CA2179196A1 - Method of preventing or treating disease characterized by neoplastic cells expressing cd40 - Google Patents

Abstract

There is disclosed a method of treating a mammal afflicted with a disease characterized by neoplastic cells that express CD40, comprising administering a therapeutically effective amount of a CD40 binding protein in a pharmaceutically acceptable buffer. CD40 binding proteins include monoclonal antibodies to CD40, and CD40 ligand. CD40 binding proteins may also be used to prevent disease characterized by neoplastic cells that express CD40, in individuals at risk for such disease.

Description

WO 95117202 PcrA)ss~ll4767 217i~1q6 ~ITLE
MEI~IOD O~ PREVENTING OR TRE~ATING DISEASE
S CHARACTERIZED BY NEOPLASTIC CELLS EXPRESSING CD4() TECHNICAL FIELD OF THE INVENTII N
The present invenlion relates IO methods of preventing or treating diseases ~ IIAIA~ t~ 1 by neoplastic cells expressing CD40. More specifically, the present invention relates to methods of treating or preventing B-cell Iymphomas.
BACKGROVND OF THE INVENTION
l,.l.llul.oblastic B-cell Iymphomas frequently arise in imm~n-,c.:""l,.u,~ised individuals such as allograft recipients and others receiving long-tem~ ; vc therapy, AIDS patients and patients with pnmary i " " " " "r~ ri~ ,r y syndromes such as ~-linked Iymphoproliferative syndrome or Wiscott-Aldrich syndrome (Thomas et al., Adl .
Cancer~es. 57:329, 1991; Straus et al., Ann. Intern. Med. 118:4~, 1993). These tumors appear to arise as a result of impaired T cell control of latent Epstein-Barr virus (EBV) infection. Similar Iylll~JIlu~ of human ongin can be induced in mice with severecombined innmllnr,ri~firir¢ncy syndrome (SCID) by inoculation of peripheral blood Iyll~ o~,yL~ (PBL) from healthy, EBV-positive individuals (Mosier et al., Nature335:256, 1988; Rowe et al., J. ~xp. Med. 173:147, 1991).
CD40, a cell-surface antigen present on the surface of both normal and neoplastic human B cells, is a peptide of 277 amino acids having a predicted molecular weight of 30,600, with a 19 amino acid secretory signal peptide comprising ~ ri~
l~y~u~ l~ùb;c amino aeids. This cell surface antigen has been shown to play an important role in B-cell l,.ul;~,~Liu.l and d;~lCll~ ;UII. A cDNA encoding CD40 was isolated from a cDNA library prepared from Burkitt Iymphoma cell line Raji (Stamenkovic et al., EMBO
J. 8:1403, 1989). CD40 is also expressed on the surface of monocytic and epithelial cells, and on some epithelial carcinomas (E.A. Clark, ~issr~e Antigens 36:33; 1990).
Activated CD4+ T cells express high levels of a ligand for CD40 (CD40L). Human CDAOL, a membrane-bound ~Iyuul~lu~ill, has recently been cloned from peripheral blood T-cells as described in Spriggs el al., J. E~p. Med. 176:1~43 (1992), and in United Slates Patent Application number 07/969,703, filed October '23, 199'2, the disclosure of which is illUUllJI ' by reference herein. The cloning of munne CD40L is described in Armitage et al., Nature 357:80, 199~. CD40L induces B-cell proliferation in the absence of an~ co-stimulus, and can also induce production of imm~nrglrh~linc in the presence of cytokines.
Monoclonal antibodies to CD40 are known in the art (see, for example, the sections dedicated to B cell antigens in LEUKOCY~E TYPll`iG 111: A.J. McMichael ed. Oxford

2 ~ 7 q 1 ~ ~ PCTIUS94114767 .
University Press Oxford, and LEUKOCYTE TYPI~G IV: Oxford Universil~ Press Oxford).
Antibodies to CD4U have been d~ ,u..a,~t to exer[ costimulatory signals on normdl B
cells, resulting in proliferative and di~r~clllidliull responses. Similarl~, CD40-L exerts protein stimulatory or rr~ I y signals to normal B cells. ,.
It has been observed that cross-linking of surface IgM on some B cell Iymphoma lines exerts inhibitory signals to the Iymphoma cells (Beckwith et al., J. Immunol.
147:2411, 1991). Similarly, exposure of malignant B or T cells to stimuli that lead to activation of normal Iyll~ o~yl~;s can result in growth arrest of the cells (Ashwell et al., Science 237:61, 1987; Bridges et al., ~. Immunol. 139:4242, 1987; Mercep et al., J.
Immunol. 140:324, 1988; Sussman et al., J. Inunultol. 140:2520, 1988; Warner and Scott, Cell. Immunol. 115:195, 1988; Page and DeFranco, J. /rrununol. 140:3717, 1988).
Garnier et al. observed that antibodies to CD40 or another B cell marker, CDA'3,showed some degree of effectiveness at inhibiting Iymphoma formation in SCID mice that had been injected with human PBL and then infected with EBV (Abstract 167, XIVth Intl.
Congress of the TrAncrlAntAti-\n Societ~, 1992). However, it was unknown in the art whether the m~rhAnicm of action involved was inhibition of binding of CD40L to CD40 by the anti-CD40 antibody, or by some other means. Therefore, there is a need in the art to determine the effects of other anti-CD40 antibodies, and of CD~0-L itself, upon B cell Iymphomas and other malignant cells that express CD~0.
SIJMMARY OF THE INVENT7()N
The present invention relates to a method of treating a mammal afflicted with a disease ,,II_lAJ,t~ by neoplastic cells that express CD40, comprising -A.l".;".~. ,;"g a lly effective amount of a CD40 binding protein in a l~ ;f Ally acceptable 25 buffer. The Ll,.~,el;~,Ally effective amount is from about 0.01 to about I mg/kg body weight. CD40 binding proteins may be selected from the group consisting of m~norlonAl antibodies to CD40, CD40 ligand, and ~ c thereof. Particula}ly preferred l~ l antibodies are hCD40m2 (deposited at the American Type Culture Collection, Rockville, MD, USA, under the terms of the Budapest Treaty, and given ATCC accession number HB11459) and hCD40m3, which are described in U.S.S.N. 08/130, 541, filed October 1, 1993. Oligomenc forms of CD40 ligand are particularly preferred, and include a soluble CD40 ligand-Fc fusion protein, and an oligomeric CD40 leucine zipper fusion protein, both of which have been described in U.S.S.N. 07/969,703, f~led October 23, 1992. The present invention also relates to a method of preventing a disease, ~ ,, f d by neoplastic cells that express CD40, in a mammal susceptible to the disease. cOmprisinc A~1minict~rin~ a therapeutically effective amount of a CD4U binding protein in al,IIA I I ~ I ~ ~ 11;~ Ally acceptable buffer, wherein th~ th~ r~re~lt;~lly effective amount is from W09s~17202 2 ~ 79 1 ~6 PCTIUS94/14767 about 0.01 to about I mg/kg body weight. Neoplastic cells tha~ express CD40 include B
Iymphoma cells~ some melanoma cells and some carcinonl;~ cell~.
BRTF.F DE~CRIPTION OF THE DRAVVlNC.f.
5 Figure I illustrates the expression of CD40 by several Iymphoma cell lines, using anti-CD40 " ,. " lf r~. " ,~l antibodies M2 and M3.
Figure 2 fi. . l ,. ~ . the inhibition of the proliferation of several Iymphoma cell lines by antibodies to CD40 (closed squares); in contrast, msIgG did not inhibit)lirc.aliO-- (open squares).
Figure 3 presents a c~ ;c~ of the effects of soluble anti-CD40 (panel A) or ;.".""l,;li"~l anti-CD40 (panel B) on Iymphoma growth.
Figure 4 ~i, ."~ , the ability of soluble CD40 ligand to inhibit Ihe growth of B-cell Iy~ la~ in vitro.
Figure 5 shows that antibodies to CD40 inhibit the growth of human melanoma cells in vitro.
Flgure 6 fl..,..."~l,. -; the ability of soluble CD40 ligalld to inhibit the growth of B-cell Iylllllllclllla~ in vivo.
DET~Tl.i~.D DESCRIPTION OF TllE INVENTION
The present invention relates to methods of treating or preventing diseases ., ;, ;I by neoplastic cells that express a cell surface molecule known as CD40. The inventive methods utilize a protein (or proteins) that specifically bind CD40 (referred to as a CD40 binding protein) in a non-covalent interaction based opon the proper c. ~ of the CD40 binding protein and CD40 itself. For example, a CD40 binding protein can 25 comprise an f Ytraf f.l1.. 1ar rcgion of a CD40 ligand. In other cases, a CD40 binding protein can comprise an antibody that binds CD40 through an antigen binding region. Additional CD40 binding proteins can be prepared through recnmhinant methods, by IJIC~ iUII of fusion proteins cfJmrri~in~ a CD40 binding region (or domain) from a CD40 ligand, or an antibody to CD40, with a second protein, for example, a human immllnf)glf~lliin Fc 30 domain.

Human CD40 anti~en (CD40) is a peptide of ~77 amino acids having a molecular weight of 30,600, and a 19 amino acid secretory signal peptide comprising ~.ledulll;l.a.-lly 35 llylllu~ bic amino acids (Stamenkovic et al., supra). A cDNA encoding human CD40 was isolated from a cDNA library prepared from Burkitl Iymphoma cell line Raji. The putative protein encoded by the CD40 cDNA contains a putative leader sequence, trans-membrane domain and a number of other features common to membrane-bound receptor .. . .. . ... . .. ... . _ . .. _ . ., . . _ _ _ . .

WO95/17202 ~ 1 7 ~ l ~ 6 PCr/US94JI4767 .
proteins. CD40 has been found to be expressed on B Iymphocytes, epithelial cel~s and some carcinoma c-ell lines.
CD40 is a member of the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family, which is defined by the presence of cysteine-rich motifs in the extracellular region (Smith et al., Science 248:1019, 1990: Mallett and Barclay, Immunolog~ Today 12:220; 1991). This family includes the Iymphocyte antigen CD27, CD30 (an antigen found on Hodgkin s Iymphoma and Reed-Sternberg cel~s), two receptors for TNF, a murine protein referred to as 4-1 BB, rat OX40 antigen, NGF receptor, and ~as antigen.
CD40 may be detected on the surface of a cell by any one of several means known in the art. For example, an antibody specific for CD4() may be used in a fluwc~
activated cell sorting technique to determine whether cells express CD40, as descnbed in Example I herein. Other methods of detectin~ cell surface molecules are also useful in detecting CD40.
CD40 Monoclonal Antibodies Monoclonal antibodies directed against the CD4() surface antigen (CD40 mAb~ havebeen shown to mediate various biological activities on human B cells. For example, CD40 mAb induce homotypic and hetetotypic adhesions (Barrett et al.,.l. Immu~ol. 1~6:172~, 1991; Gordon et al., J. Immunol. 140:1425, 1988), and increase cell size (Gordon et al., J. Immunol. 140:1425, 1988; Valle et al., Eur. J. Immunol. 19:1463, 1989). CD40 mAb also induce proliferation of B cells activated with anti-lgM, CD20 mAb, or phorbol ester alone (Clark and Ledbetter, Proc. Ivatl. Acad. Sci. CISA 83:4494. 1986; Gordon et al., LEUKOCYTE TYPING nI; A.~. McMichael ed. Oxford University Press. Oxford, p. 426;Paulie et al., J. Immunol. 142:590, 1989) or in concert with IL-4 (Valle et al., Eur. J.
Immunol. 19:1463,1989; Gordon et al., Eur, J. Immunol. ~7:1535, 1987), and produce IgE (Jabara et al., ~. ~xp. Med. 172:1861, 1990: Gascan et al.. J. Immunol. 147:8, 1991), IgG, and IgM (Gascan et al., .~. Im~nunol. ~47:8, 1991) from IL-4-stimulated T cell-depleted cultures.
In addition, CD40 nlAb have been reported to enhance IL-4-mediated soluble CD23/FcERII release from B cells (Gordon and Guy, Immunol. Toda~ 8:339, 1987;
Cairns et al., Eur. J. Immunol. 18:349, 1988) and to promote B cell production of IL-6 (Clark and Shu, J. Immunol. 145:1400, 1990~. Recently, in the presence of CDW32+adherent cells, human B cell lines have been generated from primary B cell por~ nn with ILA and CD40 mAb (Banchereau et al., Science 241:70, 1991). Furthermore, germinal center c.,lltlul,yl~ can be prevented from undergoing apoptosis if they are activated through CD40 and/or receptors for antigen (Li~ et al., Nature 342:929, 1989).
Each of the above ~ c describes CD40 mAb that stimulate a biological activity of B
cells.
.. . .

WO 9'i/17202 2 ~ 7 ~ PCrn~ss~l/14767 U.S.S.N. 08/130, 541, filed October 1. 1993~ the relevant disclosure of which i~i..cu.l,u~ d by }eference. discloses ~wo monoclon~l ;mtibodie~ to CD~0~ referred to a~
hCD40m2 and hCD40m3 Unlike other CD~() mAb, hCD4Un1~ (ATCC HB11459) and hCD40m3 bind CD40 and inhibit binding of CD4() to cells that constitutivel~ express CD40L. Greater than 95% inhibition of binding was observed with hCD40m2 or with CD4û rnAb M3, at c~ llC as low as 1~.51ag/ml~ as compared to irrelevant IgG or acontrol CD40 mAb, G28.5. hCD40m2 was also able to inhibit CD40L-induced TNF-~
production.
Additional CD40 mnnoclnn~l antibodies may be generated using conventional techniques (see U.S. Patent Nos. RE32,011, 4,9()~,614, 4,543,439, and 4,411,993 which are ;~n,V ,uu- ' herein by reference; see al.~o Mo)~oclonal An~ihodies, Hy~ridornas A New Dirnension in Biological A~lal~ses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A La~oraror~ Man~al, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also illCUI IJUI aL~d herein by reference).
Briefy, an animal is injected witll ~ form of CD40 suitable for generating an jmmune response against CD40. The animal may be ~ I as neGded until levels of serum antibody to CD40 have reached a plateau, then be given a final boost of soluble CD40, and three to four days later sacrificed. Or~ans which contain large numbers of B
cells such as the spleen and Iymph nodes are harvested and disrupted into a single cell suspension by passing the organs through a mesh s~reen or by rupturing the spleen or Iymph node " . . .1,. ,~ which r~ I ' the cells Alternatively, suitable cells for preparing monoclonal antibodies are obtained through the use of in virro ;~ techniques. Briefy, an animal is sacrificed and the spleen and Iymph node cells are removed. A singie cell suspension is prepared, and the cells are placed into a culture which contains a form of CD40, which is suitable for generating an immune response as described above. C~lhsl~t~ ntl~, the Iyl~ lu- ~ are harvested and fused as described below.
Celis which are obtained through the use of in virro i,.""....;,..l;nn or from an immunized animal as described above may be immortalized by transfection with a virus such as the Epstein bar virus (EBV) (see Glasky and Reading, Hybridorrla 8(4):377-389, 1989). Altl,l--ali~.,ly, the harvested spleen and/or Iymph node cell ~ i-",c are fused with a suitable myeloma cell in order to create a "hybridoma~ which secretes mnnnt lnn~l antibody. Suitable myeloma lines are preferably defective in the ,u~ u~,LiOI~ or expression of antibodies, and are additionally syngeneic with the cells from the immunized animal.
Many such myeloma cell lines are well knowll in the art and may be obtained from sources such as the American Type Culture Collection (ATCC), Rockville~ Maryland ~se~
Catalogue of Cell Lines & H~bridornas, 6th el., ATCC, 1988).

WO 95/17202 2 1 7 9 ~ 9 6 PCTJUS9~ 67 .
CD40 Li~and Activated CD4+ T cells express high levels of a li~and for CD4() (CD40L). Hum~l1CD40L, a membrane-bound ~ ,v~.,uL~i", has recently been cloned from peripheral blood T-cells as described in Spriggs et al., ). Exp. Med. 176:1543 (199~)~ and in United States Patent Application number 07/969,703, filed October 23, 1992, the disclosure of which is ul~Jul~L~d by reference herein. The cloning of murine CD40L is described in Armitage et al., Na~ure 357:80, 1992. CD40L induces B-cell proliferation in the absence of any co-stimulus,andcanalsoinducepJoductionof;-"-",."~ ,l,ulinsinthepresenceofcytokines.r~D40-L is a type Il membrane polypeptide having an eYtr:lr~ rr region at its C-terminus, a ,,~ region and an intracellular region at its N-terminus. Soluble CD40-L comprises an eYtr~lllllllr region of CD40-L (amino acid 47 to amino acid 261 of SEQ ID NO: I) or a fragmenI thereof. CD40-L biological activity is mediated by binding of the extracellular region of CD40-L with CD40, and includes B cell proliferation and induction of antibody secreuon (including IgE secretion).
U.S.S.N. 07/969,703 describes preparation of a soluble CD40-L/Fc fusion protein referred to as CD40-L/FC2. CD40-L/FC2 contains an eight amino acid hydrophilic sequence described by Hopp et al. (Hopp et al., Biol~echr~olog~ 6:1204,1988; referred to as Flag~), an IgGI Fc domain, a IGly4Ser]3 linker sequence (described in U.S. Patent 5,073,627), and the eYtrslr~ r region of human CD40-L. Also described in U.S.S.N.
07/969,703 is a soluble CD40-L fusion protein referred to as trimeric CD40-L., which contains a 33 amino acid sequence referred to as a "leucine zipper," the eight amino acid hydrophilic sequence described by Hopp et al. (srlpra), followed by the rYtrs~r~ r region of human CD40-L. Both oligomeric forrns of CD40-L induce human B cell ~JIulire~ iul~ in the absence of any co-stimuli, and (in rr,njl-nrtir,n with the ~tr~JIuirl;G-~ cytokine) result in the production of IgG, IgE, lgA and IgM.
The CD40-L/FC2 and the trimeric CD40-L described in U.S.S.N. 07/969,703 will be useful in the present inventive methods, as will other forms of CD40-L that can be prepared using known methods of preparing ,~( v~ ,l proteins.
Additional CD40 Bindin~ Proteins Binding proteins may also be constructed utilizing l~ " ,l ,~ DNA techniques to incorporate the variable regions of a gene which encodes an antibody to CD40. (see James W. Larrick et al., "Polymerase Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes From Single Hybridoma Cells,"
Biotechnology 7:934-938, September 1989: Reichmalln et al., "Reshaping Human Antibodies for Therapy," Nat~re 332:3~3-3~7, 19~: Roberls et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein F.ng~in~rring,"
Nat~re 328:731-734, 1987: Verhoeyen et al.. "Reshaping Hun1an Antibodies: Grafting an . _ . ,,, . , , , . . _ , ,,, . ,, .,, , . , , , , , , , . , . , . . , . . . , _ . , . _, , _ WO9S/17202 2 ~ 7 ~ ~ 9.6 PCT/IJS94114767 .
Antilysozyme Activit~," Science239:1534-1536, 1988: Chaudhnry et al.~ "A R~..,.,l.;,~ "
Immllnr~ltrlxin Consisting of Two Antibody Variable Domains Fused to PseudomonasExotoxin," Nature 339:394-397, 1989).
Briefly, DNA encoding the anti~en-binding site (or CD40 binding domain; variableregion) of a CD40 mAb is isolated, amplified, and linked to DNA encoding anotherprotein, for example a human IgG (see Verhoeyen et al., sl~pra; see also Reichmann et al., supra). Altematively, the antigen-binding site (variable region) may be either linked to, or inserted into, anolher completely different protein (see Chaudhary et al., supra), resulting in a new protein with antigen-binding sites of the antibody as well as the functional activity of the completely different protein.
ru~ -u-G, DNA sequences which encode smaller portions of the antibody or variable regions which specifically bind to m::mms~ n CD40 may also be utilized within the context of the present invention. Similarly, the CD4() binding region (extracellular domain) of a CD40 ligand may be used to prepare other CD40 binding proteins. DNAsequences that encode proteins or peptides that foml oli~omers will be p~Lrticularly useful in .p.~ iUI~ of CD40 binding proteins comprising an antigen binding domain of CD40 antibody, or an ~tr~e~ r domain of a CD4U ligand. Certain of such oligomer-fomming proteins are disclosed in U.S.S.N. 07/969,703; additional, useful oli~omer-forming proteins are also disclosed in U.S.S.N. 08/107,353, filed August 13, 1993, and in 2û U.S.S.N. û8/145,830, filed September 29, 1993.
Once suitable antibodies or binding proleins have been obtained, they may be isolated or purifled by many techniques well known to those of ordinary skill in the art (see Ant~bodies: A Laborator~ Manual, Harlo~ and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, uu. irc~Liun on protein A or protein G columns, or any Culllb;ll~Liu~
of these tcrhnifl~ Rrr~ CD40 binding proteins can be prepared according to standard methods, and tested fo~ binding specificity to the CD40 utilizing assays known in the art, including for example ELISA, ABC, or dot blot assays, as well by bioactivity assays such as those described for CD40 mAb.
SCID mouse models The term SCID (severe combined immune deficiency) mouse refers to a mutant C~B-17 strain of mouse with a ~,IIIUIIIO~UIIIC 16 deficiency that prevents correct T cell receptor and immlln-lglnblliin gene rearrangement, and is thus virtually devoid of functional - 35 B and T cells (Bosma et al., Nature 301:~57: 1983). SCID mice can be successfull~
lG~ i with human fetal Iymphoid tissues, and with adult human Iymphocytes, and have thus been useful as a model for studying human immune function in vivo (Mosier et al., Nature 335:256, 1988; McKune et al., Sc~ence 74i :163~, 1988; Kamel-~eid and Dick, , . ,,, ,, , . . , . , ., .. .. ,, . . . ., .. . , . . . , ... . . . , . . , _ .

wo 95/17202 2 1 7 ~ PCr~ss4~14767 .
Science ~4~:1707, 19~8). SCID mice reconsIituted with human peripheral blood lymphocytes (PBL) from individuala with àerologiclll evidence of l~ps[ein-Barr ~irus (EBV) infecrion often develop Iylll~JllVlllAS of B cell ongin (Mosier et al. supra; Cannon et al., J. Clin. Invest. 85:1333, 1990; Rowe et al.. J. ~xp. Mcd. 173:147, 1991; Purtilo et al., Int. J. Cancer 47:510, 1991). Veronese et al. reponed that the presence of functional T
cells in the injected PBL was absolutely necessar~ for ~,lutlt~:.;c)" of latently EBV-infected Bcellsintotumormasses(J.Exp.Med. 176:1763.199'2); Thelymphomasthatdevelopin this SCID mûuse model are highly aggressive, alld analogous to EBV-lymphomas that arise in i~ u~ c:l individuals.
n of CD40 Bindin~ Protein C~
The present invention provides ther Lpeutic ~ullliJClaiLiulls comprising an effective amount of a CD40 binding protein in a suitable diluent or carrier and methods of treating mammals using the ~ u~ ,c For therapeutic use, purified CD40 binding protein ûr a biologically active analog thereof is A.illlillisLtltcl to a p:ltient, preferably a human. for treatment in a manner al~ ul~lidL~ to the indication. Thus, for example, CD40 binding protein pl,_.."- ~ C~ lf)~ (for example, in the form of a soluble f-l~trarPlllllAr domain of CD40 ligand, or a fragment thereof, or a mnnr,cirn~l antibody to CD40) which is a l,lli.,;aLc,ed to achieve a desired therapeutic effect can be givel~ by bolus injection, 20 continuous infusiûn, sustained release from implants, or other suitable technique.
Typically, a CD40 binding protein therapeutic agent will be a.i--li-~ialt,ed in the form of a ~ l composilion comprising purified CD40 binding protein in with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to patients at the dosages and rf ncf ntr~lir,nC employed. Ordinarily, the5 ~ aLiul~ of such ~f,-"l,r,~;~i,-"c entails combining a CD40 binding protein with buffers, such as ascûrbic acid. Iow molecul:Lr weight (less than abûut 10 residues) puly~ idcs, prûteins, amino acids. carbohydrates including glucûse, sucrose ûr dextrans.
chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with conspecific serum albumin are exemplary appropriate0 diluents.
Appropriate dosages may be determined by methods that are known in the art.
Typically, Lll~,la,.l.,J~i~ally effective dosages of CD40 binding proteins will be in the range of from about 0.01 to about I mglkg body weight. Moreover, CD40 binding proteins may also be used in conjugates of, ûr combination with, drugs, toxins or radioactive35 rf mrr,llnfic Preparation of such conjugates for treatment of various diseases are known in the art (see, for example. Waldmann, Sciencc ~5~:1657. 19YI).

WO95/17202 21 79 ~ 96 PCTIUS94114767 .
PrevennQn or treatment These results presented herein indicitte thi~t CD~() billdin~ proteins may be ofsignificant clinical use not only in the treatment of B-cell Iymphomas~ but also in the prevention of EBV-induced B-cell Iymphoma that can occur after U~ ."~li, r or in other S instances of i.,,,,l~,l,-.,,,l,lJlcaa;on. such as AIDS, and which present a âignificant risk in such patient populations. Since CD40 binding proteins can inhibit various B-cellIy~ llulllds directly, it may not be necessary to use them in conjugittes of toxins or radioactive ~..,.,1,,,.,,.,l~ thereby avoiding toxicity and potential negative effects on normal B cells.
The inventive methods may be useful in prevention of immunoblastic B-cell Iy~ Illas that frequently arise in i~ n~ ullliac ;l individuals. In such preventative methods, a mammal at risk of developin~ an immunoblastic B-cell Iymphoma is adll.;,.i,t~,lc l CD40 bindin~ protein. The CD~L0 binding proteins can be ~dll~ Lc-cd for as long as the state of ;~ l"u",i,c that places the individual at risk exists.
Similarly, the results indicate that the inventive methods may be used to prevent occurrence (or .c~,c-,ul lc~lcc~ of neoplastic disease ~ a~ t.. ;~.d by other types of malignant cells that express CD40 in individuals at risk for s~lch disease. Individuals that are considered at risk in these instances include those with family history or other genetic indicating IJlc.l;spuailiull to cancers in which the neoplastic cells express 20 CD40, and individuals that develop drug-resistant neoplastic disease as a result of .1....,...Lh .~ y, in which the drug-resistant neoplastic cells express CD40.
Individuals afflicted with disease ~,llala~ ,d by neoplastic cells that express CD40 may also be treated according to the inventive methods. The term treatment, as it is generally llnrl~r~nr d in the art, refers to initiation of therapy after clinical symptoms or 25 signs of disease have been observed. The inventive methods may be used in c~,l j l l -l Li~
with other therapies appropriate for afflicted individuals. including ~ Y. rddiation therapy, and ;,,.,,,,,,.~,II.. '"1'~-The relevant disclosures of all references cited herein are specifically illcullJ~JIa~cd 30 by reference. The following examples are intended to illustrate particular ~Illbodiand not limit the scope, of the invention.
F,Y~mnl~ 1 This example describes the chala~,Lcl;~aLi.,l, of human B-cell Iymphoma cells and 35 cell lines. Cells used included RL and DB~ cell lines obtained fronl patients with diffuse~
large cell Iylll~ dS of B-cell origin (Beckwith et al., supr~ nd TU2C and CHIM6~, EBV-induced Iylll~lllulllda obtained from SCID mice that had been injected with PBL from EBV-seropositive individuals. These cells were maintained in culture under standard WO95/17202 ~ 1 7 ~ ~ 9 6 pr,~fusg4~14~6~
.
culture conditions for lesà thall six months prior to initiation of the stud~. Other cells included Raji, a cell line cultured irom a patienl wilh Burkit~'s Iymphoma. and LCL-~311 a IY" ~l~' ~ cell line generated by infecting human PBL with EBV in vilro .
All of the cell lines were posirive for CD4() expression by fiow cytometry, using S anti-CD40 m~ cl~nal antibodies M2 and M3, results are shown in Figure 1. RL, DB, and Raji cells were l-n,.,~ ul.~ in their expression of CD40, whereas the EBV-induced Iy~ Jl.ulllàs from SCID mice were l~ uc~ us in the st,~ining intensity with anti-CD40.
Lymphomas from these mice have previously been .i~ rd to be l.~ ,.u~ uus and oligoclonal (Nakarnine et al., An~ J. Pathol. 142:139, 1993), which may account for the differentiai expression of CD4(). CD20, another B cell marker, was also present on the tumor cells.
Exa '^ 2 This ex~nple describes the effect of anti-CD40 antibodies (M2 and M3) on the proliferative potential of Iymphoma cells and cell lincs ~ll vi~ro. Proliferation was rmincd using an assay s~lbclantially as described by Rowe et al. (.1. Exp. Med.
173:147, 1991). Briefiy, cell lines were split '24 hours before assays were performed.
Cells were , ~"~ lf -~ in culture medium to a concentratioll of I x 105/ml, and 100 ~1 of cell suspension was plated in 96-well, round bottom microtiter plates (Corning Glass Works, Corning NY, USA) already containing 100 ~LI of ~ ,l.",l;a;~,ly diluted reagents (mnn~clnnal anti-CD40 antibodies M2 and M3, obtained from Immunex Corporation, Seattle, WA, USA, or mouse IgG myeloma protein (mslgG) purchased from Cappel, Westchester, PA, USA). Seventy-two hours later, l ~Ci of ~3Hj-Llly~ ,c/wcll (specific activity 6.7 Ci/mmol; Ne~ Engiand Nuclear Research Products, Boston, MA, USA) was added for the final 8 to 18 hours of culture. Cultures were harvested onto glass fiber filters with a PhDCell Harvesting System (Cambridge Technology Inc., Cambridge, ~iA USA), and [3H]-thymidine uptake was assayed by liquid ~in~illa~ion using an LKB ~3-counter (LKB Illa~lulll~ a Inc., Turku, Finland). Each experiment was performed four to six timeâ, with the results of a ~cl~csG~cli\~e experiment being presented in hgure 2.
Incubation with anti-CD40 monoclonal antibodies M2 and M3 resulted in âignificant inhibition of the ,..ulirc.~,iull of RL, DB, LCL-'311 and EBV-lymphoma cell lines tested, with an optimal inhibition of 40-60~7G occurring at 1-10 llg/ml of soluble antibody, depending on the Iymphoma cell. The Raji cell line did not appear to be si~l.;r._a..lly affected by soluble anti-CD40.
The effects of soluble anti-CD40 on Iymphoma growth were then compared to those of immohili7~ d anti-CD40. Briefiy, wells were illcubated ovemight at 37C with go~t anti-mouse antibody. Monoclonal anti-CD4() antibodies M~, M3, an anti-CD20 mrnoclllnal antibody provided b~ Dr. Kevin Conlon (Laboratory of E~ .,ldl 1() ... . . .. . . .. ........ ..... .. . . . . .. .... . ... ....... .. . . . . . .

WO 95/17202 2 1 7 9 1 9 6 PcrJuss4ll4767 .
Immunology. BRMP. NCI-FCRDC, Frederick, MD. USA) or mslgG~ at a concentration of 10 ~Lg/ml, were then added to he wells. and the wells ~ere incu~ated for an addilional -L
hours at 37C. The proliferation assays were then perfornled as described above: results are shown in Figure 3. lmmohili7~tion resulted in cie~lific~nrly greater inhibition of 5 proliferation (p<0.05) by the immobili7l~d anti-CD4() antibodies as compared to soluble anti-CD40, or soluble or imn~nhili7l d anti-CD20. Thus, in contrast to it's effects on normal B cells, Stim~ tiotl of CD40 exerts inhibitory effects on EBV-induced B
Iymphomas.
F.Yzlmnle 3 This example illustrates the effect of CD40 ligand on the growth of B-cell Iymphomas in vitro. Soluble CD40 ligand (CD40-L, described in U.S.S.N. 07/969,703) was obtdined from trdnsfected COS-7 cells as supernatant fluid, and tested in a ~-ulirc-~lLiull assay used as described above, in E~:ample ~. using RL or TU2C cells. Both murine and 15 human CD40-L-containing supernatant fluid~ were ~ested. since murine CD40-L binds to human cells that express CD4(). and acts as a costimulus in the same manner as human CD40-L. Each lot of ~u~ aldllL fluid was titrated to determine the corr~ntr~ion that yielded optimal inhibirion of ,UlUI;~ lLiUll~ a 1:5 dilution yielded maximal inhibition.
Exemplary results are presented in Figure 4; values are presented as percent of 20 inhibition compared to control supernatant fluids. The soluble human ligand was inhibitory for the various Iyl--,vllu..~ds tested, with maximal inhibition seen (50-80~o) on RL and TU2C cell lines at a 1:5 dilution of the ~UI,~ lL~IlL fluid. The soluble murine CD40-L
produced similar, if not better, inhibitory effects. Control ~u~ lalll fluid from COS-7 cells transfected with vector alone actually promoted Iymphonla cell growth. Accordingly, 25 the inhibitory effects of CD40-L on B Iymphomas par~llels that of antibodies to CD40 F.~ lmDle 4 This example illustrates the effect of anti-CD4() on the growth of human B-cell Iy~ JhJlll~l~ in SCID mice. C.B- 17 scidlscid (SCID) mice were obtained from the Animal 30 Production Facility (NCI-FCRDC, Frederick, MD. USA) and were not used until 6-8 weeks of age. The mice were kept under specific-patho~en-free conditions at all times;
they housed in llliului~oldtul cages, and all food, water and bedding were autoclaved before use. Tl;~ 7~le(4omgLlilll~lllu~lillland2oon~gc~llr1~
per 320 ml) was included in suspension form in the drinkillg water given to the mice. All 35 rnice received antisera to asialo GMl (Wako Chemical. Dallas, TX, USA), a marker present on murine NK cells (Murphy et al., El~`. J. Immunol. 22:1421; 1992) av~,lluu~ly one day before cell transfer. to remove host resistance to the tumor.
Il WO 95/17202 2 1 7 9 ~ ~ ~ PcrnlSg4114~67 ., --On day 0. SCID mice were injected either in~raYenousl~ or i~ a~ iL~ àlly wiih 5 x lo6 RL or TU'C cells. The ~umor cell recipients thel~ received ei[her ~ lag of anti-CD~0, or mslgG in 0.2 ml HBSS (Hank's balanced salt solution) iniravenously every other da)~
for a period of 10 days (toial of 5 injeciions), siariillg at da~ (), 3, or 14. Mice were S monitored for tumor developmeni and progression; moribund mice were el~hr~n~ All mice were necropsied for evidence of tumor. Liver, kidney and Iymphoid organs were analyzed hi~trlogir~lly for presence of tumor cells. Both parametric (student's t test) and non-parametric (Wilcoxan rank sum test) analyses were performed io deiermine if the groups differed ~ ;r;~ ~ly (p<0,05). All eA~ had 3-10 mice per group, and 10 were performed 2-3 times. The resulis are presenled ill Table I below.
Table 1: Effeci of anii-CD40 .~ llilli~il~l~iUII on su vival in umor-bearin~ mic-1 reaiment No. of Mean Day of F~rrrimPn~ Tumor (Rouie) (Initiation) Mice Death RL (i.p.) None 3 34i0 RL (i.p.) anti-CD40 6 >138_26.5a (da~ 0) TU2C (i.p.) None 3 28+0 TU~C ~i.p.) anii-CD4() 6 >76+45,0b (day ()) 2TU2C (i.v.) l~one 6 30+6.3 TU2C (i.v.) anti-CD40 6 >38+2.6C
(d;~y 3) TU2C (i.v.) allti-CD40 6 >32_4.6C
(day 14) 3RL (i.p.) None 6 39+3.5 RL (i.p.) anti-CD40 6 >107+21.9a (day 3) RL (i.p.) anti-CD40 6 >79+34,3d (day 14) a: No deaths due to tumor; all (6J mice surviving b: Two deaths due to tumor, olher (4 oul or 6 mice) showin~ no evidence of ~umor.
c: One of six recipient mice showing no evidencc of d d: Three deaths due ~o lumor, Ihree mice survivin~, Anti-CD40 significantly (pc0.05) improved surviYal of mice receiving either RL or TU2C tumors when trealment was initiated on day 0, 3 or 14. When SCID mice were 20 treated with anti-CD40 on day 0, no evidence of tumor u~as present in the mice receiving the RL B-cell Iymphoma line after several months. However. some mice receiving the EBV Iymphoma TU2C and anti-CD40 did develop tumor several weeks after cessation of anti-CD40 treatment.

Wo 95/17202 2 1 ~ ~ ~ 9 6 PCr~S94/14767 .
Differential pqtterlls of metast~tic ~row~h ~ere observed for the different routes of tumor cell adminislratioll. Mice recei~in~ the EB v-induced Iympllomas i.p. developed peritoneal tumors with exlensive metastases in the l~mph nodes and liver. whereas mice receiving the Iymphomas i.v. primarily developed renal ml~tqctqC~ Anli-CD40 was 5 capable of significantly inhibiting tumor grown and promoting survival of recipient mice regardless of the route of tumor dd~ l;aLI ~liOII.
Treatrnent of tumor-bearing mice with, nti-CD4() also resulted in ci~nifir,qnllyimproved surviYal when treatnlent was initiated 3 or 4 days after tumor cell transfer, and even as late as 14 days. These results indicate that anti-CD40 treatment was also 10 efficacious when treatment was iniuated with rel;~tively large and extensive tumor burdens (>1 cm3) in the recipient mice.
EYqmnle ~
This example illustrates the effect of anti-CD4() on the growth of tumors in SCID
15 mice injected with PBL from EBV-seropositive individuals. SCID mice (described in Example 4 above) were given injections of Ir~.",l.;"~"l human growth hormone (rhGH:
Genentech, South San Franciso, CA. USA)~ which has beell d~ la~di~,d to promote EBV~ "~ ;c in huPBL-SClD mice (Murph)~ et al.. Brail~ Bèhal~. Immlm. 6:3~:
1992), ~ bl~ due to promotion of human T-cell ~n~,.,r~"~"L in treated mice (Murphy et al., Proc. Natl. Acad. Sci. USA 89:4481; 1992). T-cell ~ .In~ t appears to beessential for human B-cell Iymphoma formation ill the huPBL-SClD model (18). rhGH
(10 ~lg in 0.2 mi HBSS) W;IS given i.p. on day () and every other day until time of assay 4-8 weeks later. Human PBLs were obtained from h~althy donors in leukrlpq~kc Anti-asialo GM-I was alllllll;aklr~d as dea~ribed for Example 4.
Human PBL were obtained from healthy, EBV-seropositive donors in lellknpq~k~
All donors were screened for antibodies to human; " ", . .. ,~ ncy virus type I (HIV- I ) and for hepatitis B âurface antigen (HBsAg), and provided informed consent before donation. The PBL were purified by counter-current elutriation, and the Iylll~llo-_y fraction, containing ~90% Iymphocytes as assessed by flow cytometry, were obtained.
The PBL (1 x 108) were injected i.p. into recipient SCID mice on day 0.
Mice were treated with anti-CD4(), anti-CD20, or with mslgG (2 1lg/0.2 ml PBS) i.p. every other day for 20 days for a total of 10 injections. Table 2 presents results ICI!JlC~ liiV~ of three ~A~ la with ~;-8 mice per group.
~3 WO95/17202 2 1 7 q ~ 9 ~ PCTIUS94fl4767 .
Tablc ~: Effect of anti-CD40 ddlllllli~ iOII of EBV-induced B-cel Iymphoma develcPment in huPBL-SCrD micA~ chimeras Mean Da~ ~o incidence of FYIl~rimrAnt Treatment No. of Mice of Death Lymphomaa Amsl~G 6 31.4+3.~ 10()~c anti-CD40 6 >50b 0~o Bn~slgG 8 26.3+~.1C 87~7G
anti-CD40 8 >55~ 0%
CmslgG 5 33.2il.~ 100%
anti-CD40 5 >53b 0%
anti-CD?0 5 >53 0%
a: Mice were moribund wilh evidence of cx~cnsi~c lumor nodul~s in ~he pcri~oncal cavil~ and evidence of Iymphoma i~y his~ological assessmcnL
b: Trealmen~ wilh anli-CD~0 resulled in no dca~hs duc lo l)~mphoma and no evidence or Iymphoma when assayed 2-6 weeks after cessalion of an~i-CD~0 ucatmen~. An~i-CD4V or anli-CD20 significantly fp<0.001) increased survival comp~red ~o control rccipienL~.
c: One out of 8 mice surviving and showing no evidence of ~umor.
The results ~ that treatment of huPBL-SClD chin1eric mice with anti-CD40 at the ime of huPBL transfer completely prevemed the d~ ~Iv~ c.ll of human B-cell Iy~ in the mice. Although anti-CD20 had no effect on the Iymphomas ir~ vilro.
treatment of the huPBL-SClD chimeras with anti-CD2() also prevented the occurrence of Iymphoma.
FYr~mrle G
This example illustrates the effect of anti-CD4() on the r ll~ rL~ l of human T
(HLA+, CD3+) and B (HLA+, CD3-) cells in SCID mice. huPBL-SClD mice chimeras were prepared as described in Example 5 above. r,l~ L~ ,s of human T and B cellsfound in the periloneal cavity were determined, and the human immunoglobulin in the serum q~l~rrit~ -A1 by enzyme-linked ;IIIIIIUIIUSOII)~IIL assay (ELISA). Animals were aiso examined for the presence of Iymphoma: those anlmal that evidenced Iymphoma weremoribund, with evidence of extensive tumor nodules in the peritoneal cavity. Mice were assayed 4-8 weeks after huPBL transfer, with all control (mslg) treated mice to EBV-induced B cell Iymphoma at day 33.2~t1.~.
Single cell ~ of peritoneal cavity cells were obtained, and evaluated by flow cytometry (FACS). Staining was performed in the presence of ~% human AB serum (Gibco BRL, Grand Island, NY, [JSA) to saturate human and mouse Fc receptors.
Reagents used in the FACS analysis were monoclonal anti human-HLA-ABC conjugated to fluorescein isothiocyanate ~FITC: Olymp~ Lake Success. NY~ USA), and Leu~L-biotinylated anti-CD3 (Becton-Dickinsol~, Mountaill Viev., C~, USA). After primar antibody incubation, cells were analyzed using an EPICS flow cytometer.

WO 95/17202 2 ~ :~ 9 ~ ~ PCI'IUS94/14767 .
Human immllnngJlr~bulin levels were assayed by ELISA. Flat-bottom 96-wel]
microtiter plates (Corning Glass Works, Corning, NY, llSA~ were coated with goat anti-human Ig (Kirke~aard and Perry Laboratory, Gci~ bul~, MD, USA) at I llg/ml in PBS, washed twice and blocked with SG~o goat serun~. The wells were Ihen incubated with sera 5 obtained from the huPBL-SClD mice chimeras, or a titration of human IgM+lgG standard (DAKO Corp., Santa Barbara, CA, USA). After washing four times, alkaline pho5pl~t~ conjugated goat anti-human Ig (Kirkegaard and Perry Laboratory, G~IL~ ,, MD, USA) was added. The plates were incubated, and washed again. After the final wash, substrate was added, and the enzyme reaction allowed to develop. OD was 10 measured at 402 nm. Results are presented in Table 3.
Table 3: Effect of anti-CD40 on human B-cell r 1l~l .n,.,. .1l and E- V-induced ~ cl~ .- . in huPBL-SClD chim ra mice ~t Pcriloneal % cells C/o cells Human Animal Cells HLA+, HLA+ serum lg Presence o~
Expenment No. TreatmenL (x lo6) CD3- CD3+ (~g/ml) Lymphoma A Imsl~G 1.6 1,8 1().3 90 +
2mslgG 1.4 0.8 2.8 120 +
3mslgG 1.1 0.6 0.4 155 +
4msl~G 1 5 0.6 1.4 406 +
5mslgG 0.6 ~.3 10.7 500 +
6anti-CD40 0.8 2.8 6.1 2.7 7anti-CD~0 1.4 1.0 1. I ().8 8anti-CD40 1.8 2.7 5.2 2.7 9anti-CD40 1.4 6.1 10.5 5.0 10anti-CJ~0 1.'2 2.0 5.2 0.2 B 1anti-CC40 9.0 6.1 7.2 11.7 2anti-CD~0 2.1 0.3 0.9 1.0 3anti-C1~0 1.8 0.5 6.7 15.0 4anti-CD40 0.6 5.1 5.0 4.0 Santi-CD~0 2.0 0.9 2.4 5.0 C Ianti-CQ0 0.8 0.0 8.1 6.5 2anti-C~20 1.4 0,0 1.1 10 3 3anti-CG20 1.8 0.0 5.~ 4.8 4anti-CL20 1.4 0.0 0.8 7.9 Santi-CD40 1.~ 10.3 0.0 41'2.0 6anti-CJ~0 9.() 1.5 0.0 86.4 7anu-C1~0 2.1 5.1 '~.9 1 1 lo o 8anti-CD40 1.8 0.6 0.9 18.5 1~
.. . . ..... . . .. . . .. ..

WO95/17202 2 ~ 7~:t 96 PCrfUS94)14~6~
.
Trearmenl with anti-CD10 did not prevenl Ihe engriLftment of hum~n T and B cells~
as determined by FACS analysis of periloneal cells. and determination of serum levels of human immunoglobulin. In i~.xr~rjmer1I A. the extent of hum~n cell en~r~frmrnr appeared 5 quantitatively less in anti-CD40 treated animals than in animals receiving mslgG, based on the levels of human Ig in the serum. However, since the huPBL-SCID chimeric mice that did not receive anti-CD40 developed B-cell Iymphomas, the high levels of human immllnoglr)blliin detected are likely to be due to the B-cell Iymphoma. In contrast to anti-CD40, treatment with anti-CD20 appeared to inhibit ~ -c,)l of B cells, both as 10 indicated in lower l,~ of B cells and in decreased levels of human Ig in the serum.
l~m~ 7 This example illustrates the effect of anti-CD40 and mslgG on the el.~ ".l of B
cells in SCID mice. huPBL-SClD mice chimeras were prepared as described in Example 5 15 above, except that the PBLs were obtained from EBV-negative donors. The chimeras were thus not expected to develop Iymphomas, thereb)~ providing ~ more accurate indication of of normal B cells The chimeras were treated with either anti-CD40 or anti-CD20, and p;,~ L~ of human T and B cells found in the peritoneal cavity were n3~ ;ll..i and the amount of human immunoglob~ in Ihe serum quantitated as 20 described in Example 6. Results are shown ill Table 5.
Table 5: Effect of Anti-CD40 Treatment on Human T."",. .,f~ "l;" Produclion in huP-L-SCID Chimeras Scrum Animal # Tre~men~a (U~/m 1. huPBL, mslgG 125.0 2. huPBL, mslgG 150.0

3. huPBL, mslgG 1.7

4. huPBL, mslgG 94.0

5, huPBL, mslgG 112.0

6. huPBL, msI~G 18.8

7 huPBL,anli-CD4() 194.()

8. huPBL, anti-CD4(3 269.0

9. huPBL, anti-CD4() 239.() huPBL, anti-CD4() 119.() 11. huPBL, anti-CD4() 206.0 1'2. huPBL. anti-CD4() 150.() a: SCID mice received 10(1 million huPBL i.p wilh 2 ug o~ anli-CD10 or mslgG giYen i.p. every other da)~ for 20 days Micc wcre analyzed ~or serum ' levels using a hum~n ~ Iin-specirlC ELISA 3 weeks afIer huPBL Irans~cr WO 95117202 2 1 7 9 1 9 6 PCTI~S9411476'~
.
Treatment wilh anti-CD1U did promote ellgr.iftn1ellt of h~lman B cells. indicating that anti-CD40 has additional value for treatment or prevenlioll of Iy~ ull-às due to its ability to remove Iymphoma cells while sparing nonnal B cell~.
FT~ '^ 8 This example illustrates the effect of antibodies to CD40 on the growth of humanmelanoma cells in vilro. Antibody to CD40 ligand (M~) was tested in a proliferation assay cl~hct~rti~lly as described above, in Example '2, using M 16 human melanoma cells, which

10 express CD40.
The results obtained are presented in Figure 5: values are presented as percent of inhibition compared to control :>UiJ~llla~dllt fiuids. Incubation with anti-CD40 mrmorlr~n~l anubody M2 resulted in significant inhibition of the i,luli~tl~liul, of M16 human melanoma cell line tested, with as little as (). I ng/ml causing inhibition of almost 50r'~G. Increased 15 inhibition was observed with increasing ~ , "" "~ of anli-CD40.
F.Y~ '^ 9 This example illustrates the effecl of Ir~ ,ull human CD40 ligand on the growth of human B-cell Iymphomas in SCiD mice. SCiD mice were oblained, and treated 20 cllhctl~nti:~lly as described in Example 4, above. On day 0, SCID mice were injected either ;~1~"1~`.;1.",.~lly with5x 106RLorTU2Ccells. Thetumorcellrecipientsthenreceived 100 ,Lli of c~ rrl supennatant fiuid ftom cells rsd:-sfected with either a vector encoding human CD40 ligand, or vector alone (control). Two CullC~ LlaliullS of the CD40 ligand-containing ~uu~llla~alll fiuid were tested: a ten-fold ~unc, - and a two-fold uullC.,IlLlat~, 25 (10x and 2x, respectively). The concentrated supernatants were administered lly every third day for a period of 15 days (total of 5 injections), starting atday 3. Mice were monitored for tumor development and i lu~l~a~iull, moribund mice were e~th ~ni7~r~ All mice were necropsied for evidence of tumor. Liver, kidney and Iymphoid organs were analyzed histologically for presence of tumor cells. Both parametric (student's 30 t test) and non-parametric (Wilcoxan nank sum tesl) anaiyses were perfonmed to determine if the groups differed significantly (p~().05). All ~ had 7-10 mice per group, and were performed 3 times.
The results of an exemplary experiment utilizing RL cells are shown in Figure 6.Similar to the results obtained in vitro itl Example 3, IC~ullliJillr~l~ CD40 ligand inhibited the 35 fro~lh of mmm c~ In vivo in SCID mi~e WO95117202 2 ~ 7 q ~ ~ 6 PCT/US94114767 SEQUENC~ ~ ISTIN^
5 ( 1 ) GENE RAL I NF ORMAT I ON:
(i) APPLICANT: ARMITAGE, RICHARD
FANSLOW, WILLIAM
LONGO, DAN L.
MURPHy, WILLIAM
(ii) TITLE OF INVENTION: METHOD OF PREVENTING OR TREATING
DISEASE CE~ARACTERIZED BY NEOPLASTIC CELLS

(iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: IMMUNEX CORPORATION
(B) STREET: 51 UNIVERSITY STREET
(c) CITY: SEATTLE
(D) STATE: WASHINGTON
(E) COUNTRY: USA
(F) ZIP: 98101 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Macintosh (C) OPERATING SYSTEM: Apple Macintosh System 7 . l (D) SOFTWARE: Microso~t Word ~or ~acin~osh, Version #S.la (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: Decerr~er 1, 1994 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: USSN 08/172, 664 (B) FILING DATE: Dece~ber 23, 1993 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Perkins, Patricia A.
(B) REGISTRATION NUMBER: 34, 693 (C) REFERENCE/DOC}~ET NUMBER: 2818 (iX) TT~.T.r`rr)MMr~NTCATION INFORMATION:
(A) TELEPHONE: (206) 587-0430 (B) TELEFAX: (206) 233-0644 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
SS (A) LENGTH: 840 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOG`!: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
1~

(iv) A~'T~-SENS-: NC
(vi ) ORIGINAL SOURCE:
(A) ORGANISM: Homo saplens ~vii) IMMEDIATE SOURCE:
~B) CLONE: CD4 0-L
0 ~ix) FEATURE:
~A) NAME/REY: CDS
~E) LOCI~TION: 46..831 ~xi) SEQUENCE DESCRIPTION: SE~ ID NO:l:

Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu Ile Thr Gln Met Ile Gly ser Ala Leu Phe Ala val Tyr Leu His Arg Ary Leu Asp AAG ATA GAA GAT GAA AGG AAT CTT CAT GAA GAT TTT GTA ~TC ATG AAA 2 4 6 Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys ASp Ile Met TTA AAC AAA GAG GAG ACG AAG AAA GAA AAC AGC TTT GAA ATG CAA AAA 39û
Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr ATG AGC AAC AAC TTG GTA ACC CTG GA} A~.T G,,G AA~ CAG CTG A^C GTT 537 Met Ser Asn Asn Leu Val Thr Leu Glu Asn GIy Lvs Gln Leu Thr Val WO 95/17202 2 1 7 9 ~ ~ ~ PCT/US94114767 AAA AGA CAA GGA CTC TAT TAT ATC TAT GCC CAA GTC ACC TTC TGT TCC 58.
Lys Arg Gln Gly 1eu Ty~ Tyr Ile Tyr Ala Gln Val Thr Pne Cys Se-165 ~ 17C 175 AAT CGG GAA G~T TCG AGT CAA GCT CCA TTT ATA GCC AGC CTC TGC CTA 630 Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu 180 185 190 l9S

0 Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gl V~l Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu 2qS 250 255 Lys Leu 30 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE ChARACTERISTICS:
(A) LENGTH: 261 amino acids (E) TYPE: arino acid (D) TOPOLOGY: linear (ii ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N '' O.l~, Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly Leu Pro Ile Ser Met Lys Ile Phe Met Tyr L
20 25 eu Leu Thr Val Phe Leu Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Ar 35 40 qS
Arg Leu Asp Lys Ile Glu Asp Glu Ar Asn L ' A
50 55 g eu H s Glu sp Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asr. Thr Gl Glu Ar Ser Leu Ser 7 0 Y g 8 0 eu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Gl~.. Gly Phe Val Lys 85 ~ 9~
sp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu loo loS llo 7() 2l79196 WO 951172~12 PCTIUS94114767 .
Met Gln Lys Gly Asp Gln Asn P-o Gl-. ~le A;a A' a ~lS Ve' Il~ Ser 115 1~. :_-Glu Ala Ser Ser Lys Thr Tnr Ser Val Leu Gln Trr, Ala Glu Lys Gly Tyr Tyr Thr Xet Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 lS0 _ lSS 160 0 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser G~ n Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala l9S 200 205 Ala Asn Thr His Ser S~r Ala Lys Pr:i Cys Gly Gln G~n Ser Ile His 210 '1S '~
Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr G1y Phe T

ly Leu Leu Lys Leu

Claims (31)

What is claimed is:

1. A method of treating a mammal afflicted with a disease characterized by neoplastic cells that express CD40, comprising administering a therapeutically effective amount of a CD40 binding protein capable of binding CD40 and inhibiting binding of CD40 to CD40L, as determined by observing at least about 90% inhibition of the binding of soluble CD40 to CD40L, in a pharmaceutically acceptable buffer, wherein the therapeutically effective amount is from about 0.01 to about 1 mg/kg body weight and inhibits proliferation of the neoplastic cells.

2. The method of claim 1, wherein the CD40 binding protein is selected from the group consisting of monoclonal antibodies to CD40, CD40 ligand, and combinationsthereof.

3. The method of claim 2, wherein the CD40 binding protein monoclonal antibody hCD40m2 (ATCC HB 11459).

4. The method of claim 2, wherein the CD40 binding protein comprises an antigen binding domain derived from antibody hCD40m2 (ATCC HB 11459).

5. The method of claim 2, wherein the CD40-binding protein is soluble, oligomeric CD40 ligand comprising a CD40-binding peptide derived from an extracellular domain of CD40 ligand, and an oligomer-forming peptide.

6. The method of claim 5, wherein the oligomer-forming peptide is selected from the group consisting of an immunoglobulin Fc domain and a zipper peptide.

7. The method of claim 6, wherein the zipper peptide forms a trimer in solution.

8 The method of claim 1, wherein the cells expressing CD40 are selected from thegroup consisting of B lymphoma cells, melanoma cells and carcinoma ceils.

9. The method of claim 2, wherein the cells expressing CD40 are selected from the group consisting of B lymphoma cells, melanoma cells and carcinoma cells.

10. The method of claim 3, wherein the cells expressing CD40 are selected from the group consisting of B lymphoma cells, melanoma cells and carcinoma cells.

11. The method of claim 4, wherein the cells expressing CD40 are selected from the group consisting of B lymphoma cells, melanoma cells and carcinoma cells.

12. The method of claim 5, wherein the cells expressing CD40 are selected from the group consisting of B lymphoma cells, melanoma cells and carcinoma cells.

13. The method of claim 6, wherein the cells expressing CD40 are selected from the group consisting of B lymphoma cells, melanoma cells and carcinoma cells.

14. The method of claim 7, wherein the cells expressing CD40 are selected from the group consisting of B lymphoma cells, melanoma cells and carcinoma cells.

15. A method of preventing a disease characterized by neoplastic cells that express CD40, in a mammal susceptible to the disease, comprising administering a therapeutically effective amount of a CD40 binding protein capable of binding CD40 and inhibiting binding of CD40 to CD40L as determined by observing at least about 90% inhibition of the binding of soluble CD40 to CD40L in a pharmaceutically acceptable buffer, wherein the therapeutically effective amount is from about 0.01 to about 1 mg/kg body weight and inhibits proliferation of the neoplastic cells.

16. The method of claim 15, wherein the CD40 binding protein is selected from the group consisting of monoclonal antibodies to CD40, CD40 ligand, and combinationsthereof.

17. The method of claim 16, wherein the CD40 binding protein is monoclonal antibody hCD40m2 (ATCC HB11459).

18. The method of claim 16, wherein the CD40 binding protein comprises an antigen binding domain derived from antibody hCD40m2 (ATCC HB 11459).

19. The method of claim 16, wherein the CD40-binding protein is soluble, oligomeric CD40 ligand comprising a CD40-binding peptide derived from an extracellular domain of CD40 ligand, and an oligomer-forming peptide.

20. The method of claim 19, wherein the oligomer-forming peptide is selected from the group consisting of an immunoglobulin Fc domain and a zipper peptide.

21. The method of claim 20, wherein the zipper peptide forms a trimer in solution.

22. The use of a CD40 binding protein in providing a medication for administration to a mammal afflicted with a disease characterized by neoplastic cells that express CD40.

23. The use of claim 22, wherein the CD40 binding protein is selected from the group consisting of monoclonal antibodies to CD40, CD40 ligand, and combinationsthereof.

24. The use of claim 23, wherein the CD40 binding protein is monoclonal antibodyhCD40m2 (ATCC HB11459).

25. The use of claim 23, wherein the CD40-binding protein comprises an antigen binding domain derived from antibody hCD40m2 (ATCC HB 11459).

26. The use of claim 23, wherein the CD40-binding protein is soluble, oligomenc CD40 ligand.

27, A method of preparing a medication for administration to a mammal afflicted with a disease characterized by neoplastic cells that express CD40, comprising formulating a CD40 binding protein capable of binding CD40 and inhibiting binding of CD40 toCD40L as determined by observing at least about 90% inhibition of the binding of soluble CD40 to CD40L, in a suitable excipient or carrier.

28. The method of claim 27, wherein the CD40 binding protein is selected from the group consisting of monoclonal antibodies to CD40, CD40 ligand, and combinationsthereof.

29. The method of claim 28, wherein the CD40 binding protein is monoclonal antibody hCD40m2 (ATCC HB11459).

30. The method of claim 28, wherein the CD40-binding protein comprises an antigen binding domain derived from antibody hCD40m2 (ATCC HB 11459).

31. The method of claim 28, wherein the CD40-binding protein is soluble, oligomeric CD40 ligand.