274 TNF-α 1 hour 3 hours 6 hours 12 hours d1 d2 d7 Figure 4 Anlysis of serum lerne, CD46 on peripherl blood mononuler ells (PBMCs), nd serum ytokine nd hemokine levels in nimls tht reeived intrvenous (4 mg/kg). () Serum onentrtions of fter intrvenous injetion of 4 mg/kg of. (b) CD46 on PBMCs mesured by flow ytometry. The perentge of CD46+ ells before injetion ws tken s 1%. () tretment levels were tken s 1%. Interleukin (IL)-1β, IFN-γ, IL-1, IL12/23 were lso mesured but not deteted or unhnged nd therefore not grphed. IFN, interferon; MCP, monoyte hemotti protein. A1266 A1274 A1278 d21 d28 of in M. fsiulris. At dy 3 postinjetion, CD46 levels on PBMCs delined bout 5% from bseline nd returned to norml by dy 28 postinjetion (Figure 4b). Sfety Animls were observed for 28 dys. No hnges in body weight, tivity, or behvior were observed. Blood ell nd hemistry nlyses of nimls injeted with 4 mg/kg did not revel bnormlities with the exeption of modest inrese in the onentrtions of serum trnsminses t dy 1 postinjetion (Tble 2). The trnsminitis might be linked to the trnsient inrese in the serum levels of proinflmmtory ytokines (interleukin-6 nd tumor nerosis ftor-α) nd hemokines (monoyte hemotti protein-1) tht we observed during the first 24-hour postinjetion (Figure 4). The ltter ould, in turn, be the result of residul mounts of bteril endotoxin present in the preprtion nd subsequent tivtion of tissue mrophges. Considering unspeifi uptke of by liver nd spleen mrophges fter intrvenous injetion into hcd46/cd2 trnsgeni mie, similr mehnism of mrophge tivtion ould lso tke ple in mques. Notbly, t the time of neropsy (dy 9 or dy 28 fter injetion), we did not detet on tissue setions. There ws (nonsignifint) derese (P =.8) in serum onentrtions of omplement ftor C3 t week 2 nd 3 fter infusion. A omplete neropsy with gross nd histopthology ws performed t the end of the observtion period. Mild enteroolitis nd myordil fibrosis were found. These hnges re ommonly seen in the olony. None of the bnormlities found during neropsy were relted to tretment. Effiy To ssess the linil effiy of our pproh, we first tested the effets of different rituximb doses on depletion of CD2-positive B-ells. Our gol ws to find dose of rituximb tht hd limited bility to deplete peripherl blood CD2-positive ells, whih in turn, would llow us to test whether would inrese rituximb effiy. In ptients with NHL, rituximb is typilly dministered t dose of 1 mg/kg. In M. fsiulris, intrvenous injetion of 1.,.5, nd.1 mg/kg rituximb resulted in lmost 1% depletion of peripherl CD2-positive ells within 24 hours (Figure 5). All of these doses lso resulted in nerly omplete depletion of CD2-positive ells in the bone mrrow, while some dose response effet ws seen with spleni CD2-positive ells (Supplementry Figure S3). However, lower rituximb dose of.1 mg/kg hd no signifint effet on peripherl CD2-positive ell levels in three different nimls (Figure 5b). Therefore, we seleted this dose for testing the effets of on enhning depletion of CD2-positive ells by rituximb. On the bsis of the kinetis of CD46 removl from PBMC, we dministered 2 dys before rituximb (.1 mg/kg) injetion in the first two treted nimls (A112 nd A11319). Two dditionl nimls (A11314 nd A11294) were given rituximb t dy 3 fter injetion. In both ombintion therpy regimens, CD2-positive ells delined to level <4% of tht observed the dy following the injetion of the sme dose of rituximb dministered lone (Figure 5). The effet ws even more pronouned in the CD46 high subfrtion of CD2-positive ells, where the ombintion therpy depleted more thn 9% of the ells (Figure 5d). This subfrtion is more refletive of the sitution in ptients with Moleulr Therpy vol. 21 no. 2 feb

6 Enhnement of Rituximb Therpy The Amerin Soiety of Gene & Cell Therpy Tble 2 Blood ell ounts nd blood hemistry in nimls tht reeived intrvenous Complement D D1 D7 D14 D21 D28 AVE SD AVE SD AVE SD AVE SD AVE SD AVE SD C Renl/hepti pnel Sodium Potssium Chloride Gluose Ure nitrogen Cretinine Totl protein Albumin Totl bilirubin Clium Phosphte Totl holesterol Alkline phosphtse (totl) ALT AST γ glutmyl trnsferse CBC WBC RBC Hemoglobin Hemtorit MCV MCH MCHC RDW-CV Pltelet ount Neutrophils Lymphoytes Monoytes Eosinophils Bsophil Immture grnuloytes ALT, lnine trnsminse; AST, sprtte trnsminse; AVE, verge of three nimls; CBC, omplete blood ount; MCH, men orpusulr hemoglobin; MCHC, men orpusulr hemoglobin onentrtion; MCV, men orpusulr volume; RBC, red blood ells; RDW-CV, oeffiient vrition of red ell distribution width; WBC, white blood ells. NHL where CD46 is upregulted in lymphom ells. 13 The level of CD2-positive ells ws not ffeted by injetion lone (Figure 5e). In ddition, the ombintion therpy hd no effets on nontrget ells, e.g., CD21+ ells (Figure 5f). Blood ell nd hemistry prmeters of nimls injeted with plus.1 mg/kg rituximb were not signifintly different from nimls injeted with lone (Supplementry Figure S4). In nimls injeted with, serum ntibodies (IgG) ginst beme detetble t dy 7 fter injetion (Figure 6). Of note, one of the nimls (A11319) tht reeived the ombintion tretment hd lerly detetble serum ntibodies tht reted with before reeiving tretment. In spite of this, tretment enhned rituximb-medited CD2-positive ell killing. The pperne of nti vol. 21 no. 2 feb. 213

7 The Amerin Soiety of Gene & Cell Therpy Enhnement of Rituximb Therpy 12 % CD2-positive ells mg/kg 12.5 mg/kg 12.1 mg/kg.1 mg/kg M M72 6 M7183 M h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h b % CD2-positive ells A1119 A A Dy 1 Dy 2 Dy 3 Dy 4 Dy 7 % Positive ells Dy 1 Dy 2 Dy 3 Dy 4 Dy 5 Dy 6 Dy 9 Dy 14 Dy 21 CD2 Dy 28 A112 A11319 A11314 A11294 d Dy 1 CD46 high within CD2 Dy 2 Dy 3 Dy 4 Dy 5 Dy 6 Dy 9 Dy 14 Dy 21 Dy 28 e % Positive ells Dy 1 Dy 3 Dy 4 Dy 5 A1278 A1266 Dy 6 Dy 9 f Dy 1 Dy 3 Dy 4 Dy 5 Dy 6 A112 A11319 Dy 9 Figure 5 Rituximb-medited depletion of peripherl blood CD2-positive ells. () Perentge of peripherl blood CD2-positive ells. tretment levels were tken s 1%. The nimls were euthnized t dy 3 nd CD2-positive ells were mesured in the spleen nd bone mrrow. (b) A rituximb dose of.1 mg/kg did not signifintly redue the perentge of peripherl CD2-positive ells. The nimls were followed for 7 dys. ( f) enhnes rituximb-medited depletion of peripherl CD2-positive ells. Animls A112 nd A11319 reeived rituximb (.1 mg/kg) 2 dys fter injetion (4 mg/kg). For nimls A11314 nd A11294 the time intervl between the two drugs ws 3 dys. () Peripherl blood CD2-positive ells. tretment levels were tken s 1%. (d) CD2-positive ells were subgted using flow ytometry for CD46 high ells. (e,f) Experimentl ontrols: (e) CD2-positive ells in nimls tht reeived only. (f) CD21-positive ells in nimls tht reeived the ombintion nd rituximb. OD 45 b 35 3 (ng/ml) d1 d2 d3 d4 d5 d6 d7 d9 d21 d28 6 h 12 h d1 d2 d3 d4 d5 d6 d7 d9 d21 d28 A1266 A1278 A112 A1274 A11319 A11294 A11314 A1266 A1278 A112 A1274 A11319 A11294 A11314 Figure 6 nd nti- ntibody serum levels in ll nimls tht reeived intrvenous injetion. Note tht niml A11319 hd ntibodies tht reted with. In spite of this, ws funtionl (see Figure 5,d). () nti- serum ntibodies. Serum ws diluted 1:1 nd nti- ntibodies were mesured by ELISA. Shown re optil density 45 bsorption vlues. (b) onentrtions. ntibodies oinided with the inbility to detet in serum (Figure 6b). Disussion We intend to use s otherpy for rituximb in ptients with non-hodgkin lymphom. Towrds this gol, we first provided dditionl in vitro effiy dt in this study. Furthermore, using relevnt trnsgeni lymphom model, we demonstrted tht intrvenous injetion of therpeutilly effetive dose is sfe nd well tolerted. We hve lso estblished protool for sled mnufturing of. Protein tht ws produed bsed on this protool ws used in studies in NHPs. M. fsiulris is n exellent niml model to test the sfety nd effiy of in preprtion for n Investigtionl New Drug submission beuse (i) CD46 is expressed in similr pttern of distribution to humns; (ii) does not hemgglutinte mque erythroytes; (iii) rituximb rossrets with mque CD2 nd depletes B-ells fter intrvenous injetion 24 similr to the observtion in humns. Mques hve been used before in sfety studies for other CD46-trgeting therpeutis suh s intrperitonel injetion of onolyti mesles viruses. 25 Tken together, our studies in M. fsiulris provide evidene tht intrvenous injetion is sfe nd well tolerted; hs good phrmokineti prmeters; results in downregultion of CD46 on PBMCs; nd inreses the effetiveness of rituximb in depletion of peripherl blood CD2-positive ells, speifilly CD2 + CD46 high ells, i.e., subset of CD2-positive ells tht resembles lymphom ells. This outome provides dditionl Moleulr Therpy vol. 21 no. 2 feb

8 Enhnement of Rituximb Therpy The Amerin Soiety of Gene & Cell Therpy support for the potentil for to enhne the ntitumor effets of rituximb in ptients with NHL. is protein derived from n denovirus with low seroprevlene of nti-ad35 ntibodies in humns. 13 However, ntibodies will likely be generted over time fter intrvenous injetion s doumented in our study in NHPs. This is unlikely to be signifint issue in ptients with NHL who hve suppressed immune system, both relted to their disese nd its tretment. 26 In ddition, remining norml B ells will be depleted by rituximb, whih further depresses ntibody responses. 27 In ft, rituximb is used linilly to suppress ntibody prodution to prevent humorl-medited trnsplnt rejetion. 28 It is therefore unlikely tht ptients with NHL treted with nd rituximb will generte ntibodies ginst. Furthermore, previous in vitro studies hve demonstrted tht remins tive in the presene of ntibodies generted ginst it. 29 This ws ttributed to the piomolr vidity of to CD46, whih outompetes the less vid nti- ntibodies. 29 In this study, we provide further support for the tivity of in the presene of nti- ntibodies. For exmple, mintined its bility to enhne the B-ell lymphom depleting effets of rituximb therpy in hcd46/cd2 trnsgeni mie in the presene of detetble nti- serum ntibodies (see Figure 2). Furthermore, one of the mques hd preexisting serum ntibodies tht reted with, likely due to immunologil rossretivity between simin nd humn denoviruses. 3 In spite of this, enhned rituximb-medited B-ell depletion. Severl studies indite tht binding to CD46 indues signling in immune ells tht blok immune responses ginst itself. 9,21,22 This rises the question whether uses trnsient immunosuppression or ntigen-speifi tolerne, phenomen tht ould be prtiulrly detrimentl for ptients with ner. There ws no linil or lbortory evidene of immunosuppression observed in our studies in hcd46/cd2 trnsgeni mie, whih reeived intrvenous injetions. Furthermore, none of the treted mques displyed signs of opportunisti infetions during the 28-dy observtion period. The severity of enteroolitis nd myrdil fibrosis tht is present in untreted nimls of the olony ws not inresed in nimls tht reeived. Finlly, it is notble tht ptients with geneti muttions within the CD46 gene do not show evidene of inresed suseptibility to infetions. 31 The role tht omplement plys in the ytotoxi effets of rituximb hs been ontroversil In our study, we hve demonstrted tht derese of the omplement regultory protein (CRP), CD46, sensitizes CD2-positive ells to rituximb in mques, whih provides support for n importnt role of omplement nd CDC in the tivity of rituximb. A ritil role of CRPs in onferring resistne in ner tretment is further orroborted by study with reombinnt inhibitor of nother CRP, CD59, in whih Qin nd ollegues showed tht CD59 inhibitor enhned CDC triggered by oftumumb nd rituximb in B-ell NHL nd hroni lymphoyti leukemi resistnt to these mabs In this ontext, it might be benefiil to ombine, the CD59 inhibitor nd potentilly n inhibitor ginst the third key ell surfe CRP, CD55, into oktil to be used s otherpeutis. In this ontext, it is lso noteworthy tht vrious monolonl ntibodies ginst CD46 did not trigger CD46 internliztion nd did not sensitize lymphom ells to rituximb therpy. 13 We speulte tht the bility of the trimeri to tightly luster t lest three CD46 moleules s well s its piomolr vidity re ruil for its funtion to remove CD46 from the ell surfe. In this study, we provide evidene supporting the sfety nd effiy of in ombintion with rituximb in the therpy of hemtologi mlignnies. otherpy ould potentilly inrese the therpeuti effiy of rituximb nd overome resistne to tretment. Furthermore, otherpy ould redue the mounts of rituximb required for tretment, thus reduing its osts nd potentil side effets. On the bsis of the enourging results from this nd previous studies, we pln to dvne this ombintion therpy into the lini to tret ptients with B-ell mlignnies. MterilS nd Methods Animl studies in mie. All experiments involving nimls were onduted in ordne with the institutionl guidelines set forth by the University of Wshington. CB17-SCID-beige mie were from Jkson Lbs (Br Hrbor, ME). C57Bl/6-derived, humn CD46 trnsgeni mie (strin MCP-8B) were desribed erlier. 38 Humn CD2 trnsgeni mie were provided by Mrk Shlomhik (Yle University). 18 Humn CD46 nd humn CD2 mie were rossed, nd double trnsgeni (hcd46/cd2) mie were identified by genotyping using til DNA. Brest ner xenogrfts were estblished by injeting BT474-M1 ells into the mmmry ft pd of CB17 SCID-beige mie. Tumor volumes were mesured s desribed previously. 39 To estblish the mouse lymphom model, C38C13-hCD46/CD2 ells were injeted into the til vein of hcd46/ CD2 double trnsgeni mie. The end point for Kpln Meier studies ws the onset of hind leg prlysis. Animl studies in mques. All studies were performed by the Wshington Ntionl Primte Reserh Center t the University of Wshington. nd rituximb were infused intrvenously t rte of 1 ml/min. Blood smples were drwn t the time points indited. At the end of the observtion period, omplete neropsy ws performed on seleted nimls. Cell ulture. Rji, Jurkt, nd MDA-MB231 tumor ell lines were from the Amerin Type Culture Colletion nd ultured s reommended. Rji ells were sorted vi flow ytometry for subfrtion tht expresses high levels of CD52. 4 BT474-M1 is tumorigeni sublone of the Her2/neu-positive brest ner ell line BT BT474-M1 ells were ultured in Dulbeo s modified Egle s medium/f:12 with 1% fetl bovine serum, 1% Peniillin/ Streptomyin, nd L-Glutmine. Primry humn vsulr endothelil ells, ornel epithelil ells, ovrin surfe epithelil ells, nd fibroblsts were from Lonz (Allendle, NJ) nd were ultured in medi from Lonz. C38C13 ells were kindly supplied by Mrk Shlomhik (Deprtment of Lbortory Mediine, Yle University Shool of Mediine). The genertion of C38C13- hcd46/cd2 ells is desribed in the Supplementry Mterils nd Methods. Mque mononuler ells from peripherl blood, bone mrrow, nd spleen were purified s desribed elsewhere. 13 In vitro vibility ssys. Cells were inubted with phosphte-buffered sline or 25 µg/ml. After 8 hours, 15 µg/ml of the orresponding mab ws dded to ells nd inubted t room temperture for 3 minutes. Norml humn serum ws dded to finl dilution of 1:5 nd ells were inubted t 37 C for nother 3 hours. Vible ells in eh well were ounted fter trypn blue stining. Eh smple ws tested in triplite nd eh well ws ounted four times. Three independent studies were performed for eh prmeter under evlution. Flow ytometry. Blood smples were treted with BD Phrm Lyse (BD Biosienes, Sn Jose, CA), wshed, nd then inubted with F Blok nd ntibodies for 3 minutes. Cells were then wshed nd subjeted to flow vol. 21 no. 2 feb. 213

9 The Amerin Soiety of Gene & Cell Therpy Enhnement of Rituximb Therpy ytometry. Simultneous four-olor flow ytometri nlysis for CD46, CD2, CD4, nd CD21 ws performed on BD FACSSCnto Flow Cytometer (BD Biosienes, Sn Jose, CA). Smples were nlyzed in triplite. An liquot of PBMCs ws stored in liquid nitrogen for further nlyses. Cytokine detetion. Serum smples were nlyzed using the Milliplex NHPs ytokine premixed 23-plex immunossy kit from Millipore (Billeri, MA). SUPPLEMENTARY MATERIAL Figure S1. Phrmokinetis studies in hcd46/cd2 trnsgeni mie. Figure S2. Phrmokineti elimintion of. Figure S3. Perentge of CD2-positive ells in bone mrrow nd spleen t 72 hours fter rituximb injetion. Figure S4. Blood ell ounts nd blood hemistry in nimls tht reeived intrvenous (4 mg/kg) nd rituximb (.1 mg/kg). Mterils nd Methods. ACKNOWLEDGMENTS We re grteful to the following ollbortors for providing vluble mterils nd dvie: Ed Clrk (University of Wshington), Mrk Shlomhik (Yle University), Oliver ss (Fred Huthinson Cner Reserh Center), Dvid Mloney (Fred Huthinson Cner Reserh Center), Lynn Rose (Settle Children s Hospitl), nd Kim Brue (Settle Children s Hospitl). The work ws supported by Ntionl Institutes of Helth (NIH) grnts R1 CA8192 (A.L.), R1 HLA78836 (A.L.), R43 CA (D.C. nd A.L.), grnt from the Wshington Stte Life Siene Disovery Fund, grnt from the Wshington Reserh Foundtion, nd two grnts from the Institute for Trnsltionl Helth Sienes t the University of Wshington. Speimens were obtined from the Ntionl Primte Reserh Center t the University of Wshington, NIH grnt RR166 nd from the Ntionl Center for Reserh Resoures nd the Offie of Reserh Infrstruture Progrms of the NIH through Grnt Number OD I.B. is reipient of postdotorl fellowship wrd from the Deutshe Krebshilfe (18988). REFERENCES 1. Boye, J, Elter, T nd Engert, A (23). An overview of the urrent linil use of the nti-cd2 monolonl ntibody rituximb. Ann Onol 14: Molin, A (28). A dede of rituximb: improving survivl outomes in non- Hodgkin s lymphom. Annu Rev Med 59: Pulte, D, Gondos, A nd Brenner, H (212). Expeted long-term survivl of older ptients dignosed with non-hodgkin lymphom in Cner Epidemiol 36: e19 e Di Getno, N, Citter, E, Not, R, Vehi, A, Grieo, V, Snzini, E et l. (23). Complement tivtion determines the therpeuti tivity of rituximb in vivo. J Immunol 171: Goly, J, Citter, E, Di Getno, N, Mngnini, M, Mos, M, Nebuloni, M et l. (26). The role of omplement in the therpeuti tivity of rituximb in murine B lymphom model homing in lymph nodes. Hemtologi 91: Reff, ME, Crner, K, Chmbers, KS, Chinn, PC, Leonrd, JE, Rb, R et l. (1994). Depletion of B ells in vivo by himeri mouse humn monolonl ntibody to CD2. Blood 83: Bellosillo, B, Villmor, N, López-Guillermo, A, Mré, S, Esteve, J, Cmpo, E et l. (21). 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