CA2243236A1 - Anticoagulant agents useful in treatment of thrombosis - Google Patents

Abstract

Monoclonal antibodies directed against coagulation factors and their use in inhibiting thrombosis are disclosed.

Description

WO 9i~2601~ PCT~US97/0~759 ~NTICOAGUI~NT AGENrrS U~:~U~ IN T~F~M~MT OF T~O~rR~SIS

FIELD OF THE l~V~. lON

This invention relates to monoclonal antibodies (mAbs~ that bind to a human coagulation ~actor or co~actor and their use as sel~-limiting inhibitors o~
thrombosis.

R~C~C~7~OUND OF THE ~ NV~ ~lON

Under normal circumstances, an injury, be it minor or major, to vascular endothelial cells lining a blood vessel triggers a hemostatic response through a sequence o~ events commo~ly re~erred to as the coagulation "cascade " The cascade clllml~tes in the conversion o~
soluble ~ibrinogen to insoluble ~ibrin which, together with platelets, ~orms a localized clot or thrombus which prevents extravasation o~ blood components. Wound healing can then occur ~ollowed by clot dissolution and restoration o~ blood vessel integrity and ~low.
The events which occur between injury and clot ~ormation are a carefully regulated and linked series o~
reactions In brie~, a number o~ plasma coagulation proteins in inactive proenzyme ~orms and co~actors circulate in the blood. Active enzyme complexes are assembled at an injury site and are sequentially activated to serine proteases, with each successive serine protease catalyzing the subsequent proenzyme to protease activation. This enzymatic cascade results in each step magni~ying the e~ect o~ the succeeding step.
For an overview o~ the coagulation cascade see the ~irst chapter o~ "Thombosis and Hemorrhage", J. Loscalzo and A. Scha~er, eds., Blackwell Scientific Publications Ox~ord, England ~1994).
While e~icient clotting limits the loss of blood at an injury site, inappropriate ~ormation o~ thrombi in veins or arteries is a common cause o~ disability and death. Abnormal clotting activity can result in and/or W O 97/26~10 PCT~US97/00759 ~rom pathologies or-treatments such as myocardial infarction, unstable angina, atrial ~ibrillation, stroke, renal damage, percutaneous translumenal coronary angioplasty, disseminated intravascular coagulation, 5 sepsis, pulmonary embolism and deep vein thrombosis. A
The formation o~ clots on ~oreign sur~aces o~ artificial organs, shunts and prostheses such as artificial heart valves is also problematic.
Approved anticoagulant agents currently used in 10 treatment o~ these pathologies and other thrombotic and embolic disorders include the sulfated heteropolysaccharides heparin and low molecular weight (LMW) heparin. These agents are administered parenterally and can cause rapid and complete inhibition 15 of clotting by activation of the thrombin inhibitor, antithrombin III and inactivation of all of the clotting factors.
However, due to their potency, heparin and LMW
heparin su~er drawbacks. Uncontrolled bleeding as a 20 result of the simple stresses of motion and accompanying contacts with physical objects or at surgical sites is the major complication and is observed in 1 to 7% of patients receiving continuous infusion and in 8 to 14%
o~ patients given intermittent bolus doses. To m;n;ml ze 25 this risk, samples are continuously drawn to.enable ex vivo clotting times to be continuously monitored, which contributes substantially to the cost of therapy and the patient~s inconvenience.
Further, the therapeutic target range to achieve 30 the desired level of ef~icacy without placing the patient at risk ~or bleeding is narrow. The therapeutic range is approximately 1 to less than 3 ug heparin/ml plasma which results in activated partial thromboplastin time ~aPTT) assay times of about 35 to about 100 35 seconds. Increasing the heparin concentration to 3 ug/ml exceeds the target range and at concentrations greater than 4 ug/ml, clotting activity is not W O 9i/26010 PCT~US97/00759 detectable. Thus, great care must be taken to keep the patient's plasma concentrations within the therapeutic range.
Another approved anticoagulant with slower and longer lasting effect is warfarin, a coumarin derivative. Warfarin acts by competing with Vitamin K
dependent post-translational modification of prothrombin and other Vitamin K-dependent clotting factors.
The general pattern of anticoagulant action,in which blood is rendered non-clottable at concentrations only slightly higher than the therapeutic range is seen for warfarin as well as for heparin and LMW heparin.
Clearly, a need exists for an anticoagulant agent which is efficacious in controlling thrombotic and embolic disorders yet does not cause uncontrolled bleedin~ or its possibility.
SUMM~RY OF T~E lNv~NllON
Accordingly, one aspect of the present invention is a method for inhibiting thrombosis in an ~n;~l comprising administering an effective dose o~ an anti-coagulation ~actor monoclonal antibody having self-limiting neutralizing activity.
Another aspect of the invention is an anti-coagulation factor monoclonal antibody having self-limiting neutralizing activy against the coagulationfactor.
Another aspect of the invention is a monoclonal antibody having the identifying characteristics of SB
249413, SB 24g415, SB 249416, SB 249417, SB 257731, SB
257732, 9E4(2)F4 or llG4(1)B9.
Another aspect of the invention is a hybridoma cell line having the identifying characteristics of 9E4(2)F4 or llG4(13B9.
Another aspect of the invention is a neutralizing Fab fragment or F(ab')~ fragme~t thereo~, produced by deleting the Fc region of the monoclonal antibodies of the invention.

W O 9~726010 PCT~US97/~07~9 Another aspect-of the invention is a neutralizing Fab fragment or F(ab') 2 fragment thereof, produced by chain shuffling whereby the Fd heavy chain of the monoclonal antibodies of the invention is expressed in a murine light chain filamentous phage Fab display library.
Another aspect of the invention is a neutralizing Fab fragment or F(ab') 2 fragment thereof, produced by chain shuffling whereby the light chain of the monoclonal antibodies o~ the invention is expressed in a murine heavy chain filamentous phage Fab display library.
Another aspect of the invention is an ;~ml]noglobulin heavy chain complementarity det~m;n;ng region having an amino acid se~uence selected from the group consisting of SEQ ID NOs: 8, 9 and 10.
Another aspect of the invention is an immunoglobulin light chain complementarity determ;n;ng region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13 and 14.
Another aspect of the invention is an altered antibody comprising a heavy chain and a light chain, wherein the framework regions o~ said heavy and light chA;n~ are derived from at least one selected antibody and the amino acid sequences of the complementarity determining regions of each said chain are derived from an anti-coagulation factor monoclonal antibody having self-limiting neutralizing activity against the coagulation factor.
Another aspect of the invention is a chimeric antibody comprising a heavy chain and a light chain, said antibody characterized by inhibiting the function of intrinsic or common pathway coagulation factors in a self-limiting manner, wherein thrombosis is inhibited and limited modulation of coagulation is produced, wherein the constant regions of said heavy and light chains are derived from at least one selected antibody W O 97/26010 PCTrUS97/00759 and the amino acid sequences of the variable regions of each said chain are derived from an anti-coagulation factor monoclonal antibody having self-limiting neutralizing activity against the coagulation factor.
Yet another aspect o~ the invention is a pharmaceutical composition comprising the humanized antibodies or chimeric antibody of the invention and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph of experimental results demonstrating the titration of normal human plasma with the murine anti-Factor IX mAbs BCl and BC2.
Figure 2 is a graph o~ experimental results demonstrating the titration of normal human plasma with the murine anti-Factor IX mAbs 9E4(2)F4 and llG4(1)B9.
Figure 3 is a graph of experimental results demonstrating the titration of normal human plasma with the murine anti-Factor X mAbs HFXHC and HFXLC and the murine anti-Factor XI mAb HFXI.
Figure 4 is a histogram of experimental results demonstrating the effect of heparin, acetylsalicylic acid and murine Factor IX mabs on activated partial thromboplastin time (aPTT) at 60 minutes in a rat carotid thrombosis model.
Figure 5 is a histogram of experimental results demonstrating the effect of heparin, acetylsalicylic acid and murine Factor IX mabs on prothrombin time at 60 minutes in a rat carotid thrombosis model.
Figure 6 is a histogram o~ experimental results demonstrating the effect of heparin, acetylsalicylic acid and murine Factor IX mabs on occlusion of carotid artery flow in a rat carotid thrombosis model.
Figure 7 is a histogram of experimental results demonstrating the effect of heparin, acetylsalicylic acid and murine Factor IX mabs on thrombus weight in a rat carotid thrombosis model.

W O 9ii26010 PCTnJS97/007S9 Figure 8 is a histogram o~ experimental results demonstrating the e~ect o~ heparin, the murine Factor IX mab BC2, a ch;me~ic Factor IX mab and humanized ~actor IX mAbs on aPTT at 60 minutes in a rat carotid thrombosis model.
Figure 9 is a histogram o~ experimental results demonstrating the e~ect o~ heparin, the murine Factor IX mab BC2, a chimeric Factor IX mab and hl~m~n; zed factor IX mAbS on thrombus weight in a rat carotid thrombosis model.

DE'r~TT-~n DESCRIPT:i:ON OF THE lN v~ QN
The present invention provides a variety o~
antibodies, altered antibodies and ~ragments thereo~
directed against coagulation factors, which are characterized by sel~-limiting neutralizing activity.
Pre~erably, the coagulation factor is ~rom the intrinsic or common coagulation pathway. Most pre~erably, the anti-coagulation ~actor antibodies are anti-Factor IX, anti-Factor IXa, anti-Factor X, anti-Factor Xa, anti-Factor XI, anti-Factor XIa, anti-Factor VIII, anti-Factor VIIIa, anti-Factor V, anti-Factor Va, anti-Factor VII, anti-Factor VIIa or anti-thrombin. Particularly preferred are anti-Factor IX antibodies. Exemplary anti-coagulation ~actor antibodies are the humanized monoclonal antibodies SB 249413, SB 249415, SB 249416, SB 249417, SB 257731 and SB 257732 directed against human Factor IX, the ~;m~ic monoclonal antibody ch~FIX
directed against human Factor IX, the murine monoclonal antibodies BCl, BC2, 9E4~2)F4 and 11~4(1)B9 which are directed against human Factor IX and/or Factor IXa or the murine monoclonal antibodies HFXLC and HFXI which are directed against human Factors X and XI, respectively. Particularly pre~erred is the anti-human Factor IX monoclonal antibody SB 249417.

W O 9i/26010 PCTrUS97/00759 The antibodies of the present invention can be prepared by conventional hybridoma techniques, phage display combinatorial libraries, immunoglobulin chain shuffling and humanization techniques to generate novel self-limiting neutralizing antibodies. Also included are fully human mAbs having self-limiting neutralizing activity. These products are useful in therapeutic and pharmaceutical compositions for thrombotic and embolic disorders associated with myocardial infarction, unstable angina, atrial fibrillation, stroke, renal damage, pulmonary embolism, deep vein thrombosis, percutaneous translumenal coronary angioplasty, disseminated intravascular coagulation, sepsis, artificial organs, shunts or prostheses.
As used herein, the term "self-limiting neutralizing activity" refers to the activity of an antibody that binds to a human coagulation factor, preferably from the intrinsic and common pathways, including Factor IX/IXa, X/Xa, XI/XIa, VIII/VIIIa and V/Va, VII/VIIa and thrombin and inhibits thrombosis in a manner such that limited modulation of coagulation is produced. "Limited modulation of coagulation" is defined as an increase in clotting time, as measured by prolongation of the activated partial thromboplastin time (aPTT), where plasma r~m~in~ clottable with aPTT
reaching a maximal value despite increasing concentrations of monoclonal antibody. This limited modulation of coagulation is in contrast to plasma being rendered unclottable and exhibiting an infinite aPTT in the presence o~ increasing concentrations of heparin.
Preferably, the maximal aPTT value of the methods of the invention are within the heparin therapeutic range.
Most pre~erably, maximal aPTT is within the range of about 35 seconds to about 100 seconds which corresponds to about 1.5 times to about 3.5 times the normal control aPTT value. In one embodiment of the invention, aPTT is .

WO9i/26010 PCT~S97/00759 prolonged without signi~icant prolongation o~
prothrombin time ~PT).
"Altered antibody" re~ers to a protein encoded by an altered immunoglobulin coding region, which may be obtained by expression in a selected host cell. Such altered antibodies are engineered antibodies (e.g., chimeric or humanized antibodies) or antibody fragments lacking all or part o~ an immunoglobulin constant region, e.g., Fv, Fab, Fab' or F(ab')2 and the like.
"Altered immunoglobulin coding region" refers to a nucleic acid sequence encoding an altered antibody of the invention. When the altered antibody is a CDR-gra~ted or humanized antibody, the sequences that encode the complementarity det~m;ning regions (CDRs) ~rom a non-human immunoglobulin are inserted into a ~irst immunoglobulin partner comprising human variable ~ramework sequences. Optionally, the ~irst immunoglobulin partner is operatively linked to a second immunoglobulin partner.
"First immunoglobulin partner" refers to a nucleic acid sequence encoding a human ~ramework or human immunoglobulin variable region in which the native (or naturally-occurring) CDR-encoding regions are replaced by the CDR-encoding regions o~ a donor antibody. The human variable region can be an immunoglobulin heavy chain, a light chain (or both ~ n~), an analog or ~unctional ~ragments thereo~. Such C3R regions, located within the variable region o~ antibodies (immunoglobulins) can be determined by known methods in the art. For example Kabat et al. in "Sequences o~
Proteins o~ Immunological Interest", 4th Ed., U.S.
Department o~ Health and Human Services, National Institutes o~ Health (lg87) disclose rules for locating CDRs. In addition, computer programs are known which are use~ul for identi~ying CDR regions/structures.
"Second immunoglobulin partner" re~e~s to another nucleotide sequence encoding a protein or peptide to W O 9i/26010 PCTrUS97/00759 which the first immunoglobulin partner is fused in frame or by means of an optional conventional linker sequence ( i . e., operatively linked). Preferably, it is an immunoglobulin gene. The second immunoglobulin partner may include a nucleic acid sequence encoding the entire constant region for the same (i.e., homologous, where the first and second altered antibodies are derived ~rom the same source) or an additional (i. e ., heterologous) antibody of interest. It may be an immunoglobulin heavy chain or light chain (or both 5~; ns as part of a single polypeptide). The second immunoglobulin partner is not limited to a particular immunoglobulin class or isotype.
In addition, the second lm~lln~globulin partner may comprise part of an immunoglobulin constant region, such as found in a Fab, or F(ab)2 (i.e., a discrete part of an appropriate human constant region or framework region). Such second immunoglobulin partner may also comprise a sequence encoding an integral membrane protein exposed on the outer surface of a host cell, e.g., as part of a phage display library, or a sequence encoding a protein ~or analytical or diagnostic detection, e. g., horseradish peroxidase, ~-galactosidase, etc The terms Fv, Fc, Fd, Fab, Fab' or F(ab')2 are used with their standard meanings See, e . g., Harlow et al.
in ~Antibodies A Laboratory Manual", Cold Spring Harbor Laboratory, (1988).
As used herein, an "engineered antibody" describes a type of altered antibody, i. e., a full-length synthetic antibody ( e . g., a chimeric or humanized antibody as opposed to an antibody fragment) in which a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts ~rom one or more donor antibodies which have specificity for the selected epitope. For example, such molecules may include antibodies characterized by a W O 9i/26010 PCT~US97/00759 humanized heavy chain associated with an unmodified light chain (or chimeric light chain), or vice versa.
Engineered antibodies may also be characterized by alteration of the nucleic acid sequences encoding the acceptor antibody light and/or heavy variable ~om~;n framework regions in order to retain donor antibody binding specificity. These antibodies can comprise replacement of one or more CDRs (preferably all) from the acceptor antibody with CDRs from a donor antibody described herein.
A "chimeric antibody" refers to a type of engineered antibody which contains a naturally-occurring variable region (light chain and heavy ch~;n~) derived from a donor antibody in association with light and heavy chain constant regions derived ~rom an acceptor antibody.
A "humanized antibody" re~ers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the r~m~;n;ng immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulins. In addition, framework support residues may be altered to preserve binding affinity. ~ee, e. g., Queen et al ., P~oc. Natl AGad Sci USA, 86, 10029-10032 (1989), Hodgson et al., Bio/Technoloay. 9, 421 (1991).
The term "donor antibody" refers to a monoclonal or recombinant antibody which contributes the nucleic acid sequences o~ its variable regions, CDRs or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody. One donor antibody suitable for use in this invention is a murine self-limiting neutralizing monoclonal antibody designated as BC2. Other suitable donor antibodies include the murine self-limiting W O 97/26010 PCT~US97/00759 neutralizing monoclonal antibodies designated as BCl, 9E4(2)F4, llG4(l)B9, HFXLC and HFXI.
The term "acceptor antibody" refers to monoclonal or recombinant antibodies heterologous to the donor antibody, which contributes all, or a portion, of the nucleic acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner. Preferably, a human antibody is the acceptor antibody.
"CDRs" are de~ined as the complementarity det~rm1ning region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e . g., Kabat et al ., Se~uences ~f Proteins o~ Immunoloaical Interest, 4th Ed., U.S. Department of ~ealth and Human Services, National Institutes of ~ealth (1987). There are three heavy chain and three light chain CDRs or CDR regions in the variable portion of an immunoglobulin. Thus, "CDRs"
as used herein refers to all three heavy chain CDRs, or all three light chain CDRs or both all heavy and all light chain CDRs, if appropriate.
CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include analogs of the naturally occurring CDRs, which analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived.
By " sharing the antigen binding specificity or neutralizing ability" is meant, for example, that although mAb BC2 may be characterized by a certain level of self-limiting neutralizing activity, a CDR encoded by a nucleic acid sequence of BC2 in an appropriate structural environment may have a lower, or higher activity. It is expected that CDRs of BC2 in such W O 97/26010 PCTrUS97/00759 environments will nevertheless recognize the same epitope(s) as BC2.
A "functional fragment" is a partial heavy or light chain variable sequence ( e . g., minor deletions at the amino or carboxy terminus of the ;mml]noglobulin variable region) which retains the same antigen binding specificity and/or neutralizing ability as the antibody ~rom which the fragment was derived.
An "analog" is an amino acid sequence modified by at least one amino acid, wherein said modification can be chemical or a substitution or a rearrangement of a few amino acids (i.e., no more than 10), which modification permits the amino acid sequence to retain the biological characteristics, e. g., antigen specificity and high affinity, of the unmodified sequence. Exemplary analogs include silent mutations which can be constructed, via substitutions, to create certain endonuclease restriction sites within or surrounding CDR-encoding regions.
Analogs may also arise as allelic variations. An "allelic variation or modification" is an a~teration in the nucleic acid sequence encoding the amino acid or peptide sequences of the invention. Such variations or modifications may be due to degeneracy in the genetic code or may be deliberately engineered to provide desired characteristics. These variations or modifications may or may not result in alterations in any encoded amino acid sequence.
The term "effector agents" refers to non-protei~
carrier molecules to which the altered antibodies, and/or natural or synthetic light or heavy chains of the donor antibody or other fragments o~ the donor antibody may be associated by conventional means. Such non-protein carriers can include conventional carriers used ~ 35 in the diagnostic field, e. g., polystyrene or other plastic beads, polysaccharides, e . g., as used in the BIAcore (Pharmacia) system, or other non-protein W O 97/26010 PCT~US97/00759 substances useful in the medical field and safe for administration to humans and animals. Other effector agents may include a macrocycle, for chelating a heavy metal atom or radioisotopes. Such effector agents may also be useful to increase the half~ e of the altered antibodies, e. g., polyethylene glycol.
- For use in constructing the antibodies, altered antibodies and fragments of this invention, a non-human species such as bovine, ovine, monkey, chicken, rodent 10 ( e. g., murine and rat) may be employed to generate a desirable immunoglobulin upon presentment with a human coagulation factor , preferably factor IX/IXa, X/Xa, XI/XIa, VIII/VIIIa, V/Va, VII/VIIa or thrombin or a peptide epitope therefrom. Conventional hybridoma techniques are employed to provide a hybridoma cell line secreting a non-human mAb to the respective coagulation factor. Such hybridomas are then screened for binding using Factor IX/IXa, X/Xa, XI/XIa, VIII/VIIIa, V/Va, VII/VIIa or thrombin coated to 96-well plates, as described in the Examples section, or alternatively with biotinylated Factor IX/IXa, X/Xa, XI/XIa, V~II/VIIIa, V/Va, VII/VIIa or thrombin bound to a streptavidin-coated plate. Alternatively, fully human mAbs can be generated by techniques known to those skilled in the art and used in this invention.
One exemplary, self-limiting neutralizing mAb of this invention is mAb BC2, a murine antibody which can be used for the development of a chimeric or humanized molecule. The BC2 mAb is characterized by a self-limiting inhibitory activity on clotting time. Asmeasured by the aPTT assay, the ef~ect of the BC2 mAb on clot time exhibits a maximal value of about 100 seconds.
The BC2 mAb also binds Factor IXa, inhibits Factor IX to IXa conversion and inhibits Factor IXa activity.
Divalent metal cofactors are re~uired for activity, with the mAb exhibiting a greater preference ~or Ca2~ over Mn . The observed ICso in the aPTT assay is . CA 02243236 1998-07-16 W O9i/26010 PCTfUS97/00759 approximately 50 nM: The BC2 mAb exhibits a species cross-reactivity with rat and is o~ isotype IgG2a.
Other desirable donor antibodies are the murine mAbs, BCl, 9E4(2)F4 and llG4(1)Bg. These mAbs are characterized by a sel~-limiting inhibitory activity on clotting time. As measured by the aPTT assay, the e~fect o~ these mAbs on clot time exhibits a maximal value of about 90 to 100 seconds for 9E4(2)F4 and about 80 seconds ~or llG4(1)B9. The BCl mAb also binds Factor IXa, inhibits Factor IXa activity but does not inhibit Factor IX to IXa conversion. A metal co~actor is not re~uired for its activity. The observed ICso ~or BCl in the aPTT assay is approximately 35 nM. The BCl mAb is o~ isotype IgGl.
Yet another desirable donor antibody characterized by a self-limiting inhibitory activity on clotting ~ime is the murine mAb HFXLC As measured by the aPTT assay, the e~fect of the HFXLC mAb on clot time exhibits a m~;m~l value o~ about 50 to 60 seconds. The HFXLC mAb binds Factor X light chain, and inhibits Factor X/Xa activity. The observed IC50 in the aPTT assay is approximately 20 nM.
Yet another desirable donor antibody characterized by a sel~-limiting inhibitory activity on clotting time is the murine mAb, HFXI. As measured by the aPTT assay, the e~fect of the HFXI mAb on clot time exhibits a m~lm~l value o~ about 100 seconds. The HFXLC mAb binds Factor XI and inhibits Factor XI~XIa activity. The observed ICso in the aPTT assay is approximately 30 nM.
While not intending to be bound to any particular theory regarding the mechanism o~ action, these mAbs appear to regulate coagulation by a non-competitive or allosteric mechanism whereby only partial inhibition is achieved.
This invention is not limited to the use o~ the BCl, BC2, 9E4(2)F4, 11~4(1)B9, HFXLC, HFXI or their t hypervariable (i.e., CDR) sequences. Any other W O 97/26010 PCTrUS97/00759 appropriate high-a~inity antibodies characterized by a self-limiting neutralizing activity and corresponding CDRs may be substituted therefor. Identification of the donor antibody in the following description as BCl, BC2, 9E4(2)F4, llG4(1)B9, HFXLC or HFXI is made for illustration and simplicity of description only.
The present invention also includes the use of Fab fragments or F(ab')2 fragments derived ~rom mAbs directed against the appropriate human coagulation factor or cofactor. These fragments are useful as agents having self-limiting neutralizing activity against coagulation factors, preferably against Factor IX/IXa, X/Xa, Xi/XIa, VIII/VIIIa, V/Va, VII/VIIa or thrombin. A Fab fragment contains the entire light chain and amino terminal portion o~ the heavy chain. An F(ab'~2 fragment is the fragment formed by two Fab fragments bound by disulfide bonds. The mAbs BCl, BC2, 9E4(2)F4, llG4(1)B9, HFXLC and HFXI and other similar high affinity antibodies, provide sources of Fab fragments and F(ab')2 fragments which can be obtained by conventional means, e. g., cleavage of the mAb with the appropriate proteolytic enzymes, papain and/or pepsin, or by recombinant methods. These Fab and F(ab')2 fragments are useful themselves as therapeutic, prophylactic or diagnostic agents, and as donors of sequences including the variable regions and CDR
se~uences useful in the formation of recombinant or humanized antibodies as described herein.
The Fab and F(ab')2 fragments can be constructed via a combinatorial phage library (see, e . g., Winter et al., Ann. Rev. Immunol., 12:433-455 (1994)) or via immunoglobulin chain shuf~ling (see, e. g., Marks et al ., Bio/~echnology, 10:779-783 (1992), which are both hereby incorporated by reference in their entirety, wherein the Fd or v~ immunoglobulin from a selected antibody (e.g., BC2) is allowed to associate with a repertoire o~ light chain immunoglobulins, vL (or VK), to ~orm novel Fabs.

W O 9il2601~ PCT~US97/00759 Conversely, the light chain immunoglobulin from a selected antibody may be allowed to associate with a repertoire of heavy chain immunoglobulins, vH (or Fd), to form novel Fabs. Self-limiting neutralizing Factor IX Fabs can be obtained by allowing the Fd of mAb BC2 to associate with a repertoire of light chain immunoglobulins. Hence, one is able to recover neutralizing Fa~s with unique se~uences (nucleotide and amino acid) from the chain shuffling technique.
The mAb BC2 or other antibodies described above may contribute sequences, such as variable heavy and/or light chain peptide sequences, framework sequences, CDR
sequences, functional fragments, and analogs thereof, and the nucleic acid sequences encoding them, useful in designing and obt~;n;ng various altered an~ibodies which are characterized by the antigen binding specificity of the donor antibody.
The nucleic acid sequences of this invention, or fragments thereof, encoding the variable light chain and heavy chain peptide sequences are also useful for mutagenic introduction of specific changes within the nucleic acid sequences encoding the CDRs or framework regions, and for incorporation of the resulting modified or fusion nucleic acid sequence into a plasmid for expression. For example, silent substitutions in the nucleotide sequence of the framework and CDR-encoding regions can be used to create restriction enzyme sites which facilitate insertion o~ mutagenized CDR and/or framework regions. These CDR-encoding regions can be used in the construction of the humanized antibodies of the invention.
The nucleic and amino acid sequences of the BC2 heavy chain variable region a~e listed in SEQ ID NOs: 5 and 7. The CDR sequences from this region are listed in SEQ ID NOs: 8, 9 and 10.
The nucleic and amino acid sequences of the BC2 light chain variable region are listed in SEQ ID NOs: 6 W O 9i/26010 PCTrUS97/00759 and 11. The CDR sequences from this region are listed in SEQ ID NOs: 12, 13 and 14.
Taking into account the degeneracy of the genetic code, various coding sequences may be constructed which encode the variable heavy and light chain amino acid sequences and CDR sequences o~ the invention as well as - functional fragments and analogs thereof which share the antigen specificity of the donor antibody. The isolated nucleic acid sequences of this invention, or fragments thereof, encoding the variable chain peptide se~uences or CDRs can be used to produce altered antibodies, e.g., chimeric or h~ nized antibodies or other engineered antibodies of this invention when operatively combined with a second immunoglobulin partner 15It should be noted that in addition to isolated nucleic acid sequences encoding portions of the altered antibody and antibodies described herein, other such nucleic acid sequences are encompassed by the present invention, such as those complementary to the native CDR-encoding sequences or complementary to the modified human framework regions surrounding the CDR-encoding regions. Use~ul DNA sequences include those se~uences which hybridize under stringent hybridization conditions to the DNA sequences. See, T. Maniatis et al., Molecul~ Clo~;nq (A LaboratQrv Manual), Cold Spring Harbor Laboratory (1982), pp. 387-389. An example of one such stringent hybridization condition is hybridization at 4XSSC at 65~C, followed by a washing in O.lXSSC at 65~C for one hour. Alternatively, an exemplary stringent hybridization condition is 50%
formamide, 4XSSC at 42~C. Pre~erably, these hybridizing DNA sequences are at least about 18 nucleotides in length, i.e., about the size of a CDR.
Altered immunoglobulin molecules can encode altered antibodies which include engineered antibodies such as chimeric antibodies and humanized antibodies. A desired W O9ii26010 PCTnUS97/007S9 altered immunoglobulin coding region contains CDR-encoding regions that encode peptides having the antigen speci~icity o~ a Factor IX/IXa, X/Xa, XI/XIa, VIII/VIIIa, V/Va, VII/VIIa or thrombin antibody, pre~erably a high af~inity antibody such as provided by the present invention, inserted into a ~irst immunoglobulin partner such as a human framework or human immunoglobulin variable region.
Pre~erably, the ~irst immunoglobulin partner is operatively linked to a second immunoglobulin partner.
The second immunoglobulin partner is de~ined above, and may include a sequence encoding a second antibody region o~ interest, ~or example an Fc region. Second immunoglobulin partners may also include sequences encoding another immunoglobulin to which the light or heavy chain constant region is ~used in ~rame or by means o~ a linker sequence. Engineered antibodies directed against ~unctional ~ragments or analogs o~
coagulation ~actors may be designed to elicit enhanced binding with the same antibody.
The second immunoglobulin partner may also be associated with e~ector agents as de~ined above, including non-protein carrier molecules, to which the second immunoglobulin partner may be operatively linked by conventional means.
Fusion or linkage between the second immunoglobulin partners, e.g., antibody sequences, and the e~fector agent may be by any suitable means, e.g., by conventional covalent or ionic bonds, protein ~usions, or hetero-bi~unctional cross-linkers, e.g., carbodiimide, glutaraldehyde and the like. Such techniques are known in the art and are described in conventional chemistry and biochemistry texts.
Additionally, conventional linker se~uences which simply provide for a desired amount o~ space between the second immunoglobulin partner and the e~ector agent may also be constructed into the altered immunoglobulin W O 9ii26010 PCTn~S97/0~759 coding region. The-design of such linkers is well known to those of skill in the art.
In addition, signal sequences for the molecules of the invention may be modified by techniques known to those skilled in the art to enhance expression.
A pre~erred altered antibody contains a variable heavy and/or light chain peptide or protein sequence having the antigen specificity of mAb BC2, e.g., the VH
and VL c~;n~. Still another desirable altered antibody of this invention is characterized by the amino acid sequence containing at least one, and preferably all of the CDRs of the variable region of the heavy and/or light chains of the murine antibody molecule BC2 with the r~;n;ng sequences being derived from a human source, or a functional fragment or analog thereof.
In a further embodiment, the altered antibody of the invention may have attached to it an additional agent. For example, recombinant DNA technology may be used to produce an altered antibody of the invention in 2~ which the Fc fragment or CH2 CH3 domain of a complete antibody molecule has been replaced by an enzyme or other detectable molecule (i.e., a polypeptide effector or reporter molecule).
The second immunoglobulin partner may also be operatively linked to a non-immunoglobulin peptide, protein or fragment thereof heterologous to the CDR-cont~;n;ng sequence having antigen specificity to a coagulation factor, preferably to Factor IX/IXa, X/Xa, XI/XIa, VIII/VIIIa, V/Va, VII/VIIa or thrombin. The resulting protein may exhibit both antigen specificity and characteristics of the non-immunoglobulin upon expression. That fusion partner characteristic may be, e.g., a functional characteristic such as another binding or receptor domain or a therapeutic characteristic if the fusion partner is itself a therapeutic protein or additional antigenic characteristics.

W O 9il26010 PCTrUS97/00759 Another desirable protein of this invention may comprise a complete antibody molecule, having full length heavy and light ch~; n.C or any discrete fragment thereof, such as the Fab or F(ab')2 fragments, a heavy chain dimer or any mln;m~l recombinant fragments thereof such as an Fv or a single-chain antibody (SCA) or any other molecule with the same specificity as the selected donor mAb, e.g., mAb BCl, BC2, 9E4(2)F4, llG4(1)B9, HFXLC or HFXI. Such protein may be used in the form of an altered antibody or may be used in its unfused ~orm.
Whenever the second ;~mllnoglobulin partner is derived from an antibody different from the donor antibody, e. g., any isotype or class of immunoglobulin framework or constant regions, an engineered antibody results Engineered antibodies can comprise immunoglobulin (Ig) constant regions and variable ~ramework regions from one source, e . g., the acceptor antibody, and one or more (pre~erably all) CDRs from the donor antibody, e. g., the anti-Factor IX/IXa, X/Xa, XI/XIa, VIII/VIIIa, V/Va, VII/VIIa or thrombin antibodies described herein. In addition, alterations, e . g., deletions, substitutions, or additions, of the acceptor mAb light and/or heavy variable domain framework region at the nucleic acid or amino acid levels, or the donor CDR regions may be made in order to retain donor antibody antigen binding specificity.
Such engineered antibodies are designed to employ one (or both) of the variable heavy and/or light ~inq of the coagulation factor mAb (optionally modified as described) or one or more of the heavy or light chain CDRs. The engineered antibodies of the invention exhibit sel~-limiting neutralizing activity.
Such engineered antibodies may include a humanized antibody containing the framework regions o~ a selected human immunoglobulin or subtype or a chimeric antibody cont~-n;ng the human heavy and light chain constant regions fused to the coagulation factor antibody W O 9i/26010 P~TnUS97/00759 functional fragment-s. A suitable human (or other ~n;m~l ) acceptor antibody may be one selected ~rom a conventional database, e.g., the KABAT~ database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by a homology ~ to the ~ramework regions o~ the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain variable framework region ~or insertion o~ the donor CDRs. A suitable acceptor antibody capable of donating light chain variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light c~; ns are not required to originate ~rom the same acceptor antibody.
Pre~erably, the heterologous ~ramework and constant regions are selected ~rom human immunoglobulin classes and isotypes, such as IgG (subtypes 1 through 4), IgM, IgA, and IgE. However, the acceptor antibody need not col~,pLlse orlly h-~r.an imm~c~l~b~lin protein se~aences.
For instance, a gene may be constructed in which a DNA
sequence encoding part o~ a human immunoglobulin chain is fused to a DNA sequence encoding a non-;mmllnoglobulin amino acid sequence such as a polypeptide e~ector or reporter molecule.
A particularly pre~erred humanized antibody contains CDRs of BC2 inserted onto the framework regions of a selected human antibody sequence. For neutralizing humanized antibodies, one, two or pre~erably three CDRs from the Factor IX antibody heavy chain and/or light chain variable regions are inserted into the ~ramework regions of the selected human antibody sequence, replacing the native CDRs of the latter antibody.
Pre~erably, in a humanized antibody, the variable domains in both human heavy and light chains have been engineered by one or more CDR replacements. It is possible to use all six CDRs, or various combinations of less than the six CDRs Preferably all six CDRs are W O97/26010 PCT~US97/00759 replaced. It is possible to replace the CDRs only in the hllm~n heavy chain, using as light chain ~he unmodified light chain from the human acceptor antibody.
Still alternatively, a compatible light chain may be selected from another human antibody by recourse to the conventional antibody databases. The remainder of the engineered antibody may be derived from any suitable acceptor human immunoglobulin.
The engineered humanized antibody thus pre~erably has the structure of a natural human antibody or a fragment thereof, and possesses the combination of properties required for effective therapeutic use, e.g., treatment of thrombotic and embolic diseases in man.
Most pre~erably, the hllm~n;zed antibodies have a heavy chain amino acid sequence as set forth in SEQ ID
NO: 31, 52, or 89. Also most preferred are humanized antibodies having a light chain amino acid sequence as set forth in SEQ ID NO: 44, 57, 62, 74, 78 or 99.
Particularly preferred is the hllm~n;zed antibody SB
249413 where the heavy chain has the amino acid sequence as set forth in SEQ ID NO: 31 and the light chaln has the amino acid sequence as set ~orth in SEQ ID NO: 44.
Also particularly preferred is the humanized antibody SB
249415 where the heavy chain has the amino acid sequence 25 as set ~orth in SEQ ID NO: 52 and the light chain has the amino acid sequence as set forth in SEQ ID NO: 57.
Also particularly preferred is the humanized antibody SB
249416 where the heavy chain has the amino acid sequence as set ~orth in SEQ ID NO: 52 and the light chain has 30 the amino acid sequence as set forth in SEQ ID NO: 62.
Also particularly preferred is the hllm~ni~ed antibody SB
249417 where the heavy chain has the amino acid sequence as set ~orth in SEQ ID NO: 52 and the light chain has the amino acid sequence as set forth in SEQ ID NO: 74.
35 Also particularly pre~erred is the humanized antibody SB
257731 where the heavy chain has the amino acid sequence c as set forth in SEQ ID NO: 52 and the light chain has W O 9i/26010 PCTrUS97/00759 the amino acid se~uence as set forth in SEQ ID N0: 78.
Also particularly preferred is the humanized antibody SB
257732 where the heavy chain has the amino acid sequence as set forth in SEQ ID NO: 89 and the light chain has the amino acid se~uence as set forth in SEQ ID NO: 99.
It will be understood by those skilled in the art that an engineered antibody may be further modified by changes in variable ~m~; n amino acids without necessarily affecting the specificity and high affinity of the donor antibody (i.e., an analog). It is anticipated that heavy and light chain amino acids may be substituted by other amino acids either in the variable domain frameworks or CDRs or both. These substitutions could be supplied by the donor antibody or consensus sequences from a particular subgroup.
In addition, the constant region may be altered to enhance or decrease selective properties of the molecules of this invention. For example, dimerization, binding to Fc receptors, or the ability to bind and activate complement (see, e . g., Angal et al ., Mol .
Tmmllnol, 3p, 105-108 (1993), Xu et al., J. Biol. Chem, 269, 3469-3474 (1994), Winter et al., EP 307434-B).
An altered antibody which is a chimeric antibody differs from the humanized antibodies described above by providing the entire non-human donor antibody heavy chain and light chain variable regions, including framework regions, in association with human immunoglobulin constant regions for both ~h~; n.~ . It is anticipated that chimeric antibodies which retain additional non-human se~uence relative to hllm~nized antibodies of this invention may elicit a significant immune response in humans.
Such antibodies are useful in the prevention and treatment of thrombotic and embolic disorders, as discussed below.
Preferably, the variable light and/or heavy chain se~uences and the CDRs of mAb BC2 or other suitable W O 97/26010 PCT~US97/00759 donor mAbs, e . g., B~l, 9E4(2~F4, llG4(1)B9, HFXLC, HFXI, and their encoding nucleic acid se~uences, are utilized in the construction o~ altered antibodies, pre~erably humanized antibodies, of this invention, by the ~ollowing process. The same or similar techniques may also be employed to generate other embodiments o~ this invention.
A hybridoma producing a selected donor mAb, e. g., the murine antibody BC2, is conventionally cloned and the DNA o~ its heavy and light chain variable regions obtained by techniques known to one o~ skill in the art, e.g., the techniques described in Samkrook et al ., "Molecular Cloning: A Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory (1989). The variable heavy and light regions of BC2 containing at least the CDR-encoding regions and those portions o~ the acceptor mAb light and/or heavy variable domain ~ramework regions required in order to retain donor mAb binding speci~icity, as well as the r~m~;ning immunoglobulin-derived parts o~ the antibody chain derived ~rom a human immunoglobulin, are obtained using polynucleotide primers and reverse transcriptase. The CDR-encoding regions are identified using a known database and by comparison to other antibodies.
A mouse/human chimeric antibody may then be prepared and assayed for binding ability. Such a chimeric antibody contains the entire non-human donor antibody VH and VL regions, in association with human Ig constant regions ~or both chains.
Homologous framework regions o~ a heavy chain variable region from a human antibody are identi~ied using computerized databases, e.g , KABAT~, and a human antibody having homology to BC2 is selected as the acceptor antibody. The se~uences o~ synthetic heavy chain variable regions containing the BC2 CDR-encoding regions within the human antibody ~rameworks are designed with optional nucleotide replacements in the W O 9i/26010 PCTrUS97/00759 ~ramework regions to incorporate restriction sites.
This designed sequence is then synthesized using long synthetic oligomers. Alternatively, the designed sequence can be synthesized by overlapping - 5 oligonucleotides, amplified by polymerase chain reaction (PCR), and corrected ~or errors. A suitable light chain 7 variable ~ramework region can be designed in a similar manner.
A humanized antibody may be derived ~rom the 10 chimeric antibody, or preferably, made synthetically by inserting the donor mAb CDR-encoding regions ~rom the heavy and light ch~; n.~ appropriately within the selected heavy and light chain framework. Alternatively, a humanized antibody o~ the invention may be prepared 15 using standard mutagenesis techniques. Thus, the resulting humanized antibody contains human ~ramework regions and donor mAb CDR-encoding regions. There may be subsequent manipulation o~ ~ramework residues. The resulting humanized antibody can be expressed in 20 recombinant host cells, e.g., COS, C~O or myeloma cells.
Other humanized antibodies may be prepared using this technique on other suitable Factor IX-speci~ic or other coagulation ~actor-speci~ic, sel~-limiting, neutralizing, high a~inity, non-human antibodies.
A conventional expression vector or recombinant plasmid is produced by placing these coding sequences ~or the altered antibody in operative association with conventional regulatory control sequences capable o~
controlling the replication and expression in, and/or secretion ~rom, a host cell. Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences, which can be derived ~rom other known antibodies. Similarly, a second expression vector can be produced having a DNA sequence which encodes a complementary antibody light or heavy chain.
Preferably, this second expression vector is identical to the ~irst except with respect to the coding sequences W O 9i/26010 PCTrUS97/00759 and selectable markers, in order to ensure, as much as possible, that each polypeptide chain is ~unctionally expressed. Alternatively, the heavy and light chain coding se~uences ~or the altered antibody may reside on a single vector.
A selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy ~;ns, The transfected cell is then cultured by conventional techniques to produce the engineered antibody of the invention. The humanized antibody which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by an appropriate assay such as ELISA or RIA. Similar conventional techniques may be employed to construct other altered antibodies and molecules o~ this invention.
Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art. For example, the pUC series of cloning vectors, such as pUCl9, which is commercially available ~rom supply houses, such as Amersham or Pharmacia, may be used. Additionally, any vector which is capable of replicating readily, has an abundance o~
cloning sites and selectable genes (e.g., antibiotic resistance) and is easily manipulated may be used for cloning. Thus, the selection o~ the cloning vector is not a limiting ~actor in this invention.
Similarly, the vectors employed ~or expression of the engineered antibodies according to this invention may be selected by one of skill in the art from any conventional vector. The vectors also contain selected regulatory sequences (such as CMV promoters) which direct the replication and expression o~ heterologous CA 02243236 l998-07-l6 W O 9i/26010 PCTrUS97/00759 DNA sequences in selected host cells. These vectors contain the above-described DNA sequences which code for the engineered antibody or altered immunoglobulin coding region. In addition, the vectors may incorporate the ~ 5 selected immunoglobulin sequences modified by the insertion of desirable restriction sites for ready manipulation.
The expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e. g., the mammalian dihydrofolate reductase gene (DHFR). Other preferable vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro). The expression vectors useful herein may be synthesized by techniques well known to those skilled in this art.
The components of such vectors, e. g., replicons, selection genes, enhancers, promoters, signal sequences and the like, may be obtained from commercial or natural sources or synthesized by known procedures for use in directing the expression and/or secretion o~ the product of the recombinant DNA in a selected host. Other appropriate expression vectors of which numerous types are known in the art for m~mm~lian, bacterial, insect, yeast and fungal expression may also be selected for this purpose.
The present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the engineered antibodies or altered immunoglobulin molecules thereof. Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, most desirably, cells from various strains of E. coli are used for replication of the cloning vectors and other steps in the construction of altered antibodies of this invention.

CA 02243236 l998-07-l6 W O 97i26010 PCT~US97/W759 Suitable host cells or cell lines for the expression of the engineered antibody or altered antibody of the invention are preferably m~mm~lian cells such as CHO, COS, a fibroblast cell (e.g., 3T3) and myeloid cells, and more preferably a CHO or a myeloid cell. Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable m~mm~lian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al., supra.
Bacterial cells may prove useful as host cells suitable for the expression of the recombinant Fabs of the present invention (see, e.g., Pluckthun, A., Tmmllnol. Rev., 130, 151-188 (1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any recombinant Fab produced in abacterial cell would have to be screened for retention of antigen binding ability. If the molecule çxpressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host.
For example, various strains of ~. coli used for expression are well-known as host cells in the field of biotechnology. Various strains of B. subtil is, Streptomyces, other bacilli and the like may also be employed.
Where desired, strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepi~optera and viral expression systems. See, e.g.
Miller et al ., Gençt;c Enqineerin~, 8, 277-298, Plenum Press (1986) and references cited therein.
The general methods by which the vectors of the invention may be constructed, the transfection methods W O 9i/26010 PCTAUS97/00759 required to produce-the host cells o~ the invention, and culture methods necessary to produce the altered antibody o~ the invention ~rom such host cell are all conventional techniques. Likewise, once produced, the altered antibodies of the invention may be puri~ied from the cell culture contents according to standard procedures o~ the art, including ammonium sul~ate precipitation, a~inity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill o~ the art and do not limit this invention.
Yet another method o~ expression of the humanized antibodies may utilize expression in a transgenic animal, such as described in U. S. Patent No. 4,873,316.
This relates to an expression system using the ~n;m~l ' S
casein promoter which when transgenically incorporated into a ~mm~l permits the ~emale to produce the desired recombinant protein in its milk.
Once expressed by the desired method, the engineered antibody is then examined ~or in vitro activity by use o~ an appropriate assay. Presently, conventional ELISA assay formats are employed to assess qualitative and quantitative binding o~ the engineered antibody to Factor IX or to other appropriate coagulation ~actors. Additionally, other in vi tro assays may also be used to veri~y neutralizing e~icacy prior to subsequent human clinical studies per~ormed to evaluate the persistence o~ the engineered antibody in the body despite the usual clearance mechanisms.
Following the procedures described ~or humanized antibodies prepared ~rom BC2, one o~ skill in the art may also construct hllm~n;zed antibodies ~rom other donor antibodies, variable region sequences and CDR peptides described herein. Engineered antibodies can be produced with variable region ~rameworks potentially recognized as "sel~" by recipients o~ the engineered antibody.
Minor modi~ications to the variable region ~rameworks W O9~/26010 PCT~US97/00759 can be implemented to effect large increases in antigen binding without appreciable increased immunogenicity for the recipient. Such engineered antibodies may effectively treat a human for coagulation factor-mediated conditions. ~uch antibodies may also be usefulin the diagnosis of such conditions.
This invention also relates to a method for inhibiting thrombosis in an ~n ; m~ l, particularly a human, which comprises ~min~ stering an ef~ective dose of an anti-coagulation factor monoclonal antibody having self-limiting neutralizing activity. Preferably, the coagulation factor is from the intrinsic or common coagulation pathway Most preferably, the anti-coagulation factor monoclonal antibody is an anti-Factor IX, anti-Factor Ixa, anti-Factor X, anti-Factor Xa, anti-Factor XI, anti-Factor XIa, anti-Factor VIII, anti-Factor VIIIa, anti-Factor V, anti-Factor Va, anti-Factor VII, anti-Factor VIIa or anti-thrombin. The mAb can include one or more of the engineered antibodies or altered antibodies described herein or fragments thereof.
Alternatively, acetylsalicylic acid can be administered in combination with the anti-coagulation factor monoclonal antibody. In some cases, combination therapy lowers the therapeutically effective dose of anti-coagulation factor monoclonal antibody.
The therapeutic response induced by the use of the molecules of this invention is produced by the binding to the respective coagulation factor and the subsequent self-limiting inhibition of the coagulation cascade. Thus, the molecules of the present invention, when in preparations and ~ormulations appropriate for therapeutic use, are highly desirable for persons susceptible to or experiencing abnormal clotting activity associated with, but not limited to, myocardial infarction, unstable angina, atrial fibrillation, CA 02243236 l998-07-l6 W O 97/26010 PCT~US97/00759 stroke, renal damage, pulmonary embolism, deep vein thrombosis and arti~icial organ and prosthetic implants.
The altered antibodies, antibodies and fragments thereo~ of this invention may also be used in 9 5 conjunction with other antibodies, particularly human mAbs reactive with other markers (epitopes) responsible for the condition against which the engineered antibody of the invention is directed.
The therapeutic agents of this invention are 10 believed to be desirable ~or treatment o~ abnormal clotting conditions ~rom about 1 day to about 3 weeks, or as needed. This represents a considerable advance over the currently used anticoagulants heparin and warfarin. The dose and duration of treatment relates to 15 the relative duration of the molecules o~ the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health o~ the patient.
The mode o~ administration o~ the therapeutic agent 20 o~ the invention may be any suitable route which delivers the agent to the host. The altered antibodies, antibodies, engineered antibodies, and fragments thereof, and pharmaceutical compositions o~ the invention are particularly useful ~or parenteral 25 administration, i.e., subcutaneously, intramuscularly, intravenously or intranasally.
Therapeutic agents of the invention may be prepared as pharmaceutical compositions cont~;n;ng an effective amount of the engineered (e.g., humanized) antibody of 30 the invention as an active ingredient in a pharmaceutically acceptable carrier. Alternatively, the pharmaceutical compositions of the invention could also contain acetysalicylic acid. In the prophylactic agent of the invention, an aqueous suspension or solution 35 containing the engineered antibody, preferably buf~ered at physiological pH, in a form ready for injection is pre~erred. The compositions for parenteral W O 97/2601~ PCTrUS97/007~9 administration will-commonly comprise a solution o~ the engineered antibody of the invention or a cocktail thereo~ dissolved in an pharmaceutically acceptable carrier, pre~erably an aqueous carrier. A variety of aqueous carriers may be employed, e.g., 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally ~ree o~ particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques ( e. g., ~iltration). The compositions may contain pharmaceutically accepta~le auxiliary substances as required to approximate physiological conditions such as pH adjusting and bu~ering agents, etc. The concentration o~ the antibody of the invention in such pharmaceutical ~ormulation can vary widely, i. e., ~rom less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on ~luid volumes, viscosities, etc., according to the particular mode o~ administration selected.
Thus, a pharmaceutical composition o~ the invention ~or intramuscular injection could be prepared to contain 1 mL sterile bu~fered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more pre~erably, about 5 mg to about 25 mg, o~ an engineered antibody of the invention. Similarly, a pharmaceutical composition of the invention ~or intravenous in~usion could be made up to contain about 250 ml o~ sterile Ringer's solution, and about 1 mg to about 30 mg and pre~erably 5 mg to about 25 mg o~ an engineered antibody of the invention. Actual methods ~or preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, "Remington's Pharmaceutical Science", 15th ed., Mack Publishing Company, Easton, Pennsylvania.
It is pre~erred that the therapeutic agent o~ the invention, when in a pharmaceutical preparation, be W O 97126010 PCT~US97/007~9 present in unit dose ~orms. The appropriate therapeutically effective dose can be determined readily by those of skill in the art. To effectively treat a thrombotic or embolic disorder in a human or other ~n;m~l, one dose of approximately 0.1 mg to approximately 20 mg per kg body weight of a protein or an antibody of this invention should be administered parenterally, preferably i.v. or i.m. Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician during the thrombotic response.
The antibodies, altered antibodies or fragments thereof described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use This technique has been shown to be ef~ective with conventional imm.unoglobulins and art-known lyophilization and reconstitution techniques can be employed.

The present invention will now be described with reference to the following specific, non-limiting examples.

W O 9i/26010 PCT~US97/00759 - EXamD1~ 1 Pre~aration and Scr~; n~ of Anti-Factor IX ~noclonal Ant; h~ ; Q~
Female Balb/C mice were injected with human factor IX puri~ied as described in Jenny, ~. et al., Prep.Biochem. 16, 227-245 (1986). Typically, each mouse received an initial injection of 100 ug protein dissolved in 0.15 mL phosphate-buf~ered saline (PBS) and mixed with 0.15 mL complete Freund's adjuvant. Booster immunizations of 50 ug protein in 0.15 mL PBS with 0.15 mL incomplete Freund's adjuvant were given approximately biweekly over a 2-3 month period. After the ~inal boost, the mouse received 50 ug of Factor IX in PBS
three days before spleen/myeloma cell fusions. Spleen cells were isolated from an ;mmlln;zed mouse and fused with NS-l myeloma cells (Kohler, G. et al., Eur. ~.
Immunol . 6, 292-295 (1976)) using polyethylene glycol as described by Oi, V. T . et al . in "Selected Methods in Cellular Immunology," Mishell, B.B. and Shigii, S M., eds , Freeman Press, San Francisco. Following the fusion, the cells were resuspended in RPMI 1640 media containing 10% fetal calf sera and ali~uots were placed in each well o~ ~our 24-well plates cont~;n;ng 0.5 mL of peritoneal lavage cell-conditioned media. On the following day, each well received 1.0 m~ of 2 x 10-' M
hypoxanthine, 8 x 10-' M aminopterin and 3.2 x 10-5 M
thymidine in RPMI 1640 media containing 10% fetal calf sera. The cells were fed every 3-4 days by removing half o~ the media and replacing it with fresh media cont~; n; ng 1 x 10-~ M hypoxanthine and 1.6 x 10-5 M
thymidine.
Approximately two weeks later, 1.0 mL of hybridoma medium was removed from each well and tested ~or anti-Factor IX antibodies using an ELISA assay as described by Jenny, R. J. et al in Meth. Enzymol . 222, 400-416 (1993). Brie~ly, ~actor IX was immmobilized onto plastic wells of 96-well microtiter plates. Hybridoma CA 02243236 l998-07-l6 W O 97/26~10 PCT~US97/~07S9 supernatants or dilutions of purified antibody were then incubated in the wells. The wells were washed and the presence of antibody-antigen complexes detected with a goat anti-murine immunoglobulin second antibody conjugated to horseradish peroxidase and the chromogenic substrate o-dianisidine.
Wells containing anti-Factor IX antibodies were subcloned by limiting dilution and grown in 96-well plates. Supernatant from the cloned hybridoma cell cultures were screened for antibody to Factor IX by the ELISA assay described above and cells from positive hybridomas were expanded, ~rozen, stored in liguid nitrogen and then grown as ascitic tumors in mice.
Exam~l~ 2 Self-Limitin~ Effect of Anti-Coaoulation Factor Antibodie~ in Coaoulation The effect of increasing concentrations of anti-coagulation factor antibodies on activated partial thromboplastin time (aPTT) of human plasma was determined in a fibrometer (Becton-Dickinson Microbiology Systems, Cockeysville, Maryland) using saxter re~erence procedure LIsO293-J, 3/93 revision (Baxter Scientific, Edison, New Jersey).
Prior to the start of the experiment, 2 to 3 mL o~
0.02 M CaCl2 in a 5 mL tube were placed into the heating chamber of the fibrometer. Human plasma samples were either freshly drawn and kept on ice or reconstituted per the manufacturer's recommendation from Hemostasis Reference Plasma (American Diagnostics, Greenwich, Connecticut).
Unfractionated heparin from porcine intestinal mucosa (Sigma Chemical, St. Louis, Missouri), low molecular weight heparin from porcine intestinal mucosa (Lovenox~, enoxaparin sodium, Rhone-Poulenc Rorer Pharmaceuticals, Collegeville, Pennsylvania) or mAb anticoagulants were prepared as approximately 50 uM

W O 9i/26010 PCTrUS97tO0759 stock solutions and-serially diluted directly into the test plasma. A blank contA;n;ng plasma without anticoagulant was included as a re~erence.
Two fibroTube~ fibrometer cups were filled with 100 ul test plasma or 100 ul test plasma with anticoagulant and 125 ul of actin activated cephaloplastin reagent (Actin reagent, from rabbit brain cephalin in ellagic acid, available from Baxter Sc~entific), respectively and placed in the fibrometer wells at 37~C.
After one minute, 100 ul of actin reagent was transferred to a plasma-cont~;n;ng cup and the contents mixed several times with a pipette. After a 3 minute incubation, 100 ul of CaCl2, prewarmed at 37~C, was added to the plasma-actin reagent mixture using a Automatic Pipette/Timer-trigger (Becton-Dickinson). The clotting times were noted and the results in Fig. 1 are presented as clotting times as a ~unction of final concentrations of anticoagulant in the total assay volume o~ 300 ul.
The nominal concentration of Factor IX in the assay is 30-40 nM.
The results shown in Fig. 1 demonstrate the effect o~ increasing concentrations o~ the murine anti-Factor IX mAbs scl and BC2 on aPTT clotting times. Both mAbs inhibit clotting by prolonging the aPTT and both mAbs reach a final saturating effect on the aPTT. The ICso values are similar at ~35 nM and ~50 nM ~or BCl and BC2, respectively, but the difference in the maximum response to the two antibodies is marked. Saturating concentrations o~ BCl increases the aPTT by about 50~ to ~40 sec. BC2, on the other hand, increases the aPTT by 3.5-fold to about 90 sec. The therapeutic target zone used in anticoagulant therapy with heparin is highlighted. The results indicate that the two mAbs bracket the heparin therapeutic aPTT range.

W O9i/26010 PCTrUS97/007S9 The properties of mAbs BCl and BC2 are summarized in Table I. Each of the BC mAbs recognizes both the zymogen, Factor IX, as well as the active protease, Factor IXa, but only BC2 is capable o~
blocking both zymogen activation as well as protease activity. BCl and BC2 were ~ound to cross-react with Cynomologous monkey Factor IX. Additionally, BC2 also cross-reacted with rat Factor IX.
Table I. Summary o~ in vitro Properties o~ Anti-Factor IX mAbs BCl BC2 Binds Factor IX yes yes Binds Factor IXa yes yes Inhibits IX to IXa no yes conversion Inhibits IXa activity yes yes in Xase complex Co~actor re~uirementnone divalent metals Ca2t > Mn aPTTmax x 100% 150 350 aPTTnormal IC50, nM ~35 ~50 Species cross- monkey rat, monkey reactivity Isotype IgGl IgG2a W O 97126~10 PCTAUS97/007~9 The results shown in Fig. 2 demonstrate the effect of increasing concentrations o~ the anti-Factor IX mAbs 9E4(2)F4 and llG4(1)B9 on aPTT clotting times. The plasma for the assay was diluted to one-half the normal concentration, giving an initial aPTT of 45 seconds.
Both mAbs inhibit clotting by prolonging the aPTT and both mAbs reach a ~inal saturating effect on the aPTT.
Saturating concentrations of 9E4(2)F4 and llG4(1)B9 increases the aPTT to ~90 to 100 seconds for 9E4(2)F4 and to ~80 seconds for llG4(1)B9. The results indicate that the two mAbs are at the upper end of the heparin therapeutic aPTT range.
The results shown in Fig. 3 demonstrate the effect of increasing concentrations of the anti-Factor X mAbs HFXLC (vs. light chain epitope), HFXHC (vs. heavy chain epitope) and the anti-Factor XI mab HFXI on aPTT
clotting times. These mAbs were obtained from Enzyme Research Laboratories (South Bend, IN). The mAbs HFXLC
and HFXI inhibit clotting by prolonging the aPTT and both mAbs reach a final saturating effect on the aPTT.
The ICso value for HFXLC is ~40 nM; saturating concentrations increase the aPTT to ~60 seconds. The IC5~ value for HFXI is ~20 nM; saturating concentrations increase the aPTT to ~100 seconds. The results indicate that HFXLC is within the heparin therapeutic aPTT range while HFXI falls at the upper end of the heparin therapeutic range. The mAb HFXHC had no ef~ect on aPTT
clotting times.
Self-limiting prolongation of the aPTT was also observed with antibodies to Factor VIII, the cofactor to Factor IXa. For example, the anti-human Factor VIII
antibody, SAF8C-IG, purchased ~rom A~finity Biologicals, Inc , increased the aPTT to a maximum of about 65 sec.
Half-maximal prolongation of the aPTT was achieved with about 100 nM antibody.

W O 97/26010 PCTrUS97/00759 -Exam~le 3 Ef ~icac~.r o:~ murine Factor IX mAb5 in Rat Thrombus M~
In order to evaluate the e~icacy o~ anti-Factor IX
antibodies in prevention o~ arterial thrombosis, the rat carotid artery thrombosis model as reported by Schumacher et al. in ~. Cardio. Pharm. 22, 526-533 (1993) was adapted. This model consists o~ segmental injury to the carotid endothelium by oxygen radicals generated by FeCl3 solution applied on the surface o~ the carotid artery.
In brief, rats were anesthetized with pentobarbitone sodium, the jugular vein cannulated ~or intravenous injections and the le~t ~emoral artery cannulated ~or blood pressure and heart rate monitoring.
The carotid artery was isolated by aseptic technique via a surgical incision in the neck and equipped with a magnetic ~low probe ~or blood ~low measurement. A~ter a period o~ stabilization, baseline parameters were established ~or the following variables: carotid blood ~low, arterial pressure, heart rate, activated partial thromboplastin time (aPTT) and prothrombin time (PT).
Therea~ter, a premeasured Whatman ~ilter paper soaked in 50~ FeCl3 solution was placed on the carotid artery ~or 15 minutes ~or complete injury of the underlying endothelial cells. A~ter removal o~ the FeCl3 soaked paper, the experiment was ~ollowed to completion over 60 minutes. At the end o~ the experiment, the carotid thrombus was extracted ~rom the carotid artery and weighed.
All agents were administered 15 minutes prior to the onset o~ carotid injury. The ~ollowing treatments were examined and compared to the Factor IX mAb BC2.
1. Heparin: 15, 30, 60 or 120 U~kg bolus, ~ollowed by in~usion o~ 0.5, 1, 2 or 4 U/kg/min, respectively over 60 minutes 2. Acetylsalicylic acid (ASA, aspirin): 5 mg/kg bolus 3. Anti-Factor IX m~b BC2: 1, 3 or 6 mg/kg bolus, ~ollowed by in~usion 0.3, 1, or 2 ug/kg/min, respectively over 60 minutes 4. Heparin: 3OU/kg bolus + lU/kg/min + ASA at 5 mg/kg 5. Anti-Factor IX mAb BC2: 1 mg/kg + 0.3 ug/kg/min + ASA at 5 mg/kg Figs. 4 and 5 demonstrate the comparative pharmacology o~ the anti-coagulant/thrombotic regimens by showing the e~ect o~ heparin, ASA and Factor IX mAb BC2 on aPTT ~Fig. 4) and PT (Fig. 5).
The key index for bleeding diathesis, aPTT, was used as the primary criterion ~or evaluation o~ e~icacy versus bleeding liabilities o~ the anti-coagulant/thrombotic agents used in the study. The results in Fig. 4 demonstrate the dose-dependent prolongation o~ aPTT by heparin with maximal prolongation of the clotting time, beyond the test limit, at the two higher doses. ASA alone did not signi~icantly increase aPTT but in combination with heparin, a marked synergistic e~ect was observed. The Factor IX m-Abs had a modest e~ect on aPTT and even at the highest dose, the increase in clotting time did not exceed the 3-~old limit o~ standard anti-coagulant practiced clinically. Most notably, the low dose o~
Factor IX -m-Ab BC2 in co-m-bination with ASA did not change the aPTT.
In Fig. 5, the data indicate that PT was also 3Q signi~icantly prolonged by heparin, at the two higher doses, and by the ASA + heparin combination, but not by any o~ the Factor IX mAb doses alone or in combination with ASA.
The e~ect o~ heparin, ASA and Factor IX mAb on carotid artery occlusion is shown in Fig. 6 The results indicate that the carotid arteries o~ all o~ the vehicle-treated ~n;m~l S occlude in response to the W O 97/26010 PCTrUS97/00759 injury. Heparin dose dependently inhibited the occlusion o~ the carotid artery. At the highest dose, heparin completely prevented the occlusion o~ the carotid artery; at this dose however, no coagulation could be initiated. ASA alone had only a minor e~ect on carotid occlusion. ASA in combination with heparin also failed to completely prevent carotid occlusion.
Factor IX mAb completely blocked carotid occlusion at the two higher doses, which have not prolonged coagulation beyond the clinically desired target. The lower dose o~ Factor IX mAb, that largely ~ailed to secure patency alone, demonstrated complete inhibition o~ carotid occlusion when administered in combination with ASA.
The e~ect o~ heparin, ASA and Factor IX mAb on thrombus weight is shown in Fig. 7. Heparin dose-dependently reduced thrombus mass in the carotid artery. However, some residual thrombus was still ~ound in the carotid artery in spite o~ complete blockade o~
coagulation. ASA alone or in combination with heparin (30 U/kg regimen) had only a partial e~ect on thrombus weight. Factor IX mAb dose-dependently reduced thrombus mass and the high dose virtually prevented completely thrombus formation. Moreover, the combination o~ the low dose anti-Factor IX mAb and ASA, a regimen that completely prevented carotid occlusion without adversely a~ecting the coagulation indices, completely prevented thrombus formation.
The studies conducted in the rat carotid thrombosis model clearly demonstrate the e~icacy o~ Factor IX mAb in prevention o~ thrombosis in a highly thrombogenic arterial injury model. Most notably, the ef~icacy o~
the Factor IX mAb was demonstrated within the desired therapeutic anticoagulant target de~ined by the aPTT.
Furthermore, heparin, the current standard anticoagulant, reached e~icacy comparable to Factor IX
mAb only at doses that severely compromised coagulation W O 97126010 PCTrUS97/007~9 to the extent of producing non-coagulable blood.
Interestingly, the observed potentiation and synergy acquired by ASA joint treatment with heparin was also demonstrated when ASA was given with anti-Factor IX mAb.
However, unlike the combination of heparin and ASA which resulted in potentiation of both the anti-thrombotic and anti-coagulant e~fects, the combination of Factor IX mAb and ASA resulted in potentiation o~ the anti-thrombotic efficacy with no consistent e~fect on ex vivo blood coagulation parameters. Taken together, the data show a superior antithrombotic capacity of Factor IX mAb compared to heparin, ASA or a combination of heparin and ASA.

Exam~le 4 5~; n~ Electron Microsco~Y of Rat Thrombosis Model Segments of rat carotid artery were collected from sham, ferric chloride only and ferric chloride + 6 mg/kg Factor IX antibody, 3/group, 15 minutes after application of ferric chloride. The arteries were ~ixed by perfusion with formaldehyde and ligated above and below the lesioned area. Fixed arteries were dehydrated, incubated in hexamethyldisilazane and dried in a desiccator. Dried arteries were opened lengthwise, placed on Scanning Electron Microscopy (SEM) stubs and sputter coated with gold.
SEM of sham arteries revealed an essentially normal endothelium with rare scattered platelets. There were a few breaks in the endothelium, probably as a result of mechanical damage during surgery and the underlying basement membrane was covered by a carpet of platelets.
No evidence of thrombus formation was observed in the sham rats.
SEM of the arteries treated with ~erric chloride revealed large mural thrombi which occupied a large portion of the lumen of the vessel. The thrombi were composed of aggregated platelets, red blood cells and W O 9i/26010 PCTnUS97/00759 amorphous and fibrillar proteinaceous material. The proteinaceous material is consistent with fibrin. The endothelium of the arteries was mostly obscured by the large thrombi. Where visible, the endothelium overlying the region treated with ferric chloride was covered by numerous adherent platelets and amorphous proteinaceous material.
SEM of the arteries treated with ferric chloride from rats also treated with Factor IX antibody, revealed the lumen of the vessels to be largely free of thrombus.
The endothelium overlying the region treated with ferric chloride showed extensive damage and some areas were covered by adherent platelets and platelet aggregates but there was little or no proteinaceous material.
E~am~l~ 5 ~nti-Factor IX mAb BC2 Heaw and Li~ht Chain cDNA
~eouence AnalY~is Total RNA was purified by using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's protocol. RNA was precipitated with isopropanol and dissolved in 0.5% SDS
and adjusted to 0.5M NaCl. Poly A+ RNA was isolated with Dynabeads Oligo (dT)2s (Dynal A.S., Lake Success, NY) according to the manufacturer's protocol. Poly Af RNA was eluted ~rom the beads and resuspended in TE
buffer. Twelve aliquots of 100 ng of RNA were reverse transcribed with a RT-PCR kit per the manufacturer~s instructions (Boehringer M~nn~eim Cat. No. 1483-188) using a dT oligo for priming For the heavy chain, PCR
amplifications of 6 RNA/DNA hybrids were carried out for 25 cycles using a murine IgG2a hinge primer (SEQ ID NO:
1) and a heavy chain signal sequence primer (SEQ ID NO:
2~. Similarly, for the light chain, PCR amplificatons of 6 RNA/DNA hybrids were carried out for 25 cycles using a murine kappa primer (SEQ ID NO: 3) and a degenerate light chain signal sequence primer (SEQ ID

CA 02243236 l998-07-l6 W O 97/26010 PCT~US97/007~9 NO: 4). The PCR products from each of the 12 amplifications were ligated in a PCR2000 vector ~TA
cloning Kit, Invitrogen, Cat. No. K2000-01). Colonies of recombinant clones were randomly picked and minipreparations of plasmid DNA were prepared using an alkaline extraction procedure described by Birnboim and Doly in Nucl . Acids Res . 7, 1513 (1979). The isolated plasmid DNA was digested with EcoRI and analyzed on a O.8% agarose gel. Double-stranded cDNA inserts o:E the appropriate size, i.e , -700 bp for the heavy chain and ~700 bp for the light chain, were se~uenced by a modification of the Sanger method. The seguence of all 12 of the heavy and light ChA; n-~ were compared to generate a consensus BC2 heavy chain variable region sequence (SEQ ID NO: 5)and consensus BC2 light chain variable region sequence ( SEQ ID NO: 6).
Sequence analysis of the BC2 heavy chain variable region cDNA revealed a 363 nucleotide open reading frame encoding a 121 amino acid sequence (SEQ ID N0: 7). The 20 heavy chain CDRl, 2 and 3 sequences are listed in SEQ ID
NOs: 8, 9 and 10, respectively.
Se~uence analysis of the BC2 light chain variable region cDNA revealed a 321 nucleotide open reading frame encoding a 107 amino acid sequence (SEQ ID NO: 11). The 25 light chain CDRl, 2 and 3 sequences are listed in SEQ ID
NOs: 12, 13 and 14, respectively.

~x~l~ 6 ~ ~;zed Antibodie Six humanized antibodies designated SB 249413, SB
249415, Ss 249416, SB249417, Ss 257731 and SB 257732 were designed to contain the murine CDRs described above in a human antibody framework.

SB 249413 contains the heavy chain F9HZHC 1-0 and the light chain F9HZLC 1-0. The synthetic variable region humanized heavy chain F9HZHC 1-0 was designed W O 97/26010 PCTrUS97/00759 using the first three framework regions of the heavy chain obtained ~rom immunoglobulin RF-TS3'CL (Capra, J.D. et al., 3. Clin. Invest. 86, 1320-1328 llg9Q) identified in the Kabat database as Kabpro:HhclOw) and the BC2 heavy chain CDRS described previously. No framework amino acids substitutions which might influence CDR presentation were made. Four overlapping synthetic oligonucleotides were generated ( SEQ ID NOs:
15, 16, 17 and 18) which, when annealed and extended, code ~or the amino acids representing the heavy chain variable region through and including CDR3 (SEQ ID NOs:
19 and 20). This synthetic gene was then amplified using PCR primers (SEQ ID MOs: 21 and 22) and ligated into the pCR2000 vector (TA cloning Kit, Invitrogen, Cat. No. K2000-01) and isolated ~rom a ~peI, KpnI
restriction digest. A second DNA fragment coding for the campath signal se~uence including the first five amino acids of the variable region (SEQ ID NOs: 23 and 24) was made by PCR amplification of the appropriate region of a construct encoding a humanized anti-Respiratory Syncitial Virus heavy chain (SEQ ID N0: 25) with two primers (SEQ ID NOs: 26 and 27) and digesting with the restriction enzymes EcoRI and SpeI. The two fragments generated were ligated into an EcoRl, KpnI
digested pFHZHC2-6pCD mammalian cell expression vector which contained the remainder o~ a human consensus framework 4 and IgCl constant region. The vector contained a single amino acid mutation of the pFHZHC2-3pCD vector described in published International Patent Application No. W094/05690. The final residue of framework 2 (residue 49) was mutated from Ser to Ala by digesting pFHZHC2-3pCD with XbaI and EcoR5 and inserting a linker generated from two synthetic oligonucleotides (SEQ ID NOs: 28 and 29). The sequence of the F9HZHC 1-0 insert is shown in SEQ ID NOs: 30 and 31.
The synthetic variable region humanized light chain F9HZLC 1-0 was designed using the framework regions of WO 97/26010 PC~US97100759 the human light chain obtained ~rom immunoglobulin LS8'CL (Carmack et al ., ~. Exp. Med. 169, 1631-1643 (1989) identi~ied in the Kabat database as Kabpro:Hkl318) and the BC2 light chain CDRs described previously. No ~ramework amino acids substitutions which might in~luence CDR presentation were made. Two overlapping synthetic oligonucleotides were generated (SEQ ID NOs: 32 and 33) which, when annea~ed and extended, code ~or amino acids representing the light chain variable region (SEQ ID NOs: 34 and 35). This synthetic gene was then amplified using PCR primers (SEQ
ID NOs: 36 and 37~ and ligated into the pC~2000 vector (TA cloning Kit, Invitrogen, Cat. No. K2000-01), and isolated from a ScaI, SacII restriction diyest. A
second DNA ~ragment coding ~or the campath signal sequence including the ~irst two amino acids o~ the variable region (SEQ ID NOs: 38 and 39) was made by PCR
amplification o~ the the appropriate region of a construct encoding a humanized anti-Respiratory Syncitial Virus heavy chain (SEQ ID NO: 25) with the two primers (SEQ ID NOs: 26 and 40) and digesting with the restriction enzymes EcoRI and ScaI. The two ~ragments generated were ligated into an EcoRl, ~acII digested pFHzLCl-2pCN m~mm~ 1 ian cell expression vector which contained the remainder o~ a human framework 4 and kappa constant region. The vector contained a single amino acid mutation o~ the pFHZLCl-lpCN vector described in published International Patent Application No.
W094/05690. A ~ramewor~ 2 residue was mutated ~rom Ser to Pro by digesting pFHZLCl-pCN with SmaI and Kpnl and inserting a linker generated ~rom two synthetic oligonucleotides (SEQ ID NOs: 41 and 42). The se~uence o~ the F9HZLC 1-0 insert is shown in SEQ ID NOs: 43 and 44.

SB 249415 contains the heavy chain F9HZHC 1-1 and the light chain F9HZ~C 1-1. These heavy and light chain -W ~ 97/26010 PCTrUS97/00759 constructs are based on F9HZHC 1-0 and F9HZLC 1-0, respectively, however, they have framework amino acid substitutions which can influence CDR presentation.
F9HZHC 1-1 has three framework amino acid substitutions which might influence CDR presentation.
Two overlapping synthetic oligonucleotides were generated (SEQ ID NOs: 45 and 46), which when annealed and extended, code for amino acids representing the altered portion of the heavy chain variable region altered (SEQ ID NOs: 47 and 48). This synthetic gene was then amplified using PCR primers (SEQ ID NOs: 49 and 50), ligated into the pCR2000 vector (TA cloning Kit, Invitrogen, Cat. No. K2000-01) and isolated from a EcoNI, KpnI restriction digest. This fragment was ligated into EcoNI, ~pnI digested F9HZHCl-0 (SEQ ID NO:
30) vector. The sequence of the F9HZHC 1-1 insert is shown in SEQ ID NOs: 51 and 52.
F9HZLC 1-1 has four framework amino acids substitutions which can influence CDR presentation. Two synthetic oligonucleotides were generated (SEQ ID NOs:
53 and 54), which when annealed, have KpnI and BamHI
cohesive ends, and code for amino acids representing the altered portion of the light chain variable region (SEQ
ID NO: 55~. F9HZLC 1-0 -(SEQ ID NO: 43) was digested with the restriction enzymes K~nI and BamXI and ligated to the synthetic DNA. The sequence of the F9HZLC 1-1 insert is shown in SEQ ID NOs: 56 and 57.
SB 2~9416 SB 249416 contains the heavy chain F9HZHC 1-1 ldescribed above) (SEQ ID NO: 52) and the light chain F9XZLC 1-2. The light chain construct is based on F9HZLC 1-1, however, it has one additional framework amino acid substitution which can influence CDR
presentation.
Two synthetic oligonucleotides were generated (SEQ
ID NOs: 58 and 59), which when annealed, have BamXI and X~aI cohesive ends and code for amino acids representing W O 9tl26010 PCT~US97/00759 the altered portion-of the light chain variable region (SEQ ID MO: 60). F9HZLC 1-1 ~SEQ ID NO: 56) vector was digested with the restriction enzymes BamHI and XbaI and ligated to the synthetic DNA. The se~uence o~ the F9HZLC 1-2 insert is shown in SEQ ID NOs: 61 and 62.

SB 249417 contains the heavy chain F9HZHC 1-1 (described above) (SEQ ID NO: 52) and the light chain F9HZLC 2-0. A F9HZLC 2-0 synthetic variable region humanized light chain was designed using the framework regions of the human light chain obtained from immunoglobulin REI (Palm and Hilschmann, Z. Physiol.
Chem. 354, 1651-1654 (1973) identi~ied in the Kabat database as Kabpro: HKL111) and the BC2 light chain CDRs described previously. Five amino acid consensus human substitutions were introduced. Six framework amino acids murine substitutions which can influence CDR
presentation were made. Two overlapping synthetic oligonucleotides were generated (SEQ ID NOs: 63 and 64) which, when annealed and extended, code for amino acids representing the light chain variable region (SEQ ID
NOs: 65 and 66). This synthetic gene was then ampli~ied using PCR primers (SEQ ID NOs: 67 and 68), ligated into the pCR2000 vector (TA cloning Kit, Invitrogen, Cat. No.
K2000-01) and isolated from a ScaI, SacII restriction digest. A second DNA ~ragment coding for the campath signal se~uence including the ~irst two amino acids o~
the variable region (SEQ ID NO: 38) was made by PCR
amplification of the the appropriate region o~ a 3Q construct encoding a humanized anti-Respiratory Syncitial Virus heavy chain (SEQ ID NO: 25) with two primers (SEQ ID NOs: 26 and 69) and digesting with the restriction enzymes EcoRI and ScaI. A third DNA
fragment encoding the remainder o~ a human framework 4 (SEQ ID NO: 70) and having SacII and NarI cohesive ends was generated by annealing two synthetic oligonucleotides (SEQ ID NOs: 71 and 72). E9HzLC 1-0 W O 97/2601~ PCTrUS97/0~759 (SEQ ID NO: 43) was-digested with the restriction enzymes EcoRI and NarI and ligated to the three DNA
~ragments. The se~uence o~ the F9HZ~C 2-0 insert is shown in SEQ ID NOs: 73 and 74.
S~ 257731 SB 257731 contains the heavy chain F9HZHC 1-1 (SEQ
ID NO: 52) and the light chain F9HZLC 1-3, a single amino acid mutation of F9HZLC 1-2 (SEQ ID NO: 62).
F9HZLC 1-2 was PCR ampli~ied with two primers (SEQ ID
NOs: 26 and 69) and digested with the restriction enzymes EcoRI and ScaI . A 9 4 bp ~ragment (SEQ ID NOs:
75 and 76) was isolated. The ~ragment was ligated into EcoRI, ScaI digested F9HZLC 1-2 vector to produce the light chain construct F9HZLC 1-3. The se~uence o~ the F9HZLC 1-3 insert is shown in SEQ ID NOs: 77 and 78.
S~ 257732 SB 257732 contains the synthetic variable region humanized heavy chain F9HZHC 3-0 and light chain F9HZLC
3-0. Four overlapping synthetic oligonucleotides were generated (SEO ID NOs: 79, 80, 81 and 82) which, when annealed and extended, code ~or the amino acids representing the heavy chain variable region being altered (SEQ ID NOs: 83 and 84). This synthetic gene was then ampli~ied using PCR primers (SEQ ID NOs: 85 and 86~, ligated into the pCR2000 vector (TA cloning Kit, Invitrogen, Cat. No. K2000-01) and isolated from a StuI, RpnI restriction digest. The isolated ~ragment was llgated into StuI, KpnI digested F9HZHCl-l (SEQ ID NO:
52) vector. This vector was then digested with EcoRI, 3 0 SpeI to remove the signal se~uence. A DNA ~ragment coding ~or the campath signal se~uence (SEQ ID NO: 23) including the ~irst five amino acids o~ the variable region was made by PCR amplification o~ F9HZHCl-0 with < two primers (SEQ ID NOs: 26 and 87) and digesting with 35 the restriction enzymes EcoRI and SpeI. The ~ragment generated was ligated into the vector. The se~uence o~
the F9HZHC3-0 insert is shown in SEQ ID MOs: 88 and 89.
4g W O 97/26010 PCT~US97100759 Four overlapping synthetic oligonucleotides were generated (SEQ ID NOs: 90, 91, 92 and 93) which, when annealed and extended, code for amino acids representing the light chain variable region (SEQ ID NOs: 94 and 95).
This synthetic gene was then amplified using PCR primers lSEQ ID NOs: 96 and 97) and ligated into the pCR2000 vector (TA cloning Kit, Invitrogen, Cat. No. K2000-01), and isolated from a ScaI, NarI restriction digest. The isolated fragment was ligated into ~caI, NarI digested F9HZLCl-3 (SEQ ID NO: 77) vector. The sequence of the F3XZLC3-0 insert is shown in SEQ ID NOs: 98 and 99.
The humanized anti-Factor IX mAbs were expressed in CHO cells. A DG-44 cell line adapted for suspension growth in serum-free medium was grown in lOOml of protein-free medium cont~in;ng lX nucleosides and 0.05%
F58 in 250 ml disposable sterile erlenmeyer flasks (Corning) on a Inno~a 2100 plat~orm shaker (New Brunswick Scientific) at 150 rpm at 37~C in a 5% CO2, 95 air humidified incubator. These cells were passaged at 4 X 105 cells/ml twice weekly. 15 ug each of the pCN-Lc-Light Chain and pCD-Hc-heavy chain vectors were linearized by digestion with Notl, co-precipitated under sterile conditions and resuspended in 50ul of lX TE
buffer (lOmM Tris, lmM EDTA, pH 7.5). The DNA was electroporated using a sio-Rad Gene Pulser (Bio-Rad Laboratories) into the Acc-098 cells using the techni~ue of Hensley et al. in ~. Biol. Chem. 269, 23949-23958 ~1994~. 1.2 X 107 cells were washed once in 12.5 ml of ice cold PBSucrose (PBS, 272mM sucrose, 7mM sodium phosphate pH 7.4, lmM MgCl2), resuspended in 0.8 ml of PBS, added to 50ul of the DNA solution and incubated on ice for 15 min. The cells were pulsed at 380 V and 25 microfarads, then incubated on ice for 10 min. Cells were plated into 96 well culture plates at 5 X 105 cells/plate in maintenance medium for 24 hr prior to selectio~. Cells were selected for resistance to W O g7/26010 P~T~US97/00759 400ug/ml G418 (Geneticin, Li~e Technologies, Inc.) in maintenance medium. 24 hr prior to assay, the cells were ~ed with 150ul of the maintenance medium.
Conditioned medium ~rom individual colonies was assayed using an electrochemiluminescence (ECL) detection method on an Origen analyzer (IGEN, Inc.).
See Yang et al., Biotechnology 12, 193-194 (1994).
All solutions necessary for the per~ormance of the assays (assay buf~er) and for the operation o~ the analyzer (cell cleaner) were obtained ~rom IGEM. The antibodies (anti-human IgG (g-chain specific), Sigma Chemicals and F(ab') 2 Fragment to Human IgG (H+L), Kirkegaard & Perry Laboratories Inc.) were labelled with TAG-NHS-ester (IGEN, Inc.) at a 7:1 molar ratio o~
TAG:protein, while the Protein A (Sigma) was labelled with Biotin-LC-Sul~o-NHS-ester (IGEN, Inc.) at a 20:1 molar ratio Biotin:protein, both according to IGEN's recommendations. Streptavidin-coated magnetic beads (M-280) were obtained ~rom Dynal.
Immunoassays were performed using the following protocol: per sample, 50ul o~ the Streptavidin-coated beads (final concentration 600 ug/ml diluted in PBS, pH7.8, with 1.25~ Tween) were mixed with 50ul Biotin-Protein A (~inal concentration lug/diluted in PsS, 25 pH7.8, with 1.25% Tween) and incubated at room temperature ~or 15min with agitation, 50ul of the TAG
antibodies (a mixture with a ~inal concentration of 1.25 ug/ml F(ab' )2 Fragment to Human IgG (H+L) and 0.25 ug/ml Anti-Human IgG (g-chain speci~ic) diluted in PBS, p~7.8, with 1.25~ Tween) were added, the solution was then added to 50ul o~ conditioned medium and incubated with agitation at room temperature ~or 1 hr. 200ul o~ assay bu~er was added to the reaction mix and the sample analyzed on the Origen I analyzer to measure ECL. The results indicated that approximately 20-37% of the colonies assayed secrete over 15 ng/ml of the antibody with an average expression o~ about 150 ng/ml.

Humanized anti-Factor IX mAbs were purified from the conditioned media using a Procep A capture step followed by ion-exchange chromatography to reduce the DNA burden. Procep A sorbent material (Bioprocessing Ltd., Durham, England) was used to prepare a column with a 1:1 diameter to height ratio. Clari~ied conditioned media was loaded onto the column at about 150 cm/hr.
The column was washed sequentially with phosphate buffered saline (PBS), PsS containing 1 M NaCl, and finally with PBS. The bound material was recovered with 0.1 M acetic acid elution. The eluate was adjusted to pH 5.5 and was diluted (1:4) with water. The diluted solution was loaded onto an S-Sepharose column (2.5 x 13 cm) which was pre-equilibrated with 20 mM sodium acetate, pH 5.5 at 80 cm/hr The column was washed with the acetate buffer until a steady baseline was obtained and the bound protein was eluted with 20 mM sodium phosphate, pH 7.4 at 25 cm/hr. The eluted material was filtered with a 0.4 micron membrane and stored at 4~C.

Exam~le 7 ~ouse~ ~h~ --icA~ltibody 100 ng of BC2 RNA were reverse transcribed with a RT-PCR kit per the manufacturer's instructions 25 ~Boehringer M~nnheim Cat. No. 1483-188) using a dT oligo for priming, and PCR amplified with synthetic ScaI ( SEQ
ID NO: 100) and NarI (SEQ ID NO: 101) primers to produce the BC2 light chain variable region with Scal, Narl ends {SEQ ID NOs: 102 and 103). This DNA was ligated into 30 ~caI, NarI digested F9HZHC1-3 (SEQ ID 77) and digested with ScaI, NarI to produce a mouse-human ch;m~ic light chain F9CHLC (SEQ ID NOs: 104 and 105).
100 ng o~ BC2 RNA were reverse transcribed with a RT-PCR kit per the manufacturer~s instructions 35 ~Boehringer M~nnheim Cat. No. 1483-188) using a dT oligo W O 97/26010 PCT~US97/007~9 for priming, and PCR amplified with synthetic ~peI (SEQ
ID NO: 106) and NheI (SEQ ID NO: 107) primers to produce the BC2 heavy chain variable region with Spel, Nhel ends (SEQ ID NOs: 108 and 109). The campath signal se~uence was PCR amplified from the RSVHZ19 heavy chain (SEQ ID
NO: 2~) with EcoRI (SEQ ID 26) and SpeI ( SEQ ID 87) primers. These two DNA fragments were ligated into a E~oRI, NheI digested IL4cHHcpcd vector described in published International Patent Application No.
WO~/07301, replacing the IL4 variable region with the BC2 Factor IX mouse variable region, to produce a mouse-human chimeric heavy chain F9CHHC (SEQ ID Nos: 110 and 111 ) .
Co-transfection and purification of the mouse-human chimeric antibody chaFIX was accomplished as described above ~or the humanized constructs.

Exam~le 8 E~ icacY of ~ ~;zea Factor IX mAbs in Rat Thrombus MOdQ1 In order to evaluate the ef~icacy of humanized anti-Factor IX antibodies in prevention of arterial thrombosis, the rat carotid artery thrombosis model as described above in Example 3 was used. Baseline parameters were established for carotid blood ~low, arterial pressure, heart rate, vessel patency and activated partial thromboplastin time (aPTT). Fifteen minutes thereafter, carotid injury was effected for 10 minutes. The parameters were determined 60 minutes a~ter onset o~ carotid injury. Carotid thrombus was also extracted from the carotid artery and weighed.
All agents were administered intravenously 15 ~ minutes before the onset of carotid injury The following treatments were examined and compared to the anti-Factor IX mAb BC2.

-W O 97/26010 P~TAUS97/00759 1. Vehicle 2. ch~FIX: 3 mg/kg bolus 3. SB 249413: 3 mg/kg bolus 4. SB 249415: 3 mg/kg bolus 5. SB 249416: 3 mg/kg bolus 6. SB 249417: 3 mg/kg bolus 7. SB 257731: 3 mg/kg bolus 8. Heparin: 60 units/kg bolus + 2 units/kg/min in~usion The aPTT was used as the primary criterion ~or evaluation o~ e~icacy versus bleeding liabilities o~
the anti-coagulant/thrombotic agents used in the study.
The results in Fig 8 demonstrate that the humanized Factor IX mAbs SB 249413, SB 249415, SB 249416, SB
249417 and SB 257731 had a modest e~~ect on aPTT at 3.0 mg/kg which is within the clinical accepted range.
The e~ect o~ the Factor IX mAbs on thrombus mass is shown in Fig. 9. The results indicate that all o~
the humanized mAbs are equally e~ective in reducing thrombus mass.
The studies conducted in the rat carotid thrombosis model clearly demonstrate the e~icacy o~ the humanized Factor IX mAbs in prevention o~ thrombosis in a highly thrombogenic arterial in~ury model. Most notably, the e~icacy of all o~ the humanized Factor IX mAbs was demonstrated within the desired therapeutic anticoagulant target de~ined by the aPTT.

Exam~le 9 AntibodY Biochemical and Bio~hYsical ProDerties The molecular mass o~ SB 249417 was determined by MALD-MS to be 148,000Da. Analytical ultracentri~ugation of SB 249417 gave an identical value In the presence o~ Factor IX plus Ca2+, the antibodies derived ~rom B~ 2 sedimented with a mass o~ 248,0003a corresponding to the W O 97~26010 PCTrUS97/00759 combined mass of the mAb and two molecules of Factor IX.
No evidence of higher ordered aggregates was observed in the presence or absence of Factor IX.
The kinetics of Factor IX binding to SB 249417 was assessed by BIAcore analysis with antibody bound to an immobilized protein A surface. Recombinant human Factor - IX ~rhFIX, Genetics Institute) at 49 nM was used and measurements performed in the presence of 5 mM Ca2+.
The interaction was characterized by rapid association, kass = 2.0 x 105 M-1 s-1 and relatively slow off-rate, kdiss = 4.1 x 10-4 s-1. The calculated Kd for Factor IX
binding was 1.9 nM.
Table 1 summarizes the biophysical properties of SB
249417.
Tablo 1 5~ ~y o~ the Biophysical Properties of SB 249417 Isotype IgG1, kappa 20 Purity by SDS-PAGE >95% (under reducing conditions) Molecular Weight Mass Spectrometry 148,000 Da Analytical Ultracentrifugation 148,000 Da Stoichiometry of Factor IX Binding Isothermal Titration ~alorimetry 1.5 moles Factor IX: 1 mole mAb Factor IX Binding Affinity Isothermal Titration Calorimetry Kd= 4 nM at 25~C
Biosensor Kd= 2 nM
35 Factor IX Binding Kinetics Biosensor kaSS = 2.0 x 105 M-1 s 1 kdiSS =4 X 10-g s-W O97/26010 PCT~US97/007~9 Table 2 summarizes the ~actor IX binding properties o~ mAbs of the present invention. The calculated dissociation constants were essentially identical within experimental error.
Table 2 Kinetic~ o~ ~actor IX R; ~i n~ to Anti-Factor IX mAbs JQa~. k,~"~3 (L~ k~i88 ( ~~ 1 C . i~;~ (D~
SB 249417 2.0 x105 4.1x10-4 1.9 BC2 4.8x105 9.1x10-4 1.9 Ch~9 2.4 x105 3.0x10-4 1.3 SB 249413 6.5 x105 2.8x10-33.7-5.1 SB 249415 7.5 x105 1.8x10-41.1-2.3 SB 249416 5.2 x105 4.1x10-4 0.8 SB 257731 9.2 x105 9.9xlO-4 1.1 SB 257732 1.1 X106 1.2x10-3 1.5 The interactions between rhFIX and SB 249417 , BC2 and other humanized constructs were characterized by titration microcalorimetry, which measures binding interactions in solution ~rom the intrinsic heat of binding. Nine injections o~ 106 uM FIX were made into the calorimeter cont~;n;ng 2 uM mAb SB 249417. Binding was detected in the ~irst 4 injections as exothermic heats. At the last 5 injections the mAb binding sites were saturated with FIX and only background heats o~
mixing were observed. The results indicated that the e~uivalence point occurred at a molar binding ratio near 2 FIX per mAb, as expected Nonlinear least s~uares analysis of the data yield the binding a~inity.
The rhFIX a~inities o~ the mAbs were measured over a range o~ temperature ~rom 34-44~C in lOmM HEPES, lOmM
CaC12, 150mM NaCl, pH 7.4. These data allow the a~inity at 37~C to be determined directly and the W O 97126010 PCTrUS97/00759 affinity at 25~C to be calculated from the van't Hoff e~uation. The data in Table 3 indicate that the affinities of SB 249417, BC2 and its other humanized constructs are within error (a factor of 2) the same.
Table 3 Titration Calorimotry Results i~or Anti-FIX mAb8 mAb Kd, nM at 25~C Kd, nM at 37~C Molar Binding Ratio FIX/mAb BC2 10 20 1.4 SB 6 12 1.9 SB 3 7 1.7 SB 4 12 1.5 SB 4 9 1.8 The mAbs SB 249413, SB 249415, SB 249417 and SB
257732 all exhibited very similar thermal stabilities by differential 5c~nn; ng calorimetry. Their unfolding Tms ranged from 70-75~C indicating high stability against thermally induced denaturation.

Exam~le 10 M~h~ism o~ AntibodY-Meaiated Inhibition o~ Factor IX
A library of chimeric constructs composed of se~uences of Factor IX spliced into the framework of the homologous protein Factor VII was constructed and used to map the epitope for the Factor IX BC2 mAb. See ~heung et al ., Thromb. Res . 80, 419-427 (1995). Binding was measured using a BiaCore 2000 surface plasmon res3nance device. The BC2 antibody was coupled directly to the chip using the NHS/EDC reaction. Binding was W O 91/26010 PCTrUS97/00759 measured by 2 min of contact time at 20uL/min with 200 nM of each of the given constructs in 25 mM MOPS, pH
7.4, 0.15 M NaCl, 5 mM CaC12. Dissociation was monitored for 3 min using the same buffer with no protein. No binding was detected to the wild type construct in the presence of 50 mM EDTA. The data are presented in Table 4.
Tab1Q 4 S~ -~y of R;~;n~ o~ Factor IX Constructs to BC2 Antibody Construct Deyree o~ R;~;~
Plasma IXa Binds r-IX Binds Plasma VII No Binding IX LC/VII HC Binds IX-A/VII Binds VII gla/IX No Binding VII-A/IX No Binding VII gla (IX 3-11)/IXBinds VII gla (IX 3-6)/IXVery Low Binding VII gla (IX 9-11)/IXVery Low Binding IX K5A Binds These data indicate that the constructs containing the Factor IX light chain and Factor VII heavy chain (IX
LC/VII HC); the Factor IX gla and aromatic stack domains ~IX-A~VII); residues 3-11 of Factor IX gla domain within the Factor VII gla domain (VII gla (IX 3-11)/IX); and Factor IX having a lysine to alanine substitution at residue 5 (IX K5A) exhibit binding to BC2. The VII gla {IX 3-11)/IX construct exhibited BC2 binding equivalent to wild type Factor IX (plasma IXa and r-IX). Thus, the W O 97/26010 PCTrUS97/00759 .
BC2 antibody binds to an epitope contained within residues 3-11 of the Factor IX gla domain.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

CA 02243236 l998-07-l6 W 097~6010 PCTrUS97/~0759 ~Q~N~ LISTING

(l) GENERAL INFORMATION

(i) APPLICANT: Blackburn, Michael Church, William Gross, Mitchell Feuerstein, Giora Nichols, Andrew Padlan, Eduardo Patel, Arunbhai Sylvester, Daniel (ii) TITLE OF THE INVENTION: ANTICOAGULANT AGENTS USEFUL IN TREATMENT
OF THROMBOSIS

(iii) NUMBER OF SEQUENCES: 111 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKline Beecham Corporation (B) STREET: 709 Swedeland Road (C) CITY: King of Prussia (D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19406 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 1.5 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 16-JAN-1997 (C) CLASSIFICATION:

(vii) PRIDR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/029,119 (3) FILING DATE: 24-OCT-1996 WO 97/26010 PCTrUS97/00759 (viii~ ATTORNEY/AGENT INFORMATION:
(A) NAME: Baumeister, Kirk (B) REGISTRATION NUMBER: 33,833 (C) REFERENCE/DOCKET NUMBER: P50438 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-270-5096 (B) TELEFAX:
(C) TELEX:

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~xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GATTTTCARr- TGCAGATTTT C 21 CA 02243236 l998-07-l6 WO 97/26010 PCTnUS97/00759 (2) INFORMATION FOR SEQ ID NO:5:

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~xi~ SEQUENCE DESCRIPTION: SEQ ID No 5 GTTGATGACT TCAAGGGACG GTTTGCCTTC ~ ~GAAA GCTCTGCCAG CACTGCCAAT 240 (2) INFORMATION FOR SEQ ID NO:6:

~i) SEQUENCE CHARACTERISTICS:
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W O 9i/26010 PCTrUS97/00759 .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:

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lxi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Ser Ser Ala Ser Thr Ala Asn Leu Gln Ile Asp Asn Leu Lys Asp Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly G~n Gly Thr Leu Val Thr Val Ser Ala CA 02243236 l998-07-l6 W O 9i/26010 PCT~US97/00759 (2) INFORMATION FOR SEQ ID NO:8:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Asn Tyr Gly Met Asn (2) INFORMATION FOR SEQ ID NO:9:

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{xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly W O 9i/26010 PCTnUS97/00759 (2) INFORMATION FOR SEQ ID NO:l0:

(i) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:

Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr l 5 l0 (2) INFORMATION FOR SEQ ID NO:ll:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly l 5 l0 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met 3~
His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr _ CA 02243236 l998-07-l6 .

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Al~ Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg (2) INFORMATION FOR SEQ ID NO:12:

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Arg Ala Ser Ser Ser Val Asn Tyr Met His 5 1~) (2) INFORMATION FOR SEQ ID NO:13:

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CA 02243236 l998-07-l6 W O 97/26010 PCTrUS97/00759 (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:13:

Ala Thr Ser Asn Leu Ala Ser (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Gln Gln Trp Ser Ile Asn Pro Arg Thr (2) INFORMATION FOR SEQ ID NO:15:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

CAACTAGTGC AATCTGGGTC TGAGTTGAAG AAGCCTGGGG CCTCAGTG~A GGTTTCCTGC 60 CA 02243236 l998-07-l6 WO gi/26U10 PCTrUS97/0U759 (2) INFORMATION FOR SEQ ID NO:16:

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(2) INFORMATION FOR SEQ ID NO:17:

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CA 02243236 l998-07-l6 W 0 97/26010 PCT~US97/00759 , (2~ INFORMATION FOR SEQ ID NO:18:

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(A) NAME~KEY: Coding Se~uence (B) LOCATION: 2. ~37 (D) OTHER INFORMATION:

WO 97/26010 PCT~US97/00759 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr ~2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:
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) ANTISENSE: NO

CA 02243236 l998-07-l6 W O 97/26010 PCTrUS97/007~9 .
(v) FRAGMENT TYPE: internal ~vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr (23 INFORMATION FOR SEQ ID NO:21:

~i) SEQUENCE C~ARACTERISTICS:
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~xi) SEQUENCE DESCRI~TION: SEQ ID NO:21:

~2) INFORMATION FOR SEQ ID NO:22:

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(A) NAME/KEY: Coding Sequence (~) LOCATION: 27...95 ~D) OTHER INFORMATION:

~xi) SEQUENCE DESCRIPTIOM: SEQ ID NO:23:

Met Gly Trp Ser Cys Ile Ile Leu Phe l 5 CA 02243236 l998-07-l6 WO 97/26010 PCTnJS97/007S9 .

Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Val Gln Leu (2) INFORMATION FOR SEQ ID NO:24:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Val Gln Leu (2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:
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CA 02243236 l998-07-l6 W O 97/26010 PCT~US97/00759 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

~2) INFORMATION FOR SEQ ID No:26:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
-W O 97/26010 PCTrUS97/00759 .
~21 INFORMATION FOR SEQ ID NO:28:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

~2) INFORMATION FOR SEQ ID NO:29:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

WO 97/26010 PCTrUS97/00759 (2) INFORMATION FOR SEQ ID NO:30: ~

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(ix) FEATURE:

(A) NAME/KEY: Coding Sequence (B) LOCATION: l ..363 (D) OTHER INFORMATION: F9HZHC l-0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala l 5 l0 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met GGA TGG ATA AAC ACC AGA AAT GGA AAG TCA ACA TAr GTT GAT GAC TTC l92 Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 6~ 70 75 80 CA 02243236 l998-07-l6 W O g7/26010 PCT~US97/00759 .

Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser ~2~ INFORMATION FOR SEQ ID NO:31:

~i) SEQUENCE CHARACTERISTICS:
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~iv) ANTISENSE: NO
~v) FRAGMENT TYPE: internal ~vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Tyr Cys ~_ 90 95 Ala Ary Glu Gly 'sn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly CA 02243236 l998-07-l6 W O 97/26010 PCT~US97/00759 .
Gln Gly Thr Leu Val Thr Val Ser Ser (2) INFORMATION FOR SEQ ID NO:32:
~.
(i) SEQUENCE CHARACTERISTICS:
_ (A) LENGTH: 165 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
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(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

AGTACTGACA CAGTCTCCAG CCACCCTGTC ~ CCA GGGGAAAGAG CCACCCTCTC 60 (2) INFORMATION FOR SEQ ID NO:33:

~i) SEQUENCE CHARACTERISTICS:
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!xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
-CCGCGGGTTA .TACTCCACT GCTGACAGTA ATAAACCGCA AAATCTTCAG GCTCTAGACT 60 CA 02243236 l998-07-l6 .

(2) INFORMATION FOR SEQ ID NO:34:

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(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Se(auence (B) LOCATION: 2...280 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg GCC ACC CTC TCC TGC AGG GCC AGC TCA AGT GTA AAT TAC ATG CAC TGG ~7 Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met ~is Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe WO 97-126010 PCT/U$97/00759 .

Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg (2) INFORMATION FOR SEQ ID No:35:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg (2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucieic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02243236 l998-07-l6 W O 97/26010 PCT~US97/007~9 (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE- NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

GACCGCGGGT TAATACTCC~ CTGCTGA 27 (2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ~NTISENSE: NO
(v) FF~GMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

CA 02243236 l998-07-l6 ~VO 97/26010 PCT~US97/00759 (A) NAME/KEY: Coding Seguence ~B) LOCATION: 27...92 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser Glu Ile Val 2) INFORMATION FOR SEQ ID NO: 3 9:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal ~vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser Glu Ile Val (2) INFORMATION FOR SEQ ID NO:40:
-~i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid CA 02243236 l998-07-l6 W 097/260l0 PCT~US97/0075g .
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: -(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

(2) INFORMATION FOR SEQ ID NO:42:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO

-CA 02243236 l998-07-l6 W O 9i/26010 PCT~US97/00759 (v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Sequence (B) LOCATION: 1...321 (D) OTHER INFORMATION: F9HZLC1-0 Ixi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr ..

.

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr 85 90 g5 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg (2) INFORMATION FOR SEQ ID No:44:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Glr Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Thr Ser As~ Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Th- Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu , WO 97/26010 PCTrUS97/00759 .
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg l00 105 .~
(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 134 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

CTACAGATCA GCAG l34 (2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 134 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

CA 02243236 l998-07-l6 WO 97/26010 PCT~US97/00759 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

GTAATACACT GCA~ CAGCCTTTAG GCTGCTGATC TGTAGATATG CCGTGCTGAC 120 (2) INFORMATION FOR SEQ ID NO:g7: _ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Sequence (B) LOCATION: 1...225 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

Pro Gly Gln Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Ser Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala CA 02243236 l998-07-l6 W 09ii26010 PCTrU~97/00759 .

Asp Asp Thr Ala Val Tyr Tyr Cys Thr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr (2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

Pro Gly Gln Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Ser Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Tyr Cys Thr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr ~2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid ~C~ STRANDEDN_SS: single (D) TOPOLOGY: linear CA 02243236 l998-07-l6 (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

(2) INFORMATION FOR SEQ ID NO:50:

(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

CA 02243236 l998-07-l6 W O 9i/26010 PCTAUS97/00759 (ix) FEATURE:

(A) NAME/KEY: Coding Seguence (B) LOCATION: 1...363 (D) OTHER INFORMATION: F9HZHC 1-1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala TCA GTG AAG GTT TCC TGC AAG GCC TCT GGA TAC ACC TTC ACT AAC TAT g6 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Ser Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Tyr Cys Thr ~rg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly CAG G&T ACC CTG GTC ACC GTC TCC TCA 363 Gln Gly Thr Leu Val Thr Val Ser Ser CA 02243236 l998-07-l6 W 097/26010 PCT~US97/n0759 (2) INFORMATION FOR SEQ ID NO:52:

(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 amino acids (B) TYPE: amino acid (C~ STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15~er Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu~Lys Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Ser Ser Val Ser Thr Ala Tyr 80~eu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Tyr Cys 95~hr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser (2) INFORMATION FOR SEQ ID NO:53:

~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 8Z base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 97/26010 PCT~USg7/00759 (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal CA 02243236 l998-07-l6 WO 97/26010 PCTrUS97/00759 ~vi~ ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

Gln Gln Arg Pro Gly Gln Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser ~2) INFORMATION FOR SEQ ID NO:56:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Se~uence (B) LOCATION: 1...321 (D) OTHER INFORMATION: F9HZLC 1-1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala T~.r Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met CAC TGG TAC C~ CAG AGA CCT GGC CAG GCT CCC AAG CCC TGG ATC TAT 144 His Trp Tyr G'n Gln Arg Pro Gly Gln Ala Pro Lys Pro Trp Ile Tyr CA 02243236 l998-07-l6 W 0~7/26010 PCT~US97/00759 .

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser GGG TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT CTA GAG CCT C~A 240 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr TTC GGC GGA GGG ACC AAG GTG GAG ATC A~A CGA 321 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg ~2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly l 5 10 15 His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Lys P~o Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 6~
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu ~ 95 WO9ii26010 PCT~US97/00759 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr 85 90 g5 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg l00 105 (2) INFORMATION FOR SEQ ID NO:58:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

GATCCGGGTC TGGGACAGAT TACACTCTCA CGATATCCAG T 4l (2) INFORMATION FOR SEQ ID NO:59:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

CTAGACTGGA TATCGTGAGA GTGTAATCTG TCCCAGACCC G 4l -W O 9i/26010 PCT~US97/00759 (2) INFORMATION FOR SEQ ID NO:6Q:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:60:

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser (2) INFORMATION FOR SEQ ID NO:61:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cD~A
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) MAME/KEY: Coding Seguence (B) LOCATION: 1...321 (D) OTHER INFORMATION: F9HZLC 1-2 CA 02243236 l998-07-l6 WO 97126010 PCTrUS97/00759 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 lQ5 (2~ INFORMATION FOR SEQ ID NO:62:

(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 107 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETIC'AL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal CA 02243236 l998-07-l6 WO 97/26010 PCT~US97/00759 (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15~lu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg ~2) INFORMATION FOR SEQ ID NO:63:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 165 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:

TCC~AGCCT TGGATCTACG CCACTAGTAA CCTGGCTTCT GGTGT 165 CA 02243236 l998-07-l6 WO 9i/26010 PCT~US97/00759 (2) INFORMATION FOR SEQ ID NO:64:

(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 161 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:

~2) INFORMATION FOR SEQ ID NO:65:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 280 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/REY: Coding Sequence (B) LOCATION: 2 ..2~0 (D) OTHER INFORMATION:

CA 02243236 l998-07-l6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:

Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser &GT ACA GAC TAC ACC TTC ACC ATC AGC AGC CTC CAG CCA GAG GAT ATC 241 Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg ~2) INFORMATION FOR SEQ ID NO:66:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein ~iii) HYPOTHETICAL: NO
~iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal ~vi) ORIGINAL SOURCE:

WO 9t/26010 PCT~US97/00759 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:

Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg l 5 l0 15 Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg (2) INFORMATION FOR SEQ ID NO:67:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: cDNA
~iii) HYPOTHETICAL: NO
~iv) ANTISENSE: NO
~v) FRAGMENT TYPE:
~vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:

(2) INFORMATION FOR SEQ ID NO:68:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: cDNA
~2 WO 97126010 PCT~US97/00759 (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
,.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:

(2) INFORMATION FOR SEQ ID No:69:

(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

2) INFORMATION FOR SEQ ID NO:70:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:

-CA 02243236 l998-07-l6 WO 97126010 PCT~US97/00759 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:

Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala (2) INFORMATION FOR SEQ ID NO:71:

i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:

GGACGTTCGG CCAAGG&ACC AAGGTGGAAA TCAAACGGAC TGTGGCGG 48 (2) INFORMATION FOR SEQ ID NO:72:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (Dl TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:

_ _ CA 02243236 l998-07-l6 O 97/26010 PCTrUS97/00759 ~2) INFORMATION FOR SEQ ID NO:73:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Se~uence (B) LOCATION: 1...321 (D) OTHER INFORMATION: F9HZLC 2-0 (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:73:

Gln Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly &AC AGA GTG ACC ATC ACC TGC AGG GCC AGC TCA AGT GTA AAT TAC ATG 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu ~5 70 75 80 W O 97126010 PCT~US97/00759 .

Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG 32l Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg l00 105 (2) INFORMATION FOR SEQ ID NO:74:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii~ HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:

Gln Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Se~ Val Gly l 5 l0 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg l00 105 CA 02243236 l998-07-l6 WO 97/26010 PCTrUS97/00759 .
~2) INFORMATION FOR SEQ ID NO:75:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A~ NAME/KEY: Coding Sequence (B) LOCATION: 27...94 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Ile Val Leu (2) INFORMATION FOR SEQ ID NO:76:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids (3) TYPE: amino acid (C) STRANDEDNESS: single (3) TOPOLOGY: linear .

(ii~ MOLECULE TYPE: protein ) HYPOTHETICAL: NO
(ivl ANTISENSE: NO

CA 02243236 l998-07-l6 WO 97-/260}0 PCTrUS97/00759 (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7~:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Ile Val Leu (2~ INFORMATION FOR SEQ ID NO:77:

(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 401 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
HYPOTHETICAL: NO
~iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Sequence (B) LOCATION: 27...401 (D) OTHER INFORMATION: F9HZLC 1-3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
~AATTCTGAG CACACAGGAC CTCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC 53 Met Gly Trp Ser Cys Ile Ile Leu Phe TTG GTA GCA ACA GCT ACA GGT GTC CAC TCC CAG A~A GTA CTG ACA CAG 101Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Ile val Leu Thr Gln CA 02243236 l998-07-l6 WO 9t/26010 PCTAUS97/00759 TCT CCA GCC ACC CTG TCT TTG TCT CCA GGG GAA AGA GCC ACC CTC TCC 149Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser TGC AGG GCC AGC TCA AGT GTA AAT TAC ATG CAC TGG TAC CAA CAG AGA 197Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Arg CCT GGC CAG GCT CCC AAG CCC TGG ATC TAT GCC ACG AGT AAC CTG GCT 245Pro Gly Gln Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala AGC GGC GTC CCA GCC AGG TTC AGT GGA TCC GGG TCT GGG ACA GAT TAC 293Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr ACT CTC ACG ATA TCC AGT CTA GAG CCT GAA GAT TTT GCG GTT TAT TAC 341Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr go 95 10D 105 TGT CAG CAG TGG AGT ATT AAC CCG CGG ACG TTC GGC GGA GGG ACC AAG 389Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys ~2~ INFORMATION FOR SEQ ID NO:78:

(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 125 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: single (~) TOPOLOGY: linear lii~ MOL--CULE TYPE: protein (iii~ HYPOTHETICAL: NO
(iv! ANT_SENSE: NO
(v~ -RAG~SENT TYPE: internal (vi' OR-GINAL SOURCE:

WO 97126010 PCT~US97/00759 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys ~2) INFORMATION FOR SEQ ID No:79:

(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 81 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TyPE:
(vi~ ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:

(2) INFORMATION FOR SEQ ID NO:80:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs CA 02243236 l998-07-l6 WO 9il~60l0 PCT~US97/00759 .
(B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
~iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:

~2) INFORMATION FOR SEQ ID NO:81:

~i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
~v) FRAGMENT TYPE:
~vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:

(2) INFORMATION FOR SEQ ID NO:82:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TQPOLOGY: linear CA 02243236 l998-07-l6 W 097i26010 PCT~US97/00759 (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
~vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:

(2) INFORMATION FOR SEQ ID NO:83:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 278 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Sequence (B) LOCATION: 3...278 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:

Ala Ser Gly Ty~ Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg CAG GCC CCT GGA C~ GGG CTC GAG TGG ATG GGA TGG ATA AAC ACC AGA 95 Gln Ala Pro Gly Gin Gly Leu Glu Trp Met Gly Trp Il_ Asn Thr Arg W 097/26010 PCT~US97/00759 AAT GGA AAG TCA ACA TAT GTT GAT GAC TTC AAG GGA CGG TTT GTC TTC 143Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe ~ 35 40 45 TCT CTA GAC ACC TCT GTC AGC ACG GCA TAT CTA CAG ATC AGC AGC CTA l9lSer Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu AAG GCT GAG GAC ACT GCA GTG TAT TTC TGT ACG AGA GAA GGG AAT ATG 239Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr ~2) INFORMATION FOR SEQ ID No:84:

(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 92 amino acids (B) TYPE: amino acid ~C~ STRRNDEDNESS: single ~D) TOPOLOGY: linear lii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv~ ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID No:84:

Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln l 5 l0 15 Rla Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr Arg Glu Gly Asn Met Asp CA 02243236 l998-07-l6 .
Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr (2) INFORMATION FOR SEQ ID NO:85:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: cDNA
~iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMEMT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:

(2) INFORMATION FOR SEQ ID NO:86:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

~xi) SEQUENCE DESCRIPTION: SEQ ID NO.86:

W O 97/26010 PCTrUS97/00759 (2) INFORMATION FOR SEQ ID NO:87:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear tii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
~v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:

(2) INFORMATION FOR SEQ ID NO:88:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 446 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
~iii) HYPOTHETICAL: NO
~iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Sequence (B) LOCATION: 27...446 (D) OTHER INFORMATION: F9H~HC 3-0 CA 02243236 l998-07-l6 WO g7/26010 PCTtUS97/nO759 (xi) SEQUENCE DESCRIPTION: SEQ ID No:88:

Met Gly Trp Ser Cys Ile Ile Leu Phe TTG GTA GCA ACA GCT ACA GGT GTC CAC TCC CAG ATC CAA CTA GTG CAA 101Leu Val Ala Thr Ala Thr Gly Val ~is Ser Gln Ile Gln Leu Val Gln TCT GGG TCT GAG TTG AAG AAG CCT GGG GCC TCA GTG AAG GTT TCC TGC 149Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys : 40 AAG GCC TCT GGA TAC ACC TTC ACT AAC TAT GGA ATG AAC TGG GTG CGA 197Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg CAG GCC CCT GGA CAA GGG CTC GAG TGG ATG GGA TGG ATA AAC ACC AGA 245Gln Ala Pro Gly Gln Gly heu Glu Trp Met Gly Trp Ile Asn Thr Arg AAT GGA AAG TCA ACA TAT GTT GAT GAC TTC AAG GGA CGG TTT GTC TTC 293Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe TCT CTA GAC ACC TCT GTC AGC ACG GCA TAT CTA CAG ATC AGC AGC CTA 341Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu AAG GCT GAG GAC ACT GCA GTG TAT TTC TGT ACG AGA GAA GGG AAT ATG 389Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr Arg Glu Gly Asn Met GAT GGT TAC TTC CCT TTT ACT TAC TGG GGC CAG GGT ACC CTG GTC ACC 437Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser _ CA 02243236 l998-07-l6 WO 9i/26010 PCTrUS97/00759 ~2) INFORMATION FOR SEQ ID NO:89:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 140 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein ~iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr 1~~5 120 125 Tyr Trp G'y Gln Gly Thr Leu Val Thr Val Ser Ser (2) INFOR~TION FOR SEQ ID NO:90:

(i) SEQUENCE CHARACTERISTICS:
l.' LENGTH: 90 base pairs (--! TYPE: nucleic acid (.! STRANDEDNESS: single (~! TOPOLOGY: linear CA 02243236 l998-07-l6 WO 9'~7/26010 PCT/US97/00759 .

(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:

TTGCAGGGCC AGCTC~AGTG TAAATTACAT 90 (2) INFORMATION FOR SEQ ID NO:91:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 108 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:

(2) INFORMATION FOR SEQ ID NO:92:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 108 base pairs ~B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYP._. cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO

CA 02243236 l998-07-l6 .
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:

(2) INFORMATION FOR SEQ ID-NO:93:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:

(2) INFORMATION FOR SEQ ID No:94:

(i~ SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 330 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

W 0 97126010 PCT~US97/00759 .

(A) NAMEtKEY: Coding Se~uence (B) LOCATION: 2...328 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:

Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg l 5 l0 15 Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp TAC CAA CAG AAA CCT GGC AAA GCT CCC AAG CCC TGG ATC TAT GCC ACG l45 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr Ala Thr AGT AAC CTG GCT AGC GGC GTC CCA TCA AGG TTC AGT GGA TCC GGG TCT l93 Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser GGG ACA GAT TAC ACT CTC ACG ATA TCC AGT CTA CAA CCT GAA GAT TTT 24l Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Aia Ala l00 105 (2) INFOR~IATION FOR SEQ ID NO:95:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l09 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single CA 02243236 l998-07-l6 WO 97/26010 PCTrUS97/00759 .
(D~ TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal _ (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:

Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ly~ Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala (2) INFOR~ATION FOR SEQ ID No:96:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
~iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v~ FRAGMENT TYPE:
(v ) ORIGINAL SOURCE:

(x-) SEQUENCE DESCRIPTION: SEQ ID NO:96:

W O 97~26010 PCTAJS97/00759 .

(2) INFORMATIoN FOR SEQ ID NO:97:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .

(ii) MOLECULE TYPE: cDNA
(iii) ~YPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:

(2) INFORMATION FOR SEQ ID NO:98:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 412 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
tvi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Sequence (B) LOC~TION: 27...412 (D) OTHER INFORMATION: F9HZLC 3-0 W O 97126010 PCTrUS97/00759 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:

Met Gly Trp Ser Cys Ile Ile Leu Phe _ TTG GTA GCA ACA GCT ACA GGT GTC CAC TCC CAG ATA GTA CTG ACA CAG 101 Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Val CA 02243236 l998-07-l6 W O 97/26010 PCTrU$97/007~9 (2) INFORMATION FOR SEQ ID NO:99:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser Gln Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe 80~er Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu 95~ln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn 100 105 110~ro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 115 120 125~al (2) INFORMATION FOR SEQ ID NO:100:

ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02243236 l998-07-l6 W 097/26010 PCTrUS97/00759 (ii~ MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:

(2) INFORMATION FOR SEQ ID NO:101:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:

(2) INFOF~ATION FOR SEQ ID NO:102:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A~ NAME/KEY: Coding Sequence (B) LOCATION: 1...335 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly G'y Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 lQ5 110 ; ) INFORMATION FOR SEQ ID NO:103:

(i) SEQUENCE CHARACTERISTICS:
(;) LENGTH: 112 ~-nino acids (~) TYPE: amino acid (C) STRANDEDNESS: single CA 02243236 l998-07-l6 W 0 97/26010 PCTrUS97/00759 (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met 3~
His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 80~sp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr g0 95~he Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro (2) INFORMATION FOR SEQ ID NO:104.

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 318 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv~ ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KFV: Coding Se~uence (B) LOCATION: 1...318 CA 02243236 l99X-07-16 W O 9i/26010 PCTrUS97/00759 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys (2) INFORMATION FOR SEQ ID NO:105:

(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 106 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (il~ MOLECULE TYPE: protein CA 02243236 l998-07-l6 WO 9i/26010 PCTrUS97/00759 (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ile Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys (2) INFORMATION FOR SEQ ID NO:106:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) O~IGINAL SOURCE:

(xi) SEOUENCE DESCRIPTION: SEQ ID NO:10~:

W O 9i/26010 PCT~US97/00759 (2) INFORMATION FOR SEQ ID NO:107:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 ~ase pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:

(2) INFORMATION FOR SEQ ID NO:108:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 369 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Se~uence (B) LQCATION: 1 ..369 (D) OTHER INFORMATION:

CA 02243236 l998-07-l6 W O 97126010 PCT~US97/00759 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Ser Ser Ala Ser Thr Ala Asn Leu Gln Ile Asp Asn Leu Lys Asp Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser (2) INFORMATION FOR SEQ ID NO:109:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02243236 l998-07-l6 WO 97/26~10 PCT~US97/00759 (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
~v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: _ Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Ser Ser Ala Ser Thr Ala Asn 80~eu Gln Ile Asp Asn Leu Lys Asp Glu Asp Thr Ala Thr Tyr Phe Cys 95~hr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser (2) INFORMATION FOR SEQ ID NO:110:

(i) SBQUENCE CHARACTERISTICS:
(A) LENGTH: 363 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:

(A) NAME/KEY: Coding Sequence r (B) LOCATION 1...363 CA 02243236 l998-07-l6 WO 97-/26010 PCT~US97/00759 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:

_ Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Ser Ser Ala Ser Thr Ala Asn Leu Gln Ile Asp Asn Leu Lys Asp Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala (2) INFORMATION FOR SEQ ID NO:111:

(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 121 amino acids (B) ?YPE: amino acid CA 02243236 l998-07-l6 W O 9i/26010 PCTAUS97/00759 (C) STRAN3EDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:111:

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr ~ 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Arg Asn Gly Lys Ser Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Ser Ser Ala Ser Thr Ala Asn Leu Gln Ile Asp Asn Leu Lys Asp Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Glu Gly Asn Met Asp Gly Tyr Phe Pro Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala ~34

Claims (38)

1. A method for inhibiting thrombosis in an animals comprising administering an effective dose of an anti-coagulation factor monoclonal antibody having self-limiting neutralizing activity.

2. The method of claim 1 further comprising administering acetylsalicylic acid in combination with the anti-coagulation factor monoclonal antibody.

3. The method of claim 1 or 2 wherein the coagulation factor is from the intrinsic or common coagulation pathway.

4. The method of claim 3 wherein the anti-coagulation factor monoclonal antibody is an anti-Factor IX, anti-Factor IXa, anti-Factor X, anti-Factor Xa, anti-Factor XI, anti-Factor XIa, anti-Factor VIII, anti-Factor VIIIa, anti-Factor V, anti-Factor Va, anti-Factor VII, anti-Factor VIIa or anti-thrombin.

5. The method of claim 3 wherein the anti-coagulation factor monoclonal antibody is an anti-Factor IX.

6. The method of claim 5 wherein the anti-Factor IX monoclonal antibody has the identifying characteristics of SB 249413, Ss 249415, SB 249416, SB
2249417, SB 257731 or SB 257732.

7. The method of claim 5 wherein the anti-Factor IX monoclonal antibody has the identifying characteristics of SB 249417.

8. The method of claim 1 or 2 wherein aPTT is prolonged without significant prolongation of PT.

9. The method of claim 4 wherein aPTT is about 35 seconds to about 100 seconds.

10. The method of claim 1 or 2 wherein the thrombosis is associated with myocardial infarction, unstable angina, atrial fibrillation, stroke, renal damage, pulmonary embolism, deep vein thrombosis, percutaneous translumenal coronary angioplasty, disseminated intravascular coagulation, sepsis, artificial organs, shunts or prostheses.

11. An anti-coagulation factor monoclonal antibody having self-limiting neutralizing activity against the coagulation factor.

12. The monoclonal antibody of claim 11 wherein the coagulation factor is from the intrinsic or common coagulation pathway.

13. The monoclonal antibody of claim 12 wherein the anti-coagulation factor monoclonal antibody is an anti-Factor IX, anti-Factor IXa, anti-Factor X, anti-Factor Xa, anti-Factor XI, anti-Factor XIa, anti-Factor VIII, anti-Factor VIIIa, anti-Factor V, anti-Factor Va, anti-Factor VII, anti-Factor VIIa or thrombin.

14. The monoclonal antibody of claim 12 wherein the anti-coagulation factor monoclonal antibody is an anti-Factor IX.

15. The monoclonal antibody of claim 14 having the identifying characteristics of SB 249413, SB 249415, SB
249416, SB 249417, SB 257731, SB 257732, 9E4(2)F4 or 11G4(1)B9.

16. The monoclonal antibody of claim 14 having the identifying characteristics of SB 249417.

17. A hybridoma having the identifying characteristics of cell line 9E4(2)F4 or 11G4(1)B9.

18. A neutralizing Fab fragment or F(ab')2 fragment thereof, produced by deleting the Fc region of the monoclonal antibody of claim 11.

19. A neutralizing Fab fragment or F(ab')2 fragment thereof, produced by chain shuffling whereby the Fd heavy chain of the monoclonal antibody of claim 11 is expressed in a murine light chain filamentous phage Fab display library.

20. A neutralizing Fab fragment or F(ab') 2 fragment thereof, produced by chain shuffling whereby the light chain of the monoclonal antibody of claim 11 is expressed in a murine heavy chain filamentous phage Fab display library.

21. An immmunoglobulin heavy chain complementarity determining region, the amino acid sequence of which is selected from the group consisting of SEQ ID NOs: 8, 9 and 10.

22. A nucleic acid molecule encoding the immunoglobulin complementarity determining region of claim 21.

23. An immmunoglobulin light chain complementarity determining region, the amino acid sequence of which is selected from the group consisting of SEQ ID NOs: 12, 13 and 14.

24. A nucleic acid molecule encoding the immunoglobulin complementarity determining region of claim 23.

25. An altered antibody comprising a heavy chain and a light chain, wherein the framework regions of said heavy and light chains are derived from at least one selected antibody and the amino acid sequences of the complementarity determining regions of each said chain are derived from the monoclonal antibody of claim 11.

26. The altered antibody of claim 25 which is humanized.

27. The humanized antibody of claim 26 wherein the heavy chain has the amino acid sequence set forth in SEQ
ID NO: 31, 52, or 89.

28. The humanized antibody of claim 26 wherein the light chain has the amino acid sequence set forth in SEQ
ID NO: 44, 57, 62, 74, 78 or 99.

29. The humanized antibody of claim 26 wherein the heavy chain has the amino acid sequence set forth in SEQ
ID NO: 31 and the light chain has the amino acid sequence set forth in SEQ ID NO: 44.

30. The humanized antibody of claim 26 wherein the heavy chain has the amino acid sequence set forth in SEQ

ID NO: 52 and the light chain has the amino acid sequence set forth in SEQ ID NO: 57.

31. The humanized antibody of claim 26 wherein the heavy chain has the amino acid sequence set forth in SEQ
ID NO: 52 and the light chain has the amino acid sequence set forth in SEQ ID NO: 62.

32. The humanized antibody of claim 26 wherein the heavy chain has the amino acid sequence set forth in SEQ
ID NO: 52 and the light chain has the amino acid sequence set forth in SEQ ID NO: 74.

33. The humanized antibody of claim 26 wherein the heavy chain has the amino acid sequence set forth in SEQ
ID NO: 52 and the light chain has the amino acid sequence set forth in SEQ ID NO: 78.

34. The humanized antibody of claim 26 wherein the heavy chain has the amino acid sequence set forth in SEQ
ID NO: 89 and the light chain has the amino acid sequence set forth in SEQ ID NO: 99.

35. A chimeric antibody comprising a heavy chain and a light chain, said antibody characterized by inhibiting the function of intrinsic or common pathway coagulation factors in a self-limiting manner, wherein thrombosis is inhibited and limited modulation of coagulation is produced, wherein the constant regions of said heavy and light chains are derived from at least one selected antibody and the amino acid sequences of the variable regions of each said chain are derived from the monoclonal antibody of claim 11.

36. The antibody according to claim 35 wherein the constant regions are selected from human immunoglobulins.

37. A pharmaceutical composition comprising the altered antibody of claim 26 or 35 and a pharmaceutically acceptable carrier.

38. The pharmaceutical composition of claim 37 further comprising acetylsalicylic acid.