CA2222231A1 - Antibody and antibody fragments for inhibiting the growth of tumors - Google Patents

Abstract

Chimerized and humanized versions of anti EGF receptor antibody 225 and fragments thereof for treatment of tumors.

Description

W O 96,140210 PCT~US~6/'03347 ANTIBODY AND ANTIBODY FRAGMENTS FOR
ll~TRITTl~G THE GROVVTH OF TUMORS

This application is a co,.~;"~ ;on-in-part of Serial No. 08/573,289 filed December 15, 1995, which was a continn~tion-in-part of Serial No. 08/482,982 filed June 7, 1995, the diselosures of both of which are incorporated herein by reference.

FIELD O~ THE INVENTIOI~

The present invention is directed to antibodies and antibody fr~mentc useful in inhibiting the growth of certain tumor cells.

~ACKGROUND OF THE INVENTION

Recent research has uncovered the important role of growth factor receptor tyrosine kinases in the etiology and progression of human m~ n~ncies. These biological .ece~Lc,l~ are anchored by means of a tr~n~memhrane domain in the membranes of cells that express them. An extracellular domain binds to a growth factor. The binding of the growth factor to the extracellular domain results in a signal being tr~n~mittecl to the intracellular kinase domain. The tr~n~ ction of this signal contributes to the events that are responsible for the proliferation and ~liLr~ iation of the cells.

Members of the epic1erm~l growth factor (EGF) receptor family are important growth factor rcce~lor tyrosine kinases. The first member of the EGF .Gc~l Lur family to be discovered was the glycoproteill having an ~p~nt molecular weight of approximately 165 kD. This glycoprotein, which was described by Mendelsohn et al.
~ in U.S. Patent No. 4,943,533, is known as the EGF receptor (EGFR).

CA 02222231 1997~ 25 ~ 5A~J~ 9~

The binding of an EGFR ligand to the EGF receptor leads to cell growth. EGF
and transforming growth factor alpha (TGF-alpha) are two known ligands of EGFR.

Many receptor tyrosine kinases are found in ~lmls~ ly high numbers on human tumors. For example, many tumors of epithelial origin express increased levels of EGF
5 receptor on their cell mc~ es. Examples of tumors that express EGF receptors include glioblastomas, as well as cancers of the lung, breast, head and neck, and bladder. The amplification and/or o~/el~ )ression of the EGF receptors on the membranes of tumor cells is associated with a poor prognosis.

Antibodies, especially monoclonal antibodies, raised against tumor antigens have10 been inv~ti~ted as potential anti-tumor agents. Such antibodies may inhibit the growth oftumors through a number of mecl~n~ For example, antibodies may inhibit the growth of tumors immllnologically through antibody-dependent cellular cytotoxicity (ADCC) or complem~nt-dependent cytotoxicity (CDC).

Alternatively, antibodies may co",~ele with growth factors in binding to their 15 receptors. Such colllpe~ilion inhibits the growth oftumors that express the rece~lor.

In another approach, toxins are conj~ ted to antibodies raised against tumor antigens. The antibody portion directs the conjugate to the tumor, which is killed by the toxin portion. ,,;

For ,c ~1~, U.S. Patent No. 4,943,533 describes a murine monoclonal antibody 20 called 225 that binds to the EGF receptor. The patent is ~eeif~ned to the University of California and licensed exclusively to ~nClone Systems Incoll,ol~led. The 225 antibody is able to inhibit the growth of cultured EGFR c~),e~ g tumor lines as well as the growth of these tumors in viw when grown as xenografts in nude mice. See Masui et al., Cancer Res. 44, 5592-5598 (1986). More recenlly~ a tre~tm~nt regimen 25 coll,l)inil1g 225 plus doxorubicin or cis-platin exhibited therapeutic synergy against ~ED S~ET

~ CA 02222231 1997-11-25 ~ ~ 6 l~91~7 several well established human xenograft models in mice. Ra~lg~ et al., J. Natl.Cancer Inst. 85, 1327-1333 (1993).

A disadvantage of using murine monoclonal antibodies in human therapy is the possibility of a human anti-mouse antibody (HAMA) response due to the presence of 5 Imouse Ig seguences. This disadvantage can be ...;~li"-;,ed by replacing the entire constant region of a murine (or other non-human m~mm~ n) antibody vvith that of a Ihuman constant region. Repl~cPmPnt of the consl~ll regions of a murine antibodyvvith human sequences is usually lcre-lcd to as chimerization.

The chimerization process can be made more effective by also replacing the 10 variable regions - other than the hy~c~valiable regions or the compl~m~ont~rity-detcl,.unil~ regions (CDRs), of a murine antibody with the corresponding human sequences. The variable regions other than the CDRs are also known as the variable firamework regions ~Rs).
.

The repl~cPm~nt of the constant regions and non-CDR variable regions vvith 15 human sequences is usually lere-lcd to as h~ ;on. The hllm~ni7ed antibody is ~ess imml~nogenic (i.e. elicits less of a HAMA Ic~onse) as more murine sequences are replaced by human sequences. Unfortunately, both the cost and effort incl ease as more regions of a murine antibodies are replaced by human sequences.
~' Another approach to re~ çir~ the immlmog~n:~ity of antibodies is the use of 20 ~mtibody L~ For example, an article by Aboud-Pirak et al., Journal of theNational Cancer Tn~titute 80. 1605-1611 (1988), CGn~LaleS the anti-tumor effect of an anti-EGF ~~ptor antibody called 108.4 with fr~grnPnt~ of the antibody. The tumor model was based on KB cells as xenogra~s in nude mice. KB cells are derived fromhuman oral epidermoid carcinomas, and express elevated levels of EGF receptors.

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CA 02222231 1997-11-2~

Aboud-Pirak et al. found that both the antibody and the bivalent F(ab')2 fragment retarded tumor growth in vivo, although the F(ab')2 fragment was less efficient. The monovalent Fab fragment of the antibody, whose ability to bind the cell-associated receptor was conserved, did not, however, retard tumor growth.

There is, therefore, a CO.. ~ .i,,g need for improved anti-tumor agents that can be ef~lciently and inex~e~ ely produced, have little or no immlln~genicity in hllm~nc, are capable of binding to lec~lol~ that are expressed in high numbers on tumor cells, and are capable of blocking the binding of such growth factors to such receptors. An object of the present invention is the discovery of such new anti-tumor agents that 10 combine the advantageous fe~ es of monoclonal antibodies, antibody fr~gment~ and si:ngle chain antibodies.

Sl[~Ml\I~RY OF T~ '. INVhNTION

T_ese and other objects, as will be a~palent to those having or.lhl~ y skill in the art, have been met by providing a polypeptide lacking the constant region and the 15 variable light chain of an antibody, the polypeptide compri~in~ the amino acid sequenceNYGVH(SEQIDNO: l),GVIWSGGNTDYNTPFTSR(SEQ
ID NO: 2), or V I W S G G N T D Y N T P F T S (SEQ ID NO: 3). The polypeptide may be conjugated to an effector molecule, such as a molecule that inhibits tumor growth. The invention further is directed to DNA encoding such polypeptides.

The invention also includes polypeptides con~icting of the amino acid sequence NYGVH, GVIWSGGNTDYNTPFTSRorVIWSGGNTDYNTPF
T S.

The invention also includes a molecule having the constant region of a human antibody and the variable region of monoclonal antibody 225 conjugated to a cytoxic ag~ent such as doxorubicin, taxol, or cis-~ mminf~lichloropl~timlm (cisplatin). The invention further includes a method for significantly inhibiting the growth of tumor cells in a human comprising ~f1mini.ettoring to the human an effective amount of a ]polypeptide lacking the constant region of the variable light chain of an antibody, the polypeptide comprising the amino acid sequence N Y G V H, G V I W S G G N T D
5 YNTPFTSR,orVIWSGGNTDYNTPFTS. Anotheraspectofthe invention is a method for ~ignifil~ntly inhibiting the growth of tumor cells in a human comprising ~rlmini~t~ring to the human an effective amount of a polypeptide con~i~ting ofthe amino acid sequence N Y G V H, G V I W S G G N T D Y N T P F
TSR,orVIWSGGNTDYNTPFTS.

The invention further includes a method for significantly inhibiting the growth of tumor cells that express the EGF receptor in a human. The method comprises ~lmini~terin~ to the human an effective amount of a molecule having the co~ t region of a human antibody and the variable region of monoclonal antibody 225,both ïn the prescence of and, in particular, in the absence of, ~;y l~t~ic molecules, such as c hemotherapeutic agents.

]l)F~CRIPT][ON OF FIGUl~

Figure 1. Effect of 225 on the growth of established A431 tumor xenografts in nude mice. Animals were injected with 10' cells in the flank. Tre~tmentc, con~i~ting of PBS or 1 mg/animal of 225 twice weekly for 5 weeks, were begun when tumors reached an average volume of 2-300 mm3. Volurnes and Remission Index (RI) were ,termined as described in the "Examples" section.

Figure 2. Effect of 225 and ehim.ori7P-l 225 (C225) on the growth of establishedA431 tumor xenografts in nude mice. Animals were treated with lmg/mouse of PBS
twice weekly for 5 weeks. A: Average tumor volumes; B: Remission Index. The ;1P~G11~ tumor regression in the PBS control group at day 37 was due to the death of CA 02222231 1997-11-2~
W O 96/410210 PCT~US96/09847 3 out of the 10 ~nim~l~ within the group at this time and the concol-lllliLant decrease in overall tumor volume.

Figure 3. Effect of C225 on the growth of established A431 xenografts in nude mice. Animals were treated with lmg of C225 or PBS twice weekly for 5 weeks.
5 The average tumor volume of the C225 group showed statistically ~ignifi~nt biological effects colll~cd to control (see text) A: Average tumor volumes (~teri~k~
show statistical significance with respect to control); B: Remission Index.

Figure 4. Dose response of C225 on the growth of established A431 xenografts in mlde mice. Animals were treated with PBS, 1, 0.5, or 0.25 mg/animal twice weekly10 for 5 weeks as described in M~t~ri~l~ Methods. Animals treated with 1 mg/dose of C225 showed statistically significant biological effects co.l.l~a~ed to control (see text).
A: Average tumor volumes (~cteri~k~ define statistical .ci~nifi~n~e with respect to control), B: Remission Index. The drop in RI for the 250 ug dose group on day 47resulted from the re-al)pe~ re of a tumor in an a~c--l tumor-free animal. (In this 15 in~t~nre, the effect of C225 was transient.) Figure 5. Inhibition of A431 cells by C225 and by heavy chain CDR-l and heavy chain CDR-2 of monoclonal antibody 225 Figure 6. Inhibition by C225-Doxorubicin conjugate of A431 cells in vivo as a fimction of c~ nr~ntration.

Figure 7. FACS analysis of EGFR cx~.cssion on human prostatic carcinoma cell lines. LNCaP (~uman prostatic carcinoma, androgen-dependent), DU 145 and PC-3 (human prostatic carcinoma, androgen-independent), and A431 (human epidermoid e,~.;".o...a) cells were removed with EDTA from the growth flask3 and stained with C'225. Data are p~e3ellLcd as MFI (Mean Fluorescence Intensity), an indirect measure CA 02222231 1997-11-2~

W O 96/40210 PCT~US9~1~5317 of antigen ~xl~lession. The results shown in this figure are ~ s~nlali~e of at least 5 .;, . ,ent~

Figure 8. Inhibition of EGF-ind~lcecl phosphorylation of the EGFR by C225.
LNCaP, DU 145, and PC-3 monolayers were ~tim~ ted with EGF in the presence or 5 absence of C225. Cells were lysed, subjected to SDS PAGE, blotted, and screened with a mouse monoclonal antibody to PTyr (UBI, Lake Placid). Lane A: no additions (basal level of EGFR phosphorylation); Lane B: stim~ tion of EGFR with 10 ng/ml EGF for 15 mim-les at room temperature in the absence of C225; Lane C: stimlll~ti~n of EGFR with EGF in the presence of 10 ug/ml of C225.

Figure 9. Growth inhibition of established DU 145 xenografts by C225. One million DU 145 cells in matrigel were innoculated into nude mice (males, nu/nu).After tumors reached an average volume of approximately 100 mm3 (day 20), ~nim~l~
were randomized (10 ~nim~l~ per group) and treated with either PBS (control) or C225 (0.5 mg/dose, 10x). Animal were treated for 35 days and followed for an 15 additional 3 weeks. Mice that were tumor-free or carrying small tumors were m~int~inecl for an additional 3 months. Significance (shown by astericks in Figure 3A) was determined by a Student's T-test and a p value < 0.5 was considered sipnifie~nt A: average tumor volume; B: growth characteristics for tumors in thePBS group; C: growth char~ct~ri~tics for tumors in the C225-treated groups.

Figure 10. Effects of C225 on tumor elimin~tion and surviva . The complete elimin~tion of tumors during the course of the study was defined by a }~emi~ion Index (RI). Animal mortality during the study was considered a tre~tment failure and included in the analysis. A: Remission Index; B: Survival curve. The empty and filled circles in Figure 10 have the same me~nin~s as in Figure 9.

CA 0222223l l997-ll-2~

W O 96/4~210 PCTAUS96/09847 Figure 11. Schem~tic l~resen~lion of the pKN 100 m~mm~ n expression vector used for the expression of the kapp light chains of the chimeric C225 andreshaped human H225 antibody.

Figure 12. Sçh~ tic repres~nt~tion ofthe pGlD105 m~n~m~ n t;~les~ion S vector used for the t;~ ssion of the heavy chains of the chimeric C225 and reshaped human H225 antibody.

Figure 13. DNA (SEQ ID NO: 4) and peptide (SEQ ID NO: 5) sequences of the kappa light chain variable region of the M225 antibody. The PCR-clones from which this information was obtained were amplified using the degenerate primer MKV4 10 (SEQ ID NO: 6)(7).

Figure 14. DNA (SEQ ID NO: 7) and peptide (SEQ ID NO: 8) sequences of the heavy chain variable region of the M225 antibody. The PCR-clones from which thisin formation was obtained were amplified using the degenerate primer MHV6 (SEQ
ID NO: 9)(7).

Figure 15. DNA (SEQ ID NO: 10) and peptide (SEQ ID NO: l l) sequences of the kappa light chain variable region of theC225 antibody.

Figure 16. DNA (SEQ ID NO: 12) and peptide (SEQ ID NO: 13) sequences of the heavy chain variable region of the C225 antibody.

Figure 17. DNA (SEQ ID NO: 14) and peptide (SEQ ID NO: 15) sequences of th~e kappa light chain variable region of the C225 antibody with the modified leader sequence from the kappa light chain of L7'CL antibody (28).

CA 0222223l l997-ll-25 W O 96/40210 PCT~US~/09847 Figure 18. Typical example of the results of a cell ELISA to measure the bindingaffinty of chimeric C225 and reshaped human H225 (225RKA/225RHA) antibodies to epidermal growth factor receptor expressed on the surface of A431 cells.

Figure 19. DNA (SEQ ID NO: 16) and peptide (SEQ ID NO: 17) sequences of ~ 5 the first version (225RKA) of the kappa light chain variable region of the reshaped human H225 antibody.

Figure 20. DNA (SEQ ID NO: 18) and peptide (SEQ ID NO: 19) sequences of the first version (225RHA) of the heavy chain variable region of the reshaped human H225 antibody.

Figure 21. Arnino acid sequences of the two versions (225RKA and 225RKB) of the kappa light chain variable region of the reshaped human H225 antibody (SEQ ID
NO: 20), (SEQ ID NO: 21), (SEQ ID NO: 22), (SEQ ID NO: 23). ~idlles are numbered according to Kabat et al. (20). Mouse framework residues conserved in the reshaped human frameworks are highlightç-l in bold.

Figure 22. Amino acid sequences ofthe five versions (225RHA, 225RHB, 225RHC, 225RHD, 225RHE) of the heavy chain variable region of the reshaped humanH225 antibody (SEQ ID NO: 24), (SEQ ID NO: 25), (SEQ ID NO: 26), (SEQ ID NO:
27), (SEQ ID NO: 28), (SEQ ID NO: 29), (SEQ ID NO: 30). R~id~les are numbered according to Kabat et al. (20). Mouse framework residues conserved in the reshaped human frameworks are highli~htçcl in bold.

CA 02222231 1997-11-2~
WO 96/40210 PCT~US96/~847 nFTAr~,Fn I)ESC~2TPTION OF T~F INVFNTION

In one aspect of the invention, a polypeptide lacking the constant region and the variable light chain of an antibody comprises the first and second heavy chain complement~rity determinin~ regions of monoclonal antibody 225. These regions 5 have the following amino acid sequences:

CDR-1 NYGVH(SEQIDNO: 1) CDR-2 GVIWSGGNTDYNTPFTSR(SEQIDNO:2) The peptide comprising the first and second complement~rity ~1~l.. ini,.~ regions 10 mentioned above may be obtained by methods well known in the art. For example, the polypeptides may be expressed in a suitable host by DNA that encodes the polypeptides and isolated. The DNA may be synth~si7~d ch~mir~lly from the four nucleotides in whole or in part by methods known in the art. Such methods include those described by Caruthers in Science ~Q, 281-285 (1985).

The DNA may also be obtained from murine monoclonal antibody 225, which was described by Mendelsohn, et al. U.s. Patent No. 4,943,533. This antibody wasdeposited in the ~m~ric~n Type Culture Collection, Bethesda, Maryland on June 7,1995. (Accession number 11935). Methods for obtaining the variable heavy chain region of antibodies are known in the art. Such rnethods include, for example, those 20 described in U.S. patents by Boss (Celltech) and by Cabilly (Genente~l-). See U.S.
Patent Nos. 4,816,397 and 4,816,567, respectively.

The DNA encoding the protein of the invention may be replicated and used to express recombinant protein following insertion into a wide variety of host cells in a wide variety of cloning and ~ es~ion vectors. The host may be prokaryotic or 25 eukaryotic.

CA 02222231 1997-11-2~

The polypeptide may contain either N Y G V H, G V I W S G G N T D Y N T P F
r s R, or V I W S G G N T D Y N T P F T S. Alternatively, the polypeptide may contain the sequence N Y G V H, and either of the sequences G V I W S G ~ N T D
YNTPFTSR,orVIWS~GNTDYNTPFTS.

The polypeptide may also be conjugated to an effector molecule. The effector molecule performs various useful functions such as, for example, inhibiting tumor growth, perrni~in~ the polypeptide to enter a cell such as a tumor cell, and directing the polypeptide to the a~lo~liate location within a cell.

The effector molecule, for example, may be a cytotoxic molecule. The cytotoxic molecule may be a protein, or a non-protein organic chemotherapeutic agent. Someexamples of suitable chemotherapeutic agents include, for example, doxorubicin, taxol, and cisplatin.

Some additional examples of effector molecules suitable for conjugation to the polypeptides of the invention include signal transduction inhibitors, ras inhibitors, and cell cycle inhibitors. Some examples of signal tr~n~ln-~tion inhibitors include protein tyrosine kinase inhibitors, such as quercetin (Grazieri et al., Biochim. Biophs. Acta 714~ 415 (1981)); lav~-n~ tin A (Onoda et al., J. Nat. Prod. 52, 1252 (1989)), and herbimycin A (Ushara et al., Biochem. Int., 41, 831 (1988)). Ras inhibitors include inhibitors of ras farnesylation, such as the benzodiazepine peptidomimetics described by James et al. in Science ~Q 1937 (1993), which have the formula shown below:

N ~
R~N~N ~ ~

CA 02222231 1997-11-2~
W O 96/410210 PCTnJS~6/0~8~7 in which R is H or CH3; and X is Methione, Serine, Leucine, or an ester or amidederivative thereof.

Proteins and non-protein chemothela~uLic agents may be conjugated to the polypeptides by methods that are known in the art. Such methods include, for S example, that described by Greenfield et al., Cancer Research 50, 6600-6607 (1990) for the conjugation of doxorubicin and those described by Arnon et al., Adv. Exp.
~Ied. Biol. ;~ , 79-90 (1991) and by Kiseleva et al., Mol. Biol. (USSR) ~, 508-514 (]i 991) for the conjugation of platinum compounds.

The invention further includes a modified antibody having the constant region of a human antibody, and the hypervariable region of monoclonal antibody 225. These modified antibodies are optionally conjugated to an effector molecule, such as acytotoxic agent. The variable region other than the hypervariable region may also be derived from the variable region of a human antibody. Such an antibody is said to be hr-m~ni7erl Methods for making hn,.,~ e~ antibodies are known in the art.
~[ethods are described, for example, in Winter, U.S. Patent No. 5,225,539.

The most thorough method for hum~ni7~tion of the 225 antibodies is CDR-grafting. As described in Fx~mrle IV, the regions of the mouse antibody that aredirectly involved in binding to antigen, the complement~rity .1~ . ",i~ g region or CDRs, are grafted into human variable regions to create "reshaped human" variable 20 regions. These fully ~ d variable regions are then joined to human constant regions to create complete "fully h~ ed" antibodies. In order to create a fully hlllllnll;,~cl antibody that binds well to antigen, it is eeePnti~l to carefully design the reshaped human variable regions. The human variable regions into which the 225 arrtibodiesCDRswill be grafted must be carefully selected, and it is usually neceee~ry 25 tc make a few amino acid changes at critical positions within the framework regions (FRs) of the human variable regions.

. CA 02222231 1997-11-25 ~ ~ S ~ g ¦ nq 8 4 7.
~S 7 ~ 7 .
The reshaped human H225 vaî iable regions, as desi~neA include up to a single amino acid change in the FRs of the sele~,led human kappa light chain variable region and as many as twelve amino acid r~ xes in the Fl~ of the s~l~ed human heavy chain variable region. The DNA sequences coding for these resl.aped human H225 5 heavy and kappa light chain variable region genes are joined to DNA sequences coding for the human y 1 and human lC CQI~ region genes, le;.~,e~ ely. The ~ haped human H225 antibody is then e A~lessed in ~ .""~!~Prl cells and tested, in CO~ ~ison with mouse M225 antibody, and chimeric C225 antibody for binding to human EGF
receptor cAIvlessed on the surface of A431 cells.

The variable region of the antibody outside of the hypervariabl~ region may also be derived from monoclonal antibody 225. In such case, the entire variable region is derived from murine monoclonal antibody 225, and the antibody is said to be cl~ill,eli~ed, ;.e., C225. Methods for making cl~ilue.iLed antibodies are known in the art. Such methods incl~lde, for e.~ ~le, those de3~ d in U.S. patents by Boss (Celltech) and by Cabilly (Gçn~ ecl-). See U.S. Patent Nos. 4,816,397 and 4,816,567, les~e.;~i~ely.

The COllS~I~ region of the modified antibodies may be of any human class, i.e., IgG, IgA, IgM, IgD, and IgE. Any subclass of the above classes is also suitable, e.g., IgGl, IgG2, IgG3 and IgG4, in which IgG1 is pl~f,.l~,d.

Any of the c~i~or molecules mentioned above in conl-ecl;ol- with conjrlgrtion to a polypeptide can also be conjugated to ~;Liln~,.ic or h~ ed antibodies ofthe invention. Doxorubicin, taxol, and ~ Gl;~. are p,efe,l~.

The polypeptides and antibodies of the invention ~ignific~n~ly inhibit the growth of tumor cells when r Iminictered to a human in an effective amount. F~ 'es oftumorcells that can be treated with the polypeptides and antibodies of the invention include glioblastomas, as well as cancers of the lung, breast, head, neck and bladder. The optimal dose can be dete. ""ned by physicians based on a number of pa,~m~le, AlAENDED SHEET

~ CA ~2222231 1997-11-25 ~S9065/09~4~

ingredient being a-imini~t~red, and the route of a-1mini~tration. In general, a serum concentration of polypeptides and antibodies that permits saturation of EGF receptors is desirable. A concentration in excess of app, o~i"lately 0.1 nM is normally sufficient.
For example, a dose of 100 mg/mZ of C225 provides a serum concentration of S approximately 20 nM for applux;~ tely eight days.

As a rouph ~lideline, doses of antibodies may be given weekly in amounts of 10-300 mg/m2. Equivalent doses of antibody fr~gm~nts should be used at more frequent intervals in order to ~ a serum level in excess of the concentration that permits saturation of EGF receptors.

Some su;table routes of a~1minietration include il,lr~v~,lous, subc~lt~neous, and intr~m-lsele ~lminietration Intravenous ~lmini~tration is p,ere"ed.

The peptides and antibodies of the invention may be ~(lminiet~sred along with additional pharm~ce~ltic~lly acceptable ingredients. Such ingredients include, for example, immllne system sfimlll~tors and che,llolllerapeutic agents, such as those 15 mentioned above.

It has now s~ isillgly been found that, unlike the murine 22S antibody, the chimeric and hl~ .;,ed antibodies ei nific~ntly inhibit tumor growth in l"~."",~le, even in the abse.lce of other anti-tumor agents, incl~tling other ch~ulllc~ elltic agents, such as ~iepl~tin, doxorubicin, taxol, and their derivatives. Signific~nt 20 inhibition may mean the shrinkage of tumors by at least 20%, pr~re,~bly 30%, and more pr~rel~bly 50%. In optimal cases, 90% and even 100% ~hrink~ge of tumors is achieved. Alternatively, si nific~nt inhibition may mean an RI greater than 0.3,p,ere,~bly greater than 0.4, and more preferably greater than 0.5.

'~ ~E~
.

CA 02222231 1997-11-2~

WO 96,t40210 PCT/US!~6~'~5W7 The significant inhibition of tumor growth and/or increase in RI manifests itself in numerous ways. For example, there is an increase in life expectency and/or a stabilization of previously aggresive tumor growth.

In cases where the side effects of chemothel~culic agents are too severe for a ~ 5 patient to continue such tre~tment~ C225 may be substituted for the chemoth~ ld~C;UtiC
agents, and achieve comparable results.

For example, the results shown in Example III-l indicate that, while the in vi~ro inhibitory lJlV~t;l Lies of 225 and C225 are comparable, the in vivo effects of the antibodies differ considerably. Antibody isotype does not play a significant role in lhe differences seen between 225 and C225 (e.g., mouse IgGl vs. human IgGl). A
recent report indicates that neither 225 nor C225 in~ eed complement me~ te(l lysis l:o any degree and the ADCC reactivity of these antibodies ~pea ed to be speciesspecific. N~ d et al., Tmmunol. Tmmlmr~ther. 37, 343-349 (1993). Thererore, if iinhibition of A431 xenografts was m~ tecl through immlme responses, 225 should lbe the more potent antibody because of its ability to activate the murine effector cells iinvolved in ADCC. The opposite is, in fact, the case.

In addition, there were differences in the way individual ~nim~l~ within a groupresponded to treatment with either 225 or C225. It appeared that C225 alone was very effective in in~ cinp complete tumor remission at the 1 mg dose whereas 225 at this ,dose level showed marginal effects. In Experim~ntc 2 and 3 of Example III-l, about 40% of the ~nim~l~ were tumor free at the end of each study. The ~nim~l~ responding in those groups usually had smaller tumors at the beginning of the ke~tmt-nt protocols, once again indicating that initial tumor burden plays a role in the biological efficacy of C225. Significantly, ~nim~le keated with either 225 or C225 showed greater survival char~ctpri~tics compared to the PBS conkol group in all studies.

CA 02222231 1997-11-2~
W O 96/40210 PCT~US~G/0~3~7 As demonstrated in Example III-2, prostatic carcinoma is also an a~plupliate target for anti-EGFR immunotherapeutic intervention with C225. Since the metastatic prostatic carcinoma cells coexpress TGF-o~ as well as the EGFR, late stage prostatic carcinoma is an especially ~l lo~liate target.

Example III-2 describes the biological effects of C225 on the activation of the EC;FR in cultured human prostatic carcinoma cells and the growth of prostate xenografts in nude mice. The in vitro experimenl~ were ~l~ci~n~ to determine theexpression levels of the EGFR on three human prostatic carcinoma cell lines and the ability of C225 to block the functional activation of the receptor. Figure 7 shows the results of a FACS analysis co. l .~ EGFR ~ ssion on A431 cells to levels seen OIl LNCaP (androgen-dependent) and PC-3 and DU 145 (androgen-independent) cells.Both PC-3 (MFI = 135) and DU-145 (MFI = 124) cells expressed about 7 fold less receptor than A431 cells (MFI = 715). Since MFI is an indirect measure of antigen density, both PC-3 and DU 145 cells would appear to express about 105 receptors each. LNCaP cells, on the other hand, ~ ,lessed very low levels of surface receptor (MFI= 12).

As shown above, the EGFR expressed by A431 cells can be stim~ te~l by exogenously added ligand (EGF) and C225 can abrogate activation of the receptor.Figure 8 shows the results of similar studies with the prostatic lines. The addition of EGF to LNCaP, PC-3, and DU 145 in.1llcecl phosphorylation of the EGFR that was blocked by C225 with high efficiency. These data in~1ir~te that C225 effectivelyinhibits ligand-activated EGFR ~ign~llin~ pathways, and has anti-tumor activity when EGFR activation is required for growth in vivo.

The ability of C225 to inhibit tumor growth in vivo was tested against established DU 145 xenografts in athymic nude mice. DU 145 cells were innoculated at 106 cells per ~nim~l~ in combination with matrigel. Tumors developed in 100% of the ~nim~l~
within 20 days. Prelimin~ry t;~ ent~ had shown that a dose level of 1 mg (lOx) CA 0222223l l997-ll-2~

W O 96/'40210 PCTrUS96/09847 in~luce(l significant tumor inhibition. For these studies, C225 was injected at a 0.5 mg ~lOx) dose level.

As shown in Figure 9, C225 alone was effective in significantly inhibiting the growth of established DU 145 xenografts (p < 0.5). The overall therapeutic effect was 5 ~pa~ by day 34 and significant with respect to the control group by day 36 (Figure ~A). All tumors in the sham-injected group continued to grow throughout the course of the study (Figure 9B) but the anti-tumor effect of the antibody was seen throughout the study (Figure 9C). Although spontaneous remissions in PBS-treated ~nim~i~ were never seen in this model, 60% of the C225 treated ~nim~l~ were tumor free by day 60 10 (Figure lOA) and rem~inecl turnor-free for an additional 90 days after l~ . ,.,i"~lion of the antibody injections. In addition, tumors that did not disappear in the C225 group grew extremely slowly after trç~tment was stopped (day 55; Figure 9C) suggesting a long-lived effect of the antibody. There was no ~i nific~nt dirr.,.~ ce in the survival curves during the course oftre~tm~nt (Figure lOB).

Example III-2 clearly shows that C225 was capable of inhibiting the growth of established, EGFR-positive DU 145 xenografts and could induce long-lived tumor remissions in a high ~elcelll~ge of treated ~nims~l~ These results could not be predicted from the in vitro data.

Not all cell lines that express EGFR at levels similar to those seen in DU 145 cells 20 respond to C225 in vivo. For example, KB cells (human epidermoid carcinoma) express about 2 x 105 EGFR per cell and activation of the receptors by EGF was blocked by C225 in vitro. However, KB xenografts did not respond to a tre~tml ntregimen including a 1 mg dose (xlO) of C225, a level able to induce complete remissions in 100% of ~nim~l~ carrying established A431 tumors. As surprisingly 25 shown in Example III-2, tre~tm~nt of mice innoculated with DU 145 tumor cells with C225 alone at a 0.5 mg dose (xlO) led to significant tumor regressions in all treated ~nim~i~ Sixty percent of the mice were in complete remission following t~ ic)n WO 96/40210 PCT~US96/09847 of the treatment. Blockage of receptor activaton by C225 also has clinical innplications for the tre~tment of metastatic prostatic carcinoma in hllm~nc, especially during the late stages of the disease.

S F,X~MpT,~

F,~ample I. Materi~l~

F,~m~ple I-l. Cell T,ine~ ~n(l Media A43 1 cells were routinely grown in a 1:1 mixture of Dulbecco's modified Eagle'sm~edium and Ham's F-12 supplPmented with 10% fetal bovine serum, 2mM L-10 L~lu~ le, and antibiotics.

The androgen-independent and ~1epçnflent hunnan prostatic carcinoma cell lines U 145, PC-3 and LNaP) were obtained from the ATCC (Rockville MD) and routinely m~int~ined in RPMI 1640 medium (Sigma, St. Louis, MO) supplçmelltPd with 10% fetal bovine serum (Intergen, Pu~cl1ase NY) and 2 mM L-gl~l~tmin~
15 (Sigma). Cells were checked regularly for the presence of mycoplasma.

F,~ml?le I-2. Pl~ lion ~n-l pnrification of M~ n~l C2~

The 225 antibody was grown as ascites in pristane primed Balb/c mice. Ascites fluid was purified by HPLC (ABX and Protein G) and determined to be >95% pure bySDS PAGE.

Human clinical grade C225 was grown in proprietary serum free medium in 300 liter lots. After clarification, the conce~ ed broth was purified on a series of W O 96/40210 PCT~US96/09847 chromatographic columns and vialed under asceptic conditions. Purity was d~ i-led to be >99% by SDS PAGE.

Fx~nu?le I-3. Pl~,~al~lion of Doxorubicin-C225 Conju~ates ~ C225 doxorubicin conjugates (C225-DOX) were ~ ed using a modification of ~he method described by Greenfield et al., Cancer Research 50, 6600-6607 (1990).Briefly, Doxorubicin was }eacted with the cro~linkin~ agent PDPH (3-[2-pyridyldithio~propionyl hydrazide) (Pierce Chemical Co.) to form the acyl hydl~one derivative doxorubicin 13-[3-(2-pyridyldithiol) propionyl] hydrazone hydrochloride.
C225 was thiolated with the reagent N-succillilllydyl 3-(pyridyldithio) propionate and reacted with doxorubicin hydrazone to form a conjugate co"l;~ a hydrazide as well as a disulfide bond. The complex was purified by gel filtration at neutral pH.
lhe C225-doxorubicin conjugate was stable at neutral to ~lk~line pH (pH 7-8) andwas stored at 4C. The conjugate was readily hydrolyzed at pH 6, releasing activeDoxorubicin.

F,xample I-4. Chimerization of Antibody 225 F~n~le I-4~. Clonin~ of H and L Ch~in cDNAs The media col~ the 225 mouse hybridoma cell line was e~p~n-lecl to one liter in tissue culture flasks. Total cell RNA was ~i~aied by lysing washed cells in 20 g~l~ni~line isothiocyanate co~ 2-mercaptoethanol, ~he~ring the solution in a dounce homogenizer to degrade cell DNA and layering the pL~dlion on a 10 ml cesium chloride cushion. After centrifugation at 24,000 rpm for 16 hr. the pellet was l;e~u~ ded in Tris-EDTA (TE) buffer and precipitated with ethanol. The poly A(+)mRNA fraction was isolated by binding to and elution from oligo dT cellulose. A
cDNA library was prepared using the poly A (+) mRNA as template and oligo dT as - the primer. The second strand was synthe~i7P(i by nick translation using RNase H and CA 02222231 1997-11-2~
W O 96/40210 PCTAUS96~3~17 DNA polymerase I. The double-stranded DNA was passed through a 2 ml Sepharose G75 column to remove oligo dT and small entities. The purified DNA was then ligated into a polylinker with the sequence:

5' -AATTCTCGAGTCTAGA -3' S (SEQ ID NO: 31) which encodes an Eco RI four base sticky end for ligation to the cloning vector, and the restriction sites for Xho I and Xba I for subsequent manipulations of the cDNAs.
The ligated cDNA was then size-selected by electrophoresis on a 5% polyacrylamide gel. The d~plopliate size fractions (~ 1500 bp for H chain and -900 bp for L chain 10 cDNA) were electroeluted from gel slices and ligated to Eco RI-digested lambda gtlO
phage DNA. Libraries were generated by p~ck~ping the ligation products in vitro and pl,~ting recombinant phage on lawns of E. coli strain C600 HFL. Phage co-.l;.;~.i,.~ H
and L cDNAs were identified by phage filter lifts that were hybridized with radiolabeled oligonucleotides of the mouse kappa and gamma con~t~nt region. The 15 identified phage were restriction mapped.

Isolates with the longest cDNA inserts were subcloned in a plasmid vector (Eco Rl-Bam HI fr~gment~ for heavy (H) chain V regions and Eco RI-Hpa I fr~gm~nt~ forlight (L) chain variable (V) regions) and DNA sequenced. The subcloned fr~gm~nt~contained the complete V region and a small portion of associated mouse constant (C) 20 region. A total of eight L chain cDNAs were sequenced and ~ ;Sc;lll four dirr~
mL~NAs. Three full-length H chain cDNAs were sequenced encoding the same V
region and a portion of the correct gamma 1 C region. Three other isolates col.t~ g gamma 2a sequence were also icl~ntified but were not studied further. To identify the correct L chain cDNA, a sample of mouse 225 antibody was sequenced by automated 25 Edman degradation after first sep~dlillg the H and L chains by SDS reducing gel electrophoresis and blotting to membranes.

CA 0222223l l997-ll-2~

W O 96/'40Z10 PCT~US96/09847 The sequence obtained for the L chain matched one of the cDNAs. This isolate ~as rearranged to J5 and was found to be 91% homologous with Vk T2. The H chain V region was found to be 96% homologous with VH 101 subgroup VII- 1.

l~x~ ?le I-4B. Adaption of cDNAs and Construction of Fxpression Vectors S
The V regions were adapted for expression by li~tin~ the body of each to a synthetic DNA duplex encoding the sequence between the closest unique restriction site to the V/C junction and the exact boundary of the V region. To this was ligated a second, short intron sequence which, when joined, restores a functional splice donor 10 site to the V region. At the end of the intron for the L chain is a Bam HI site and at the end of the H chain intron is a Hind III site. The adapted L Chain V region was then isolated as a Xba I-Bam HI fragment (the Xba I site was in the original linker used for cDNA cloning) while the adapted H chain V region was isolated as a Xho I-Hind III fr~gm~nt The c~ ion vector pdHL2, co,.~ ,g human kappa and human gamma 1 constant regions, was used for insertion of the adapted L chain V region. The resnltinf~: plasmid, pdHL2-Vk(225), was then digested with Xba I and Bam HI and used for the insertion of the adapted L chain V region. The resnlting plasmid, pdHL2-Vk(225), was then digested with Xho I and Hind III and used for the insertion of the adapted H chain V region. The final vector was identified by restriction mapping and identified as pdHL2-ch225.

Fx~m~l?le I-4C. Expression of Chimeric 225 in T~ re~iled Hybridoma Cells The pdHL2-ch225 plasmid was introduced into hybridoma Sp2/0 Agl4 cells by protoplast fusion. The bacteria harboring the plasmid were grown to an optical density of 0.5 at 600 nm at which time chlor~mph~nicol was added to arrest growth - and amplify the pl~mid copy number. The following day the bacteria were treated W O 96/40210 PCTAJS~6/0~17 with Iysozyme to remove the cell wall and the resulting protoplasts were fused to the hybridoma cells with polyethylene glycol 1500. After fusion, the cells were grown in antibodies to kill any surviving bacteria and were plated in 96-well plates. Theselection medium (co.~ methotrexate (MTX) at 0.1 ~uM) was added after 24-48 5 hours to allow only the transfected cells to grow, by virtue of their ex~re~iOn of the rnarker gene (dehydrofolate redllct~ee) present on the ex~les~ion plasmid.

After two weeks, several MTX-resistant clones were obtained that were then tested for antibody ex~l. s~ion. Culture ~u~ x were added to wells coated with an anti-human Ig (Fc-specific) antibody as the capture reagent. The detection system 10 was an HRP-conjugated goat anti-hurnan kappa antibody. The majority of cloneswere found to be secreting human antibody det~rrnin~nte and the three highest producers were further adapted to grow at 1 ~aM and then 5 ,uM methotrexate. Two of the lines, ~lesi~n~te-l SdER6 and SdER14, continued to grow well at the higher levels of MTX and were subcloned by limiting dilution. The productivity of the subclones 15 was tested by seeding cells at 2 X 105 cells per ml in grow~h medium and m~enring the accumulated a3~tibody on day 7. The two highest producers from the first subcloning were lines SdER6.25 and SdER14.10. These were subcloned a second tirne and the final three candidate lines were tleei~n~ted SdER6.25.8, SdER6.25.49, and SdER14.10.1. Clone SdER6.25.8 was selected based on ex~ sion of antibody.

Fx~mple I-5. An~l,ysis of C225 F~ ssed from SdER6.25.8 Studies with antibody produced from the clone SdER6.25.8 were cl n~lnct~l to char~ct~ri7P the nature of the antibody. Culture supPrn~t~nte from the transfected cell clones c;x~e~si"g C225 antibody were tested for their ability to bind human tumor 25 cells t;X~ S~illg di~ levels of EGF receptor. A431 epidermal carcinoma cells (high expressors) were intensely stained while M24 mel~nom~ cells (ex~lessing 10-fo]d fewer receptors) were moderately stained. A neuroblastoma line, IMR-32, which does not express EGF receptor, was not stained.

W O 96./40210 PCT~US96/09847 lFx~mple I-6. Fffects of Chimeri7ing the C225 Antibody The a~ ent Kd was found to be 0.1 and 0.201 nM for C225 and 1.17 and 0.868 nM for 225, using ELISA and SPR methods, respectively (Table 1). These results were similar to published data for C225 (Kd - 0.39 nM) and 225 (Kd = 0.79 nM, Kd5 = 1 nM) as shown in Table 1. The antibodies were found to inbibit the proliferation of cultured A431 cells to the same extent (Table 2). In addition, 225 and C225 were able to block EGF-in~ ced phosphorylation of the EGFR in A431 cells. These results indicated tha~ chimeri7~tion of 225 did not affect the biological properties of the .mtibody and increased the relative binding affinity of C225 for EGFR.

10 F~x~nu; le II. Methods and Assays Fxam~le II-l. Relative ~ffinity Mea~u~ ents by FT T~A

The relative binding affinity of the antibodies was (iet~rrninlo~l using an ELISA
protocol previously described by Lokker et al. J. Tmmunol. 146, 893-898 (1991).
]3riefly, A431 cells (104 or 105 per well) were grown in 96 well microtiter plates overnight at 37~C. Cells were fixed with 3.7% neutral buffered formalin for 10 .,.i.ll-(es at room lelll~ UlC. After washing three times with PBS, wells were blocked with 1% bovine serum albumin in Hank's b~l~n~e(l salt solution for two hours at room temperature. C225 or 225 were added to the wells at various conc~ntr~tions (serial dilutions starting at 50 nM). After a two hour inc~b~ti~n at 37~C, plates were 20 extensively washed with PBS and in~uh~te-l with goat anti-human antibody (Sigma, '3t Louis MO, 1:1000) for one hour at 37~C. Plates were washed and the chromogenTMB (E~irkeg~rd and Perry, C'T~ithPrsburg MD) added for 30 minl-tes in the dark.The color reaction was stopped with 1 N sulfuric acid and the plates read in an ELISA
reader at 450 nm. The relative binding affinit,v is defined as the concentration giving 25 1he half m~xim~l OD.

CA 02222231 1997-11-2~
W O 96M0210 PCT~US96/09847 ~xam~le II -2. Affinity Constants of 225 and C225 us;n~ Surface Plasmon Reson~nce ~'echn--lo~y (SPR) The d~ lL binding affinities of M225 and C225 were also deterrnined using the InAcoreTM (Pharmacia Biosensor, Piscdldw~y NJ, m~nllf~rtllrer's application note 301 and O'Sh~nnl?~sy et al., Anal. Biochem. 212, 457-468 (1993). Briefly, soluble recombinant EGFR was immobilized on sensor chips via amino groups as described by the m~nllf~rtllrer. Real time binding parameters of 225 and C225 to EG~R was established at various antibody concentrations and the d~-paLel.L Kd was calculated from the binding rate col~L~-l~ obtained via non linear fitting using BiaevaluationTM

2.0 Software.

Fxample JT-3. In vitro Inhibition of Cell Grow~ with 225 and C225 The in vitro inhibitory activity of 225 and C225 was determinecl by plating A431cells (300-500 per well) in 96 microtiter plates in complete growth medium. After adding C225 or 225 in various concentrations (4 replicates per concentration), plates vvere incubated for 48 hours at 37DC followed by a 24 hour pulse with 3H-thymidine.
Cells were harvested, collected on filter mats and counted in a Wallace Microbeta scintill~til-n counter to determine percent inhibition. Percent inhibition COlllpdl'CS the decrease in 3H thymidine incol~u,dlion of antibody-treated cells with cells grown in tlhe absence of antibody.

Fxample TT-4. ~nim~l Studies Athymic nude mice (nu/nu; 6-8 weeks old females) were obtained from Charles River Laboratories. Animals (10 mice per tre~tment group) were innoculated in the right flank with 107 A431 cells in 0.5 ml of Hank's balanced salt solution. Mice were observed until tumors were visible (about 7-12 days) and had reached an average volume of 150-300 mm3. At that time, antibody therapy was begun. The therapy W O 96/40210 PCT~US9~ 17 included twice weekly hllld~ iloneal injections (varying concentrations in 0.5 ml of PBS) over 5 weeks. U 1 ~nim~l~ received injections of PBS. Tumors were measured two times per week and volumes calculated using the following formula: 7~/6 x larger diameter x (smaller diameter)2. Animals were followed at least 3 weeks after the final 5 antibody tre~tment (8 weeks after the start of therapy) at which time U 1 and test ~nim~l~ with extremely large tumors were e~-th~ni7e~1 Tumor free ~nim~l~ and animal with small tumors were followed for an additional 2-3 months. St~ti~tir~lanalysis of tumor growth in each of the studies was done using a two tailed Student's T-test.

In addition to demonstrating growth inhibitory effects of the antibodies, many ~nim~l~ were found to be in complete remission (i.e., tumor free). This biological effect was quantified as a Remission Index (RI), defined as the number of tumor free mice/total ~nim~l~ within a tre~tment group. Termin~tion occured at the time of e lth~n~ei~ for ~nim~l~ with large tumors, and 2-3 months later for other 5mim5-l~
15 Animals that died during trP~tment were excluded from this analysis. For example, one complete remission among eight surviving ~nim~l~ equals an RI of 0.125.

Fx~m~le III. F~iological Activit,v of C225 Fx~ml?le ITT- 1. The Capacitv of the Antibodies to Inhibit the Growth of A43 1 Xeno~rafts ;n Nude Mice Animals were innoculated in the flanks with A43 1 cells. Tumors of 150-300 mm3 a~pealed by day 7-10. Refering to Experiment~ 1~ in Table 3, ~nim~l~ were then r~n-l- mi7~1 and injected with PBS or 225 (Exp 1), PBS, 225, or C225 (Exp 2); and PBS or C225 (Exp 3 and 4). In Experiment~ 1-3, ~nim~l~ received injections of 1 mg of antibody (in 0.5 ml PBS) twice weekly over 5 weeks for a total dose of 10 mg of ~ 25 antibody per arlimal. In Exp 4, ~nim~l~ received one ofthree possible doses: 1, 0.5, WO 96/40210 PCT~US~6J~17 and 0.25 mg/injection for total doses of 10, 5, and 2.5 mg, respectively. Tumors were m~easured twice weekly over the course of tre~tmçnt Tumor-free ~nim~l~ and animals with small tumors c-ntin~led to be monitored for 2-3 months following the sacrificing of animals with large tumors.

Figure 1 shows the effect of 225 on the growth of A431 b~mors in nude mice (Exp 1)~. The average tumor volumes of the experimental and U 1 groups were similar (Figure lA) and only one complete b~lmor remission was observed (Remission Index(Pl) of 0.17; Figure lB and Table 3). A co~ on of 225 and C225 is shown in Figure 2 (Exp 2 in Table 3). Although there was no significant difference in average bumor size between the groups, ~nim~lc treated with C225 had an RI of 0.44 (i.e., 4/9 complete remissions) compared to an RI of 0.11 for 225 (Figure 2B and Table 3). The ~,~c,.l tumor regression for the PBS U 1 group at day 37 (Figure 2A) was abtributable to the death of 3/10 ~nim~l~ at this time and the conc(-.. iils1.. l decrease in overall bLlmor volume. A similar RI for C225 was seen in Exp 3 (Figure 3B, RI =
0.4). In addition, inhibition of bLlmor growth by C225 was also found to be significant when co~ ~ed to the growth of xenografts in PBS-treated mice (Figure 3A; p < 0.02 following day 32).

Because a number of ~nim~l~ receiving C225 showed tumor regressions at the 1 mg/injection level, the lowest biologically effective dose was definP~l Figure 4 shows the results of the dose .~;~ollse ~ (Exp 4). All ~nim~lc receiving 1 mg/injection underwent complete remission and r~m~int?cl tumor free for over 100days following te~ l ion of the antibody injections (Figure 4A and B; Table 3).
These results are highly sigruficant with p values varying from p < 0.006 on day 33 to p < 0.0139 on day 59. In Experiment~ 2 and 3, about 40% ofthe ~nim~l~ receiving the 1 mg dose of C225 underwent complete remission although C225 showed si~rnific~nt tumor regression in Exp 3 (Figure 3). The increased efficacy of the 1 mg dose in Experimentc 3 and 4 in significantly reducing average tumor volume versus U
1 may have occured because mice carrying smaller tumors were used at the start of the W O 96/40210 PCTAJS~6/0~47 re~tm~nt protocols in these c~c.;, . .ent~ (l 52 mm3 [Exp 4] and l 85 mm3 [Exp 3] vs.
267 mm3 [Exp 2]). These data suggest that the clinical effectiveness of C225 may be r elated to tumor burden.

At the 0.S mg dose in Exp 4, the overall inhibition of tumor growth was not - 5 statistically significant because of the large variations in tumor volume among :~nim~1s of both the PBS and the 0.5 mg groups. However, the RI was high for the 0.5 mg group (RI = 0.63; Figure 4b and Table 2) indicating that the antibody in~ cecl ;mti-tumor responses in individual ~nim~ e~ gly, the 0.5 mg dose group in :Exp 4 had a higher RI than the l mg dose group in Exp 3. This result may be attributed to the effects of tumor burden. Although the average starting volume for hlmors in the Q5 mg dose group was l 60 mm3, there was great variability in tumor size among individual ~nim~1~ A number of ~nim~l.c carried smaller tumors (<l00 mm3) that are most ~usc~lible to the biological effect of C225. At 0.25 mg dose,average tumor growth ~e~cd to be greater than the PBS U l. This was due to the l 5 inclusion within this group of two ~nim~1~ with large tumors (760 and l 140 mm3) at the start of the tre~tment~ which resulted in an increase in average tumor volume during the course of Exp 4. Overall, there is no significant difference between these groups but it is hlh.e;~ling to note that one animal (l/8) at the 0.25 mg dose was tumor free at the end of the study (RI = 0. l 3). At day 47, there a~ealcd to be a drop in the RI. At this time, a tumor reappeared in one mouse that had ~ nlly undergone a complete rerni~sion In this single case, C225 had a tr~n~jent biological effect. This animal is not included in Table 3. As with the l mg dose group, tumor-free ~nim~ in the 0.5 and 0.25 mg groups rem~ine-l tumor free a ., lillillllllll of 2-3 months after the PBS control mice were sacrificed.

Table 1. DISSOCIATION CONSTANTS (Kd) FOR 225 AND C225 AS DETERMINED BY VARIOUS METHODS

Kd(nM) M[ETHOD~ RECEPTOR 225 C225 REFERENCE
FORM
Scatchard A431 Lysates 1 nd CancerRes.
53, 4322-4328 (1993) Sc~lcllal-l M24met cells 0.78 0.39 Tmmlmnl.
Tmmlln~ ther.
37, 343-349 (1993) E]LISA Fixed A431 1.17 0.147 cells S]'R Soluble 0.868 0.201 .-,cel,lol * Scatchard results are ~ ssed as Kd, SPR results as al)p~._.ll Kd, and ELISA data 20 as the a~al~..L affinity, a relative measure of the Kd. See ~tl?ri~l~ and Methods for description of the gene.dLion of the ELISA and SPR data.

W O 96/'40210 PCT~US96/09847 l'able 2. IN VITRO INHIBITION OF A431 CELLS

% INHIBITION
ng/ml of Antibody C225 The results shown in Table 2 represent a typical ~ .;l"ent in which the ability of 225 and C225 to inhibit the growth of A431 was tested invitro. Details are described above. Percent in~nibition is defined as the decrease in 3-H thymidine incorporation of antibody-treated samples (4 replic~tes/concentration) versus cells growing in the absence of antibody.

Table 3 represents a co",pa,;son of complete tumor remissions in athymic nude nnice carrying established A431 tumors following trç~tm~nt with PBS, 225, or C225 t.wice weekly for 5 weeks. Animals were treated with 1 mg of antibody in 0.5 ml of PBS by the i~ d~filoneal route except for study 4, which is a dose l~onse ~:x~.hl~ent in which mice were given 1, 0.5, or 0.25 mg/injection. Tumor 20 nn~asu,~.,lents were done as described above. This chart describes the RI at the time vvhen the ~nim~l~ (PBS control and test) carrying large tumors were e~lth~ni7P~l All z~nim~l~ showing complete remissions or small tumors were followed for an additional 2-3 months. The diLr~iellces in total number of ~nimsll~ results from death of mice vvithin these tre~tment groups during the course of the C;x.~L~ nt~

WO 96t40210 PCT/US96/09847 Table 3. REMISSION INDICES FOR ANIMALS INNOCULATED
WlTH A431 CELLS AND TREATED WITH 225 OR C225 EXP TREATMENT # REMISSIONS/ REMISSION
TOTAL* INDEX**
225 1/6 0.17 2 225 1/9 0.11 C225 4/9 0.44 3 C225 4/10 0.40 PBS 0/3 ~

4 C225: 1 8/8 1.0 C225: 0.5 5/8 0.63 C225: 0.25 1/8 0.13 PBS 0/4 ~

* Tumor free ~nim~l~/total number of surviving ~nim~l~ Differences in the numberof ~nim~l~ presented are the result of mice dying during the five week course of the various trç~trnent regim~n~, and these were not included in the st~ti~tic~l analysis.
20 *"' The Remission Index (RI) is defined as the fraction of mice that were tumor free on the day when the PBS control mice and test ~nim~l~ with large tumors were euth~ni7P~l A complete remission at the 0.25 mg dose level showed a subsequent recurrance of tumor (day 47).

~.xample III-2. Inhibition of Growth of Established Human Prostatic Carcinom~
:~e~ografts in Nude Mice FxAnl,~le III-2A. FACS Analysis of C225 Bindin~ to DU 145.PC-3 and LNCaP

The relative t;x~le3sion levels of EGF receptor on DU 145, PC-3 and LNCaP cells was ~letermin~d by FACS analysis. Cells were grown to near confluency in complete mediD, removed from the flasks with non-enzymatic dissociation buffer (Sigma), and resll~p~onflç-l at 5-10 x 105 per tube in 100 ul of cold H-BSA (Hanks balanced salt solution co~ 1% BSA). Ten micrograms C225 or an irrelevant myeloma-derived human IgGl (Tago, Bllrlin~Amc CA) were added to the tubes and incubated 10 on ice for 60 mimltes After washing with cold H-BSA, goat anti-human IgG
conjugated to FITC (Tago, B--rlin~me CA) was added for an additional 30 ",i""~;son ice. Cells were washed 2 times with cold H-BSA, resuspended in 1 ml of H-BSA,and analyzed using a Coulter Epics Elite cell sorter (Coulter, Hialeah FL). Baseline fluroescçnce was determin~ocl using the FITC-labelled secondary antibody alone and 15 non-specific flurorescence was defined by the irrelevant isotype control. Data is presented as the Mean Fluroescence Intensity (MFI), which is an indirect measure of antigen density. MFI is defined as the mean channel fluorescenre multiplied by the pt,~;~nl~ge of positive cells for each sample.

F~ e ITT-2B. Ph~s~horylation Assays on PC-3. DU 145. and LNCaP Cell~

Phosphorylation assays were perforrned on PC-3, DU 145, and LNCaP cells to cletennine if the EGF receptors expressed by these cells were functional and inhibited by C225. A ssays and Western blot analysis were performed as previously described by Gill et al., Nature 293, 305-307 (1981). Briefly, DU 145, PC-3, and LNCaP cells were grown to 90% confluency in complete medium and then starved in DMEM-0.5 calf serurn 24 hours prior to t;~.;, . ,entAtion. Cells were stimlllAt~d with EGF in the ~resence or absence of C225 for 15 ~ s at room te",~t,~ue. Monolayers were then washed with the ice cold PBS co~ .;"~ 1 mM sodium orthovAnA-lAte Cells CA 02222231 1997-11-2~
W O 96/40210 PCT~US96/09847 were Iysed and subjected to SDS PAGE followed by Western blot analysis. The phosphorylation patterns were determined by probing the blot with a monoclonal antibody to phosphotyrosine (UBI, Lake Placid NY) followed by detection using the ECL method (~mer.~h~m).

5 Fx~nu?le III-2C Anim~l Studies Athymic nude mice (nu/nu; 6-8 weeks old males; Charles River Labs, Wilmington ~/[A) were innoculated subcutaneously in the right flank with 1 o6 DU 145 in 0.2 ml of Hank's b~l~nred salt solution mixed with 0.2 ml of matrigel. Mice were observed w1til tumors were visible (about 14-20 days post challenge) and had reached an 10 average volume of about 100 mm3. Animals were weighed and randomly divided into tre~tment groups (10 ~nim~l~ per group). Antibody therapy, which included twice weekly LL a~. ;lon~l injections of 0.5 mg of C225 over 5 weeks, was begwn.
Control ~nim~l~ received injections of PBS. Preliminary studies established that there was no significant difference between the growth of DU 145 xenografts in ~nim~lc15 treated with polyclonal, DU 145-absorbed human IgG compared to PBS. Tumors w,ere measured two times per week and volumes calculated using the following formula: ~/6 x larger ~ meter x (smaller diameter)2. Animals were followed for at least 3 weeks following the final antibody injection (8 weeks after the start ofth~erapy), at which time control ~nim~l~ were ~ P~1 Tumor free ~nim~l~ and 20 mice with small tumors were followed for an additional 2-3 months. Statistical analysis of tumor growth in each study was (letermined with a two tailed Student's T-test using the colll~uL~l program SigmaStat (Jandel, San Rafael CA). A p value of <
0.05 was considered ~ignificz~nt CA 0222223l l997-ll-25 W O 96/40210 PCTAUS96~ 7 F,xam~le III-3. Biological Activity of Pe~tides Co..l~ CDR Re~ions of 225 This example demonstrates that peptides constructed using 225-CDR sequences had biological activity against cell lines that express EGF rect;l!lol~. A series of six peptides were generated with the following sequences:
-5 Heavy Ch~in CDR-l NYGVH

CDR-3 RALTYYDYEFAYW (SEQ ID NO: 32) ht Chain CDR-l RASQSIGTNIH (SEQ ID NO: 33) CDR-2 YASESIS (SEQ ID NO: 34) CDR-3 QQNNWP (SEQ ID NO: 35) These peptides were dissolved in PBS at a concentration of 1 mg/ml. A431 cells were plated at 1000 cells per well in 96 well plates. Peptides were added at various 15 ,concentrations. The chimeric C225 antibody and an irrelevant, isotype- m~tch~cl immllnoglobulin were used as a positive and negative U ls, respectively. Plates were incubated for 72 hours at 37~C and pulsed overnight with 3H-thymidine. Cells were 'harvested and counted in a liquid scintill~ti-n counter. Percent inhibition is defined as the decrease in 3-H thymidine incorporation of antibody or peptide treated cells20 co~ d to cells grown in the absence of antibody or peptide.

As can be seen in Figure 5, A431 cells are inhibited by C225 and by heavy chain CDR-l and heavy chain CDR-2 of monoclonal antibody 225. In contrast, isotype-m~trl~l?cl irrelevant antibody and U 1 peptide did not inhibit A431 cells. These results intlic~te that heavy chain CDR-l and -2 are able to inhibit the growth of A431 cells by 25 ihlL~r~,Lillg with the binding of ligand to the EGFR.

Ex~ml?le III-4.13iolo~ical Activity of C225-Doxorubic;n Cor~ju~ate fC225-DOX) The biological activity of C225-DOX was evaluated in vitro using EGFR
S~illg cell lines A431, KB and MDA-468 as well as EGFR non-G~les~ing cell lines Molt-4 and SK-MEL-28. EGF receptor e~les~ion was verified by FACS
analysis using C225 and C225-DOX conjugate. Assays were conducted over a 72h incubation period using 3[H]-thymidine and WST-l as a read out. In all assays with EGFRc t;~ ;ssing cell lines, i.e., A431, KB and MDA-468 cells, C225-DOX
exhibited high inhibition of cell proliferation when compared to no tre~tment orhIgGl U 1 s. Comp~rieons of equimolar concentrations of C225-DOX with doxorubicin alone or mixtures of C225 and doxorubicin showed a 4-5 fold higher inhibition using the C225-DOX conjugate. Inhibition of cell proliferation by C225-DOX was also seen in EGFRc non~xl~c;s~illg cell lines at higher doses. The C225-DOX inhibition in EGFRc-negative cell lines was 5-15 fold lower than EGFRc-positive cell lines and was similar to inhibition seen with equimolar concentrations of doxorubicin alone. Representative results are shown in Figure 6 for activity of C225-DOX on 431 cells.

E~ml?le IV. Hnm~ni7~tion of M225.
F.x~m~ e IV-l . Abbrevi~tiorl~
Dulbecco's Modified Eagles Medium (DMEM); Foetal Calf Serum (FCS);
ribonuceic acid (RNA), mesc~nger RNA (rnRNA); deoxyribonucleic acid (DNA);
double-stranded DNA (ds-DNA); polymerase chain reaction (PCR); enzyme linked immuno~hsorbant assay (ELISA); hour (hr); minute (min); second (sec); human cytomeg~lf)virus (HCMV); polyadenylation (poly(A)+); immnnt~globulin (IgG);
monoclonal antibody (mAb); complemPnt~rity ~1et~rmining region (CDR); frarneworkregion (FR); Tris-borate buffer (TBE); bovine serum alburnin (BSA); phosphate buffered saline (PBS); room le~ e~dlul~ (RT); nanometre (nm~; epiclrrm~l growth far.tor ~c;c~lor (EGFR);

W O 96./40210 PCT~US96/09847 Fxample IV-2. Materials Media components and all other tissue culture m~teri~l~ are obtained from Life Technologies (UK), except for FCS which is purchased from JRH Biosciences (USA).The RNA isolation kit is obtained from Stratgene (USA) while the 15' strand cDNA- 5 synthesis kit is purchased from Pharmacia (UK). All the constituents and eqllipment for the PCR-reactions, including AmpliTaq(~)DNA polymerase, are purchased from Perkin Elmer (USA). The TA Cloning(E~ kit is obtained from Invitrogen (USA) and the Sequenase~ DNA sequencing kit is purchased from Amersharn Tntern~tional (UK). Agarose (UltraPureTM) is obtained from Life Technologies (UK). The WizardTM
PCR Preps DNA Purification Kit, the MagicTM DNA Clean-up System and XLlBlue competent cells are purchased from Promega Corporation (USA). All other molecular biological products are purchased from New Fngl~n(1 Biolabs (USA). Nunc-Tmmlmo Plate MaxiSorpTM immun~plates are obtained from Life Technologies (UK). Both thegoat anti-human IgG, Fcy fragment specific, antibody and the goat anti-human IgG(H+L) / horseradish peroxidase conjugate are purchased from Jackson Tmmlln~Research Laboratories Inc. (USA). TMB substrate A and substrate B are obtained frolm Kirk~ rd-Pery (USA). All other products for both ELISAs are obtained from Sigma (UK). Microplate Manager~) data analysis software package ispurchased from Bio-Rad (UK). The molecular modelling package QUANTA is obtained from the Polygen Corporation (USA) and the IRIS 4D v~olk~lalion is ~wchased from Silicon Graphics (USA).

Fx~mRle IV-3. PCR clonin~ and sequenc;ng of the mouse variable re~ion genes The mouse M225 hybridoma cell line is grown, in suspension, using DMEM
suppl~m~nt~cl with 10% (v/v) FCS, 50 Units/ml penicillin / 50~Lg/ml ~L.~Ioll,ycin and 580 ,ug/ml L-glllt~mine Approxim~t~ly 108 viable cells are harvested, while the su~ from ~e hybridoma cells is assayed by ELISA to co~firm that they are producing a mouse antibody. From the 1 o8 cells total RNA is isolated using a RNA

CA 02222231 1997-11-2~
W O 96/40210 PCT~US~6/09847 Isolation kit according to the m~nl-f~cturers instructions. The kit uses a guanidinium thiocyanate phenol-chloroform single step extraction procedure as described by Chomczynski and Sacchi (6). Also following the m~nnf~ lrers instructions, a 1st Strand cDNA Synthesis kit is employed to produce a single-stranded DNA copy of S the M225 hybridoma mRNA using the NotI-(dT)l8 primer supplied in the kit.
A.pproximately 5 llg of total RNA is used in a 33 ~1 final reaction volume. The completed reaction mix is then heated to 90 ~C for 5 min, to denature the RNA-cDNA
duplex and inactivate the reverse transcriptase, before being chilled on ice.

To PCR-amplify the mouse variable region genes the method described by Jones and Bendig (7) is followed. Fesçnti~lly, two series of degenerate primers, one series ~lPsi~n~(l to anneal to the leader sequences of mouse kappa light chain genes (i.e.
~IKVl-l l; Table 4) and one series designed to anneal to the leader sequences ofmlouse heavy chain genes (i.e. MHV1-12; Table 5), are used in conjunction with pLimers tleei~ned to anneal to the 5'-end of the mouse kappa light chain constant region gene (MKC; Table 4) and the 5'-end of the mouse y l heavy chain constant region gene (MHCGl; Table 5), respectively, to PCR-clone the mouse variable region genes of the M225 antibody. Separate reactions are prepared for each of the MKV and MHV degenerate primers, with their respective col~La~lL region primer. The PCR-reaction tubes are loaded into a Perkin Elmer 480 DNA thermal cycler and cycled (after an initial melt at 94 ~C for 1.5 min) at 94 ~C for 1 min, 50 ~C for 1 min and 72 ~C for 1 min over 25 cycles. At the completion of the last cycle a final extension step at 72 ~C for 10 min is carried out before the reactions are cooled to 4 ~C. Except for between the ~nnP~ling (50 ~C) and extension (72 ~C) steps, when an e~rtPn-led ramp tirne of 2.5 min is used, a 30 sec ramp time between each step of the cycle is ernployed.

20,ul aliquots from each PCR-reaction are run on agarose gels to det~rrnine which have produced a PCR-product of the correct size. Those PCR-reactions that do appear to amplify full-length variable domain genes are repeated to produce independent -W O 96/40210 PCT~US96/09847 PCR-clones and thereby minimi~e the effect of PCR-errors. 6 ,ul aliquots of those E'CR-products of the correct size are directly cloned into the pCR TMII vector, provided by the TA Cloning~) kit, and transformed into INVaF' competent cells asclescribed in the m~n11f~1rers instructions. Colonies co,.~ g the plasmid, with a 5 correctly sized insert, are identified by PCR-screening the colonies using the pCRTMII
~ E~orward and pCRTMII Reverse oligonucleotide primers described in Table 6 according to the method of Gussow and Clackson (8). The putative positive clones i~l~ntifie~l are finally double-stranded plasmid DNA sequenced using the Sequenase~)DNA
',eq11enring kit according to the method of Redston and Kern (9).

10 F x~mple IV-4. Construction of chimeric genes The cloned mouse leader-variable region genes are both modified at the 5'- and 3'-ends using PCR-primers to create restriction enzyme sites for convenient insertion into 1he t;xl~le;,~ion vectors, a Kozak sequence for efficient eukaryotic tr~n~1~tion of the mRNA encoding the ~c~ccli~e immun~)globulin chains (lO) and a splice-donor site l 5 fior the correct RNA splicing of the variable and con~t~nt region genes. A HindIII site is added to the 5'-end of both mouse variable region genes, however, dirr~,c.ll r estriction sites are ~tt~l~h~cl to the 3'-end of the mouse variable region genes i.e. a BamHI site at the 3 '-end of the VH gene and a XbaI s;te at the 3 ' -end of the VK gene.

PCR-reactions are prepared according to the method for the construction of 20 chimeric genes in Kettleborough et al. (l l), using the primers C225VH5' and C225VH3' for the heavy chain, and C225VK5' and C225VK3' for the kappa light chain (Table 7). Following an initial melting step at 94 ~C for 90 sec the mixes are PCR-,~mp1ifie-1 at 94 ~C for 2 min and 72 ~C for 4 min over 25 cycles. This two step PCR-cycle, as opposed to the more usual three step cycle, is possible because each of the 25 primers is clecign~l to anneal to the template DNA over 24 bases which allows them to anneal at the relatively high tcln~ dLulc of 72 ~C. A 30 sec ramp time is used between ~each step and at the end of the last cycle, the PCR-reactions are completed with a final CA 02222231 1997-11-2~
WO 96/4L0210 PCT~US96/09847 e~.~tension step at 72 ~C for 10 min before cooling to 4 ~C. The PCR-products are column purified using a WizardTMPCR Preps DNA Purification kit according to the n1l~nllf~ct-lrers instructions, digested with the a~ ol).iate restriction enzymes, as is pl!asmid plJC19, and separated on a 1% agarose / TBE buffer (pHg.8) gel. The heavy 5 and kappa light chain variable region genes are excised from the agarose gel and purified using a Wizard' PCR Preps DNA Purification kit. The pUCl 9 is also excised from the agarose gel and purified using the MagicTMDNA Clean-up System as per the m~nllf~ctllrers instructions. The heavy and kappa light chain variable region genes are then separately ligated into the purified pUCl9 to produce plasmids PUC C225VH and 10 PUC-C225VK, ~c~ccli~ely~ and transformed into XLlBlue conl~c;L~ cells. Putative positive colonies co..~ ;..g the a~plop.;ate plasmLid are then id~ntifie~l by PCR-screening, using oligonucleotide primers RSP and UP (Table 6) and finally ds-DNAsequenced both to confirm the introduction of the sequence modifications and also to prove that no ullw~led changes to the DNA sequence have occured as a consequence15 of the PCR-reactions.

To modify the signal peptide sequence at the 5'-end of the kappa light chain variable region PCR-mutagenesis is used, according to the protocol described by Kettleborough et al. (11). PCR-primers C225VK5'SP and C225VK3'SP (Table 7) are used on PUC C225VK template DNA to create the modified gene (C225VKSP) using 20 the modified two step PCR amplification protocol. The PCR-product is then column purified before tlipesting both the purified PCR-product and PUC-C225VK with H,indIII and PstI. The PCR-fragment and the plasmid DNA are then agarose gel-purified, ligated together and cloned to create plasmid PUC-C225VKSP. As before,p~Ltative positive tran~ro....~ are id~ntifiçcl via a PCR-screen (using the RSP and UP
25 primers) and then ds-DNA sequenced to confirm both the presence of the modified signal peptide and the absence of PCR-errors.

The adapted mouse kappa light and heavy chain leader-variable region genes are then directly inserted, as a HindIII-BamHI fragment in the case of the mouse VH and W O 96~'40210 PCT~US96/09847 as a HindIII-~aI fragment in the case of the mouse V~c, into vectors cleei~n~-l to express chimeric light and heav,v chains in mRmmRli~n cells. These vectors contain the HCMV enhancer and promoter to drive the transcription of the imml-n~globulinchain, a MCS for the insertion of the immunoglobulin variable region gene, a cDNA
5 clone of the a~lo~,iate hurnan kappa light or heavy chain constant region, a synthetic poly(A)+ sequence to polyadenylate the immllnoglobulin chain mRNA, an artificialsequence designed to terminRte the trRn~çrir~tion of the immlm~globulin chain, a gene such as dhfr or neo for selection of transformed stable cell lines, and an SV40 origin of replication for transient DNA replication in COS cells. The human kappa lightchain mRmm~ n e,-~les~ion vector is called pKN100 (Figure 11) and the human yl heavy chain mRmmRliRn ~re~ion vector is called pGlD105 (Figure 12). Putative positive colonies are both PCR-screened, using primers HCMVi and New.HuK for therhimeric kappa light chain vector and primers HCMVi and HuCy 1 for the chimeric heavy chain vector (Table 6), and undergo restriction analysis to confirm the presence 15 of the correct insert in the ~ies~ion vector constructs. The new constructs co~
the mouse variable region genes ofthe M225 antibody are called pKN100-C225VK
(or pKN100-C225Vlcsp ) and pGlD105-C225VH, respectively.

FxRmple lV-S. Molecular modelling of mouse M225 antibody variable region~

To assist in the design of the CDR-grafted variable regions of the H225 antibody, a 20 molecular model of the variable regions of the mouse M225 antibody is built. Modelling the structures of well-characterized protein families like immllnoglobulins is achieved using the established method of modelling by homology. This is done using an IRIS 4D
workstation running under the UNIX op~dlillg system, the molecular modelling package QUANTA and the Brookhaven crystallographic ~1RtRbR~e of solved protein structures 25 (12)-CA 02222231 1997-11-2~
W O 96/410210 PCT~US96/09847 The FRs of the M225 variable regions are modelled on FRs from similar, structurally-solved immunoglobulin variable regions. While identical arnino acid side chains are kept in their original c,fie.ll~lion, mi~m~tch-?d side chains are substituted using the maximum overlap procedure to m~int~in chi angles as in the original mouse M225 antibody. Most 5 ol~the CDRs of the M225 variable regions are modelled based on the c~nonic~l structures far hypervariable loops which correspond to CDRs at the structural level (13-16).
However, in cases such as CDR3 of the heavy chain variable region, where there are no known canonical ~I~U~;LUIeS~ the CDR loop is modelled based on a similar loop structure present in any structurally-solved protein. Finally, in order to relieve unfavourable atomic 10 contacts and to optimize Van der Waals and electrostatic interactions, the model is subjected to energy ,-,;"i",i~l;on using the CHARMm potential (17) as implemented in QlUANTA-The FE~s from the light chain variable region of M225 antibody are modelled on the FE~s from the Fab fragment of mouse monoclonal antibody HyHel- 10 (18). The FRs from 15 the heavy chain variable region are modelled on the FRs from the Fab fragment of mouse monoclonal antibody Dl.3 (19). Those amino acid side chains which differ between the mouse M225 antibody and the variable regions upon which the model is based are first substituted. The light chain of Fab HyHel-10 antibody is then su~ .lposed onto the li~ht chain of Dl.3 by ~ .-hi~g residues 35-39, 43-47, 84-88 and 98-102, as defined by 20 Kabat et al., (20). The purpose of this is to place the two heterologous variable regions, i.e. the HyHel-10-based kappa light chain variable region and the Dl.3-based heavy variable region, in the correct orientation with respect to each other.

CDRl ~1) ofthe light chain variable region of mAb M225 fits into the Ll canonical group 2, as proposed by Chothia et al. (14), except for the presence of an isoleucine, 25 instead of the more usual leucine, at canonical residue position 33. However, this substitution is considered too conservative to merit significant concern in ~igning a ç~nonic~l loop structure to this hypervariable loop. The Ll loop of mouse Fab HyHel-10 is identical in amino acid length and m~tches the same canonical group - with a leucine W O 96140210 PCT~US96/09847 Jlt position 33 - as the Ll loop of M225 mAb. Consequently this hypervariable loop is used to model the Ll loop of M225 kappa light chain variable region. Similarly, CDR2 ~L2) and CDR3 (L3) of the M225 mAb both match their respective canonical group 1]Loop structures. In addition, the corresponding hypervariable loop structures of the S lHyHel-10 Fab fi~gment are also both group 1. Accordingly, the L2 and L3 loops of the ]M225 kappa light chain variable region are modelled on L2 and L3 of Fab HyHel-10.

Likewise, CDRl (Hl) and CDR 2 (H2) hypervariable loops of the heavy chain variable region of mAb M225 both fit their respective canonical group 1 loop structures as defined by Chothia et al. (14). Moreover, the co~l~onding Hl and H2 hypervariable loops of mouse D1.3 Fab fr~gment also match their respective canonical group 1 loop structures. Consequently, as with the light chain, these hypervariable loops are modelled on the Hl and H2 loops of the heavy variable region upon which the model is based. To identify a m~t~hing loop structure to the CDR3 (H3) hypervariable loop of the heavy chain variable region of M225 the Brookhaven ti~tt~hzt~e is searched for a loop of idt?ntic~l length and similar arnino acid sequence. This analysis found that the H3 loop of the mouse Fab 26/9 (21) exhibited the closest match to the H3 loop of M225 mAb and is consequently used as the basis for this hypervariable loop in the mouse M225 variable region model. After adjusting the whole of the model for obvious steric clashes it is finally subjected to energy ~,~it~ ion, as imp!en-ttontçd in QUANTA, both to relieve unfavourable atomic contacts and to optimiGc~ van der Waals and electrostatic interactions.

Fx~tn~le IV-6. De~i~rt of the reshaped hllm~tn H225 stntihody variants.

The first step in clecigrting the CDR-grafted variable regions of the H225 antibody is the selection of the human light and heavy chain variable regions that will serve as the basis of the hl-m~tni7~1 variable regions. As an aid to this process the M225 antibody light and heavy chain variable regions are initially colll~;d to the con~n~ sequences of the four subgroups of human kappa light chain variable regions and the three CA 02222231 1997-11-2~
W O 96/40210 PCT~US96/09847 subgroups of human heavy chain variable regions as defined by Kabat et al. (20). The mouse M225 light chain variable region is most similar to the consensus sequences of both human kappa light chain subgroup I, with a 61.68% identity overall and a 65.00%
identity with the FRs only, and subgroup III, with a 61.68% identity overall and a 68.75%
S identity with the FRs only. The mouse M225 heavy chain variable region is most similar to the consensus sequence for human heavy chain subgroup II with a 52.10% identity overall and a 57.47% identity between the FRs alone. This analysis is used to indicate which subgroups of human variable regions are likely to serve as good sources for human variable regions to serve as t.omrl~tes for CDR-grafting, however, this is not always the case due to the diversity of individual sequences seen within some of these artificially constructed subgroups.

For this reason the mouse M225 variable regions are also co~ cd to all the recorded examples of individual sequences of human variable regions publically available. With respect to human antibody sequences, the mouse M225 light chain va~riable region is most similar to the sequence for the human kappa light chain variable region from human antibody LS7'CL (22) - which is not related to the mouse L7'CLsequence. The kappa light chain variable region of human LS7'CL is a member of subgroup III of human kappa light chain variable regions. The overall sequence identity belween the mouse M225 and human LS7'CL light chain variable regions is calculated to lbe 64.42% overall and 71.25% with respect to the FRs alone. The mouse M225 heavy chain variable region is most similar to the sequence for the human heavy chain variable region from human antibody 38Pl'CL (23). Surprisingly, the heavy chain variable region of human 38P1'CL is a member of subgroup III and not subgroup II of the human heavy chain variable regions. The overall sequence identity between the mouse M225 andhuman 38Pl'CL heavy chain variable regions is calculated to be 48.74% while the identity between the FRs alone is 58.62%. Based on these comp~n~on~, human LS7'CL
lig~lt chain variable region is selected as the human FR donor template for the design of reshaped human M225 light chain variable region and human 38Pl'CL heavy chain CA 02222231 1997-ll-2~

variable region is selected as the human FR donor template for the design of reshaped human M225 heavy chain variable region.

As is commonly seen, the human light and heavy chain variable regions that are selected for the hllnn~ni7~tion of the M225 antibody are derived from two different ~ 5 human antibodies. Such a selection process a]Llows the use of human variable regions which display the highest possible degree of similarity to the M225 variable regions. In addition, there are many s~lccessfiul examples of CDR-grafted antibodies based on variable regions derived from two dirr~ human antibodies. One of the best studied examples is reshaped human CAMPATH-l antibody (24). Nevertheless, such a strategy alLso requires a careful analysis of the interdomain packing residues between the kapp light chain and heavy chain variable regions. Any mis-packing in this region can have a dramatic affect upon antigen binding, irrespective of the conformation of the CDR loop structures of the rç~ih~pe~l human antibody. Consequently, the amino acids located at the ~j'K/VH int~rf~ce, as defined by Chothia et al. (25), are checked for nnllsll~l or rare resi(l~-es Any residues so identified are then considered for mllt~g~?n~si~ to an amino acid rmore commonly seen at the specific residue position under investigation.

The second step in the design process is to insert the M225 CDRs, as defined by K;abat et al. (20), into the selected human light and heavy chain variable region FRs to create a simple CDR-graft. It is usual that a mouse antibody that is hllm~ni7~cl by a simple CDR-graft in this way, will show littLe or no binding to antigen. Consequently, il: is irnportant to study the amino acid sequences of the hu~nan FRs to ~letermin~ if any of these amino acid residues are likely to adversely infLuence binding to antigen, either directly through int~etions with antigen, or indirectly by a]Ltering the positioning of the C'DR loops.

This is the third step of the design process where decisions are made as to which amino acids in the human donor FRs shou]Ld be changed to their CO~l~ ~onding mouse ~ ~225 rsidues in order to achieve good binding to antigen. This is a difficult and critical CA 02222231 1997-11-2~
W O 96/40210 PCT~US96/09847 step in the hnm~ni7~tion procedure and it is at this stage that the model of the M225 variable regions becomes most useful to the design process. In conjunction with the model the following points are now addressed.

It is of great importance that the canonical structures for the hypervariable loops (13-S 16) are conserved. It is therefore crucial to conserve in the hnm~ni7~d H225 variableregions any of the mouse FR residues that are part of these canonical structures. It is allso helpful to compare the sequence of the M225 antibody to similar sequences from other mouse antibodies to lletermine if any of the amino acids are unusual or rare as this may indicate that the mouse residue has an illl~Ol L~lL role in antigen binding. By studying the 10 model of the M225 variable regions, it is then possible to make a prediction as to whether any of these amino acids, or any other residues at particular positions, could or could no influence antigen binding. Co.,.p~.;..g the individual human donor sequences for the kappa light and heavy chain variable regions to the con~n~n~ sequence of human va~riable regions subgroups to which the donor sequences belong, and identifying amino acids that are particularly lmllc~l is also illlpol~ll. By following this design process a n~lmber of amino acids in the human FRs are identified that should be changed from the arnino acid present at that position in the human variable region to the amino acid present at that position in the Mouse M225 variable region.

Table 8 describes how the first version (225RKA) of the reshaped human H225 kappa light chain variable regions is ~eCi~rn~rl T_ere is only one residue in the reshaped human FRs where it is considered n~ce~s~ry to change the arnino acid present in the human FRs to the amino acid present in the original mouse FRs. This change is at position 49 in FR2, as defined by Kabat et al. (20). The tyrosine found in human LS7'CL kappa light chain variable region is changed to a lysine, as found in mouse M225 kappa light chain variable region. From the model it appears that the lysine in M225 is located close to CDR3 (H3) of the heavy chain variable region and may be h~ d~;Lillg with it. The residue is also pc-siti~ nP~1 adjacent to CDR2 (L2) of the kappa light chain variable region and is rarely seen at this location amonst the members of mouse kappa light chain subgroup V, as CA 02222231 1997-11-2~

W O 96/'40210 PCT~US~G~ 7 defined by Kabat et al. (20), to which the M225 kappa light chain variable region belongs. For these reasons it is felt prudent to conserve the mouse lysine residue in A second version is also made of the reshaped human kappa light chain (225RKB) ~hich reverses the FR2 modification made in 225RKA, by replacing the Iysine at position 49 with the original human tyrosine amino acid. Consequently, this version of the r eshaped human kappa light chain will contain no mouse residues in the FRs whatsoever.

With respect to the design of reshaped human H225 heavy chain variable region, Table 9 shows the first version (225RHA). In all there are eight residues in the reshaped ]human FRs where it is considered nPcess~ry to chamge the amino acid present in the human 38Pl'CL FRs to the amino acids present in t]he original mouse M225 FRs (i.e.
A24V, T28S, F29L, S30T, V48L, S49G, F67L and R71K). At positions 24, 28, 29 and 30 in FRl the amino acid residues as present in the rnouse sequence are retained in the reshaped human H225 heavy chain variable region because they ~ sc;lll some of the canonical residues important for the Hl hypervariable loop structure (14). Sincec~nc-~ic~l residues are so critical for the correct orientation and structure of hypervariable loops that they are gt-n~r~lly always conserved in the reshaped variable region. Moreover, residue positions 24-30 are considered part of the Hl hypervariable loop itself and so are even more critical to the correct col~fu""~lion and orientation of this loop and justifying their conservation even more strongly. Similarly, residue position 71 in FR3 is another position in the heavy chain variable region which has been identified by Chothia et al.
(14) as one of the locations important for the correct orientation and structure of the H2 hypervariable loop and, as such, is one of the canonical amino acids of CDR2.
Consequently, the lysine in the mouse will replace the arginine in the hw~nan at this residue position. At positions 48 and 49 in FR2 and 67 in FR3, the valine, serine and phenyl~l~nin~ residues (respectively) present in the human 38Pl'CL VH sequence are ~h~nped to leucine, glycine and leucine (,~e~;Li~lely) as present in the mouse M225 VH
sequence. This descision is made on the basis of the model which shows that all three CA 02222231 1997-11-2~
WO 96/40210 PCT~US96/09847 residues are buried underneath the H2 loop and so could influence the conformation of the hypervariable loop and hence interfere with antigen binding. These are then the mouse residues conserved in the first version of the reshaped human H225 heavy chain variable region.

Version B of the reshaped hurnan H225 heavy chain variable region (225RHB) incorporates all the substitutions made in 225RHA and, in addition, contains a further mouse residue. At position 41 in FR2 the human threonine residue is replaced by proline wllich is invariably seen at this position in the mouse subgroup IB and is also very commonly seen in human subgroup III. In co~ d~7l, threonine is not usually seen at this location in the human subgroup III (only l l/87 times) and from the model it is appears that the residue is located on a turn located on the surface of the M225 VH region. What effect this may have on hypervariable loop structures is unclear, however, this version of the reshaped human H225 heavy chain variable region should clarify this.

Version C of the resh~recl human H225 heavy chain variable region (225RHC) incorporates all the ,u~lilulions made in 225RHA and, in addition, contains a further two mouse residues located at position 68 and 70 in FR3. From the model of the mouse M225 variable region, both the serine at position 68 and the asparagine at position 70 appear to be on the surface and at the edge of the antigen binding site. Since there is a possibility that either or both amnio acids could directly interact with EGFR, both the threonine at position 68 and the seine at position 70 in the hurnan FRs are replaced with thecorresponding mouse residues in 225RHC.

Version D ofthe 1~ pecl human H225 heavy chain variable region (225RHD) simply inco~ .ld~ all the mouse FR substitutions made in 225RHA, 225RHB and 225RHC to cl~l~l, . .i ,~e the combined effect of these changes.

Version E of the reshaped human H225 heavy chain variable region (225RHE) incol~oldles all the ~ m~ made in 225RHA and, in addition, incorporates another W O 96,~40210 PCTrUS96105~17 residue change at position 78 in FR3. From the model there is some evidence to suggest that the mouse amino acid (valine) at position 78 could influence the col~,l"~lion of the Hl hypervariable loops from its location buried ~ln~l~rne~th CDRl. Consequently, the human residue (leucine) is replaced by the mouse arnino acid in 225RHE.

S 1 ~xaml~le IV-7. Construction of the h~lm~ni7ed antibody variable region ~enes The construction of the first version of the reshaped human H225 VK region ~225RKA) is carried out e~nti~lly as described by Sato et al. (26). In essence, this involves ~nn~ling PCR-primers encoding FR modifcations (Table 10) onto a DNA
template of the chimeric C225VK gene using the two step PCR-amplification protocol to synthesi7e the reshaped human variable region gene. As a consequence, the FR DNAsequence of the chimeric C225VK is modified by the primers to that of the le~lla~ed ~uman kappa light chain variable region gene 225RKA. The newly synth~i7~od reshaped variable region gene, follo~,ving column pllrifi~ ~tion~ is digested with HindIII and.XbaI, ;agarose gel-purified and subcloned into pUCl9 (digested and agarose gel-purified in an identical manner). The new plasmid construct, pUC-225RKA, is then transformed into XLlBlue competent cells. Putative positive clones are identified by PCR-screening (using primers RSP and UP) and then finally ds-DNA sequenced, both to confirm their illL~;~iLy and discount the presence of PCR-errors. From the c~ nfirm~d postive clones an individual clone is selected and directly inserted, as a HindIII-XbaI fr~gment into the human kappa light chain m~nnm~ n t;~ cs~ion vector (pKN100) to create the plasmid pKNl 00-22SRKA. The 1 .lLe~iLy of this vector construct is confirm~ d via PCR-screening (using primers HCMVi and New.Hulc) and restriction digest analysis.

Version B of the reshaped human H225 VK (225RKB) is constructed using oligonucleotide primers 225RKB.K49Y and APCR40 (Table 11). A 100 1ll PCR-reaction mix comrri~ing 65.5 111 of sterile distilled/deionized water, 5 ~1 of 2 ng/~Ll plasmid pUC-225RKA termplate DNA, 10 ~1 of 10 X PCR buffer II, 6 1ll of 25 mM MgCl2, 2 ~Ll each of the 10 mM stock solutions of dNTPs, 2.5 111 aliquots (each of 10 IlM) of primers CA 02222231 1997-11-2~

2~5RKB.K49Y and APCR40 and 0.5 1ll of AmpliTaq~DNA polymerase is overlayed with 50 ~Ll of mineral oil and loaded into a DNA thermal cycler. The PCR-reaction is PCR-amplified, using the two step protocol over 25 cycles, and the PCR-product column purified before it is cut with MscI . Plasmid pUC-225RKA is also cut with MscI and both 5 the digested PCR product and the plasmid fragment are agarose gel-purified. The PCR-product is then cloned into pUC-225RKA, to create pUC-225RKB, before being k~msformed into XLlBlue co~ el~lll cells. Putative positive kansforrnant are first identified, using primers 225RKB.K49Y and UP in a PCR-screening assay, and then confirm~cl via ds-DNA seqll~onrin~ A selected individual clone is finally sublconed into p~N100 to produce the plasmid pKN100-225RKB, whose correct construction is confirmed both by using primers HCMVi and New.Hulc (Table 6) in a PCR-screening assay and reskiction analysis.

The construction of the first version of the reshaped human H225 VH region (225RHA) is also carried out ess~nti~lly as described by Sato et al. (26). In the case of the reshaped human 225RHA gene this involves ~nn.o~linp PCR-primers (Table 12) onto both a DNA template of a prevoiusly hnm~ni7~d mAb, to create the 5'-half of the reshaped human kappa light chain variable region gene, and the chimeric C225VH gene, to synth~si7P the 3'-half of the reshaped human kappa light chain variable region gene.
Again, the two step PCR-~mplifi~tion protocol is used and the ~ ped variable region 20 gene created is cloned into pUCl9 vector, as an agarose gel-purified HindIII-BamHI
fr,lgm~nt, to create plasmid pUC-225RHA. Putative positive clones identified by PCR-screening (using primers RSP and UP) are finally ds-DNA sequenced both to confirm the D1~A sequence and prove the absence of PCR-errors. From the confirmto~l positive clones an individual clone is selected and directly inserted, as a HindIII-BamHI fiagment, into 25 the human yl heavy chain m~mm~ n ~.cssion vector pGlD105 to create plasmid pGlD105-225RHA. The construction of this plasmid is then confirm~ both by using primers HCMVi and ~AS (Table 6) in a PCR-screening assay and restriction analysis.

CA 02222231 1997-11-2~

W O 96.140210 PCT~US96/09847 Versions B of the reshaped human H225 VH (225RHB ) is synthe~i7~d in a two step PCR-mutage~esis procedure in ~he following manner. Two separate 100 1ll PCR-reaction mixes are first prepared by combining 65.5 111 of sterile distilledldeionized ~vater, 5 ~11 of 2 ng/~ll plasmid pUC-225RHA template DNA, 10 ~1 of 10 X PCR buffer II, 6 ~Ll of 25 mM MgCl2, 2 1ll each of the 10 mM stock solutions of dNTPs, 2.5 ,ul ~ aliquots (each of 10 IlM) of primers APCR10 and 225RHB.T4 lP-AS in the first PCR-reaction, and primers APCR40 and 225RHB.T41P-S in the second PCR-reaction (Table 13), and finally 0.5 1ll of AmpliTaq g)DNA polymerase. Each of the two PCR-reaction mixes are overlayed with 50 ~11 of mineral oil, loaded into a DNA thermal cycler and PCR-amplified using the two step protocol over 25 cycles. The two PCR-products are then agarose gel-purified, to separate them from any template DNA
rem~ining in the PCR-reaction, before being ~c:iu~cnded in 50~1 of distilled/deionized water and their concentration dc~., . .;,.~1 In a second PCR-reaction 20pmol aliquots of each of the two PCR-products from 1he first PCR-reaction (equivalent to 8 111 ofthe APCR10/225RHB.T41P-AS PCR
product and 10 ~11 of the APCR40/225RHB.T41P-S PCR-product) are added to 57.5,ulof sterile distilled/deionized water, 10 ,ul of 10 X PCR buffer II, 6 ~11 of 25 rnM
MgCl2, 2,ul each of the 10 mM stock solutions of dNTPs and 0.5 ~1 of AmpliTaq~)DNA polymerase. This PCR-reaction is overlayed with mineral oil and PCR-amplified using the two step protocol over 7 cycles only. A third PCR reaction is then ~ d comprising 1,ul of the product of the second PCR-reaction 69.5,L~l of sterile distilled/deionized water, 10,ul of 10 X PCR buffer II, 6,ul of 25 mM MgCl2, 2 1 each of the 10 mM stock solutions of dNTPs, 2.5,ul aliquots (each of 10 ~lM) of the nested primers RSP and UP and 0.5,ul of AmpliTaq~DNA polymerase. The PCR-reaction is overlayed with mineral oil and amplified using the two step protocol fior a final 25 cycles. This PCR-product is then column purified, isolated as an agarose gel purified IIindIII-BamHI fr~gm~nt, subcloned into HindIII-BamHI digested and agarose gel -purified plasmid pUCl9, and finally transformed into XLlBlue competent cells. Putative positive tran~r~ are first identified and then confirmed CA 0222223l l997-ll-2~
W 096/40210 PCT~US~G/OS~7 as described previously. A selected individual clone is then sublconed into pGlD105 to produce the plasmid pGlD105-225RHB - which is confirmed using primers H~CMVi and yAS (Table 6) in a PCR-screening assay and by reskiction analysis.

Version C of the reshaped human H225 VH (225RHB ) is synth~si7~d in a similar manner to 225RKC. A 100,ul PCR-reaction mix co~ 65.5,ul of sterile distilled/deionized water, 5 ~l of 2 ng/lll plasmid pUC-225RHA template DNA, 10,~41 of 10 X PCR buffer II, 6,~l of 25 mM MgCl2, 2,ul each of the 10 mM stock solutions of dNTPs, 2.5,ul aliquots (each of 10 M) of primers APCR40 and 225RHC.T68S/S70N (Table 13) and 0.5,~41 of AmpliTaq~DNA polymerase. The PCR-reaction is overlayed with mineral oil PCR-amplified, using the two step protocol over 25 cycles, and column purified prior to digestion with SalI and BamHI.
Plasmid pUC-225RHA is also cut with with Sall and BamHI and both the digested PCR product and the plasmid are agarose gel-purified. The PCR-product is then cloned into pUC-225RHA, to create pUC-225RHC, before being kansformed into Xl,lBlue competent cells. Putative positive kansformant are first illentifiPfl using primers RSP and UP in a PCR-s-;Lcel~ig assay, and later confirmed via ds-DNA
seq~lencing A selected individual clone is then sublconed into pGlD105 to produce the plasmid pG1 D 105-225RHC. The correct construction of this vector finally proven both by using primers HCMVi and yAS (Table 6) in a PCR-s-;lecnillg assay and restriction analysis.

Version D of the reshaped human H225 VH (225RHD) is a product of the changes incorporated into versions B and C of the reshaped human heavy chain of H225 antibody. Fortuitously, it is possible to ~m~lg~m~te the changes made to these heavy chain variable region genes by digesting both pUC-225RHB and pUC-225RHC with Sall and BamHI. The 2.95 kb vector fragment from pUC-225RHB and the approximately 180 bp insert fragment from pUC-225RHC are then agarose gel-purified before being ligated together and transfor~ned into XLlBlue competent cells.
Positive trar~rollll~ll are identified and ds-DNA sequenced before a selected .

CA 0222223l l997-ll-25 W O 96~40210 PCT~US~6/0~8~7 individual clone is sublconed into pGlD105 to produce the plasmid pGlD105-2,25RHD. The correct construction of this vector is finally confirmed as described previously.

Version E of the reshaped human H225 VH (225RHE) is a derivative of 225RHA
and is synthesi7e~1 in an identical manner to 225RHC using primers APCR40 and 2,25RHE.L78V (Table 13). A selected 225RHE clone from plasmid pUC-225RHE is then sublconed into pGlD105 to produce the vector pGlD105-225RHE - the correct construction of which is proven in the usual manner.

Example IV-8. Transfection of DNA into COS cells The method of Kettleborough et al. (11) is followed to transfect the m~mm~ n e~lcs~ion vectors into COS cells.

F,x~m~l le IV-9. Protein A purification of recombinant 225 antibodies Both the chimeric C225 antibody and the various reshaped human H225 antibody constructs are protein A purified according to the protocol described in Kolbinger et al. (27).

F,xam,ple IV-10. Mouse Antibody F,T,T~A

Each well of a 96-well Nunc-Tmml-no Plate MaxiSorpTM imml-noplate is first coated with 100,ul aliquots of 0.5 ng/,ul goat anti-mouse IgG (y-chain specific)antibody, diluted in coating buffer (0.05 M C~hl,onal~-bicarbonate buffer, pH 9.6), and incubated overnight at 4 ~C. The wells are blocked with 200,ul/well of mouseblocking buffer (2.5% (w/v) BSA in PBS) for 1 hr at 37 ~C before being washed with 200,L~l/well aliquots of wash buffer (PBS / 0.05% (v/v) tween-20) three times. 100 ,ul/well aliquots of the t;~ .;, . ,ent~l samples (i.e. harvested media from the M225 CA 02222231 1997-11-2~
W O 96/41)210 PCTAJS96/09847 hylbridoma cell line - spun to remove cell debris) and 1:2 sample dilutions, diluted in sarnple-enzyme conjugate buffer (0.1 M Tris-HCl (pH 7.0), 0.1 M NaCl, 0.02% (v/v) tween-20 and 0.2% (w/v) BSA), are now dipensed onto the immllnoplate. In addition, a purified mouse IgG standard, serially diluted 1:2 from a starting concentration of 1000 ng/ml, is also loaded onto the immlmc)plate. The imml-noplate is incubated at 37 ~C for 1 hr and washed three times with 200 ,ul/well of wash buffer. 100 ,~bl of goat anti-mouse IgG/horseradish peroxidase conjugate, diluted 1000-fold in sample-enzyme conjugate buffer, is now added to each well, following which the imlmnnoplate is inrllbRted at 37 ~C for 1 hr before it is washed as before. 100 ,~41 aliquots of TMB peroxiodase substrate A:peroxidase substrate B (1:1) are now added to each well and inr~lbRtecl for 10 min at RT in the dark. The reaction is halted by dispensing 50 ,ul of 1 N H2SO4 into each well. The optical density at 450 mn is finally determined using a Bio-Rad 3550 microplate reader in conjunction with Microplate ManagerTM.

FxRmple IV-I 1. Ouantification of whole hllmRn yl/lc Rntibody via FT T~A

Each well of a 96-well Nunc-Tmml~n- Plate MaxiSorpTM immunoplate is first coated with 100 ,ul aliquots of 0.4 ng/,ul goat anti-human IgG (Fcy frRgment specific) antibody, diluted in coating buffer (0.05 M Carbonate-bicarbonate buffer, pH
9.6), and incubated overnight at 4 ~C. The wells are then each blocked with 200 ~1 of human blocking buffer (2% (w/v) BSA in PBS) for 2 hr at RT before being washed with 200 ,ul/well aliquots of wash buffer (PBS / 0.05% (v/v) tween-20) three times.
100 ,L41/well aliquots of the ~e, ;~,~entRl samples (i.e. harvested cos cell supern~tent~
- spun to remove cell debris) and 1:2 sample dilutions, diluted in sample-enzymeconjugate buffer (0.1 M Tris-HCl (pH 7.0), 0.1 M NaCl, 0.02% (v/v) tween-20 and 0.2% (w/v) BSA), are now dipensed onto the immunoplate. In addition, a purified hulnan ~y 1/K antibody, which is used as a standard and serially diluted 1:2, is also loaded onto the immunt)plate. The immunoplate is incubated at 37 ~C for 1 hr before being washed with 200 ,ul/well of wash buffer three times. 100 ,ul of goat anti-human CA 02222231 1997-11-2~

W O 96/40Z10 PCT~US961'0~17 kappa light chainlhorseradish peroxidase conjugate, diluted S000-fold in sample-enzyme conjugate buffer, is added to each well, following which the immunoplate is incubated at 37 ~C for 1 hr before it is washed as before. The rem~inrl~r of theprotocol is idPntic~l to the mouse antibody ELISA.

5 Fx~n~le IV-12. A431 Cell FT TSA for the detection of FGFR antigen bindin~

The procedure is based upon the one provided by ImClone Systems Inc. to eterrnine the relative binding affinity of the recombinant 225 antibody constructs, to EGFR expressed on the surface of A431 cells. The A431 cells are plated onto a 96-well flat bottomed tissue culture plate and inellb~teA overnight in DMEM media with 10% (v/v) FBS at 37 ~C and 5% CO2. The following day the media is removed, the cells are washed once in PBS and then fixed with lOO ,ul/well of 0.25% (v/v) gluteraldehyde in PBS. This is removed and the plate is washed again in PBS before it is blocked with 200,ul/well of 1% (w/v) BSA in PBS for 2 hr at 37 ~C. The blocking solution is removed and lOO ,~LVwell aliquots of the ~.t;.;...ental sarnples (i.e.
harvested cos cell supern~tçnt.c - spun to remove cell debris) and 1 :2 sample dilutions thereof (diluted in 1% (w/v) BSA in PBS) are dispensed onto the tissue culture plate.
In addition, 80,uVwell aliquots of purified human yl/lc antibody, which is used as a standard and serially diluted 1 :5 from a starting concentration of 20,ug/ml, is also loaded onto the plate. The plate is in-ub~t~cl at 37 ~C for 1 hr and then washed with 200 ~Vwell of 0.5% (v/v) tween-20 in PBS, three times.100,ul of goat anti-human IgG (H+L)/horseradish peroxidase conjugate, diluted 5000-fold in 1% (w/v) BSA inPBS, is now added to each well, following which the plate is incubated at 37 ~C for 1 hr before being washed first with 200,uVwell of 0.5% (v/v) tween-20 in PBS (three times) and then distilled deionized water (twice). The r~m~in-ler of the protocol is idçntic~l to the mouse antibody ELISA.

CA 02222231 1997-11-2~

Fx~le IV-13. Clonin~ and sequenc;n.~ of the variable re~ions of the M225 ~ntibody The presence of mouse antibody in the media from the M225 hybridoma cells at the point of harvesting the cells for RNA purifica~ion was proven using the mouse 5 antibody ELISA. Following 1 st strand synthesis the single stranded cDNA template was PCR-amplified with two series of degeneldle primers, one series specific for the k~ppa light chain signal peptide/variable region genes (Table 4) and the second series splecific for the heavy chain signal peptide/variable region genes (Table 5). Using these primers both the V~c gene and the VH gene of the M225 antibody were 10 successfully PCR-cloned from the M225 hybridoma cell line.

The M225 kappa light chain variable region gene was PCR-cloned, as an ~,~,ro~ .ately 416bp fr~gment, using primers MKV4 (which annealed to the 5' end of the DNA sequence of the kappa light chain signal peptide) and MKC (-lç~i~n~cl toarmeal to the 5 'end of the mouse kappa constant region gene). Likewise the M22515 heavy chain variable region gene was PCR-cloned, as an al~ruxilllately 446bp fr.~ment using the MHV6 (which annealed to the 5' end of the DNA sequence of theheavy chain signal peptide) and MHCGl (~lçcign~ to anneal to the 5 ' end of the CH
domain ofthe mouse yl heavy chain gene) prim~rs To ...i..i...i,~ the possibility of introducing errors into the wild-type sequences of 20 the mouse M225 variable region genes, either caused by AmpliTaq~) DNA
polymerase itself or changes introduced by reverse transcriptase (which has an error frequency approximately l/lO that of AmpliTaq~), a strict protocol was followed. At ledst two separate PCR-products, each from a di~ .~,.ll total RNA pr~d~ion and subsequent 1 st strand cDNA synthesis reaction, were PCR-cloned and then completely 25 DNA sequenced on both DNA strands for both the kappa light chain and heavy chain variable region genes of M225 mAb.

CA 02222231 1997-11-2~

W O 96./40210 PCT~US96/09847 From DNA sequence analysis of several individual clones from each of these :PCR-reactions the mouse M225 antibody VK and VH genes were determined as shown iin Figures 13 and 14, respectively. The amino acid sequences of the M225 VK and VH
regions were compared with other mouse variable regions and also the consensus 5 sequences of the subgroups that the variable regions were subdivided into in the Kabat ~ ~t~b~ce (20). From this analysis the M225 VK region was found to most closely match the con~t~n~ sequence of mouse kappa subgroup V, with an identity of 62.62% and a ~imil~rity of 76.64% to the subgroup. However, the kappa light chain variable region also displayed a close match to mouse kappa subgroup III with a 61.68% identity and a 76.64% ~imil~rity to its con~n~n~ sequence. When only the FRs of the M225 kappa light chain variable region (i.e. without the amino acids in the CDRs) were co~ ~cd to mouse subgroups III and V the identity increased to 66.25%for both subgroups while the similarity rose to 78.75% for subgroup III and to exactly 80.00% for subgroup V. Similar analysis of the M225 VH region found that it 15 exhibited the closest match to the con~n~ sequence of mouse heavy chain subgroup IB in the Kabat ~l~t~b~e (20). Identity between the mouse heavy chain variable region amino acid sequence of M225 and the consensus sequence of subgroup IB was measured at 78.15% while the similarity was calculated to be 84.87%, with no other con~çn~llc sequence coming even remotely near these values. These results confirm 20 that the mouse M225 variable regions appear to be typical of mouse variable regions.

F~ml?le IV-14. Corl~truction and ~,ici,~ion of chimeric C225 ~ntibody The PCR-products from the two PCR-reactions prepared to construct the C225 VK
and VH genes were s~dLely subcloned into pUCl9 as HindIII-BamHI fi~gment~ and then PCR-screened to identify putative positive tran~rol,l,~ll~. Those transformants so 25 i(lentified were then ds-DNA sequenced, to confirm their synthesis, and then subcloned into their le~c~ e m~mm~ n c;~ ion vectors. The DNA and amino acid sequences of the chimeric C225 kappa light chain and heavy chain variable CA 02222231 1997-11-2~
WO 96/413210 PCT~US96/09847 regions are shown in Figures 15 and 16, respectively. Once the integrity of the expression vectors had also been confirmed, by PCR-screening and restriction allalysis to confirm the presence of the correct insert, the vectors were co-transfected into COS cells. After 72 hr incubation, the medium was collected, spun to remove cell S debri and analysed by ELISA for antibody production and binding to EGFR.
Unfortunately, no chimeric antibody could be detected in the supernatent of the COS
cell co-transfections.

An analysis of the leader sequence of C225V,K established that it was llnlleu~l,compared to the leader sequences of other kappa light chain variable regions in mouse 10 kappa light chain subgroups III and V (20). To try and find a more suitable leader squence, the Kabat ~l~t~b~e was analysed to identify an individual kappa light chain which both matched C225VK amino acid sequence and whose signal peptide sequence was known. This search identified the kappa light chain of mouse antibody L7'CL
(Z8) which exhibited a 94.79% identity and a 94.79% similarity to the C225VK region 15 and a perfect match with resepct to FRl, which play an important role in the excision ofthe signal peptide during secretion. The amino acid sequence ofthe L7'CL kappalight chain signal peptide (i.e. MVSTPQFLVFLLFWIPASRG (SEQ ID NO: 36)) displays all the characteristics thought important in a such a signal sequence - such as a hydrophobic core - and so it was decided to replace the signal peptide of the PCR-20 cloned 225VK with this new sequence. Another point of interest was that thedifferences between the M225VK and the L7'CL signal peptides nearly all occured atits 5'-end where the MKV4 primer ~nn.o~lPd (i.e. the first 33 bases which is eqiuvalent to the first 11 amino acids of the signal peptide) when the M225VK gene was originally PCR-cloned. Thus, these differences could well be primer in~ ce-l errors in 25 the DNA sequence of the signal peptide. PCR-mtuagenesis of the C225VK template produced an a~loxilllately 390bp product. The HindIII-PstI digested and purifiedfiagment was then subcloned into i~lPntic~lly digested and agarose gel-purified plasmid PUC-C225VK and lldll.,rolllled into XLlBlue competent cells. Putative positive l~ rollllants were identified and then ds-DNA sequenced. The C225VKSP

CA 0222223l l997-ll-2~

W O 96/40210 PCT~US96/09847 gene (Figure 17) was subcloned into pKN100 and the resulting ~x~les~ion vector (pKN 100-C225VKSP) PCR-screened and restriction digested to confirm the presenceof the correct insert. This vector was finally co-transfected into COS cells with pGlD105-C225VH and after 72 hr incubation, the mediurn was collected, spun to 5 remove cell debri and analysed by ELISA for antibody production and binding to~ EGFR. This time chimeric C225 antibody was detected in the supern~tent of the COS
cell co-transfections at an approximate concentration of 150 ng/ml and this antibody bound to EGFR in the cell ELISA. Figure 18 shows a typical example of one such experiment.

F~ml?le IV-15. Construction and ex~i4ssion ofthe reshaped H225 antibody C--225R--A/225R ~¦A) The construction of the first version of the reshaped human H225 kappa light chain variable region (225RKA) produced an approximately 416bp product that was then subcloned into pUCl 9 as a HindII-BamHI fr~gm~nt Putative positive 15 transformants were identified using the PCR-screening assay and then ds-DNA
sequenced. The 225RKA gene (Figure 19) was subcloned into pKN100 and the rçsnltin~ ~x~L~ic;,~ion vector (pKN100-225RKA) PCR-screened and restriction digested to collfillll the p..3e,lce of the correct insert. Likewise, the construction of the first version of the ~4clls~ped human H225 heavy chain variable region (225RHA) produced 20 an ~proxi",ately 446bp product which was then subcloned into pUCl9 as a HindII-BamHI fr~gm~nt Putative positive l~ rol.~lants were again identified in the PCR-screen and then ds-DNA sequenced. The 225RHA gene (Figure 20) was subcloned into pGlD105 and the reslllting t;~ ;s~ion vector (pGlD105-225RHA) PCR-screenedand restriction digested to confirm the presence of the correct insert.

These vectors were then co-transfected together into COS cells and after 72 hr incubation, the medium was collected, spun to remove cell debri and analysed by ELISA for antibody production and binding to EGFR. The concentration of reshaped CA 02222231 1997-11-2~

hurnan antibody in the COS cell supernatents was slightly higher than those following transient ~x~r~ssion of the C225 chimeric antibody (approximately 200 ng/ml). Inaddition, a significant level of binding to EGFR was shown in the cell ELISA. Figure 8 shows a typical example of one such experiment which appears to show that the S reshaped human H225 antibody (225RKA/225RHA) bound to EGFR t;x~l~ssed on thesurface of A43 1 cells with about 65% of the relative affinity of the chimeric C225 antibody.

The amino acid sequences of the two versions of the kappa light chain reshaped human H225 variable regions constructed are shown in Figure 21, while the amino acid sequences of the five versions of the heavy chain reshaped human H225 variable regions constructed are shown in Figure 22.

CA 0222223l l997-ll-2~

W O 96,/40210 PCTrUS96/'~17 F~ le IV-16. References l. Mendelsohn, J. (1988). In: Waldmann, H. (ed). Monoclonal antibody therapy.
Prog. Allergy Karger, Basel, pl47.
2. Aboud-Pirak, E., Hurwitz, E., Pirak, M.E., Bellot, F., Schlessinger, J., and Sela, ~ 5 M. (1988).J. Natl. CancerInst.80:1605.
3. Masui, H., Kawarnoto, T., Sato, J.D., Wolf, B., Sato, G., and Mendelsohn, J.
(1984). Cancer Research 44:1002.
4. Mueller, B.M., Romerdahl, C.A., Trent, J.M., and Reisfeld, R.A. (1991). Cancer Research ~1:2193.
10 5. Rodeck, U., Herlyn, M., Herlyn, D., Molthoff, C., Atkinson, B., Varello, M., Steplewski, Z., and Koprowski, H. (1987). Cancer Researc~ 47:3692.

6. Chomczynski, P., and Sacchi, N. (1987). Anal. Biochem. 162: 156.

7. Jones, S.T., and Bendig, M.M. (1991). Bio/Technology 9:88.

8. Gussow7 D., and Clackson, T. (1989). Nucleic Acids Res. 17:4000.

9. Redston, M.S., and Kern, S.E. (1994). Biotechniques 17:286.

10. Kozak, M. (1987). J. Mol. Biol. 196:947.

11. Kettleborough, C.A., S~ nh~ J., Heath, V.J., Morrison, C.J., and Bendig, M.M.
(l991).Brotein Eng 4:773.

12. Bçrn~tein, F.C., Koetzle, T.F., Willli~m~, G.J., Meyer, E.F., Brice, M.D., Todgers, J.R., Kennard, O., Shimanouchi, T. and Tasumi, M. (1977). J.Mol. Biol.
112:535.

13. Chothia7 C., and Lesk, A.M. (1987). J. Mol. Biol. 196:901.

14. Chothia7 C., Lesk, A.M., Tramontano, A., Levitt, M., Smith-Gill, S.J., Air, G., Sheri~, S., Padlan, E.A., Davies, A., Tulip, W.R., Colman, P.M., Spinelli, S., Alzari, P.M., and Poljak, R.J. (1989). Nature 34:877.

15. Tramontano, A., Chothia, C., and Lesk, A.M. (1990). J. Mol. Biol. 215:175.

16. Chothia7 C., Lesk, A.M., Gherardi, E., Tomlinson, I.M., Walter, G., Marks, J.D., Llewelyn, M.B., and Winter, G. (1992). J. Mol. Biol. 227:799.

CA 0222223l l997-ll-2~
W O 96/40210 PCTAUS96t~17 1 7. Brooks, B.R., Bruccoleri, R.E., Olafson, B.I:~., States, D.J., Sw~min~th~n~ S., and Karplus, M. (1983). J: Comp. Chem. 4: 187.
13. Padlan, E.A., Silverton, E.W., Sheriff, S., Cohen, G.H., Smith-Gill, S.J., and Davies, D.R. (1989). Proc. Nat. Acad. Sci. USA 86:5938.
5 19. Fi~rhm~nn, T.O., Bentley, G.A., Bhat, T.N., Boulot, G., Mariuzza, R.A., Phillips, S.E.V., Tello, D. and Poljak, R.J. (1991) J.Biol.Chem. 266:12915.
20. Kabat, E.A., Wu, T.T., Perry, H.M., Gottecm~n, K.S., and Foeller, C. (1991).Sequences of proteins of immunological interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office.
21. Schulze-G~hmen, U., Rini, J.M., and Wilson, I.A. (1993). J.Mol.Biol. 234:1098.
2:2. Silberstein, L.E., Litwin, S., and (~ r~ C.E. (1989). J. Exp. Med. 169: 1631.
23. Schroeder Jr., H.W., Hillson, J.L., and Perlmutter, R.M. (1987). Science 238:791.
24. Rierhm~nn~ L., Clark, M., Waldmann, H., and Winter, G. (1988). Nature 322:21.
25. Chothia, C., Novotny, J., Bruccoleri, R., and Karplus, M. (1985). J. Mol. Biol.
186:651.
26. Sato, K., Tsuchiya, M., S~ nh~, J., Koishihara, Y., Ohsugi, Y., Kishimoto, T., and Bendig, M.M. (1993). Cancer Research 53:851.
27. Kolbinger, F., S~ nh~ J., Hardman, N., and Bendig, M.M. (1993). Protein Eng. 6:971.
2$. Pech, M., Hochtl, J., Schnell, H., and 7~rh~ll H.G. (1981). Nature 291:668.

CA 02222231 1997-ll-2~

WO 96,/40210 PCT~US96/0~47 Fx~m~le IV-17. Tables Table 4. Degenerate and specific PCR-primers used in the cloning of the M225 kappa light chain variable region genes.

Table 5. Degenerate and specific PCR-primers used in the cloning of the M225 heavy S chain variable region genes.

Table 6. Primers for PCR screening transformed colonies Table 7. Primers for co~ ;Lillg t~himPric C225 antibody kappa light chain and hea~vy chain variable region genes and also for modifying the signal peptide sequence of the C225 antibody kappa light chain.

10 Table 8. ~li nmPnt of amino acid sequences leading to the design of the first version of the reshaped human H225 antibody kappa light chain variable region (225RKA).

Table 9. ~lignmPnt of amino acid sequences leading to the design of the first version of the reshaped human H225 antibody heavy chain variable region (225RHA).

Table 10. Primers for constructing reshaped human antibody H225 kappa light chain 15 variable region gene 225RKA-Table 11. Primers for constructing reshaped human antibody H225 kappa light chainvariable region gene 225RKB-Table 12. Primers for constructing reshaped human antibody H225 heavy chain20 variable region gene 225RHA.

CA 0222223l l997-ll-25 W O 96/40210 PCT~US96J'05~,17 Table 13. Primers for constructing reshaped human antibody H225 heavy chain variable region genes 225RHB, 225RHC, 225RHD and 225RHE.

CA 02222231 1997-11-2~

W O 96140210 PCT~US~ 7 Table 4. Degenerate and specific PCR-prirners used in the cloning of the M225 kappa light chain variable region genes.

Name Sequence (S' - 3') MKVla(30mer) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG
S MKV2(30mer) ATGGAGACAGACACACTCCTGCTATGGGTG

MKV3 (30mer) ATGAGTGTGCTCACTCAGGTCCTGGCGTTG

~KV4(33mer) ATGAGGGCCCCTGCTCAGTTTTTTGGCTTCTTG
A A C AA
MKV5 (30mer) ATGGATTTTCAGGTGCAGATTATCAGCTTC
A T
MKV6(27mer) ATGAGGTGCCCTGTTCAGTTCCTGGGG
T TT C G C T A
10 ~KV7(31mer) ATGGGCATCAAGATGGAGTCACAGACCCAGG
T TTTT T
MKV8(31mer) ATGTGGGGACCTTTTTTCCCTTTTTCAATTG
T G C AA
MKV9(25mer) ATGGTATCCACACCTCAGTTCCTTG
G T G
MKV10(27mer) ATGTATATATGTTTGTTGTCTATTTCT
MKV11 (28mer) ATGGAAGCCCCAGCTCAGCTTCTCTTCC
15 MKCb (20mer) ACTGGATGGTGGGAAGATGG

a MKV in~ tec primers that hybridize to leader sequences of mouse kappa light chain variable region genes.
b MKC indicates the primer that hybridizes to the mouse kappa constant region gene.
.

CA 02222231 1997-ll-2~
WO 96/4~D210 PCT~US96/098~7 Tal~le S. Degenerate and specific PCR-primers used in the cloning of the M225 heavy chain variable region genes.

Name Se~ e (5' - 3') MHVla(27mer) ATGAAATGCAGCTGGGGCATCTTCTTC

5 MEIV2 (26mer) ATGGGATGGAGCTGTATCATGTTCTT
A CC
MEIV3 (27mer) ATGAAGTTGTGGTTAAACTGGGTTTTT
A

MHV4(25mer) ATGAACTTTGGGCTCAGCTTGATTT
G T G
MHVS (30mer) ATGGACTCCAGGCTCAATTTAGTTTTCCTT
MHV6(27mer) ATGGCTGTCCTAGGGCTACTCTTCTGC
T G C G
MHV7 (26mer) ATGGGATGGAGCGGGATCTTTCTCTT
A T G A
MHV8 (23mer) ATGAGAGTGCTGATTCTTTTGTG
MHV9(30mer) ATGGATTGGGTGTGGACCTTGCTATTCCTG
C A
M~IV10(27mer) ATGGGCAGACTTACATTCTCATTCCTG
MHVl l (28mer) ATGGATTTTGGGCTGATTTTTTTTATTG
lS MHV12(27mer) ATGATGGTGTTAAGTCTTCTGTACCTG

MHCGlb(21mer) CAGTGGATAGACAGATGGGGG

a MHV indicates primers that hybridize to leader sequences of mouse heavy chain variable region genes.
b MHCG indicates primers that hybridize to mouse constant region genes.

W O 96/40210 PCT~US96/09847 Table 6. Primers for PCR sc . eenil.g transfonTIed colonies Name Sequence (5' - 3') pCRTMIFol~ardPrimer(18mer) C T A G A T G C A T G C T C G A G C
pCR~IIReverse Primer(21mer) T A C C G A G C T C G G A T C C A C T
A G
S :RSP ~Reverse Sequencing Primer) A G C G G A T A A C A A T T T C A C A
(24mer) C AGGA
UP (IJniversal Primer) (24mer) CGCCAGGGTT T T C C C A G T C
ACGAC
yAS (20mer) ACGACACCG T C A C C G G T T C
G
HC~i (28mer) GTCACCGTCCTTGACAC G C
GTCTCGGGA
~0 New.~u~ (25mer) G TTGTTTGCGCATAATCAC
A GGGCA
Huyl(17mer) T TGGAGGAGGGTGCCAG
225~B.K49Y(60mer) C AGC AAAGACCTGGCCAGG
CTCCAAG G C T T C T C A T A T A
TTATGCTTCTGAGTCTATC
TCT

SUBSm~TESHEET(RULE26) CA 0222223l l997-ll-25 W O 96/40210 ' PCT~US96/09847 Table 7. P;imers for constmcting chirneric CX25 antibody kappa light chain and heavy chain variable region genes and also for modif~ing the signal peptide sequence of the C225 antibody kappa light chain.

Name Sequence (5' 3') C225VHS'(36mer) AAGCTTGCCGCCACCATGG
CTGTCTTGGGGCTGCTC
C225VH3' (34mer) GGATCCACTCACCTGCAGA
GACAGTGACCAGAGT
C225VK5' (36mer) AAGCTTGCCGCCACCATGG
TATCCACACCTCAGAAC
C225VK3' (40mer) T C T A GAAGGATCCACTCAC
GTTTCAGCTCCAGCTTGGT
C C
C225VK5'SP (99mer) AAGCTTGCCGCCACCATGG
TATCCACACCTCAGTTCCT
TGTATTTTTGCTTTTCTGG
ATTCCAGCCTCCAGAGGTG
ACATCTTGCTGACTCAGTC
TCCA
~O C225VK3'SP (21mer) AGAGATAGACTCAGAAGCA
TA

SUBS 111 UTE SHEET (RULE 26) WO 96,~40210 PCT~US96/09847 Table 8. Alignment of amino acid sequences leading to the design of the first ve~sion of the reshaped human H225 antibody kappa light chain variable region (225RKA).

Kabat # FR or Mouse MouseHumsn Human 225 Surface Commcnt CDR C225 -V -mdonor RKA or LS7'CL Buricd FRI D D E E E Surface 2 2 1 I I l* I I (~ ~ ' ~A for L1 loop ~3 3 I L Q V* V Y Surfsce [1676]L~6/270 (~Linked to ~831 rlO75] 101+39R+40T+41N) in L 1/116 ir;human -m.
4 4 I L M L L L L ~W276 in mouse ~c-V.
5 I T T* T* T T
6 6 l Q Q* Q Q Q
7 7 I S S S~ S S
8 8 I P P P* P P
9 9 I V S G A ~ Surface [1621] Vsl notseen in mouse [Sl 1466] K-V.
V-1/107 in human -m.
10 I I S T* T I Surfsce [1396] I~4/286 (~~ linked to ~185] .1177] mouse K-V
lle not seen in human -m.
11 11 I L L L* L L
12 12 I S S S~ S S
13 13 I V A L L L Surface [1616]V 47/276inmouse [677] 187] IC-V.
V~17/106 in human -m.
14 14 I S S S* S S
15 I P L P* P P P~26/286 in mouse ~-V.
16 16 I G G* G* G G

17 17 I E D E E E E~95/~76 in mouse lC-V.

19 19 I V V A A ~ Buried V-10/98 in human -m.

SUBS I H UTE 5HEET (RULE 26) WO 96/40210 PCT/US96/03~17 T:lble 8 - Continued Knbat # FR or Mousc Mouse Human Human '725 Surface Comment CDRC225 -V -mdonor RKA or LS7'CL Buried 20 20 I S T T T T Half- S=77/299 in mouse K-V.
buried S~1/97 in humsn -m.

21 21 I F I L* L L Buried [1595]F~5/301 inmouse~-llS] [234] V. Phe notseen in human -m.

22 22 I S T S* S S S~l 16/296 in mouse K-V.

23 23 ~i~l C C* C* C C

25 25 1 A A~ A* A A ~ AA rorLl loop.

26 26 l S S* S* S S

27 27 l Q Q Q Q Q

27E I - - - . -29 29 1 I I V V I C~ 'AA forLl loop.

33 33 1 I L L L I C~ 'AA forL1 loop.

34 34 CDR1 H N A A H Pucking A~

35 35 FR2 W W~ W* W W

36 36 I Y Y Y* Y Y Puci;ing AA.

37 37 l Q Q* Q Q Q

38 38 I Q Q Q Q Q Pucking AA.

39 39 I R K K R R R-10/252 (~9L iinked to 3L+IOI+40T+41N) in mouse 1C-V.

SUBS i 11 UTE SHEET ~RULE 26) W O 96/40210 PCTrU5961~3317 Table 8 - Continued Kabat # FR or Mouse Mouse Human Human 2: Surface Comment CDR . C225 -V -m donor Ri or LS7'CL Buricd 40 I T P P* P P Surface [1301]T~5/2S5 (~ linked to [~~] [1080] 3L+101+39R+41N) in mousc K-V.
Thr not sccn in human -m.

41 41 I N G G* G G Surface tl-~671N~5/246 ~ linked to [5] [1009] 3L+IOI+39R+40T) in mousc K-V. Asn not secn in human -m.

42 42 I G G Q Q Q Surface G~1/67 in human -m.

43 43 I S S A A ~ Surface S~2/66 in human -m.

44 44 I P P P* P P Corc packin g AA.

45 I R K R* R R R=34/250 in mousc K-V.
(Possible link to AA3, 10, 39-41).

46 46 I L L L* L L Pacl;ing AJ~.

47 47 I L L* L* L L

48 48 1 I I I* I I C ' 'AA forl2 Loop.

49 49~2 K Y Y . Y K Buricd ClosetoH3 loop& maybe 1l9] [1042] ~ ~ with it. (~1) [1234] K--13/249 (~c linked to 581) in mouse K-V.
(Possible link to AA3, 10, 39-41.). Lys not sccn in human -m.

51 51 1 A A A A A ~ '~ for12 loop.
52 52 I S S S* S S f~-- ' ' AA for L2 loop.
53 53 I E R S N E~
54 54 I S L R* R S
55 1 I H A* A
56 56CDR2 S S T* T S
57 57FR3 G G* G* G G
58 58 1 I V I* I I I~34/23'~ (~ linked to 49K) in mousc K-V. (Possiblc link to AA 3, 10, 39-41.) 59 59 l P P* P* P P
60 I S S D A _ Surfacc Ser not sccn in human -m.
61 61 I R R* R* R R

SU8STITUTE S~ EET (RULE 26) Table $ - Continued Kabat # FR or Mousc Mouse Human Humsn 225 Surfnce Comment CDR . C225 -V -m donor RKA or LS7'CL Buried 62 62 I F F* F* F F
63 63 I S S S* S S
64 64 I S G* G* G S C ~ '~A for L2 loop.
65 65 l S S* S* S S

67 67 l S S* S* S S
68 68 I G G* G* G G
69 69 I T T T* T T

71 71 I F Y F* F F Canonical ~A for 11 loop.
F-83/243 (,~; unked to 72T) in mouse l~-V.
7Z 72 I T S T* T T T-73/246 (~ linked to 71F) in mouse lC-V.
73 73 I L L L* L L
74 74 I S T T* T I Half- [1220]S-29/247 in mouse 138] ~837] buried lC-V.
~ Ser not seen in human -m.
75 75 1 I I* I*
76 76 I N S S* S S Surface [12111N-34/242inmouse [681 [880] l~-V.
N-2/64 in human -m.
77 77 I S N R S S S~l 13/236 in mouse 7c-V.
78 78 I V L L* L L Buried V~67/245 inmouseK-V.
V~1/65 in human -m.

80 80 ¦ S Q P P ~ Surt'ace [1181]S~56/243inmouse [94] [166l IC-V. S~8/65 in human -m.

82 82 I D D* D* D D
83 83 1 I I F* F E Surface [1176]Denotseeninhuman [98] [150] -m.
84 84 1 A A A* A A

85 85 I D T V* V _ Half- D=36/243 in mouse l;-V, buried Asp not seen in human -m.
86 86 l Y Y* Y* ~ Y
87 87 I Y F Y* Y Y Packing AA.
Y-109/237 in mouse ~c-V.

SUBSTtTUTE SHEET (RULE 2~i) WO 915/40210 PCTrUS96/09847 Table 8 - Continued KsbAt # FR or Mouse Mouse Human Human 2~5 Surface Comment CDR -. C225 -V -m donor RKA or LS7'CL Buried 88 88FR3 C C* C' C C
89 89CDR3 Q Q Q~ Q Q Paclcing AA.
90 I Q Q Q~ Q Q C ' ' A~ for L3 loop.
91 91 I N G Y R N Packing AA. (U~usual AA) 95 I P P P P P ~'- ' 'AA forL3 loop.

96 96 I T R G L T Core p~cl;ing AA.
97 97CDR3 T T~ T T T C ' ' AA for L3 loop.
98 98FR4 F F* F' F F Core pacl;ing A~.
99 99 I G G~ G' G G
100 100 1 A G Q G _ surface A~26/215 in mouse K-V.
Ala not seen in humnn -m.
101 101 I G G* G* G G
10'~ 102 I T T~ T* T T
103 103 I K K* K K K
104 104 I L L* V V _ buried L~15/56 in human-m.
105 105 I E E' E~ E E
106 106 I L I I* I I bulied L~'~3/176 in mouse lC-V.
L~1/56 in humnn -m.

107 107 FR4 K K~ K' K K

SUBSTITUTE SHEET (RULE 26) WO 96/4al210 PCT~US96/09847 Table 8 - Continued Co~ ; ofAA Mouse Mouse Human Human '25 Comment Vnnnblc Re~ion AA C225 1C-V ~c-m Donor RH~
Sequences to M~25 LS7'-CL

PERCENTlDENllTY 100.0 6~.6'~ 61.68 65.4'~ 79.~4 Comment:Therenr~2:2sminoncid in thc L~ . ' betwcen F~AMEWORKS 100.0 66.15 68.75 71.25 72.50 thevnriablercgionsoftheresh~ped ONLY knppa light chnin ~2SRK~ nnd the mouse M225 knppa light chnin.
PERCENT 100.0 76.64 72.90 77.57 87.85 Candidnte AA for furthcr mut~tion Sl~lLA:RlTY include rcsidue~ ant positions 39-45 FRA~WORKS 100.0 80.0 80.0 8'~.50 83.75 (which ~re unusunl) nnd a bnck ONLY mutntion at ~osition 49 i.e. K49Y.

Legend: (*) invariant residues as defined either by the Kabat conse.,:,us sequences i.e. 95% or greater occurrence within Kabat subgroup (Kabat et al., 1991) (in the case of columns 5 and 6) or as part of the c~nonic~l structure for the CDR loops (in the case of column 8) as defined by Chochia et aL, (1989); (BOLD) positions in Frs and CDRs where the human amino acid residue was replaced by the corresponding mouse residue (uNDFRr rNF) positions in Frs where the human residue differs from the analogous mouse residue number; (8) nulllb.,.i.~g of ch~ngPs in the human Frs; (mouse C225) amin~ acid sequence of the VL region from chimeric C225 antibody; (mouse -V) consen~.ls sequence of mouse kappa VL regions from subgroup V
(Kabat et al., 1991); (human -m) consensus sequence of human VL regions from subgroup m (Kabat et aL, 1991); (Human Donor LS7'CL) amino acid sequence from human LS7'CL
antibod y (Silbe, :"ein, L.E. et aL, 1989); (Surface or Buried) position of amino acid in relation to the rest of the residues in both chains of the antibody variable regions; (225RKA) amino acid seq~lenre ofthe first version ofthe re~l,aped human mAb H225 VK region; (Core packing AA/Packing AA) amino acids located at the VL/V~ interface as defined by Chothia et al.
(1985); (Canonical AA) amino acids defined by Chothia and Lesk (1987), Chothia et al.
(1989), Tramontano et al. (1990) and Chothia et al. (1992) as being important for CDR loop co~ "dLion SUBSTITUTE SH EET ~RULE 26) CA 0222223l l997-ll-25 WO 96/40210 PCT~US~6/0~317 Table 9. A~lignment of amino acid sequences leading to the design of the first version of the reshaped human H225 antibody kappa light chain variable region (225RK,~).

Kabat # FR orMouseMouseHumanHuman 225 Surfacc Commcnt CDR C225 IB m donor PKA or 38PI Buried FRI Q Q* E E ~ Surface Q--13/l~in humanm.
2 2 I V V* V V V

4 4 I L L* L* L L
5 I K K* V V V Surface 11446]Lysnotscenin 199I 1499] human m.
6 6 I Q E E E ~ Buried Q=15/84 (~ linked to 13Q+40S+80F+84S+85N+
89I) in mouse IB.
Q=0/164 in human m.
7 i I S S* S* S S
8 8 I G G* G' G G
9 9 I P P* G* G _ Surface Pro not seen in human I G G G G G
Il 11 I L L* L L L
12 12 I V V* V . V V

14 14 I P P* P* P P
I S S* G* G G Surface Sernotseeninhumanm.
16 16 I Q Q* G G _ Surface Gin not seen in human m.
17 17 I S S* S* S S
18 18 I L L* L* L
19 19 I S S* R R _ Surface Ser not seen in human m.
1 I I* L L L Buried I~1/143 in humanm.
21 21 I T T* S* S S Surface Thrnotseeninhumanm.
22 ~ I c c* c* c c 23 23 I T T* A A ~ Surface T~1/128 in human m.
24 24 I V V* A A V Buried C ' ' A~ for Hl loop.
V=9/132 in human m. (O
2~ I S S* S* S S
26 26 I G G* G G G C ~ ' AA for Hl loop.
27 27 I F F* F* F F C - ' ~A for Hl loop.

SUBS 111 UTE SHEET tRULE~ 26) WO 96/4'D210 PCTAJS96/09847 Table 9 - Continued Kabat # FR or Mousc Mouse Human Human 225 Surface Comment CDR. C225 IB mdonorRKA or 38P1 Buried 28 ;!8 I S S* T T S CanonicalAAforHI loop.
S-6/104 in human m. (o2) 29 29 I L L* F F L ~~ ~ l A~ for Hl loop.
L~1/108 in human m. (Zi3) 30 30 FRI T T S S T Canonical AA forHI loop.
T 1/103 in humAn m. (~) 34 34 I V V M M V C ~ ' A~ for H1 loop.
35 35 I H H S H H Pacl;ing AA.
35a 1 - x 35b CDRl - S
36 36 FR2 W W* W* W W
37 37 I V V V* V V Pslcl;ing AA.
38 38 I R R* R* R R
39 39 I Q Q* Q*~ Q Q Pacl~ng ~.
40 40 I S P A A ~ Half- S~12/97 (~ linked to buried 6Q+13Q+80F+84S+85N+89 I) in mouse IB.
S~l/9 1 in human m.
41 41 I P P* P T T Surface [1382]P-7S/87inhumanm.
[12-~3] [1 1]
42 42 I G G* G* G G
43 43 I K K* K* K K
44 44 I G G* G G G
45 45 I L L* L* L L Core pacldng ~.
46 46 I E E* E E E
47 47 I W W* W* W W Pslcl;ing ~.
48 48 I L L* V V L Buried L-2/86 in human m.
Undemeath H2 loop (~5) 49 49 FR2 G G S S G Buried [1390] G~21/86 in human [985] [58] m. Undemeath H2. (~6) 51 51 1 I I* I ~ I

52 52 I W W* S G W

SUBSTITIJTE SHE~T tRULE 26) WO 96/40210 PCT/U5~oll~ 7 Table 9 - Continued Knbnt # FR or Mouse Mouse Human Humnn 2''5 Surface Comment CDRC225 IB mDonorRHA or 38PI Buried 52a 1 - - G - -52b 1 - - K
52c 1 - - T

55 55 i G G~ G G G ~ ~ ' AA for H2 loop.

57 57 I T T'' T T T

59 59 I Y Y~ Y Y Y

60 60 I N N~ A P N

63 63 I F L V~ V F
o4 64 I T M K ~ K T
65 6S CDR2 S S~ G~ G S
66 66 FR3 R R~ R~ R R
67 67 I L L- F~ F L Buried Leu not seen in human m.
(o7) 68 68 I S S T T I Surface Edge of bindin~ site. Ser not seen in humnn m.
69 69 1 I I'' I~ I I
70 70 I N S S~ S ~ Surface [1478] Veryedgeofbinding rl8l 16621 site. N-1/107 in mouse IB.
N-1/86 in human m.
71 71 I K K~ R~ R K Buried r ~ ~ A~ ror H2 hop.
Lys is not seen in human m.
(o8) 72 72 I D D~ D E ~ Half- [1457] D~71/85 in human [13441 [311 buried m.

74 74 I S S~ S A A Surface S~75/84 in human m.

SUBSTITUTE SHEET(RULE 26) WO 96/40210 PCT/US~GI~17 Table " - Continued Knbat # FR or Mouse Mouse Human Human 225 Surface Commont CDRC225 ~3 m Donor RHA or 38PI Buried 76 76 ¦S1800] S N N N Half S~8/85 (~ possibly linked to buried 49G) in humsn m.
Conserve if binding poor7 77 77 I Q Q* T S ~ Surface [1419] Gin not seen in [199] [51] humanm.
78 78 I V V* L L L Buried V~3/84 in human m.
79 79 I F F* Y Y y H~lf- Phe not seen in human m.
buried 80 80 I F L L* L L Buried [1490] F~22/112 (~ linked t24] [857] to 6Q+13Q+40S+84S+85N+
891) in mouse IB. Phe not Seen in humnn m.
81 81 I K K* Q Q Q Surfnce K~22152inhumnnm.
82 82 I M M M* M M
82a 83 I N N N N N
82b 84 I S S* S S S
82c 85 I L L L L* L
83 86 I Q Q R R B Surface [ 1482] Q~4/93 in humnn m.
[1 18] .[415]
84 87 I S T A A A Surface S~4/116 (~ possibly linked to 6Q+13Q+40S+80F+85N+
89I) in mouse IB. Ser not seen in human m.
85 88 I N D E G _ Surfnce [1503] N~l 1/116 (~ linked [121 [1_44] [9] to 6Q+13Q+40S+80F+84S+
89I) in mouse IB. Asn not seen in human m.
86 89 I D D* D D D
87 90 I T T* T T T
88 91 1 A A* A* A A
89 92 1 I M V V y Half- I~24/113 (~ possibly linked buried to 6Q+13Q+40S+80F+84S+
85N) in mouse IB. I~1/94 in human m.
90 93 l Y Y* Y* Y Y
91 94 I Y Y* Y* Y Y Pncl;ing AA.
92 95 l C C* C* C C
93 96 1 A A~ A A A PnckiDg AA.
94 97 I FR3 R R R~ R Cnnonicnl ~ for Hl loop.
95 98 CDR3 A D G S A Pacl;ing AA. (Unusual AA) ~SlT~E SHEEr (RUL~ 2f;) WO 96/40210 PCT/US9~,J'1)9317 Table 9 - Continued Kaba.t # FR or . Mouse Mouse Human Human 225 Surfacc Comment CDRC2-'5E~ mDonor RHA or 38PI Buried 99 102 I Y x X T Y

100a 104 I Y Y L D Y
IOOb 105 I E D S A E
IOOc I - P G
I OOd I - D x 100e 1 - K Y
100f l - Y Y
IOOg I - F Y
IOOh I - T Y
100; 1 - L H
lOOj I - W Y
IOOk 106 I F F F F F Core pJd;illg AA.

103 109FR4 W W~ W~ W W Core pJCkil~g AA.
104 110 I G G~ G' G G
105 111 l Q Q' Q Q Q
106 112 I G G* G~ G G
107 113 I T T~ T' T T
108 114 I L L L M M [1020]L~59/76 in human m.
[349] [28]
109 115 l V V V' V V
110 116 I T T~ T' T T

111 117 l V V' V' V V
112 118 I S S' S~ S S
113 119FR4 A S S' S S A~28n6 in mouse IB.
Ala no~ seen in human m.

SUBSTITUTE SHEET (RULE 26) CA 02222231 1997-11-2~

W O 96/41D210 PCT~US96/09847 T~ble 9 - Continued Complmsoll of AA Mou~e Mouse Humall Human '~ Comment Vanable ~'egion AA C'~5 IB m Donor I~H"
Seq~~n~ to M:~25 38PI

PERCI~NTIDENTITY 100.0 78.15 55.46 48.74 7~.47 Ther~are'~6amino~cid ' ' in the FR betwcen the FRAME:'WORI~S 10(~.0 88.51 63.~ 58.6'~ G7.8~ valiableregionsofthereshapedheavy ONLY chain H~'~5RH" and the mouse M225 heavy chain PERCEr~T 100.0 84.87 71.43 67.'~3 84.87 ''-75RHB=''''SRHA+T41P
SIMILAIRITY ~')5RHC = '~25 REIA+ T68S +S70N

FR~MEWORKS 100.0 93.10 79.31 75.86 79 31 '~ ~SRHD = ~S RHu + 225RHc ONLY ''~SRHE = ''5 RHA + L78V

Legend: (*) invariant residues as dèfined either by the Kabat consensus sequences i.e. 95% or greater occurrence within Kabat subgroup (Kabat et Ql.,1991) (in the case of colurnns 5 and 6) or as part of the canonical structure for the CDR loops (in the case of column 8) as defined by Chothia et al., (1989); (BOLD) positions in Frs and CDRs where the human amino acid residue was replaced by the corresponding mouse residue (UNDFRI~INE) positions in Frs where the human residue differs from the analogous mouse residue number; (o) numbering of .h~n~s in the human Frs; (mouse C225) amino acid sequence of the VH region from chimeric C225 antibody; (mouse IB) consensus sequence of mouse VH regions from subgroup IB
(Kabat et al., 1991); (human III) consensus sequence of human VH regions from subgroup III
(Kabat et al., 1991); (Human Donors: 38P 1) amino acid sequence from human antibody 38P1'CL (Schroeder Jr et al. 1987); (Surface or Buried) position of amino acid in relation to the rest of the residues in both chains of the antibody variable regions; (225RH,~) amino acid sequenc,e of the first version of the reshaped human mAb H225 VH region. (Core packing of the first version of the reshaped human mAb H225 VH region (Core packing AA/Packing AA) amino a.cids located at the VL/VH interface as defined by Chothia et al. (1985); (Canonical AA) amino a.cids defined by Chothia and Lesk (1987), Chothia et al. (1989), Tramontano et al.
(1990) .and Chothia et a~. (1992) as being important for CDR loop conformation.

SUBSTITVTE StlEET (RULE 26) CA 02222231 1997-11-2~

W O 9/5/40210 PCTAJS96~'~5~,~7 Table 10. Primers for constructing reshaped hum~ antibody H225 kappa light chain variable region gene 225RKA-Name Sequence (5' - 3') 225RKA.LEAD(88mer) CTGGAGACTGAGTCAG TACG
A T T T C A C T T C T G G A G G C TC G
A A T C C A G A A A A G C A A A AAT A
C T T G G T T C T G A G G T G T G G A T
A C C A T G G T
225RKA.FR1 (80mer) T C G T A C T G A C T C A G T C T C C A
G C C A C C C T G T C T T T G A GTC C
AGGAGAAAGAGC CAC C CTC T
C C T G C A G G G C C A G T C A GAG T
225R~KA.FR2a (74mer) G A G A T A G A C T C A G A A G C A T A
C T T T A T G A G A A G C C T T GGA G
C C T G G C C A G G T C T T T G C T G A
TACCAGTGTATGT T
225RJKA.FR3 (71mer) G G C T T C T C A T A A A G T A TGC T
T C T G A G T C T A T C T C T G GAA T
C C C T G C C A G G T T T A G T GGC A
G T G G A T C A G G G
225RlKA.FR3a(77mer) T T T TGT TGACAGTAA T AAAC
T G C A A A A T C T T C A G G C T C C A
C A C T G C T G A T G G T A A G AGT A
AAA T C T G T C C C T GA T C C
225RKA.CDR3 (33mer) G A T T T T G C A G T T T A T T A C T G
T C AAC AAAA T AA T
225R]KA.FR4a (68mer) T C T A G A A G G A T C C A C T C AC G
T T T CAGC T C CAC C T T G GTC C
C T C C A C C G A A C G T G G T TGG C
C A G T T A T T
225RKA.V78L(42mer) A C T C T T A C C A T C A G C A GTC T
GGAGCCTGAAGATTT T GCAG
T T
225RKA.L108I(57mer) TCTAGAAGGATCCAC T CACG
T T T G A T C T C C A C C T T G GTC C
CTCCACCGAACGTGG T T

WO 96/41)210 PCT/US96/09847 TablelO.contin~

225~A.LS7 leader AAGCTTGCCGCCACCATGGA
(99mer) AGCCCCAGCTCAGCTTCTCT
TCCTCTTGCTTCTCTGGCTC
CCAGATACCACCGGAGAAAT
CGTACTGACTCAGTCTCCA

W O 91S/~0210 PCT~US96/09847 Table 11. Primers for constructing reshaped human antibody H225 kappa light chain variable region gene 225RKB-Name Se~ e (S' - 3') 225~B.K49Y(60mer) CAGCAAAGACCTGGCCAGGC
TCCAAGGCTTCTCATATATT
ATGCTTCTGAGTCTATCTCT
APCR40 (25mer) C T G A G A G T G C A C C A T A T G C G
GTGTG

CA 02222231 1997-ll-2~
W O 96/4~210 PCT~US96/0~8~7 Table 12. Primers for constructing reshaped human antibody H225 heavy chain variable region gene 225RHA.

Name Sequence (~' - 3') 225RHA.FRl (37mer) GGTGCAGCTGGTCGAGTCTG
GGGGAGGCTTGGTACAG
225~A.FRla (50mer) GGCTGTACCAAGCCTCCCCC
AGACTCGACCAGCTGCACCT
CACACTGGAC
225RHA.CDRla (64mer) C C C AGTGTACACCATAGTTA
GTTAATGAGAATCCGGAGAC
TGCACAGGAGAGTCTCAGGG
ACCC
225RHA.FR2 (63mer) TTAACTAACTATGGTGTACA
CTGGGTTCGCC A G G C T A C A G
GAAAGGGTCTGGAGTGGCTG
GGA
225RHA.FR3a (74mer) C TGTTCATTTGCAGATACAG
GGAGTTCTTGGCATTTTCCT
TGGAGATGGTCAGTCGACTT
GTGAAAGGTGTATT
225~A.FR3 (73mer) C T C C C TGTATCTGCAAATGA
ACAGTCTCAGAGCCGGGGAC
ACAGCCGTGTATTACTGTGC
CAGAGCCCTCACC
225RHA.FR4a (65mer) GGATCCACTCACCTGAAGAG
ACAGTGACCATAGTCCCTTG
GCCCCAGTAAGCAAA

W O 96i/40210 PCT~US96~3q7 Table 13. Primers for constructing reshaped hurnan antibody H225 heavy chain variable region genes 22SRHB, 225RHC~ 22SRHD and 225RHE

Name Sequence (5' - 3') 225RHB.T41P-S (35mer) G G G T T C G C C A G G C T C C A G G A
AAGGGTCTGGAGTGG
225RHB.T41P-AS (30mer) T C C T G G A G C C T G G C G A A C C C
AGTGTACAC C
225RHC.T68S/S70N (46mer) C A C A A G T C G A C T G A G C A T C A
ACAAGGAAAATGCCAAGAAC
T C C C T G
225RHE.L78V (72mer) C A C A A G T C G A C T G A C C A T C T
T CAAGGAAAATGCCAAGAAC
T C C G T T T A T C T G C A A A T G A A
CAGTCTCAGAGC
APCR10 (25mer) T A C G C A A A C C G C C T C T C C C C
GCGCG
APCR40 (25mer) C T G A G A G T G C A C C A T A T G C G
G T G T G
RSP (Reverse Sequ~n~ing Primer) A G C G G A T A A C A A T T T C A C A C
(24mer) A G G A
UP(U~ el~lPrimer)(24mer) C G C C AGGG T T T T C C C AG T C A
C GAC

Claims (91)

WHAT WE CLAIM IS:

1. A polypeptide lacking the constant region and the variable light chain of an antibody, the polypeptide comprising the amino acid sequence G V I W S G G N T DY N T P F T S R or V I W S G G N T D Y N T P F T S.

2. A polypeptide according to Claim 1, comprising amino acid sequences N Y G V Hand G V I W S G G N T D Y N T P F T S R or V I W S G G N T D Y N T P F T S.

3. A polypeptide consisting of the amino acid sequence N Y G V H or G V I W S G
G N T D Y N T P F T S R.

4. A polypeptide consisting of the amino acid sequence N Y, G V H or V I W S G GN T D Y N T P F T S.

5. A polypeptide according to Claim 1 conjugated to an effector molecule.

6. A polypeptide according to Claim 5 wherein the effector molecule inhibits tumor growth.

7. A polypeptide according to Claim 5 wherein the effector molecule is cytotoxic.

8. A polypeptide according to Claim 5 wherein the effector molecule is doxorubicin.

9. A polypeptide according to Claim 5 wherein the effector molecule is cisplatin.

10. A polypeptide according to Claim 5 wherein the effector molecule is taxol.

11. A polypeptide according to Claim 5 wherein the effector molecule is a signal transduction inhibitor.

12. A polypeptide according to Claim 5 wherein the effector molecule is a ras inhibitor.

13. A polypeptide according to Claim 5 wherein the effector molecule is a cell cycle inhibitor.

14. DNA encoding a polypeptide lacking the constant region and the variable light chain of an antibody, the polypeptide comprising the amino acid sequence N Y G VH, G V I W S G G N T D Y N T P F T S R or V I W S G G N T D Y N T P F T S.

15. DNA encoding the polypeptide of Claim 14 comprising amino acid sequences N
Y G V H and G V I W S G G N T D Y N T P F T S R or V I W S G G N T D Y N T P
F T S.

16. DNA encoding a polypeptide according to Claim 14 conjugated to an effector molecule.

17. DNA encoding a polypeptide according to Claim 16 wherein the effector molecule inhibits tumor growth.

18. A molecule having the constant region of a human antibody and the hypervariable region of monoclonal antibody 225 conjugated to an effector molecule.

19. A molecule according to Claim 18 wherein the effector molecule is a cytotoxic agent.

20. A molecule according to Claim 19 wherein the cytotoxic agent is doxorubicin.

21. A molecule according to Claim 19 wherein the cytotoxic agent is taxol.

22. A molecule according to Claim 19 wherein the cytotoxic agent is cisplatin.

23. A molecule comprising a constant region of a human antibody; a variable region other than the CDRs of a human antibody, the variable region comprising a kappa light chain and a heavy chain, and the CDRs of monoclonal antibody 225.

24. A molecule according to claim 23 wherein the constant region has an amino acid sequence of an IgG.

25. A molecule according to claim 24 wherein the IgG is IgG1.

26. A molecule according to claim 23 that is reshaped according to Example IV.

27. A molecule according to claim 23, wherein the heavy chain has at least one amino acid, according to the Kabat numbering system, at an amino acid position selected from the group consisting of 24, 28, 29, 30, 41, 48, 49, 67, 68, 70, 71 and 78, substituted with a murine amino acid selected from the corresponding Kabat aminoacid position.

28. A molecule according to claim 23, wherein the kappa light chain has an aminoacid, according to the Kabat numbering system, at position 49 substituted with amurine amino acid selected from the corresponding Kabat amino acid position.

29. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence selected from 225RKA or 225RKB.

30. A molecule according to claim 23 wherein the heavy chain variable region has an amino acid sequence selected from 225RHA, 225RHB, 225RHC, 225RHD, or 225RHE.

31. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 225RHA.

32. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 225RHB.

33. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 225RHC.

34. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 225RHD.

35. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 225RHE.

36. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid sequence 225RHA.

37. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid sequence 225RHB.

38. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid sequence 225RHC.

39. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid sequence 225RHD.

40. A molecule according to claim 23 wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid sequence 225RHE.

41. A molecule according to Claim 23, wherein the molecule is attached to an effector molecule.

42. A molecule according to Claim 39, wherein the effector molecule is a cytotoxic agent.

43. A molecule according to Claim 40, wherein the cytotoxic agent is doxorubicin.

44. A molecule according to Claim 40, wherein the cytotoxic agent is taxol.

45. A molecule according to Claim 40, wherein the cytotoxic agent is cisplatin

46. A method for significantly inhibiting the growth of tumor cells in a human comprising administering to the human an effective amount of the polypeptide according to Claim 1.

47. A method for significantly inhibiting the growth of tumor cells in a human comprising administering to the human an effective amount of the polypeptide according to Claim 3 or Claim 4.

48. A method for significantly inhibiting the growth of tumor cells in a human comprising administering to the human an effective amount of a molecule having the constant region of a human antibody and the variable region of monoclonal antibody 225.

49. A method for significantly inhibiting the growth of tumor cells in a human comprising administering to the human an effective amount of a molecule having aconstant region of a human antibody; a variable region other than the CDRs of a human antibody, the variable region comprising a kappa light chain and heavy chain, and the CDRs of monoclonal antibody 225.

50. A method according to claim 47, wherein the kappa light chain variable region has an amino acid sequence selected from 225RKA or 225RKB.

51. A method according to claim 47, wherein the heavy chain variable region has an amino acid sequence selected from 225RHA, 225RHB, 225RHC, 225RHD, or 225RHE.

52. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 225RHA.

53. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 22SRHB.

54. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 225RHC.

55. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 225RHD.

56. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKA and the heavy chain variable region has amino acid sequence 225RHE.

57. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid Sequence 225RHA.

58. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid sequence 225RHB.

59. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid sequence 225RHC.

60. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid sequence 225RHD.

61. A method according to claim 47, wherein the kappa light chain variable region has amino acid sequence 225RKB and the heavy chain variable region has amino acid sequence 225RHE.

62. A method according to any of Claims 44-47, further comprising administering a cytotoxic agent.

63. A molecule according to Claim 60, wherein the cytotoxic agent is doxorubicin.

64. A molecule according to Claim 60, wherein the cytotoxic agent is taxol.

65. A molecule according to Claim 60, wherein the cytotoxic agent is cisplatin

66. The method according to Claim 44 or Claim 45, wherein the polypeptide is conjugated to an effector molecule.

67. The method according to Claim 46 or Claim 47, wherein the molecule is conjugated to an effector molecule.

68. The method according to Claim 64, wherein the effector molecule is cytotoxic.

69. The method according to Claim 64, wherein the effector molecule is doxorubicin.

70. The method according to Claim 64, wherein the effector molecule is cisplatin.

71. The method according to Claim 64, wherein the effector molecule is taxol.

72. The method according to Claim 64, wherein the effector molecule is a signal transduction inhibitor.

73. The method according to Claim 64, wherein the effector molecule is a ras inhibitor.

74. The method according to Claim 64, wherein the effector molecule is a cell cycle inhibitor.

75. The method according to Claim 65, wherein the effector molecule is cytotoxic.

76. The method according to Claim 65, wherein the effector molecule is doxorubicin.

77. The method according to Claim 65, wherein the effector molecule is cisplatin.

78. The method according to Claim 65, wherein the effector molecule is taxol.

79. The method according to Claim 65, wherein the effector molecule is a signal transduction inhibitor.

80. The method according to Claim 65, wherein the effector molecule is a ras inhibitor.

81. The method according to Claim 65, wherein the effector molecule is a cell cycle inhibitor.

82. The method according to any of Claims 44-47, wherein the tumor cells are prostatic tumor cells.

83. The method according to Claim 80, wherein the prostatic tumor cells are late stage prostatic tumor cells.

84. A nucleic acid molecule that encodes a molecule comprising: a constant region of a human antibody, a variable region other than the CDRs of a human antibody, thevariable region comprising a kappa light chain and a heavy chain, and the CDRs of monoclonal antibody 225.

85. A vector comprising the nucleic acid molecule claim of 84.

86. A vector according to claim 85, wherein the vector is an expressible vector.

87. A vector according to claim 86, wherein the vector is expressible in a prokaryotic cell.

88. A vector according to claim 86, wherein the vector is expressible in a eukaryotic cell.

89. A prokaryotic cell comprising the expressible vector of claim 87.

90. An eukaryotic cell comprising the expressible vector of claim 88.

91. A pharmaceutical composition, comprising the molecule of claim 23 and a pharmaceutically acceptable carrier.