PCTIUS 9 21 0~ 5 45 ;
: RO / US 2 t ~ Jr 1992 `
A COMBINATION OF ANTI-cr~B-2 MONOCLONAL æNTIBODIE5 AND ~ET~OD OF USING
The pre~ent invention relate~ to a combination of monoclonal antibodi-~ capable of preventing and treating tumors. More ~p~cifically, the monoclonal antibodies are single chain monoclonal antibodie~ which are capable of treating and prev~nting tumor~.
Amplification ~nd/or overexpre~ion of the erbB-2 gene (al~o ~alled HER-2 or neu) r-~ult~ in overexpre~ion of erbB-2 mRNA ~nd prot~in~ and hA~ be~n demon~trated in 20-30%
of adenocarcinoma~ o~ th~ brea~t (1-5), ovary (3), lung (6) and ~tomach (73. Two lin~ o evidence implicate erb3-2 overexpr~s~ion in th~ pathog~n~ of human neoplasi~.
First, ov~roxpr~ion ha~ n linkod with poor progno~is in brea#t (~-11), ovarian (12), stomach (13), and lung cancer (14), indicating th~t over~xpr~-ion alt~r~ the cancer cell.
Second, arti~ici~l ovQr~xpr~s~ion of erbB-2 induce~ a tran~ormod phQnotyp- in NIH~3T3 fibroblasts (15, 16) as w~ in mamm~ry ~pithelial cell~ (17) ~ugge~ting that ovar~xpro~sion can contrl~uto directly to the development of the mal~gnant ph-notyp~.
Du~ to th- oxten~iv~ homology botw~n gpl85er~ 2 and tho p~d~rm-l growth factor recoptor (EGFR), it i~ wid~ly a~um~d th~t th~ir activation might proc~-d through ~imilar mQchani-m~. On- ~uch m-ch~ni~m involve~ receptor dim riz~tion/oligomor$zation which i5 thought to be an import~nt ~t~p in the activation of the EGFR intrin~ic tyro~in~ kina~e function ~1~, 19). Int-rferiny with receptor-r-ceptor interaction ha~ b~on evaluatRd as a potontial ther~poutt c approach to treatment of canc~r3 with erbB-2 ov~rexpre~ion. Pr~vtou~ ~tudl~ hav~ evaluated the u~o of ~ingl~ monoclonal antibodies dir~cted again~t ~rbB-2 PCTI~JS 9 2 / 0 8 5 4 5 R0/US 2~ GG, 1992 (20) and the r~lated Epidermal Growth ~actor Receptor (EGF~) ~protein (21) a~ pot~ntial therapeutic agent~ for the treatment of canc~r.
Use of 9ingle monoolonal antibodie~ directed against erbB-2 which have been evaluated a~ potential therapeutic agents have resulted in increased qp185erbB-2 autophosphorylation, resulting from increases in the activity o tyro~ine kina~e. Single anti~ody agents have shown promi~e a~ potential anticancer therapie~l (20, 27).
SUMMARY OF THE _~NVENTION
One ob~ct of Appli~ant'~ inv~ntion relates to a combination of ~t l~a~t two monoclonal anti~odie~ cap~ble of treatin~ or pr~v~nting hum~n malignaneieR wherein the malignant c~118 over~xpre~ gpl85~rbB 2. Th- combination compriJ~ 2t l~a~t a fir~t and ~econd ~ntibody ~ach of which r~cogniz~3 th- gp1~5 extrac~llular domaln o erbB-2. The activity demon~rat~d by th~ combination antibody tr~atment ha~ ~hown gr~at~r activlty than expected by the ~um cf the indi~idu~l ~ntibodi~ at th~ ~me o~erall antibody concentr~tion.
Anoth-r ob~ct of tho pre~ent invQntion provide~ for th~ u~o of ~onoclonal antl~odi~J which ar~ ~ingle chain monoclon~l a~tibodi-~. Th9 ~ingl- chain antibodie~ can b~
u~d to form a bi-pocific ~nti~ody.
DETAILED DESCRIPTION OF THE DRAWINGS
Flgur~ lA. Sp~ci~icity o monoclonal antibodieq #21 an~ #23. Subconfluent SK-~r-3 monolay~r~ wore metabolically`
la~-lod with 35S-Cy~ (~p~c. açt. 1000 Ci/mmol). ~Total cell protein~ were immunoprecipitated with 10 ~g of th- indicated antibodi~. The immune compl~xe~ w~re recov-red by Protein G Agaro~- (G~n-x, Gaither~bur~, MD) and analyzed by SDS-PAGE
PCT/IJS 92/ 0~545 .
R0/US 2`t G~JT 1992 on an 8-16% Tri~-Glyoine gel. The gel was exposed to film at -70C overnight with an intensifying screen.
Figur~ lB. gp185 ov~rexpres~ion in the gastric cell line N87 and a tumor from N87 mou#e xenografts compared to high and low gpl85erb~ 2 overexpres~er~. Cells or tumo were lysed in ~ample buffer which contained 0.125 M
Tris-HCl, 4% SDS, 0.002% bromophenol blue, and 15% glycerol.
5~ ~-mercapto~thanol was addod after the protein concentration wa~ d~t~rmined. Samples (lO ~g total prote~n) wer~ boil~d for 3 min, fractionat~d by SDS-PAGE on 8-16%
Tris-Glycine g~l and tran~forred to nitrocellulose Detection of gpla5~rb~ 2 wa~ p-rformed with a monoclonal antibody to th~ c-terminal portion of th~ protein.
Figuro lC. South~rn blot analy~i~ of the erbB-2 gen~
in N~7 (ga8tric), SK-Br-3 (br~Jt)~ and SK-OV-3 (ovarian~
cell line~ ~nd human plac~nta. DMA wa~ extracted from cell lines and human plac~nta ti3~ue u~ing ~u~nidine thio~yanate and c~sium gr~di~nt c~ntrifugation. DNA ~lS ~g) was cleaved with re~triction ~nzym~ HinDI r I, ~parated by electrophor~ on ~ lX agaro~ gel, tran~ferred to nitrocollulo~, and prob-d with radioactive erbB-2 cDNA
probe a~ proviou-ly d~crlb~d (26). The cDNA probe corre~pondJ to th~ entir~ erbB-2 protein eoding rogion.
Figur- Z. Effect of Ab#21 and Ab#23 on th- growth of ~uman N~7 ga-tric tumor cell~ in a monolayer MTT ~rowth aJ~y. ~ ~ingl- ~11 suspen~ion of 10,000 c~ /well was plat-d ln ~h~mically defined medium consi3ting of R~MI-1640 ~uppl~nted with In~ulin (5 ~g/ml) , human tranYf~rrin ( 10 ~g/ml) , 17-B-~tradiol (10 nM), sodium ~elenite (5 nM), and 10 mM H~pe~. PBS, Ab#21, Ab#23 or a combination of Ab#21 and Ab#23 at th~ indicated conc-ntratlon w~r~ then ~ddod. The plate~ wer- ~rown at 37C in a Sx C02 humidifi-d atmo~ph~r~. After 7 days, 50 ~1 .. .. .... . . . .. . .. . .. . ............. . . . . .. . .
;-- PCT/US 92/ C~545 R() ~ US 12 1 OCI 1~2 o 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (0.1 mg) w~re added and allowed to incubate for 4 hours at 37C. 90% of the media wa3 then removed and the cry~tals ~olubilized in 0.175 ml ~M50. Optical densltles were measured at 540 nm in a Molecular Devices Vmax kinetlc microplate reader. Re~ult~ are th~ average of ei~ht well~
with standard deviation~ noted. Under th~ conditions used, the ~ell number i~ directly proportional to MTT reductlon.
Figure 3A. Effect~ of tr-atm~nt with Ab#21 ( ), Ab#23( ), a combination of Ab#21 and Ab#23 ( ), or PBS
( ) on th~ growth of N87 tumor x~nograft~ in BNX mice.
Tumor c~ (5 X 10 /mou~e) were ~ubcutaneously inje~ted into th~ 1ank~ of BNX (b~ig~, nudo, xid) mice. Tr~atment begun on day 1 consist~d of four trial group~ (3 ml~e p~r qroup) ~ach giv~n 0.2 ml intrap~riton~2l inj~ctions twice a week o eith~r P~S ( ) , Z00 ~ purifi~d Ab#21 ~0), 200 ~q purifi~d Ab#23 ( ), or a mixtur~ of 100 ~g purified Ab#21 and 100 ~g of purifiod Ab#23 ( ) for thr~ we~ks. Tumor ~rowth i~ report~d a~ an average r-latlv~ tumor volume, ~.e.m. ~15X. Two repoat~ of th~ exp-rim~nt gave the -~ame re~ultJ .
Figur~ 3B. E~f~ct of tr~atm~nt aft~r tho formation of ~mall tumors C~ w~re in~-cted u~ing th~ ~am~ tr-atmont protocol a~ abov~ exc-pt or tho fact th~ tr~atmen~ wa~
b~gu~ 4 ~ay- ft-r c~ll in~ctlon in~t~ad of 1 day after Anlm~l Gar- WaJ in accordanc~ with in~titutional guideline~
Flgur~ 4A Eff~ct o~ antibody binding on erbB-2 prote~n turnov~r Subconfluant N~7 cell monolayer~ were pul~-lab~l~d 1 h with 20 ~Ci 35S-Cy~t~ln- and then ~hased with 5 mM Cy~ in the pr-~-nce o~ Ab#21 alone, Ab#23 alone, or a 1 1 combination o Ab#21 and Ab#23 (10 ~g/ml)for 24 h Total c~llular prot~in wa- lmmunopr~cipltated a~ de~ribed in Figur~ 1 u~ing a monoclonal antibody dir~ctod again~t the `- RO/US` 21 OCT1992 :
c-terminu~ of gpl~Serb~ 2 coupled to Sepharose and analyzed by SDS-PAGE. The gel wa~ expo~ed to film at o70C over~lgh' with an int~nsifying Rcreen.
Figur~ 4B. Measurement of tyro~ine phosphorylation of ~pl85erbB 2 after incubation with an~ibody combination.
Cells were plated as in Figure 4A. After 1 h cells were proces~ed a~ in Figure lB. The protein~ were electroblot~ed onto nitrocellulo3~ paper and incubated with anti-pho~photyro~ine IgG (polyelonal, Up~tate Biotechnology, Inc.) and immunod-t~ct~d u~ing an ECL w~tern blotting detoction ~y~tem (Amer~ham). The fil~ wa~ expo~ed for S min at room t~mp~rature.
Figur~ 5. Eff~ct~ of Ab#21 and Ab#23 on th~ growth of human Calu-3 lung ~denocarclnom~ tumor cell~ in a monolay~r MTT growth a~ay. A ~$ngle c~ u~pen~ion of 10,000 cell~/well wa~ plat~d in a chemic~lly defined medium consi~ting of RPMI-1640 ~uppl-m-nt-d with In~ulin (5 ~g/ml), hum~n tr~nsf-rrin ( 10 ~g/~l ) , 17-B-~tradiol (10 nM), sodium s-l-nito (5 nM), ~nd 10 mM H-pe~. PBS, Ab#21, Ab#23 or a combination of Ab#21 2nd Ab#23 at the indicated conc-ntr~tion w~r~ thon add~d. Th- plate~ were grown at 37C in ~ 5% C02 humidifi~d ~tmosphero. Aftor 7 day~, 50 ~1 of 3-(4,5-d$m~thylthiazol-2^yl)-2,5-diph-nyl tetrazolium bromid- (0.1 mg) w~r~ add2d and allowed to incubate for 4 hour~ at 37C. 90X o th~ m~dia was th~n removad and the cry-t~ olubiliz~d in 0.175 ml DMSO. Optical densitie~
w-r- m~urod at 540 nm in a Molocular D~vic~ Vmax kinetic micropl~ta r~ador. Re~ult~ ar~ th~ av~rag- of eight wells withi~tandard d-viation~ noted. Und-r the conditions used, tho c~ll numb~r i~ dir-ctly propor~ional to MTT reduction.
Figur~ 6. E~f~ct~ of Ab#23 and Ab#94 on tho growth of human Calu-3 lung adonocarcinoma tumor cell~ ln a monolayer MTT ~rowth ~a~ay. A ~inglQ c~ u-pon~ion of 10,000 PCThJS 9 21 0 ~ 5 4 5 R O / U S ~ 1 0 ~ ~ 1992 cells/well was plated in a ~hemically defined med~um consisting of R8MI-1640 supplemented with Insulin (5 ~g/ml), human tran~ferrin ( 10 ,ug/ml ), 17-B-eRtradiol ( 10 nM), sodium selenite (5 nM), and 10 mM Hepe~. PBS, Ab#23, Ab#94 or a combination of Ab#21 and Ab#23 at the indicated concentration were then addod. The pla es.were grown at 37C in a 5% C02 humidifiod atmo~ph~re. Aft~r 7 days, 50 ~1 of 3-(4,5-dimethylthiazol-2-yl)-2,5-diph~nyl tetrazolium bromide ~0.1 mg) were added and allow~d to incubate for 4 hours at 37C. 90% of the m-dia wa~ then remov~d and the cry~tal~ solubiliz~d in 0.175 ml DMSO. Opti.caL densities wer~ m~a~ured at 5~0 nm in ~ Mol~cular D~vice~ Vmax kinetic microplat~ r~ader. R~ult~ ar~ th~ average of eight wellc with ~tandard deviation~ not~d. Und~r the ~ondition~ used, tho ~ell number i~ dir~ctly proportional to ~r reduction.
Figure 7. The cDNA ~egu~nc~ for th~ ~ingle chain anti-erba2 anti~ody, Ab#23.
Figur~ a. Th~ cDNA s~guence for the ~single chain anti-er~B2 antibody, Ab#21 (-22).
DETAIL~D DESCRIPTION OF THE INVENTION
. On~ obj-ct of the pre~nt invention i8 a combination of at l~t two monoclonal antibodie~, which i capable of preventing and treating human malignanc~ 03, wherein th~
m~lignant cell~ overoxpros~ gpl~S~rb8 2 and wh~rein said at l~a~t ~wo diff~r-nt antibodio~ each r~cognize a different ~pitop~ of th~ gp185 expr~ion product of ~rbB-2, th~refore the antibodlo~ do not cro~ r~act with each other. An embodiment; of ~h~ pr~ent invention provides for the combin~tion to compri~- fir~t and J-cond antibodie~ which ar~ prof~rably combined ~uch that th~ re~ulting ratio of the 1r~t to second i~ effoctive for decrQ~sing the exprc~sion product of th- orbB-2 gene. ~ conveni~nt method for PCTIIJS 921 0~545 RO/US 2~ ocr 1992 measuring the expression product of erbB-2 qene may be .ou.~d in Figur~ 4A. Th~ decrea~e in the expression of the erbo-2 gene product is the result of the combination decreasing the half life of er~B-2 prot~in in the cell. In another embodiment of the present invention, the combination o~ ~he antibodie~ haa the characteristic trait of essentially not increasing the tyro~ine pho3phorylation of qp185 expr~sslon product.
An example of a fir~t to 3econd antibo~ie~ ratio ha~n~
the acti~ity nec~s~ary to decr~a~ the expre~sion product of th~ erbB 2 gen~ compri~e~ a ratio of rom about 1:2 to about 2:1. Preferably, such a ratio i 1:1. The present in~ention, how~ver, i~ not intended to be limited to the antibody ratio3 di~cus~ed herein. The fact that other ratio are effectiv~ ænd may yield higher ~ctivity than the 1:1 ratio used as an oxampl~ i~ rRcognized and acknowledged by th~ inv~ntor~ as being within the ~cope of this invention.
Th~ activity of thi~ combination iq ex~mplified in Figur-~ lA-C, 2 and 3A, B as follow~. Figure lA-C
demon~trato th~t th~ N87 c~lls overexpres~ the qp185 erbB-2 protoin a~ a r~ult of orbB-2 ~n~ ampiification. Fiyure 2 show~ that a combination of Ab#2 1 and Ab#23 inhibi t3 the growth of N87 c~ in vitro. Similar re~ult~ have been d-mon~trat~d u~ing th~ combination of A~#23 and Ab#g4 as w~ll a~ Ab#23 and Ab*21, on the growth of human Calu-3 lung ~d*noc~rcinoma ~ S~e Figuro~ S and 6 ) . Figure 3A and B show the a~tivity of combination~ of Ab#21 and Ab#23 inhibi~in~
and r~ r~irg tho growth of N87 c~lls growing as tumors in immunod~icient mico. Tho~- r-~ults indicate the generaL
naturo of th- application of combin~tion~ of antibodi~s.
Tho antibodio~ a~ain~t the erbB2 gene encoded product u~ed in t~i- invontion can bo de~l~n-d as chimeric - PcTllJs 92/0~^3545 ~:
RO/US 21 OCTt992 `
antibodies. Chimeric antibodies have variable re~ ons (antigen binding regions) of nonhuman (e.g., murine) or:gi~
and con~tant regions of human origin. Because they are predominantly human, chimeric antibodies are less immunogenic in humans, whioh can help overcome problems associated with administering foreign proteins to humans.
In addition, the antibodies of the present inven~ion may be produced through genetic recombination or the Koh1er-Milstein hybridoma method for production of antibodie~. It is also recogniz~d that fragments, analogues or derivative of the antibod~es th~m~elve~-can be utilized in thi~ inv~ntion in place of th- entire anti~ody.
Another obj-~t of th~ pre~cnt inv~ntion provides for antibodiet againJt erb3-2 ~@n~ ~ncod~d product which are de~ignod a~ ~in~le chain antibodie~. A ~ingle chain antibody i~ one in which th~ light and heavy variable regions of th~ antibody are link~d tog~th~r to form a sin~le chain antibody. It i5 contemplated in thi~ application that a combination of th~e antibodie~ include antibodies which ar~- combinod a~ an admixturo a~ discu~od above and antibodi~ whlch are combinad to form a bi~pecific antibody.
A bi~poci~ic ant1body i~ an artificially produced antibody u~ually compri~ed of two ~inglo chain antibodies oach of whlch i~ r~cogniz-~ a different antigen binding ~ite.
Th- followin~ are examples of ~ome of the human malignancioJ ~hic~ may ~ treated or pr~vented usinq the pro~nt inv~nt~on; adonocarcinona~ o the breast, ovary, lung and ~tomach.
Anoth-r embodiment of applicant~' invontion provide a method or proventing and erad$catins tho human malignancie~
de~cribed a~oYo-~-- The m~thod ln~rolve~ admini tering~ to a patient an ~-~octiv~ do~o of a combination of anti-erba-2 _~_ PCT1tJS 92/ 08545 RO/US ~1 ocr l99 antibodies to achieve an effective concentration of 'he antibody combination at the tumor site; for example, a concentration of at least l~g/ml. Preferably the concentration at the tumor site does not exceed abou lO~g/ml. In general, in order to achieve the des1red concentration of the combination at the tumor site the combination i~ admini tered in a dose from about .1 ms/ks to about 10 m~/kg of body weight.
Another embodiment of Applicant~' invention pro~ides for the antibody combination to b~ u3ed in pas~iv~ tumor therapy, wherein an effecti~ do~e of the antibody combination i~ admini~t~red in or with a pharmaceutieally acceptabl~ v~hicl~ to a pati~nt afflicted with a human mali~nancy ovor-xpre.~ing gpl85erbB 2. ~xampl~ of vehicl~s ar~ non-toxic buff~rq, phy~iological ~lina, etc. :
Applicant3' lnv~ntion al30 provides for at Least one of the antibodi~ of tha antibo~y combination to ~ used as a compon~nt of an immunotoxin. For immunotoxin therapy, a~
loa~t ona antibody of tho combination can bo linked to an anti-canc~r pharmaceuti~al or a cytotoxin to form an lmmunotoxin. V~rious pharmaceutical or cytotoxic a~ents can b~ ch~lcally or ~en~tically coupled to th~ combination.
Exampl~- o som~ usoful therap~utlc- agent~ include:
radio~ctlv~ compound~ (~.g., isotopeQ of Boron and Rhenium);
ag~nt~ which bind DNA, ~uch a~ ~lkylating ag~nt~ or various Antibodi~ (o.g., daunomy~n, adriamycin, chlorambueil);
ntl-m-t~boliteJ (~.g., mQthotrex~te~; and inhibitors of .-prot~in ~ynth~ (e.g., diphth~ria toxin and toxic plant protoin~. Th- u~- of the combination, wh~r~in at l~ast one of th- antibodi-~ in t~- comb1nation ia couplod to an immunotoxln will lncreasa tho afficacy of tha therapy.
Admlnlstratlon to a p~tient o an effective dose of the combination of antibodie~ d-~crib~d herein may be PÇTI~JS 9 2 / 0 8 5 4 5 -R()/US ~1 CCT 1992 `~
accomplished via chronic intraveneous administration for a period of time sufficient to result in the re~ression or eradication of the human maliqnancy bein~ treated.
Administration o the combination may also be accomplished in a patient by direct inj~ction or delivery of the combination to the tumor ~ite. Such administ~ation would be of sufficient duration and concentration to result in eradication or reduction of the tumor.
Although th~ ~cope of tho prasent invention is not intended to be limited to any theoretical rèa~oning, the following theoriet may explain ~ m~chani~m by whi~h down regulation and protection from human malignant cells overexpre Ying gpl85~rb~ 2 i~ achie~ed by the two antibody combination.
Ono mechani~m which ha~ b~en po~tulated i~ that the two antibody combination act-~ by conJtraininq gplSSerbB 2 into an activat~d conformation thu~ mimicking an agoni3t li~and.
If tho two antibody combination mimic~ the ligand, then ~r~atmont u~ing the combination ~ould ro~ult in increased gpl~5erbB-2 autophosphoryl~tion. Anti-pho~photyrosine immunoblot~ wero u8ed to te~t thi~ hypothe~i~. A~ ~hown in Flgur~ 4B, -no increa~e in tyro~ine pho~phorylation of gplaS~rb3 2 from ~87 c~119 wa~ observ~d 1 or 2 hour~ after tho addition of th- antibody combination or up to 24 h of tro~tm-nt. Thi~ ~ugge3t~ that tho antibody combination doe not incro~-~ th~ autopho5phorylation of gpl85~rbB 2 and th~rofor- doe~ not act to inhibit tho acti~ity of the tyro~in~ ~inas~.
Th~ re~ult~ demon trate that a combination of`-anti-roceptor antibodie~ l~ad~ to different and more potent anti-tumor activitio~ than ~inglo antibodies~ More ~p-cifically, re~ult~ indicat~ that the combinatton ant~ody thorapy i~ a use~ul approach to tr~atmont of human RO/~S 21 ~C~ ~992 malignancie~ overexpressing qplaS . This approach may be particularly important in the treatment of gastric cancer, a disease which re~ponds poorly to current system;~
ohemotherapies.
The present invention is further illustrated ~y the following Example~ which are not intended to limit the scope of the invention.
-PreDaration of Antibodies At a ~ourc~ of human erbB-2 protein we used a NIH~3T3 cell engineered to ~xpr~ th~ human er~-2 prot~in on its surface (N/erbB-2). Membrane preparation~ of these cells were pr~pared by hypotonic ly~i8 in 2mM Hepe~ pH 7.4, removal of nucL~i by centrifu~ation at 5,000 x g and isolation of membrana~ by centrifugation at lO0,000 x g.
Mic~ wer- immunizod with lOO~g of N/erbB-2 membrane preparatlon in a 50:50 mix of ad~uvant in 200~1. Adjuvant wa~ ~r~und'~ compl~te or the fir~t injection followed by Freund'~ incomplet~ ad~uvant. Mlco were given 4 intraperiton~al in~ection~ over 4 w~ek~. On~ w~Qk following the la~t boo~t ~ra wa8 o~tainQt and an anti-erbB-2 immune re~pon~o wa~ det0rmin-d to axi~t by immunoprecipition analysi~ o~ gpl85 ~rbB-2 protein from metabolically labeled cell An immun~ mou~ w~ then ~olected and boosted with lOO~g of N/~rb~-2 membrane preparation and fu~ion wi'h Ag8.6~3 my~lom~- cell~ eonducted according to standard m-thod~. Sol~ction of hybrid clone~ wa-~ by re~istence to hypoxanthin~, am~nopterin, and thymin~ (HAT) contalning media ag~in according to ~tand~rd m~thod~. Id~ntification of hybridoma~ s-creting an ant~-erbB-2 monoclon-l antibody wa~ ~y E~ISA u~lng a~ antlgen N~erb8-2 membrane protein attached to 96 w~ll di~he~. EL~SA r~action wa~ developed PCT/US 9 2/ 0 8 5 4 5 ` -R O ~ US 2 ~ ocr 1992 ",~,, . .
using peroxidase coupled goat anti-mou~e antibod~ and etandard methods. Hybridoma culture~ secretin~ an anti-erb~-2 antibody were subjected to two rounds of single cell cloning and identification of positive subclones by ELISA a~ de~cribed above.
The above procedure i3 used to produce three monoclonal antibodie~ de~ignated a~ Ab#21; Ab#23; and Ab#94.
Monsclonal antibodioY dir~cted against the extracellular domain of gpl85erbB 2 wer~ te~ted for spec~fic reaction to N/erb8-2 cell membran~J in an E~ISA a_say. Two of tha~e de~ignated Ab#21 and ~b#23 after ~creenin~ in growth a~says exhibit~d the hi~h~t biological activity and were u~ad in thi- study. Antibod~e~ were i~olatad in large amount~ rom aJciteJ 1uid and purifiad by HPLC with a Gammabind Ultra column (Gen~x, Gaither~burg, MD). Standard SDS-~AGE gol al-ctrophore-1~ was run und~r non-reducing conditionJ u~ing CoomaJ-~ blue staining with a single ~and of 130 kd observod indicating a >98% purified preparation (data not shown).
Both ant~bodia- ~pecifically immunoprecipitated a ~inql- 35S-labelod prot~in of MW 185,000 from SK-Br-3 cellQ
(~ bre~-t cancor call l$n~ which overexpro~e~ gpl~5erB 2 prot-in) t22) ~ ~hown in Figuro lA. No immunoprecipltation wa~ d~t-ctod in cell~ which do not overexpre~ the gpl~S-r~B 2 protain (e.~. MDA-MB-468, data not shown).
. .
- - - - Th~ offect of the~ anti~odia~ on coll proliferation - w~ tudied on a gaJtric call line, N87, which overexpres~es gpl85~r~8 2 at l~vel~ commen~urat~ with SK-Br-3. An ! ' I , immunoblot of tho N87 ccll line and a nude mou~e tumor - xenograft from N87 i~ ~hown in Fig~re lB compared to th-brea~t cell llne~ SX-8r-3 (high lov-l o gpl85~rbB 2 -= - overexpre~lon) and MDA-M~-231 (low L~sl of gpl85erbB 2 ` ov~r~xpreJ~ion) Tho lovel~ of erb8-2 geno amplification in .
PCTIUS 92/ ~545 RO/US ~ 1 OCT ~992 N87 as shown in Figure lC surpa~sed those found i~ the wel characterized SK-ar-3 and SK-OV-3 cell lines (22).
Effect of Antibodies on Tumor Growth In Vitro The effect of these antibodies on ~rowth was first studied in ~itro u~ing a ~emiautomated colorimetric MT-assay. A ~ingl~ cell ~u~pension of 10,000 cells/well was plated in a chemically defined media consi~ting of RPMI-1640 supplemcnted with in~ulin, h~man tran~ferrin, 17 -estradiol, sotium elemita and H~pe~ bu~fer. PBS, Ab#21, Ab#23 or a combination of Ab#21 and Ab#23 were th~n added. The cells were allowed to grow at 37C in a 5% CO2 humidified atomo~phere. After 7 day~ (4,5-dimethylthiazol-2-y)-2,5-diphenyl totrazolium bromide (MTT
reagent~ wa~ add-d and allowed to incubate for 4 hours at 37~C, 90% of the med~a wa~ then removed and the cryqtals ~olubilized $n DMSO. Optical d-n~itie~ were mea~ured at 540 nm in a molecular devic~J Vmax kinetic microplate read~r. A
do~e re~pOn#- an~ly~i~ of th- effect~ of the antibodies on N87 cell prollf-ration i~ ~hown in Figure 2. Antibodie~
Ab#21 or Ab#23 admini-t~red individually had no effect on tho monolay-r growth o~-cell~ up to a concqntration of 10 ~lg/ml (6 I~M). Admini~tration o a 1:1 combination of Ab#21 and Ab#23, how-ver, markedly affected cell proliferation at do--~ a~ low ~ g~mL-~- Fab fragment~ prepared from both antibodi~ al~o had- no ef~ct on cell growth alone or in combinat~on (data not ~hown). In analogou~ experiment~ with thr~e oth-r g~tric. cell line~ di~playinq little or no o~erexpr-J~lon by immunoblot analy~1~, no inhibition o growth even at the highe~t do~e wa~ ob~erv-d with the antlbody comblnat~on or-the antlbodle~ alone.
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PCTIUS 92/ 08545 `~
~O i US 2 1 OCI 1992 -Preventive Combination AntibodY TheraD~
The efficacy of combination antibody therapy was testedon the growth of N87 tumor xenografts. One inoculation of five million N87 cells w~re injected qubcutaneously into nude mice produce rapidly ~rowing tumors. wi~h a short latency. Tumor growth at the injection site was easily quantitated. A~ ~hown in Figure 3A, the N~7 cells did not form tumor~ in the animal~ treated twic~ a week for three wee~ with a total of 200 ~g of antibodie~ per injection with the com~ination of Ab#21 and Ab#23. In ~harp contra~t they w~re pot~ntly tumorigenic in anim~l~ treated with the singlQ anti~odie~ or PBS and th~ tumor gr~w to ov~r 1 cm3 in tumor volum~ ovor th~ p-riod m~a~ur~d. In contra~t to in vitro experim9nt~, ~ach monoclonal antibody alone may have limited activity to partially re~tri~t the rate of tumor growth. ~owov-r, th~ activity exhibited by the ~ombination far excood~d th- cumulativ~ ~ffect ~xp~cted from the combination.
To dot~rmino if tho ~ombin~d th~rapy with Ab#21 and Ab#23 wa~ abl~ to er~dicat~ ~tabli~hod tumors, an ~xp~rim~nt waJ p~rform~d in which tumor~ were allowed to grow to mo~urablo ~iz-~ prior to ~ntib-ody treitment. The ro~ult~ ~ro illuJtratod in Figuro 3~. In animal group~
randomi2-d ~o that tho starting ~iZ~ of the tumor~ was near th- 8~m volum- (100 mm3), th- tumor~ contlnuad to grow when th- anim-l~ w-ro givon ~inglo ant~body tr~tmont of Ab#21 or Ab#23 (200 ~g/in~ection, 2 injection/weak, 3 w~ak~, 6 mic~).
In contraJt, ln tho animal~ givan two anti~ody combination tr~atmont o Ab#21 and Ab#23, ro~uLt~ ~hown ar~ the avera~e of 6 anim~l~, tumor~ complotaly rogr-~s-d ~ftar ll day~ (4 treatment~ of 200 ~g of total antibody) ~ T~-i5 i~ the first report-d ob~-rvation of tumor x~nogr~ft re~re~ion induced PCT/~JS 92/ 0~545 RO/US ~I CCT1992 :.
by a combination o anti-erbB-2 monoclonal antibodies Previou~ studie~ have shown that two anti-neu antibodies can inhibit the growth o tumors by murine cellc transformed by the mutationally a~tivated neu onco~ene (23) The activation of the murine neu oncogene is accomplished by point mutation as evidenced by qualitative interference in the ~tru~ture and function of the ncu gene, whereas the human erbBo2 oncogene i8 activated by overexpression Oc erbB-2, a quantitative interf~rence of the apparently normal prot~in which r~ ult~ in tumor formation.
Since the mechani~m~ for tumor growth are qo different b~tween murin~ and human, it i~ totally unexpect~d that ~imilar mechanism~ of neutrslization of the g~n~ involved would b~ effectiv~. Thi~ aff~ct i~ al50 ~een with the inhibition of leuk~mic tumor cell growth using anti-transf~rrin monoclonal antibodie~ (24~. -EXAMP~E 4 Ant~Droliferatlve_Eff~ct~ of AntibodY Combin~tion To inve~tl~at- the mol~cular baJi~ for the antiproliferativo eff~ct~ o~ Ab#21 and Ab#23, we mea~ured th~ rato o gpl85~rbB 2 turnover in the pre~onco or ab-enco of antibodio~. N87 c~ wer~ pu~ b~lod with 35s-cy~
and th~n cha-~d for ~ariou~ tim-J in the presenc~ of ~ingl~
anti~ody or tho Ab#21/*23 combination. Tho ro~ultJ of 24 h cha~e are ~hown in Figure 4A. The`
anti~ody gpl85~rb8 2 combination induced rapid degradation of gpl85-rbB 2 whil~ the individual antibody treatment had littlo or no ff-ct. Thu~, tho antiproliferative effect of ~b#21/Ab#23 treatm-nt might likely bo explained by th~ir ability to incr~aJo th~ turnov~r of gpl85~r~B 2, EXAMP~E 5 .
PCT/US 9 ~ / O ~ 5 4 5 RO~US 21~ ocr 1992 ;
Combinatlon Antibodv Treatment Effect~ the Growth of Calu-~Cells In order to demonstrate that the effect of combination of anti-erb~-2 antibodie~ Ab #21 and Ab #23 is not limited to effect on the N~7 ga~tric cancer c~ll line, we investigated the human lung adenorarcinoma cell line Calu-3.
This cell overexpresse~ the gp185 erbB-2 protein as det~rmined by immunoblot analy3i~ (data not shown). In experiments very ~imilar to that de3cribed above, the combination of Ab #21 and Ab ~23 ~how dramntic inhibition of cell growth as mea~ured in an MrT a~ay (figure 5). In this experim~nt, a ingle c~ll suYpon ion o 10,000 cell~/well wa~ plated in a ch~mic~lly defined media con~i~ting of RPMI-1640 3upplamented with in~ul~n, human tran~ferrin, 17-e~tradlol. ~odium s~lenite, and E~p~ buffer. PBS, Ab~21,Ab#23 or a combination of Ab#21 and Ab#23 w~re then added.
The cells wer~ allowed to ~row at 37C in a 5% C02 humidified atmo~ph~re. ~ft~r 7 day~, MTT reagent wa~ added and allowed to ineubat~ or 4 hour~ at 37C. 90% of the media wa~ thon remov~d and tho cry~tal~ ~ol~biliz~d in DMSO.
Optical don~iti~ w~ro maa~ur-d at S40 nm in a Molecular Devic~s Vm-x kin~tic microplat~ reader. A do~e re~pon~o analy~is of th~ ~fect~ of antibody tr~ nt i~ ~hown in figur- 5. This r~ult indicat~ that combination antibody th-rapy i~ not limited in off~ctiven~ to N87 cell~ or .... .
g~stric cancer c~ . It al30 indicat~ that combination antlbody thQrapy ma~ havo effectiven-~s in the tr~atment of - adenocar~inoma of the lung.
Effectivene~ of Other _ Combination~ o Anti~odie in ~ ~nhibitlna C~ll Growth RO/US 210C~1992 In order to determine if Ab#21 and Ab#23 are unique in their ability to combine to ~ause qrowt~ inhibition, we inve~tigated th~ combination of Ab#94 and Ab#23 on the growth of Calu-3 cells in vitro. An MTT aqsay of cell growth was conduct-d as d~cri~ed in ~XAMoeLE 5. As ~hown in figure 6, the combination of anti~odie~ inhibits cell growth and th~ individu~l Antlbodio~ do not~ Thi~ indicate~ that the a~illty to combine ~ntibodi~ to produce a more profound growth inhibition i~ not limited to a particular an~ibody com~ina~ion.
Antibodi~ #21; Ab#23; an~ Ab#94 h~v~ ~e~n d~po~it~d at the American Typ~ Cultur- Collection, 12301 Parklawn Drive, Rockville, Md. 20as2, USA. Ab#21 wa~ depo~ited on _ and giv~n ATCC # _ . Ab#23 wa~
deposit~d on ~ and giv~n ATCC ~ . A~#94 wa~ d~po~itod on ~ and giv~n ATCC #_ . .
Whil~ th- pr~ent inv~ntion h~ bo~n de~cribed in ~onjlanction with ~p~clfic ~m~odim-ntJ thoreo, it i9 evident that many alt~rn~tlv~, modific~tionJ and v~ri~tion~ will be apparent to tho~e ~kill-d in th~ art in vlew of the for-going d~-crlption . Accordingly, th- inventlon i ~ ~
int~nded to ~m~r-c~ all ~uch alt~rn~ti~t~J, modification~ and Yariation~-in follow~nq within tho broad~-t ~cope and ~pirit of th- fol10wing elaim~. ;
Exu~ 7 G'~n-r~t~on of ~ in~l~ ch~in ~Fv) ro~ mAb 23 - ~ol~ i RNA w~ extract~d from hybridom- cell~ u~inq oligo dT affinity chrom~tography ( In vitrog~n) . cDNA was pr~p~r-d using rand~m prim~r (N6) (~o-rhinqcr Mannh~im~.
unoglo~lin light and he~vy chain clon~ w~r~
i olat-d u~ing PCR and th- prim~r~: light ch~in, S' CAC GTC
GAC~AS~ ~AG CTG ACC CAC TCT CCA and GAT G~A TCC AGT TGG TGC
AGC ATC3'; he~vy chain S ' C GGA ATT TCA GGT TCT GCA G~A GTC
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RO/US 2~ocr 1992 WGG3' and 5' AGC GG~ TCC AGG GGC CAG TGG AT~ GAC3' ~G,A,C,T
stand for ~tandard nucleot~des; I for ins~ine, W for A or T3. Thn products of th~ PCR reaction wre cloned into PUC18.
Linkag~ i~to a SC(Fv) wa~ by PCR giving the individual li~ht and h~avy cDNA clon~ and 4 oli~onucLeotides 5' - cg~at~a~tccagctgaccc~gtctc 5' - ga~gatttac~gaaccagaggtaga~ccttttatttcca~cttgga S' - ctggttct~gta~atctt~tg~qgtaa~ggtgtgcagctgcaqga~
S' - cgagtgc~agcttagg~gacgqtg~ccgt .
The light and heavy chaln ~oding r~g~ons w~re joined by a ~ynthetlc linkor GSTSGSGXSSEGKG ~p~cified by overlapping oligonucleot~d-~ a~ de~crib~d. Th~ intact scFv coding region was in~rt~d in fram~ w~th~ an E.coli OMPA leader ~quonc~ ~nd~r d~r~ction of th- la~bda PL promoter.
~nduction of prot-in and b~ct~rial ly~$s and refoldinq was a. pr~viuo~ly do~crib-d (28). ~cFv was purified as a ~inqle pe~k ~o~ CM chrom~tography ~nd ~udged to b~ >70~ by S~S gel el~ctrophor~
EX~mP1e 8 G~nor~tion o~ ~ ~cFv from ~ ~21 Poly ~ RNA wa- oxtract~ fro~n hybridoma cell~ u~inq oligo ~T af~i~lty chrom~togr~phy ~ In ~ritrogon) . cDNA wa~
pr-par-d u~ing r~ndom prim-r (N6) (80~rhin~-r Mannh~im).
Th~ aunoglo~ulln liqht and hoavy chain clone~ were i-olat-d u~ing PCR ~nd t~ prim~r~ ht ch~in, S ' CAC GTC
GAC ATT CAG CTG ~CC C~C TCT CCA and - GAT GGA TC:t: AGT TGG TGC
AGC ATC3 '; h~vy ch~ln 5 ' C GGA ATT TCA GGr TC~ GCA GIA GTC
WGG3 ' ~nd 5 ' AGC GGA TCC AGG GGC CAG TGG ATA G~C3 ' ~ G, A, C, T
~tand for ~t~nd~rd nuclootld~; I for ino~in~, W for A or T]. Th- product- of t~ PCR reactlon wr- clon~d into PUC1~3.
Linkage into ~ ~cFv w-~ by- PCR qivinq th~ individual light and h-avy cDNA clone~ aAd 4 oliqonuc}ootlda~
5' - cg~g~tga~tce~gctgacccagtctc PCTltJS 9 2 ~ 0 8 5 4 5 -~O~US 2 1 OC~ 1992 :
5' - gaagatttaccagaaccagaggtagaaecttttatttccagctt~ga 5' - ctggttctggtaaatcttctgaaggtaaa~tgtgcagctgcaqga~
5' - cgagtgcaagcttaggagacggtgaccgt.
The light and hea~y chain coding regions were joined by a synth~tic linker GSTSGSGKSSEGKG ~pecified by overlapping oligonucleotide~ aJ d~crib~d. Tho intact scFv coding region was in~-rt~d in f ram- with an E.coli OMPA leader ~eguence under d~rection of the lambda PL promoter.
Induction of protein and bacterial ly~i~ and refolding was as previuosly d-~cribed (28). ~cFv wa~ purified as a single peak from CM chromatography ~nd ~udgod to bo >70~ by SDS gel electrophorocis.
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PCTlUS 92/ 08545 ~:
- RO/US 2~1 CCT ~992 ~;
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