AP583A - Recombination IL4 antibodies useful in treatment of IL4 mediated disorders. - Google Patents

Abstract

Chimeric and humanized il4 mabs derived from high affinity mabs, pharmaceutical compositions containing same, and methods of treatment are provided.

Description

Field of the Invention

The present invention relates generally co the field of fusion proteins, and to proteins useful in treatment and diagnosis of conditions mediated by ILA and excess IgE production, and mote specifically to chimeric and humanized HA antibodies.

Background of the Invention

Atopic allergic diseases range from the relatively minor, such as seasonal rhinitis and conjunctivitis, to the more serious, such as atopic dermariria and atopic asthma, and life threatening, such as anaphylactic shock Linking these conditions is the immune response of the body to allergens, which response involves the production of immunoglobulin £ (IgE) antibodies in generically predisposed individuals (atopy). Inhibition of IgE production has long been a goal in specific immunotherapy of allergic disease using detensidzatioo vaccines. However, in recent yean the safety and efficacy of vaccine therapy have been questioned, but the desire to reduce IgE levels has not waned.

Interlculdn 4 (ILA) is a protein mediator in the lymphoid system. Stutfiesof lymphocytes fhxnatopic individuals have revealed the presence of higher titan normal nambers of T lymphocytes with the ability to secrete IL4 in response to stimulation, and larger quantities of HA secreted following stimulation.

Anti-IL4 antibody has been found to inhibit IgE, but rax IgG, or IgG,, (Finkebnan ex al, Ann. Rev. Immunol.. J:303 (1990)], and the production Of Π-5 secreting Tcelh [Maggie of. UBB0HA&.1&2]42 (1992)]. Further, recent data suggests that ILA may affect eosinophil accumulation in tissues. See, t.g. Tappers* al. Call. £:457 (1990); Tepperet al, CdL flJ03 (1989).

There remains a need in the an for a high affinity ILA antagonist, which would reduce eosinophil inflammation both by reducing the proliferation of H_5 secreting cells, and by inhibiting an adherence mechanism whereby eosinophils may be accumulating in tissues, and can be used to treat, prevent or diagnose allergic reactions.

AP/P/ 96/00782

nail *rctrvto	07 MAR 1996
D'-ETTOR GENERAL
ACTION OFFICER
FILE

IC

In a first aspect the present invention provides a fusion protein having a binding affinity for human interieulrin-4 which comprises complementarity determining regions (CDRs) derived from a non-human neutralizing monoclonal antibody (MAb) characterized by a dissociation constant equal to or less than 2x10 ¹⁰ M for human HA. and a first fusion partner in which at least oae, and preferably all complementarity determining regions (CDRs) of the first fuaon partner are replaced by CDRs from the non-human monoclonal antibody (MAb). The noohuroan neutralizing monoclonal antibody may be selected from the group consisting of 339 and 6A1 as described more fully in the Detailed Description. Preferably, the fusion protein is operatively linked to a second fusion protein as well, which comprises all or a pan of an immunoglobulin constant chain.

In a related aspect, the present invention provides CDRs derived from nonhuman neutralizing monoclonal antibodies (MAb) characterized by a dissociation constant equal to or less than 2 χ 1 O'’* M for human IL4, and nucleic acid molecules encoding such CDRs.

In another aspect, the invention provides humanized antibodies having ax least one. and preferably six, complementarity determining regions (CDRs) derived from non-human neutralizing monoclonal antibodies (MAb) characterized by a dissociation coaeam equal io or less than 2x 10*’* M for human IL4.

In stin another aspect, there ia provided a chimeric antibody containing human heavy and fight chain constant regions and heavy and light chain variable regions derived from non-human neutralizing monoclonal antibodies (MAb) characterized by a dinoriarino constant equal to or less than 2 x 10*“ M for human OA.

In still another aspect, the present invention provides a pharmaceutical compostioo which contains one (or more) of the above-described fusion proteins or MAbs (e.g., humanized, chimeric, etc.) and a pharmaceutically acceptable earner.

In a further aspect, the present invention provides a method far treating andfor preventing allergic conditions in humans by administering to said human an effective amount of pharmaceutical composition of the invention.

In yet another aspect, the present invention provides methods for, and components useful in, die recombinant production of the fusion proteins, MAbs (e g., humanized, chimeric, etc.), CDRs thereof, a Fab, or F(ab)j, or analog thereof which is derived from non-human neutralizing monoclonal antibodies (MAb) characterized by a dissociation constant equal to or less than 2 x ICT¹⁰ M for human

AP/P/ 96/00782

-21L4. These components include iscia:ed nucleic acid sf lenccs cncocir; same, recombinant plasmids containing the nucleic acid sequences under the ccatrol of selected regulatory sequences capable of directing the expression thereof in host cells, and host cells (preferably mammalian) transfected with the recombinant plasmids. The production method involves culturing a transfecttd host all line of the present invenooo under conditions such that an antibody, preferably a humanized antibody, is expressed in said cells and isolating the expressed product therefrom.

In yet another aspen of the invention is a method to diagnose allergies and other conditions associated with excess immunoglobulin E production in a human which comprises contacting a sample of biological Quid with the fuaoc proteins, MAbs (e.g.. humanized, chimeric, etc.) and Faba of the instant invention and assaying for the occurrence of binding between said fusion protein, MAb or Fab and human interleukin 4.

In another related aspect is provided a method for screening monoclonal antibodies which have a high titer for human interkukia 4 which comprises: (a) preparing a hybridoma cell line characterized by secretion of a monoclonal antibody to human interleukin 4; and (b) screening said hybridoma ceU line with aldehyde coupled human iaterieuldn-4 or biotinylated human interieukin-4. Preferably, the hybridoosa cell Bee ia screened with biotinylated human interleukin-4.

Also provided is a neotrahzing MAb having high affinity tot TLA. a Fab fragment or a Hab¹), fragment thereof, produced 'by screening a library of hydridoma products withildehyde-coupled human interieukin-4 or biotinylated human QA

In another aspect, the present invention provides rodent neutralizing monoclonal antibodies specific fcr human teterieukin-4 and having a binding affinity characterized by a dissociation constant equal to or less than about 2 χ Iff* M. Exemplary of such moaocionai antibodies is the murine MAb, 3B9, and the rat MAb, 6AI and other MAbs have the same identifying characteristics (ie., binds io the sameeptopeCs) as 3B9or 6A1 with a specificity for human 1L4 and a dissociation constant equal to or fess than atom 2 x Iff ^mM). Another aspect of the invention is hybridan» 3436AUC1B9.

Other aspects and advantages of the present invention are described farther in the following detailed description of the preferred embodiments thereof.

AP/P/ 9 6 / 0 0 7 8 2

-3Fig. 1 [SEQ ID NOS: 1 ar.d -] Locates the light chain variable region (amino acids 21-132) for die murine IL4 antibody 3B9, and ±e human/murine 3B9 chimeric antibody is well as the native signal sequence (amino acids 1-20). The underlined portions indicate the CDRs [SEQ ID NOS. 15 and 16; SEQ ID NOS: 17 and 18; and SEQ ED NOS: 19 and 20).

Fig. 2 [SEQ ID NOS; 3 and 4) illustrates the heavy chain variable region (amino adds 20-140) of the murine 3B9. and the native signal sequence (amino acids 1-19). The underlined portions indicate the CDRs [SEQ ID NOS: 21 and 22;

SEQ ID NOS: 23 and 24; and SEQ ID NOS: 25 and 26].

Fig. 3 [SEQ ID NOS: 9 and 10) Ulustraies the heavy chain variable region (amino adds 21-141) of the butnan/oenne 3B9 chimeric antibody and its signal sequence (amino adds 1-19; SEQ ID NOS: 5 and 6). The underlined portions indicate the CDRs derived from 3B9 [SEQ ID NOS: 21 and 22; SEQ ED NOS: 23 and 24; and SEQ ID NOS: 25 and 26],

Fig. 4 [SEQ ID NOS: 11 and 12] illustrates the heavy chain variable region (amino adds 20-141) of the humanized 3B9 antibody and a signal sequence (amino acids 1-19: SEQ ID NOS: 5 and 6). The underlined portions indicate the CDRs derived from 3B9 [SEQ ID NOS: 54 and 22; SEQ ID NOS: 55 and 24; and SEQ ID

NOS;56and26)Fig. 5 [SEQ ID NOS: 13 and 14] illustrates the light chain variable region (amino adds 21-131) of the humanized 389 antibody and a signal sequence (amino adds 1-20; SEQ ID NOS: 7 and 8). The underlined portions indicate the CDRs derived from 3B9 (SEQ ID NOS: 53 and 16; SEQ ID NOS: 17 and 18; and SEQ ID

NOS: 27 and 28].

Fig. 6A [SEQ ID NOS: 5 and 6] is a heavy chain signal sequence used in Example 4 below.

Fig. 68 [SEQ ID NOS: 7 and 8] is a tight chain signal sequence used in Example 4 below.

Rg. 7 is a schematic drawing of plasmid pIL4chhc3-pcd employed to express a dmaeric 114 heavy chain » mammalian cells. The plasmid contains a beta lactamase gene (BETA LAC), in SV-40 origin of replication (SV40). a cytomegalovirus promoter sequence (CMV), a signal sequence, the chimeric variable heavy'chain of SEQ ID NOS: 9 and 10, a human heavy chain constant region, a poly A signal from bovine growth hormone (BGH). a betaglofcin promoter (beta glopto), a dihydrofolate reductase gene (DHFR), and another BOH sequence poly A signal ut a pUCl9 background.

8 L C 0 / 9 6 ,’d/dV

Fig 8 ϋ a schematic drawing o: piasnuc pIL4chic pc^ employed to express the chimeric 1L4 light chain variable region of SEQ ID NOS; 1 and 2 in rnarrm^lj jj] cells. The plasmid differs from that of Fig. 7 by containing a chimeric light chain variable region rather than that of the chimeric heavy chain, a human light chain constant region and a neomycin gene (Neo) in addition to DHFJL

Fig. 9 is a schematic drawing of plasmid pEL4hxhc-l-pcd employed to express the synthetic IL4 heavy chain variable region of SEQ ID NOS: 11 and 12 in mammalian ceils. The plasmid differs from that of Fig. 7 by containing a humanized heavy chain variable region rather than that of the chimeric heavy chain

Fig. 10 is a schematic drawing of plasmid pEAbzlcl-O-Pca employed to express the humanized ILA light chain variable region of SEQ ID NOS: 13 and 14 in mammalian cells. The plasmid differs from that of Fig. 8 by containing a humanized light chain variable region rather than (hat of the chimeric light chain and does not encode the DHFR gene.

Derailed Description of the Invention

The present invention provides a variety of antibodies, fragments thereof, and fusion proteins pameuiariy humanized antibodies, which are characterized by human EL4 binding specificity, neutralizing activity, and high affinity for human

HA as exemplified in murine MAb3B9 or the ratMAbdAl. These prodocts are useful in therapeutic and pharmaceutical eoopcMiwxtt far treating HA-mediated and IgE-mediated allergic reactions. These products are also useful in die of an ILA mediated coudidon by measurement (e.g^ by enzyme linked immonosobent assay (ELISA)) of circulating, endogenous ΠΑ levels in humans.

/. DsfieidouFuxkn protein* refers io a protein encoded by * fusion molecule, which may be obtained by expression in a selected host cell. Such fusion proteins are engineered antibodies, e.g^ chimeric or humanized antibodies, or antibody fragments lacking all or pan of an immunoglobulin constant region, e.g., ^v. Fab. or

Hab), and the like.

Fusion molecule* refen io a add sequence encoding the complementarity determining regions (CDRs) from a non-human immunoglobulin that are inserted into a first fusion partner comprising human variable framework sequences. Optionally, the first fusion partner is operatively licked to a second fusion partner

First fusion partner” refers to a nucleic add sequence encoding a human framework or human immunoglobulin variable region in which the native (or

AP/P/ 96/00782

-5CDRs are r;placed oy f.i CDRs of a doner Tie human var.aoie region can be ar. immunczloculir. heavy chair., a heh; cram ;or bom chains), an ana.cg or functional fra goer is thereof. Such CDRs or CDR regions, located within the variable region of antibodies (immunoglobulins) can be determined by town methods in the an. For example Rabat et ah, fSeac-nces of Proteins of Iremcnological Interest. 4th Ed.. U3. Department of Health and Human Services, National Insdtues of Health (1987)], disclose rules far locating CDRs. In addition, cocnpaisr programs are known which are useful for identifying (IR regiona/structures.

The tens ’high titer” refen to an antibody having a binding affinity characterized by a K, equal to or less than 2 χ ΙΟ^-* M for human H4.

By binding specificity for human IL4 is meant a high titer (or affinity) for human, not bovine or murine. 1L4.

Second fusion partner** refers to another nucleotide sequence encoding a

IS protein or peptide to which the first fusion partner is fused in frame or by means of an optional conventional linker sequence (i.e., operatively linked). Preferably it is an hnmunoglognlin. The second fusion partner may include a nucleic add sequence encoding the entire constant region for the same (Le., homologous - the first and second fusion proteins are derived from the same source) or an additional (ix..

heterologous) antibody of interest It may be as immunoglobulin heavy chain or light chain (or both chain* as part of a single polypeptide). The second fusion panner is not faded» a particular immunoglobulin class or isotype. In addition, the second fusion partner may comprise pan of an inmimoglobuiro constant region, such as found in a Fab, or Ffab), (Le-, a discrete part of an appropriate human constant region or framework region). Such second fusion partner may also comprise a sequence encoding an integral membrane protein exposed on the outer surface of a host cell. e.£, as part of a phage display library, or a sequence encoding a protein for analytical or diagnostic detection, eg., horseradish peroxidase, βgalactosidase, etc.

The terms Fv. Fc. Fab, or F(ab)j are used with their standard meanings (see, βψ, Hariow et aL, Antibodies A Laboratory Manual. Cold Spring Harbor Laboemry,(19U)).

As used herein, an engineered antibody describes a type of fusion protein, i.e., a synthetic antibody (e.g-. a chimeric or humanized antibody) ir. which a portion of die light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts from one or more donor antibodies which have specificity for the selected epitope. For example, such molecules may include

AP/P/ 96/00782

-610 at.di>odi=s chaiarxs.ixid by a hurr-mized heavy ;dxn associated w'~ ar. cnmodificd light chau; (or chimenc light chair.), or v.ce versa. Engineered antibodies may also be characterized by alteration of the nucleic acid sequences encoding the acceptor antibody light and/or heavy variable domain 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» a type of engineered antibody which contains naturally-oecannnf variable regioa (light chain and heavy chains) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody.

A humanized antibody’ refers to a type of engineered antibody having its CDRs derived from a noo-human doctor immunoglobulin, the remaining immunogiobufin*derived parts of the molecule being derived from one (or more) human immunoglobulin. In addition, framework support residues may be altered to preserve binding afBninty (see, eg., Queen et aL, Proc. Natl Acad Set USA.

2£: 10029-10032 (1989k Hodgson β al.. Bia/Technology. 2:421 (1991)).

The term donor antibody refers co an antibody (polyclonal, monoclonal, or recombinant) which contributes the nucleic acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first fusion power, so as to provide the fusion molecule and resulting expressed fusion protein with die

One donor antibody suitable for me in this invention is a noo-htanan neutralizing monoclonal antibody (Le., murine) designated as 3B9. The antibody 3B9 is defined as a Mgh titer, hnman-ILX specific (Le, does tat recognize bovine or murine 1L4), neutralizing etibody of isotype IgQ, having die variable fight chain DNA and amino arid sequences of SEQ ID NOS: 1 and 2, and the variable heavy chain DNA and amino arid sequences of SEQ ID NOS: 3 and 4 on a suitable murine IgG constant regioa.

AP/P/ 96/00782 nr recombinant) heserologona to the donor aadbody. which comributee all (or nay portion, but preferably aU) ef the nucleic add sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to ±e second fusion partner. Preferably a human antibody is the acceptor antibody.

CDRs are defined as the complementarity determining regioa amino acid sequences of an embody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g.. Rabat et al., Seocences of Proteins of

Ιγ-γγ.· - l-terest. 4:,-. Ed.. $ ucpirjuMit or Kea-Li and H.xjr, Services, \3c0.nal Insctues of Health (1937). There are three heavy chain aee three light chain CDRs (or CDR regions) in the varioie 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 ««rig»» 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 3B9 may be characterized \ry a certain level of antigen affinity, and a CDR encoded by a nucleic acid sequence of 3B9 in an appropriate structural environment may have a lower or higheraffinity.it is expected that CDRs of 3B9 in such environments will nevertheless recognize the same epitope/s) as 3B9. Exemplary heavy chain CDRs of 3B9 include SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID. NO: 26; and exemplary light chain CDRs of 3B9 include SEQ ID NO: 16; SEQ ID NO: 18; and SEQ ID NO: 20.

A functional fragment is a partial heavy or light chain variable sequence (e.g, minor deletions U die amino or carboxy terminus of the hnnnmoglobiui variable region) which retains the came antigen binding specificity and/or neutralizing ability as the antibody from which the fragment was derived.

An analog is an amino add sequence modified by at least one amino add, wherein said modification can be chemical or a substitution or a rearangement of a few amino acids (Le^ no more than 10), which modification permits the amino add sequence to retain the biological characteristics, e.g^ antigen specificity and high titer or affinity, of the unmodified sequence. For example, silent nutations can be constructed, via subsdtions, to create endonuclease restriction rites within or surrounding CDR regions.

• I

Analogs may also arise as allelic variations. Ah allelic variation or nxxfificadoo to an aheratioo in die nucleic add sequence encoding the ammo add or peptide sequences of the invention. Such variations or modifications may be due to degeneracies 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. For example, the amino arid sequences of the fight chain CDR SEQ ID NO: 16 are identical for the native • 8 *

AP/P/ 96/ 0 0782

Ο ³⁰ rnurmc and humuuxd 3B> antibody. However, ±is CDR sequence is t oded by both SEQ ID NO: 15 and SEQ ID NO: 33. Simiuxiy, CDR SEQ ID NO: 22 is encoded both by SEQ ID NO: 21 and SEQ ID NO: 54; CDR SEQ ID NO: 24 is encoded both by SEQ ID NO: 23 and SEQ ID NO: 55; and CDR SEQ ID NO: 26 is encoded both by SEQ ID NO: 25 and SEQ ID NO: 56.

The terra 'effector agents refers to non-protein carrier molecules to which the fusion proteins, and/or natural or synthetic light or heavy chain of the donor antibody or ocher fragments of the donor antibody may be associated by conventional means. Such non-protein earners can Include conventional cereiea used in the diagnostic field, e.g., polystyrene or other plastic beads, polysaccharides, e.g., as used in the BLAeare [Pharmacia! system, or other noo-protein subsances 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 m increase the half-hfe of the fusion proteins, e.g., polyethylene glycol..

II. High Affinity ILA Monoclonal Antibodies

For use in constructing die antibodies, fragments and fusion proteins of this invention, a non-human species (for example, bovine, ovine, primate, rodent (e.g-, murine and mt), etc.) may be employed to generate a desirable immunogiobalio upon presentment with native human 1L4 or a peptide epitope therefrom. Oonventional bybridoma techniques are employed io provide a hybridoma cell line secreting a nan-fcuman MAb io IL4. Such hybridomas are then screened Bring IL4 covalently attached to 96-weH plates or alternatively with biotinylated T1A for use la a screening assay, as described in detail in Example 2 below. Thus one feature of the instant fcroaion is a method to detect MAbs for human DL4 in which me assay systems avoid denaturing of 114. fo soch a manner. It was discovered that high tiw (or high affinity) MAbs to human IL4 can be detected.

As one example, the production of a high titer, neunfizing MAh from a murine donor is disclosed for the first time. MAb 3B9, which is a desirable murine (donor) antibody for use in developing a chimeric or bumamsed antibody, la describnd in tieofifn Example 1 below. Th· 3B9 MAb is characterized by aa antigen binding specificity for human IL4, with a & of less than 2Λ * Iff* M (about 1.8 x Iff* M) far HA. The K. for IL4 of a Fab fragment of this 3B9 is less than about 3 x Iff” M. The epitope of this antibody could not be mapped to H-4 with linear peptides, and hence the epitope is considered to bind co a non-contiguous epitope. Thepaueraof binding suggests a binding site at the B-C loop (residues 6069) -> C helix (residues 70-93) region. These regions refer to the map designations

AP/P/ 96/00782

-9:rovicec in Cccl: et al. J. Mo?, Bio!.. 2 i <675-6<Ί9ν'.Uisr -: ai. Biol Chem., 267:20371-20376 Π992). Wiodaveret al, FESS 1 «r.. 109:55-6-1 (1992), Redfield et al. Biochem,. 30:11029-11035 (1991), Smith etal. J. Mol, Biol.. 22x^99-904 (1992), Garten « al, (1992), and Powers et al. Biochem.. 21:43345 4346 (1992) and Science. 25fr 1673-1677 (1992), incorporated by reference herein.

Another desirable donor antibody is the rat MAb, 6AI. The production of this MAb is provided below in Example 7. This MAb is characterized by being isotype IgG^ and having a dissoriarion constant for hIL4 of less than 2.0 x 10r^u M (about 1,6 x IO⁴* M). As with 3B9. the target epitope of this 6A1 does not map with IL4 linear peptides, and the epitope is therefore considered to be noncontiguous and three diaeosionaL The pattern of binding to IL4 tnutcins and its biological activity indicates binding in the D helix region of human IL4 (amino acid residues 109-127), most likely around the tyrosine at amino add residue #124.

This invention is not limited to the use of the 3B9 MAb, the 6A1 MAb, or its hypervanable (Le., CDR) sequences. Any other appropriate high titer ILA antibodies characterized by a dissociation constant equal or less than 2.0 x 1(7¹⁰ M for human Π-4 and corresponding anti-IL4 CDRs may be subsumed therefor. Wherever in the following description the donor antibody is identified as 3B9 or 6A1. this designation is made for illustration and simplicity of description only.

///. Antibody Fragments

The present invention also includes the use of Fab fragments or Ffab'X fragment! deived from MAb· directed against human DLA. These fragments are useful as agents protective In vfwo against IL4- and IgE- mediated conditions or in vitro as part of an EL4 diagnostic. A Fab fragment contains the entire light chain and amino teremoal portion of the heavy chain; and an Ffablj fragment is the fragment formed by two Fab fragments bound by disulfide bonds. MAbs 3B9.6A1, and other similar high affinity, IL4 binding antibodies, provide sources of Fab fragments and F(ab*)j fragments which cu 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 RabOj fragments are useful tbcaaaelves as ihaapiuiilc, prophylactic or diagnostic agents. and as dooms of equenoes indndiag the variable regions and CDR sequences useful in the formation of recombinant or humanized antibodies as described herein.

IV. Αηή-lbt Amino Acid and Nucleotide Sequences of Interest

The MAb 3B9 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

AP/P/ $6/00782

-10r.-jcicic acid sequences encoding them, useful ir. designing end obtaining vane s fusion proteins (including engineered antibodies) which are characterized by the antigen binding specificity of the doner antibody.

As one example, the present invention thus provides variable light chain and 5 variable heavy chain sequences from the DL4 murine antibody 3B9 and sequences derived therefrom. The heavy chain variable region of 3B9 is characterized by amino acid residues 20 to 140 of SEQ ID NO. 4. The CDR regions are indicated by underlining is Rg. 2 and are provided in SEQ ID NO: 22; SEQ ID NO: 24; and SEQ ID NO; 26. The light chain clone variable region of 3B9 is characterized by amino add residues 21 to 132 of Fig. 1 (SEQ ID NO: 2]. The CDR regions are from amino add residues 44-58 [SEQ ID NO; Id); 74-80 [SEQ ID NO; 18]; and 113-121 [SEQ ID NO: 20J.

Chimeric heavy chain variable region and signal nucleotide and axnino add sequences are provided. These sequences are identical to the 3B9 heavy chain with

IS the exception of the signal sequence. The chimeric heavy chain signal sequence is provided in SEQ ID NOS; 5 and 6. The CDR regions are indicated by underlining in Rg 3 and are identical in amino acid sequence to the native murine CDRs [SEQ ID NOS: 21-26]. The chimeric light chain variable region nucleotide and »minr> acid sequences are identical to the unmodified 3B9 sequences («minn acid residues

21-132 of SEQ ID NO: 2), making use of the natural mouse signal sequences (amino add residues 1-20 of SEQ ID NO: 2).

A hBmaalaed heavy drain variable region and signal sequences are illustrated in Rg. 4 (SEQ ID NO: Il sod 12). The signal sequence is also provided in SEQ ID NO: 5 and & Other suitable signal sequences, known to those of drill in

Ae an, may be substituted for the signal sequences exemplified herein. The CDR amino add sequences of this construct are identical to the native marine and chimeric heavy chain ORs and are provided by SEQ ID NO: 22 (encoded by SEQ ID NO: 54), SEQ ID NO: 24 (encoded by SEQ ID NO: 55), and SEQ ID NO: 56 (encodes SEQ ID NO. 26).

An exemphuy (synthetic) humanized fight chain variable seqoence is flfasntedta Rg. 5 [SEQ ID NOS: 13 and 14). The signal sequence spans amiao addrcridnes I»19 of SEQ ID NO: 8. The CDR sequences of thia figure are designated by underlining and differ from the CDR of the native murine CDR by a single amino acid of SEQ ID NO: 20. Thus, the CDRs of the humanized light chain are provided by SEQ ID NO: 53 and 16. SEQ ID NO: 17 and 18. and SEQ ID NO:

and 28. This difference is described in detail in Example 3.

AP/P/ 9 6 / 0 0 7 8 2

-11 Της nu:'·;:; acid sequences of this invention, or fragments thertnf. encafing the variable ugh: chain and heavy chain peptide sequences are used in unmodified form or are synthesized to introduce desirable modifications, e.g., restriction sites.

The isolated nannally-occurring or alternatively synthetic nucleic add sequences, which are derived from MAb 3B9 or from other desired high titer IL4 antibodies may optionally contain restriction sites to facilitate insertion or Egation into a suitable nucleic acid sequence such as encoding a desired antibody framework region, Egarion with mutagenized CDRs or fusion with a nucleic acid sequence encoding a selected second fusion partner.

Taking into account the degeneracy of the genetic code, various coding sequences may be constructed which encode the variable heavy and light chain aanno add sequences, and CDR sequences of the invention as well as functional fragments and analogs thereof which sham the antigen specificity of the donor antibody. The isolated nucleic acid sequences of this invention, or fragments thereof, encoding the variable chain peptide sequences or CDRs can be used to Γ produce fusion proteins, chimeric or humanized antibodies, or other engineered antibodies of this invention when operatively combined with a second fusion partner.

These sequences are also useful for mutagenic introduction of specific changes within the nucleic acid sequences encoding the CDRs or framework rqpocs. and for incorporation of the tesuhing modified or fusion nucleic add sequence into a pUsnrid for expression. For example^ silent substitutions in the nucleotide sequence of the framework and CDR-eacoding regions were used to create restriction enzyme sites which facilitated insertion of mutagenized CDR (and/or framework) regions. These CDRregiaos were used in the construction of a humanized antibody of this invention.

It should be noted that in addition to isolated nucleic add sequences encoding portions of the fusion proein and antibodies described herein, other such _r - nucleic acid sequences may be employed, such as those complementary to the native sequences. Useftil DNA sequences include those sequences which hybridize under e stringent hybridization conditions (see, T. Maniatb et iL MntaEUtatflaDUgjifi

Laboratory MsaaiD. Cold Spring Harbcr Laboroory (1982), pages 387 to 389] to the DNA sequences. An example of one such stringent hybridization condition is hybridization at 4XSSC at 65°C, followed by a washing in 0-1XSSC at 65^SC for an hour. Alternatively an exemplary stringent hybridization condition is in 50¾ formamide, 4XSSC at 42*C. Preferably, these hybridizing DNA sequences are ai least about 18 nucleotides in length, i.e, about the size of a CDR.

AP/P/ 9 6 / 0 07 8 2

-12V. Fusion Molecuies and Fusion Proteins

Fusion molecules can encode fusion proteins which includes engineered antibodies such as, chimeric antibodies, and humanized antibodies. A desired fusion molecule contains CDR sequences encoding peptides having the antigen specificity of an ILA antibody, preferably a high affinity antibody such as is provided by the present invention inserted into a first fusion partner (a human framework or human immunoglobulin variable region).

Preferably, the fiat fusion partner is operatively linked to a secood fusion partner. The second fusion partner is defined above, and may include a sequence encoding a second antibody region of interest, far example an Fc region. Second fusion partners may also include sequences encoding another immunoglobulins to which the tight or heavy chain constant region is fused in frame or by means of a hater sequence. Engineered antibodies directed against functional fragments or analogs of JIA may be designed to elicit enhanced binding with the same antibody.

_r 15 The sccood fusion partner may also be associated with effector agents as defined above, indudiag non-protein carrier molecules, to which the second fusion parser may be operatively linked by conventional means.

Fusion or linkage between the second fusion partners, e.g.. antibody sequences, and the effector agent may be by any suitable means, e.g., by oomentioital covalent or ionic bonds, protein fusions, or hetero-btfbnctiooal croes* Bakers, e-g^ caorbodfimide, glutaraldehyde, and the tike. Such techmqoes an known in die an and readily described in conventional chemistry and biochemistry mats.

Additionally, conventional Baker sequences which simply provide far a desired amount of space between the second fusion partner and the effector agent may also be conssocred into the fusion molecule. The design of aoefa linkers is well known to those of skill in the an.

In addition, signal sequences for the molecules of the invention may be modified to enhance expression. As one example a desired fusion protein having as amino acid sequence of the murine heavy chain sequence, which is identical to the chimeric variable heavy chain (Va) of Hf. 2 [SEQ ED NO: 4], had the original signal peptide replaced with another signal sequence (amino add residues 1-20) [SEQ ID NO: ¢).

An exemplary fusion protein contains a variable heavy and/or light chain peptide or protein sequence having the antigen specificity of MAb 3B9, e.g., the V_H [amino acid residues 21-141 of SEQ ID NO: 9 and 10] and V_L chains [amino add residues 21-132 of SEQ ID NOS: 1 and 2], Still another desirable fusion protein of this invention is characterized by the amino acid sequence containing at least one,

-13AP/P/ 9 6 / 00 7 8 2

G ³⁰ χ-ς preicra. t alt of fr.c CDRs ·' t>.e '.-ariaxi rtr.zc. of if.e r.cav> x-.i'cr hg.-,» chaiss of me murine ar.sbody molecuie 555 with the remalmag sequences being derived from a human source, or a functional fragment or analog thereof. See, eg., me humanized V_H and V_t regions of SEQ ID NOS; 11 and 12 and SEQ DD NOS; 13 and 14 (Figs. 4 and 5).

la still a further embodiment, the engineered anobody of the invention may have attached co it an additional agent. For example, the procedure of recombinant DNA technology may be wed to produce an engineered antibody of the invention in which the Fc fragment or CH3 domain of a complete antibody molecule has been replaced by an enzyme or other detectable molecule, (te^ a polypeptide effector or reporter molecule)

The second fusion partner may also be operatively linked to a noonnmonoglobulin peptide, protern or fragment thereof heterologous to the CDR cootaining sequence having the antigen specificity of murine 3B9. The resulting protein may exhibit both anti-114 antigen specificity and characteristics of the nootinmuRoglobulin upon expression. That fusion partner characteristic may be, rig.. 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.

Another desirable protein of this invention may comprise a complete antibody molecule, having full length heavy and light chains, or any discrete fragment thereof, such as the Fab or F(ab% fragments, a heavy chain tinner, or any minimal recombinant fragments thereof such as an F. or a single-chain antibody (SCA) or any other molecule with the same specificity as the selected donor MAb, C-g., MAb.3B9 or 6AI. Such protein may be used in the fora of a fusion protein, or may be used in tea unfused fora.

Whenever the second fusion partner is derived from another antibody, e.g., any isotype or class of immunoglobulin framework or constant region, aa engineered antibody results. Engineered antibodies can comprise immunoglobulin (Ig) constant legions and variable framework regions from one source, e-g., the acceptor antibody, and one or mote (preferably all) CDRs from the donor antibody, e.g^ die aad IL4 antibody described herein, fo addition, alterations, e.g, deletions, substitutions, or aiditions, 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 chains of the Π4 MAb (optionally modified as

- 14AP/P/ 9 6 / 0 0 7 8 2 cescr.tied'i or cr.e or mors of the bdow-identitied heavy or light chain CDRs (see Example 3). The engineered antibodies of the invention are neutralizing, ie., they desirably block binding to die receptor of the ILA protein. For example, the engineered antibody derived from MAb 3B9 is directed against a specific tertiary protein epitope of human IL4 believed to be at the B-C loop -> C helix region, as described above.

Such engineered antibodies may include a humanized antibody containing die framework regions of a selected human immunoglobulin or snbtype, nr a chimerin antibody containing the human heavy and Hght chain constant regions fused to the ILA antibody functional fragments. Λ suitable human (or other animal) acceptor antibody may be one selected from a conventional diabase, e,g~ die RABAT® diabase, 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 die framework regions of the donor antibody (on an amino add basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of die donor CDRs. A acceptor antibody capable of donating light chain constant or variable framework regions may be seleoed in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.

Derintiy the hereroiogous framework and constant regions are relecred from human immonoglofaoBn classes and isotypes, soeb aa IgG (subtypes 1 through 4), IgM, IgA, sad IfE- However, the acceptor antibody need not comprise only human immunoglobulin protein sequences. For instance a gene may be constructed in which a DNA sequence encoding pan of a human immunoglobulin chain is fused to a DNA sequence encoding a noo-immnnoglobulin amino Kid sequence such as a polypeptide effector or reporter molecule.

One »»·ηψΐ« of g particularly deniable humanized antibody contains CDRs of 3B9 inserted onto the framework regions of a selected human antibody sequence.

For neutralizing horamaed antibodies one, two or preferably three CDRs from the ILA antibody heavy chain andfer fight chain variable region are inserted into the framework regions of the selected human antibody sequence, replacing the native CDRs of the latter antibody.

Preferably, 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 replaced. It is possible to replace the CDRs only in the

AP/P/ 9 6 / 0 0 7 8 2

- 15Ar .

human heavy , nn, using as light chain the unmodified ugh: chain from the humaacccptor antibody. Still alternatively, a compatible light chain may be selected free 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 preferably has the structure of a natural in man antibody v a fragment thereof, and possesses the comhinan^ of properties required for effective therapeutic use. ¢.^ treatment of Π-4 mediated inflammatory diseases in man. or for diagnostic toes.

As another example, an engineered antibody may contain three CDRs of the variable light chain region of 3B9 [SEQ ID NO: 16,18,20 and 28] and three CDRs of the variable heavy chain region of 3B9 (SEQ ID NO: 22,24 and 26]. The resulting humanized antibody is characterized by the antigen binding specificity and high affinity of MAb 3B9.

It will be understood by those skilled in the an that an engineered antibody may be farther modified by changes in variable domain amino acids without necessarily affecting the specificity and high affinity of the donor antibody (Le„ an analog). For example, humanized monoclonal antibodies have been constructed wherein the light chain amino add residue at position 120 was an arginine (SEQ ID

NO. 13 and 14) or threonine [SEQ ID NOS:57 and 58). It is antiapamd that heavy and light chain imino adds may be mbedmaed by ocher amino adds efaher fa the variable domain frameworks or Q3Ra <r both.

In addition, the constant region may be altered to enhance or decrease selective properties of the molecules of the instant invention. For example, dimerizaixA, binding to Fc receptors, or the ability to bind and activate ««pt»* (see, e.g„ Angal et al., Mbt. famuBML 30:105-108 (1993), Xn et aL, J. Biol. Chem 252:3469-3474 (1994), Winter et aL, EP 307,434-B).

A fusion protein which is a chimeric antibody differs from the hnmanfaed antibodies described above by providing the entire non-human donor antibody heavy chain and light chain variable regions, including framework regions, fa association with human immunoglobulin constant regions far both chains. It is anticipated that chimeric antibodies which retain additional non-human sequence relative to humanized antibodies of this invention may elicit a significant immune response in humans.

5 Such antibodies are useful in the prevention and treatment of IL4 mediated allergic disorders, as discussed below.

AP/P,' 56/00782

- 16Ah

V/. Production of Fusion Proteins and Ensir^red Antibodies Preferably, the variable light and/or heavy chair, sequences and the CDRs cf

MAb 3B9 (SEQ ID NO: 16, 18,20, 22,24 and 26] ar other suitable donor MAbs (e.g.. 6AI), and their encoding nucleic acid sequences, are utilized in the construction of fusion proteins and engineered antibodies, preferably humanized antibodies, of this invention, by the following process. The same or similar techniques may also be employed is generate other embodiments of this invention.

A hybridoma producing a selected donor MAb, e.g-, the murine antibody 3B9, is cocvenrionally dotted, and the DNA of its heavy and light chain variable regions obtained by techniques known to ooe of skill in the art, eg., the techniques desenbed in Sambrook et t>L. Molecular Cloning (A Laboratory Manuall 2nd edition. Cold Spring Harbor Laboratory (1989). The variable heavy and tight regions of 3B9 containing at least the CDRs and those portions of the acceptor MAb light and/or heavy variable domain framework region required in order to retain donor MAb binding specificity, as well as the renaming immunoglobulin-derived parts of the antibody chain derived from a human immunoglobulin are obtained using polynucleotide primers and reverse transcriptase. The CDRs are identified using a known database and by comparison to other antibodies.

A mouse/human chimeric antibody may then be prepared and assayed far binding ability. Such a ddmoic antibody cootiiiis the entire non-buman donor antibody V_a and V_Lregjona, ia association with human lg coostant regions for both chains.

Homologous framework regions of a heavy chain variable region from a human antibody were identified using computerized databases, e.g, ΚΑΒΛΤΦ. and a human antibody having homology io 3B9 wu setecsed as die acceptor antibody.

The sequences of synthetic heavy chain variable regions containing the 3B9 CDRs within the human antibody frameworks were designed with optional nucleotide replacements in the framework regions β incorporate restriction sites. This designed sequence is then synthesized by overlapping oligonucleotides, amplified by polymerase chain reaction (FCR), sad oanectcd for errors.

A snbafaie tight chain variable framewuik region was designed in a similar manner.

A humanized antibody may be derived from the chimeric antibody, or preferably, made synthetically by inserting the donor MAb CDRs from the heavy and light chain* appropriately within the selected heavy and light chain framework. Alternatively, a humanized antibody of die invention nude be prepared using standard mutagenesis techniques. Thus, the resulting humanized antibody contains

-17AP/P/ 9 6 / 0 0 7 8 2 huoan framework rca. ns ir,d dor.or MAb CDRs There may be s.bsequen: manipulation of framework residues. The resulting humanized antibody can be expressed in recombinant host cells, e.g., COS or CHO cells. Additional details of this procedure are provided in Example 4. Other humanized antibodies may be prepared using this technique on other suitable IL4-specific, neutralizing, high titer, non-human antibodies.

A conventional expression vector or recombinant plasmid is produced by placing these coding sequences far the fusion protein in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal seqaences, which can be derived from ocher known antibodies. Similarly, a second expression vector is ptodneed having a DNA sequence which encodes a complementary antibody light or heavy chain. Preferably this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are ooacerned so oo ensure as far as possible that each polypeptide chain is functionally expressed.

A selemed host cell is co-aansfecwd 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 chains The transfected cell is then cultured by conventioaal techniques to produce die engineered antibody of the invention. The humanized antibody which indades the association of both the recombinant heavy chain and/or tight chain is screened ftom culture by appropriate aasay, such as ELISA or RIA. Similar conventional techniques may be employed to construct other fusion proteins and molecules of this invention.

Suitable vecaon for the dotting and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in die art. FOr example, the conventional pUC scries of cloning vectors, may be used. One vector used is pUCl9, which is commercially available from supply houses, as Amerehaot (Buckinghamshire, United Kingdom) or Phanada (Uppsala, Sweden). AddStionafly, any vector which is capable of rephearing rai&Iy, has an abundance of cloning sites nd marker genes, and is easily manipulated may be used for cloning. Thus, the selection of the cloning vector is not a limiting factor in this invention.

Similarly, the vectors employed for expression of the engineered antibodies according »this invention may be selected by one of skill in the an from any conventional vector. The vectors also contain selected regulatory sequences which

AP/P/ 96/00782

-18Ar .

arc in operaave association w;m . 'e DNA coding sequences of the immunogloouim regions and capable of directing the replication and expression of heterologous DNA sequences ia selected host cells, such as CMV promoters. These vectors contain the above described DNA sequences which code for the engineered antibody or fusion molecule. Alternatively, the vectors may incorporate the selected immunoglobulin sequences modified by the insertion of desirable restriction sites for ready manipulation.

The expression vectors may also be characterized try marker genes suitable for amplifying expression of the heterologous DNA sequences, e-g., the mammalian dihydrofolate reductase gene(DHFR) or neomycin resistance gene (neo¹). Other preferable vecuar sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobm promoter sequence (beuglopro). 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. rcplkons, selection genes, enhancers, promoters, signal sequences and the like, may be obtained from natural sources or synthetired by known procedures for use io directing the expression and/or secretion of the product of the recombinant DNA in a selected host Other appropriate expression vectors of which numerous types are known in the an for mammalian, bacterial, insect, yeast and fungal expression may also be selected for dus purpose.

The present inveatkn also encompasses a cell line transfected with a recombinant plasmid comainiag the coding seqnencca of die engineered antibodies or fusion molecules hereof Host cells useful for the cloning and other manipulations of these dotting vecus are also conventional. However, most desirably, cells from various strains of £. ceO ere used forrepticadon of die dotting vecton and other steps in the construction of fusion proteins of this Invention.

Suitable host ceils or cell lines for die expression of the engineered antibody or fusion protein of die invention are preferably a eukaryotic cell such as CHO,

COS, a fibroblast cell (e.g.313). and myeloid ceils among others, and most preferably a mammalian ceiL sack as a CHO cdl era myeloid cell. Human cells may be used, thus enabling the molecule ao ba modified with bnman jtycosyhtion patterns. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells and methods for transformation, culture.

amplification, icreening and product production and purification are knowr. in die an. See, e.g., Sam brook ere/, cited above.

AP/P/ 9 6 / 00 7 8 2

-1910

Bacterial cells ma, ,rovc useful as host cells suiu^'e for the expressioc of the recombinant MAbs of the present invention. However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or ic a non-glycosylated form, any recombinant MAb produced in a bacterial cell would have to be screened for retention of antigen binding abiliry. If the molecule expressed 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 expressiou are well-known u host cells in the field of biotechnology. Various strains of B. subtilis, Streptomyces, other bacflli and the like may also be employed in this method.

Where desired, strains of yeast cells known so those skilled in the art are also available as host cells, as wefl as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller et al., Genetic Engineering. £:277-298, Plenum Press (1986) and references cited therein.

The general methods by which the vectors of the invention may be constructed, transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the fusion protein or engineered antibody of the invention from such host cell are all conventional techniques. Likewise, once produced, the fusion proteins or engineered antibodies of the invention may be purified from the ceQ culture contents according io standard procedures of the art, including ananonfaan suttee precipitation, affinity columns, column chromatography, gel electrophoresis and the tike. Such techniques ate within the skill of the art and do not fenit due invention.

Yet another method of expression d the humanized antibodies may utilize expression in a raugcnic animal, neb as described in U. S. Patent No. 4J73 J16. This relates to an expression system using ihe animal's casein promoter which when oansgenically incorporated into a mammal permits the female to produce the desired recombinant protein in its milk.

Once expressed by the desired method, the engineered antibody is then examined for m vitro activity by use of an appropriate assay. Presently ¹ conventional ELISA assay formats are employed io taett qualitative aad quantitative binding of the engineered antibody io an 1L4 epitope. Additionally, other in vitro assays, e.g. BIAcore [Pharmacia], may also be used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the engineered antibody in the body despite the usual clearance mechanisms.

AP/P/ 9 6 / 0 0 7 8 2

Follower,g the procedures described for njraanxzca antibodies creparet .-ora

3B9, one of Skill in the an may also cocsaract humanized antibodies from other donor EL4 ana bodies, variable region sequences and CDR peptides described herein.

Engineered antibodies can be produced with variable region frameworks potentially recognized as “self by recipients of the engineered antibody. Minor modifications to the variable region frameworks can be implemented to effect large increases in antigen binding without appreciable increased immunogenicity for the recipient. Such engineered antibodies can effectively treat a human for ΠΑ mediaaed conditions. Such antibodies may also be useful in the diagnosis of such conditions.

VII. TherapeubdPropkytaaic Uses

This invention also relates to a method of treating humans experiencing an allergic disorder which comprises administering an effective dose of antibodies including one or mare of the engineered antibodies or fusion proteins described herein, or fragments thereof.

The therapeutic response induced by the use of the molecules of this invention is produced by the binding to human IL4 and thus subsequently blocking IgE release. Thus, the molecules of the present invention, when in preparations and formulations appropriate for tiierapeutic use, are highly desirable for those persons experiencing an allergic response, such as an allergic rhinitis, conjunctivitis, atopic dermatitis, atopic asthma, and anaphylactic shock.

The forion prawans, antibodies, engineered antibodies or fragments thereof of tins invention may also be used to conjunction with other antibodies, particularly human MAbs reacore with other makers (epitopes) responsible for the condition against which the engineered antibody of the invention is directed. Similarly MAbs reactive with epitopes responsible for the condition ba selected animal against which the antibody of the invention is directed may also be employed in veterinary compositions.

The therapeutic agents tf this invention are believed to be desirable for treatment of allergic conditions far from about 2 days to about 3 weeks, or as needed. For example, longer treatments may be desirable when treating seasonal

AP/P/ 9 6 / 0 0 7 8 2 infosba protocol with prior an treatments of ΠΑ mediaaed disorders. The dose and duratk» of treatment relates to die relative duration of die molecules of die present invention is the human circulation, and can be adjusted by one of skill in die an 35 depending upon the condition being treated and the general health of ±e patient.

The mode of adminismtion of the therapeutic agent of the invention may be any suitable route which delivers the agent to the host The fusion proteins.

annbccics, engineered anobt,. ^:es, and fragments thereof, and pharmaceutical compositions of the invention arc particularly useful for parenteral atimiaistration.

i.C., subcutaneously, intramuscularly, intravenously, or intranasally.

Therapeutic agents of the invention may be prepared u pharmaceutical 5 compositions containing an effective amount of the engineered (e.g., humanized) antibody of the invention as an active ingredient io a pharmaceutically acceptable earner. Ia the prophylactic agent of the invention, an aqueous suspensioa or solution containing the engineered antibody, preferably buffered at physiological pH. in a form ready for injection is preferred. The compositions for parenteral odaicissation will commonly comprise a solution of the engineered antibody of the invention or a cocktail thereof dissolved ia an pharmaceutically acceptable carrier, preferably an aqueous earner. 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 free of particulate maser. The» solutions cay be sterilized by conventional, well known sterilization techniques (e.g.. filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antibody.of the invention in such pharmaceutical formulation can vary widely, te^ from less than about (15%, usually at or at least aboat 1% to as mochas 15 or 20% by weght and will be selected primarily based on fluid vuimca, viscosities, etc^ acoocdfog tothe particular mode of sdznitrfsoatioe selected.

Thns, a phamaeeudcal composition of the invention for iacraixmscalar injection could be prepared to contain 1 mL sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of an engineered antibody of the invention. SimOariy. a pharmaceutical comporitiou of the invention for intravenous infttsion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 to about 30 and preferably 5 mg to about 25 mg of an engineered antibody of the invention. Actual methods far preparing parennally administrable compositions ate well known er will be apparent to dtoae dolled in the ut and are described in more detafl in, for —mpt*, RentinggnA Pharmaceutical Science, 15th etL Mack Publishing Company, Easton. Pennsylvania.

It is preferred that the therapeutic agent of the invention, when in a phxrmaceuccel preparation, be present in unit dose forms. The appropriate therapeutically effective dose can be determined readily by those of skill tr. the art To effectively treat an inflammatory disorder in a human or ocher animal, one dose of approximately 0.1 mg to approximately 20 mg per 70 kg body weight of a protein

AP/P/ 96/00782

-22Ar .

- or an ano body of this invcntios should be administered parent-rally’, preferably i.rn.

(intramuscularly). Such dose may, if necessary, be repeated u appropriate time intervals selected as appropriate by a physician during the inflammatory response.

The invention also encompasses the administration of the TLA fusion proteins 5 of this mvendoo concurrently or sequentially with other antibodies or fusion proteins characterized by anti-IL4 activity, such as anti-tumor necrosis factor activity or other pharmzceotical activities compatible with the UA receptor binding ability of die fusion proteins of this invention. Such other antibodies are available commercially or can be designed In a mvuier similar to that described herein.

The fusion proteins and engineered antibodies of this invention may aiso be used io diagnostic regimens, such as for the detenmnatioo of 1L4 mediated disorders or tracking progress of treatment of such disorders. As diagnostic reagents, these fusion proteins may be conventionally labelled for use in ELISA's and other conventional assay formats for die measurement of Π-4 levels in serum, plasma or other appropriate tissue. The nature of the assay in which the fusion proteins are used are conventional and do not limit this disclosure.

The antibodies, engineered antibodies or fragments thereof described herein can be lyophilized for Stonge and reconstituted in a suitable carrier prior to use.

This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution techniques can be employed.

The following examples illustrate various aspects of this invention including the coostraction of exemplny engineered antibodies and expressiou thereof In suitable veewrs and boa cells, and are act to be construed as Smiting the scope of this invention. All amino adds are identified by conventional three letter or single letter codes. AH necessary restriction enzymes, plasmids, and other reageaa and materials were obtained from commercial sources unless otherwise ΑΠ general cloning ligation and other recombinant DNA methodology were as performed fat T. Marians er aL, died above, or the second edition thereof (19S9),

C. eds. Sambrook a aL by the same publisher CSauabrookeroL”).

$ ³⁰

AP/P/ 9 6 / 0 0 7 8 2

-23A Η . J - ύ

EvtT.pie 1 - krC iTtiC of MAb 3B

A. Immunization procedure

Four mice (FI hybrids of Balb/c and C57BU6) were immunized subcutaneously with 50 gg recombinant £. coli human IL4 in Freunds complete adjuvant and 4 weeks later boosted intnperitoncally ftp.) with 50 gg IL4 in Freunds incomplete adjuvant. On the basis of a good serum antibody titre to 114 one mouse received further immunizations of 200 gg IL4 (tp. in saline) at 8 weeks, two days later with 100 ggIL4(Lp. in saline) and two days later with 50 gg IL4 (Lp. fa saline). Two days foQowing the final immunization a splenectomy was performed.

B. Fusion Procedure and Screening System

Mouse spleen cells were used to prepare hydridouaas (by standard procedures. e.g. as described by Kohler et al, Nature. 256:495 (1975)) from which >250 clones ot cells were screened far secretion of antibody to IL4, using (be commercially available BIAcore system, and ELISA assays u described below, for £L4 binding. Five wells gave a positive response. Only 1 done from mice, 3B9, was strongly positive. All secondary clones derived from 3B9 were positive.

Example 2 - ELBA Assays and Affinity Constants

A. ELISA

The screening assay, performed as fofiowa, was designed to measure affinity for native human Π4. For experiment 1 aldehyde activated 96-wefl plates were coated with 114 at 1 ggAnL, 100 gl/well in 0.1M borate buffer, pH L5, and incubated overnight at RT. The UL4 was covalently attached to the plate. IL4 solution was aspirated end ooo-specific binding (NSB) sites were blocked with 1% bovine serum albumin (BSA) in TBS buffer (50 mM Tris, 150 mM NtG, 1 mM MgQ„ 0.02% NaN„ pH 7.4) far 60 minutes at 37*C. Following this tad each of the following steps, the plate was washed 4 times ia wash buffer (10 mM Dis. 150 mM Nad, 0.05% Tween 20,0.02% NaN,, pH 7.4). Following this, 50 gL hybridoma medium (or purified 3B9 or Fab fragments) and 50 gL assay buffer (0.5% bovine gaaaaa gtoboUn in TBS buffer) was added and the plates were incubated for 60 mfaramset37*C. One hundred gL of biotinylated anti-mouse antibody was added per weQ in assay buffer and incubated as above. One hundred gL of alkaline phosphatase conjugated streptavidin was added per well and incubated (30 minutes at 37^eC). One hundred gUwell PNP substrate was added and incubated 30 minutes at 37*Q Readings were taken at an optical density of 405 nm.

AP/P/ 96/00782

-24For experiment 2. screpuvidin-coa:«j plates (IOC jiL/well. 1 gg/nL in phosphate buffered saline (PBS)) were incubated overnight at 4*C and were assayed as follows. Streptavidin soluuoa was aspirated, NSB sites blocked with 1 % BSA in TBS buffer (SO minutes at 37*C). Following this step, and each of the steps which follow, the plates were washed four times in wash buffer. Fifty tiL biotinylated ΠΑ was added with SO pL assay buffer and incubated for 30 minutes at 37°C Following this, 50 yL purified 3B9 IgG or Fab fragment (or hyfaridoma medium) plus SO pL assay buffer was added, incuhued 60 minutes it 37*C One hundred gL anti-mouse IgG aBcihae phosphatase conjugate was added and incsbated far ¢0 minutes at 37*C Ooe hundred pL PNP substrate was added and incubated 30 minutes at 37*C The readings wee taken as above.

B. Calculation of3B9 Affinity for 0,-4

Using the results of the experiments described above, and summarized as follows, the K. for 3B9 was calculated as described in Beatty et *1, J.Immqnol,

Methods. 100:173-179 (1987):

Kwr* — ,1........

K2(Ab*J-{AbJ)

Ab' concentration of Ab bound at 150 ng/ml biotinylated hIL4 Ab concentration of Ab bound at 300 ng/ml biotinylated hIL4

Dissociation constants, K*. «era calculated from the relationship:

<s-_J_

Ktf

Experiment 1: ELISA assay on a streptavidin coated 96-well plate (100 ngAraO). IQ -12 x Iff Μ (3B9 Fab)

Experiment 2: ELISA assay on a streptavidin coated 96-weU plats (100 ogAreO). IQ * 1.4 x 1C^W Μ (3B9 IgG)

C Spedfidty

MAb 3B9 reougntaa human QA bur does not recognize bovine or marine ELA Ooe way β determine this is as follows. An ELISA can be performed usings

96weflpla»eoeiod with sad-moore IgG. and subsequently blocked with bovine scram tfane, open wWch 50|tL3B9 (100 ngfotij, 25 pLaf aao-human ΠΑ sad 25 yL biotin-IL4 were ioCobated far ¢0 mrautes at 37*C, followed by a wash, streptavidin conjugated alkaline phosphaoue and PNP.

Similarly, MAb 6A1 was found not to recognize bovine or murine ILA.

AP/P/ 96/00782

-23* > 1 *4 Γ

F.xa.Ti?:- ? - Humanized Antibody

One humanized antibody was designed to contain murine CDRs within a human antibody framework. This humanized version of the EL4 specific mouse antibody 3B9, was prepared by performing the following manipulations.

A. cDNA Cloning cDNA dooes were made of the 3B9 heavy and light chains from mRNA extracted oat of the 3B9 hybridoma cell line (Example 1] using a Boehringer Mannheim Idt Primers tpeeiBe far either the mouse hinge region or kappa constant region were used far first strand synthesis.

The kappa chain primer is (SEQ ID NO: 29]:

f-CTAACKCTCATrCCnTrnjAAGCrCTTCACAATGGG-3·

The gamma heavy chain primer is [SEQ ID NO*. 30]:

yCTACATATGCAAGGCTTACAACCACAATC 3'.

The double stranded cDNA was cloned directly into plasmids pGE\i7f+ [Promega] that were then transformed into £. coli L)U5~<i [Bethesda Research Labs].

B- DNA Sequencing

Eight heavy and one light chain murine cDNA denes from Pan A 20 above were aeqaenced. The results of sequencing of die variable regions of these dooes are shown in SEQ Π> NO: 1 and 2 and 3 and 4. Each done contained amdno acids known to be oonaerved among moose heavy chain variabto regions or light chain variable regions, and murine signal sequences. The GDR amino add sequences are listed below.

The CDR regions for the heavy chain are SEQ ID NO*. 22,24 and 26.

(amiao adds 50-56,71-86 and 119-129 of SEQ ID NO: 4). See Fig. 2. These sequences are encoded by SEQ ID NO: 21. SEQ ID NO: 23, and SEQ ID NO: 25. respectively. The CDR regions for the tight chain are SEQ ID NO: 16, 18 and 20 (amino adds 45-58,74-80, and 113-121 of SEQ ID NO: 2). See Fig. 1. There sequences are encoded by SEQ ID NO: 15.17, and 19. respectively. ¹

C Selection of Henan ftatnawotks

Following the cloning of 3B9. the anaao add sequences of the variable region (amino acids 21-132 of SEQ ID NO: 2 and amino acids 20 to 140 of SEQ ID NO: 4) were compared with the human immunoglobulin sequence database using the KABAT& and the SWISS databases in order to identify a human framework for both the heavy and light chains which would moat closely match the murine parent in sequence homology. In addition to these searches for sequence homology, the

AP/P/9 6/00782

-26I heavy a-d light chains were also evaluated against a positional '.iraoasc generated from itruutural models of the Fab domain to assess potential conflicts due to amine acid substitutions which might influence CDR presentation. For the present case, co obvious conflicts were detected in the structural search; hence, the DNA coding deduced from the amino add sequence homology searches was used.

The heavy chain framework regions of an antibody obtained from a tumen myeloma immunoglobulin (COR) was used [Ε. M. Press and Ν. M. Hogg, Biochem. £, 112:641-660 (197(0]. This sequence was found to be approximately 77% homologous (04% ideatfry) io the 3B9 variable chain region at the amino add level

For a suitable light chain variable framework region, the light chain variable framework sequence of the human antibody identified in H. G. Klobeck a al, NncL Acids Res.. 12:6515-6529 (1955) was used. The human antibody sequence was found to be approximately 802% homologous (72.0% identity) to the murine 3B9 variable light chain region at the amino acid level.

Given the murine 3B9 CDRs [SEQ ID NO: 15-26] and the sequence of the human antibody, a synthetic heavy chain was made and PCR performed to fill in and amplify the DNA. These sequences were synthesized by the following overlapping oligonucleotides and amplified by PCR. SEQ ID NO: 31-37 provides five overlapping oligos and 2 PCR primers. Oligo 1 [SEQ ID NO: 31] is found spanning bases 5-121. OGgo 2 [SEQ ID NO: 32} is found spanning bases 122-241, and oligo 3 [SEQ ID NO: 33] ia found spanning bases 242-361. Therwobooom strand primers SEQ ID NO: 34 and SEQ ID NO: 35 span bases 134-110 and bases 253-230. Any errors m the mapped sequence which were inserted by PCR were corrected. PCR was again performed using as the 5* primer nucleotides 1-25 SEQ ID NO: 36 and as the 3’primer nucleotides »1-341 SEQ ID NO: 37.

The synthetic «viable region was ligated into the expression vector pCD along with the synthetic signal sequence SEQ ID NO: 5 and 6 from the chimeric heavy chain construction along with an IgGj human constant region. The synthetic

V„ and signal sequence nucleotide and amino add sequences are provided in Fig. 4 [SEQ ID NOS: 11 and 12]. The «tian add sequences of the CDRs [SEQ ID NOS: 22,24 and 26] are identical K the murine 3B9CENU. However, the coding sequences for tbcae CDRs (SEQ ID NOS: 54,55 and 56] differ from the marine 3B9 coding sequences [SEQ ID NOS: 21,23 and 25]. The resulting expression vector, IlAhzbcl-l-Pcd is shown in fig. 9.

AP/P/ 96/00782

-27The CDR gene regions of a pre -jsnng ii*ht cnajr. framework were rastriccoc digest removed and replaced with the following synthetic IL-4 CDR genes, which were synthetically made.

For CDR 1:

SEQ ID NO: 38: 5CTAGCTGTGTCTCTCGG0GAGAGGGCCACCATCAAC TGCAAGG3’

SEQ ID NO: 39: CCTTGCAGTTGATOGTGGCCCTCTOGCCCAGAGACACAG SEQ ID NO: 40:TOGAGAGGCCTCCCAAAGTGTTCATTATGATGGTGATAG TTATATQAACTOCTATCAOCAGAAAOCC

SEQ ID NO: 41:

GGGTrTCTGCTGATACCAGTTCATATAACTATCACCATCATA

ATCAACACTTTGGGAGGCCTC

ForCDR2:

SEQ ID NO*. 44:

IS GGGCAGCCrCCTAAGTTGCTCATTTACGCTGCATCCAATCTA GAATCTOOGGTAC SEQ ID NO: 45:

CCGAGATTCTAGATTGGATGCAGCGTAAATCAGCAACTTAGG

AGGCTCCCC

FcrCDR3:

SEQ ID NO: 42:

ATACTACTOTCAGCAAAGTAATCAGGATCCTCOGAGGTTCGG

CGGAGGGAC

SEQ ID NO: 43:

CTTGGTCCCTCCGCCGAACCroGGAGOATCCTCATTACTTTG CTGACAGTAGT

The synthetic V_Land signal sequence nucleotide and amino add sequences are provided in Hg. 5 (SEQ ID NOS: 13 and 14J. The amino add sequences of die first two CDRs (SEQ ID NOS: 16 and 18] ate identical to the corresponding murine

3B9 CDRs. However, the coding sequence for the first CDR [SEQ ID NO: 53] differs from the marina 3B9 codrng sequence [SEQ ID NO: 15J. Farther, b the last CDR. two hnmaafaed constructs of the 3B9 amino acid sequence ware ematruemd. One, [SEQ ID NO: 28]. differs by a single amino add (SEQ ID NO: 20] fiom the native murine 3B9 sequence. SEQ Hf NO: 28 is encoded by SEQ ID NO: 27. The synthetic variable light regions were ligated into the expression vector along with de signal sequence (SEQ ID NOS: 7 and 8). One of the resulting expression vectors, IL4hzlcl-O-Fcn is illustrated in Rg. 10.

AP/P/ 9 6 / 0 0 7 8 2

- 28 · >.cse $yftL,cac vanablc light and/or heavy chain sequence arc employed in the consrucuoa of a humanized antibody.

Example-4 -Expression of Homanized MAb in COS and CHOcelh pUC18 subclonea for the V. were made » add a signal sequence originally obtained &raa a human antibody SEQ ID NO*. 5. For the V_L, pUCl 8 snbdonei were made to add a signal sequence SEQ ID NO. 7.

The humanized heavy chain, derived from an IgG, isotype, exhibits 893% boooiogy (843% identity) at the amino acid level with «he marine heavy chain bom 3B9. This synthetic V„ is provided in amino acids 20-141 of SEQ ID NOS: 11 and 12.

The humanized light chain, a human kappa chain, sbowi 92.0% homology (86.6% identity) with 3B9 at the amino acid level. This synthetic V_L (amino acids 21 to 131 of SEQ ID NOS: 13 and 14] containing the 3B9 Q3Rs was designed and synthesized as described above for the synthetic heavy chains.

The DNA fragments containing their respective signal United to either the humanized heavy or light variable regioes were inserted into pUCl9*based mammalian eell expression plasmids containing CMV promoters and the hnmaa heavy chain or human light chain constant regions of the chimera produced in Example 5 below, by conveotiooal methods [Maniatis et ni, dteti above] to yield the plasmids IL4hafacl«Utd (heavy chain) (Rgnre 9) and IL4hzlcl-o-PcnX)ight chain) [Figure 10J. The HZHC and HZLCphaztids are cotransfected into ODS cdls and sopenatants assayed by the EUSA described immediately above for the presence ofbumanized antibody after three and five days. Another humanixed antibody Wttcoutnicted bar with an lgG4 isotype.

The above example describes the preparation of an exemplary engineered antibody. Similar procedures may be followed for the development of other engineered antibo&s, using other anti-IL4 antibodies (e-g, 6A1 * Example 7) developed by conventional means.

AP/PI96/00782

Pwmyfc < - rfOrfroate Aerfhnrf»

A- A chimeric heavy chain wu constructed by iaoiadng the amine variable heavy chain regioe from die original moo» MAb 3B9 as ΰ EcaRl-fisEQ restriction fragment. A small DNA ohgotner was designed and synthesized to link the mouse variable region with the human IgGl constant region (BmEn * Apal):

5* primer SEQ ID NO: 50: GTCACCGTCTCCTCAGCTAGCACCAAGGGGC 3' primer SEQ ID NO: 51: CTTGGTGCTAGCTOAOGAGACG

-29Λ r

These two fragments were i._o :d into piasnud pCD (See Fig. 7;(digestcd with EcoRI and Apal) that already encodes the human IgGi constant region. This clone did not express; therefore, the wild-type 517TR and signal sequence were deleted and replaced with SEQ ID N03 and 6.

Because a convenient restriction endonuclease site was not available at the 3* end of the signal sequence, a BstFTT site was introduced (i.e., a silent mutation) via PCR. The following PCR primers were used;

SEQ ID NO: 48: J* primer 5’CAGGTTACCCTGAAAGAGTC 3’

SEQ ID NO: 49:3' primer 5GAAQTAGTCCTTGACCAG 3*

A BsrPTT. Pal restriction fragment was then isolated from this plasmid. A new signal sequence and 5TTTR were then designed and synthesized having EcoRI and BstEH ends.

SEQ ID NO: 46: S' primer AATTCGAGGACGCCAGCAACATGGTGTTGCA GACCCAGGTCTTCATTTCrCTOTTOCTCTGGATCTCTGGTOCXTACCOGC

AG

SEQ ID NO: 47; 3' primer GTAACCTGCCCGTAGGCACCAGAGATCCAGA

GCAACAGAGAAATCAAGACCTGGGTC7GCAACACCATGTTGCTGG0GTC

CTCG

The chimeric light chain was constructed by applying the PCR technique to the original murine 3B9 light chain that wu cloned into pGEM72f(+) [Promegal· The primers affixed were the comnedally available pUCll universal reverse primer at the 5* end (EfipKI) and a 3* primer fiat introduces a 22si she [TCATCTAOATCCCO CCQCCACΛ<3TACOTTTCA^CΓCCAGCTTOGTOCC3^, SEQ ID NO: 52], nsed to fuse the mouse variable region to the human constant regiou. This variable region was then ligated into the expression vector pCDN (EcoRI £2gd) (Hg. 8) that already contains the human kappa region.

Media stiperaaants were collected three and five days later and assayed by die ELISA described as follows: ELISA plates were coated with 0, Ipg of a goat antibody specific for the Fc region of human antibodies. The media supernatants ware added for one hour. A horseradish peroxidase conjugated goat anribodjrqpedflcfcr an entire human IgG antibody was added. TWa was followed by wfcfirioa of ABTS peroxidase snbamte (Kfafeegaard fi Perry Laboratories Ine, Gaithersburg. MD) for one hour. Expression of the chimeric antibody was detected. In a second ELISA the COS cell supernatants containing the chimeric antibody bound specifically to recombinant human ILA protein. This result confirmed that genes coding for an antibody specific for ΠΑ bad been cloned.

APIPi 96/00782

-30ft. • 1 Λ’. }

B. A humanized heavy chain can aljc be obtained frora this chimeric heavy chain. The humanized heavy chain was designed from by inserting the murine CDRs into a human framework. The chosen human framework was as described above, the most homologous protein sequence in the Swiss protein data based to the murine 3B9 V„ (amino acids 20-140 of SEQ ID NO 4). This humanized heavy chain sequence (EcoRI ^oal) was made synthetically and PCR performed to fill in and amplify DNA as described above. This synthetic variable regioo was ligated into the die expression vector pCD (EcoRI ΔθλΠ together with the synthetic signal sequence SEQ ID NOS: 5 and 6 from the chimeric heavy eh^p construction and an IgG_t human constant region.

Simfiariy, a humanized light chain can be derived from the chimeric light chain as described for the heavy chain. This gene (EcoRV jjjgrf) was also made synthetically. The humanized V_L was ligated into the expression vector pCN, digested with Ecn&Z Hart along with a signal sequence (EccRI Eco^V). The expression vector provided the human kappa constant region.

Examole-6 - Ratification and Thermodynamics. Humanized MAb

Purification of CHO expressed chimeric and humanized 3B9 can be achieved by conventional protein A (at G) affinity chromatography followed by ion 20 exchange and molecular sieve chromatography. Similar processes have been soooessflgdty employed for (be purification to >95» parity of other MAba (t-g., m respiratory syncytial vires and malaria circumsporozoite antigens).

The affinity and detailed thermodynamics of ILA binding to humanized MAb 3B9 and marine 3B9 (Example 1) were determined by titration Qticrocalorimetry.

This medtodmeasoras hinting reactions by virtue of their intrisic beats of reaction (see, a,g., Wiseman «aL Anil. Biochem. 122:131-137 (1919). The affinity of both MAbs was foood to be too tight to measure directly at ambient temperature. Thus, a thermodynamic approach was taken: i) the affinity was measured at 60*C, where it is weak enough to be measured directly, and (ii) the temperature-dependence of the binding enthalpy was measured from 30-60*C Together, these daa allow ealettiadoa of Ae affiahy over a wide range of tampemnn using the Gibb·· Helmhoiz equation.

A summary of the IL4 binding thermodynamics of the humanized and murine 3B9 antibodies are presented in Table 1. Based upon the changes is free energy, enthalpy, entropy and heat capacity of the two MAbs, the binding tharoodymaitics are indistinguishable.

AP/P/ 9 6 / 0 0 7 8 2

-31i.nencodynamiis of hIL-4 binding to Kunwuncd 3B9 and .\jnne 339 at pH

7.4. 150 mM Nad, and 25’C

mAb	Kd picomolar	AG kcal/ mol DL4	ΔΗ kcal/ mol HA	-TAS kcal/ molIL4	AC cal/mol ΠΛΓΚ
humanized 3B9	11	-13A±0.6	-21.02	82*2.1	-580*160
murine 3B9	19	-133*0.6	-205*1	7.2*1.2	-660*200

0.-4 affiaitiw of fconanzied 389 tad marine 3B9 woe measured ή» quadruplicate and duplicate, rcspcaiwly.

Examnle 7 - Pmrincnon and Characmrizarion of Rat MAb / j MAb 6A1, chosen for high affinity binding, wis derived from an immunized rat, using the same immunization protocol as described for the moose in Example 1. 10 6A1 was selected from hybridomas (specifically, hybridoma 3426AHC1B9) prepared from rats immunized with human ΏΑ.

The Ki Set 6A1 wu calntlaard as described in Beatty g al. J. hnmmol·

Methods. 100:173-179 (1987)» be 2 X 10*M.

Hybridoma 3426Λ11C1B9 was deposited October 6,1993 with the

European Collection of Animal Cell Cultures (ECACC), Public Health Laboratory

Service Centre for Applied Microbiology A Research. Ponou Down, Salisbury,

Wiltshire, SP4 0X3, United Kingdom, under accession number 93100620, and has been accepted as a patent deposit, in aceotdanoe with the Budapest Treaty of 1977 governing the deposit of microorganisms for the purposes of patent procedure.

Exzrrmle 8 - Biological Activity of MAbs: 3B9 /hnmairircriY 3B9 /Morine) and fiAl ¹ _ The following assays were performed using the procedures described below.

A. Binding» Glycosylated ihIL4

The above-identified antibodies were raised to non-glycosylared recombinant 25 human IL4 (rhIL4) which was produced in £. catt. Because native human IL4 is glycosviated, it was important to confirm binding to material secreted by a

AP/P/ 9 6 / 0 0 7 8 2

-32maraaaiian cell line. 3? - binds ccualiy well to both glycosylated and nonglycosylaxed human recombinant EL4, and is not therefore directed to an epitope that would be masked on natural human ILA.

B. Inhibition of IL4 Binding to Receptor

The ability of 3B9 to inhibit the binding of ILA to its receptor was studied using “I-rhQA binding to the gibbon cell line, MLA (ATCC TTB201], that bears approximately 6000 receptors per cell. MLA cells were incubated with “’l-QA for 30 miautes at 37*C Uptake of radioactivity was determined la a gamma coantn after separation of cell bound “*Ι-Ι1Α by centrifugation through an oil-gradient.

Non-specific binding was determined by incubating in the presence of a 100-fold molar excess of onlabelled HA (Park et ah J. Ftp. Med.. 2^6=476-488 (1987)]. The IC* value for onlabeled UA is this assay was 22 pM when the amount of (added)

HA was 83 pM. For intact murine (IgG) 3B9 the 1C» was 63 pM, and 93 pM for the Fab fragment At another concentration of QA (218 pM), the assay amount for marine (IgG) 3B9 was 109 pM,

C. Inhibition of Lymphocyte Proliferation

Using the method described in Spits et al, J. Immunol.. 222=1142-1147 (1987), human peripheral blood lymphocytes are incubated for three days with phytohemaggiutinin, a T cell mitogen, to opreguiate the ΠΑ receptor. The resultant blast cells are then stimulated for three days farther with QA. Proliferation is measured by the incorporation of *H thymidine. Bce& proliferation was measured by the assay Of Callari et ah foLwmholtiiiMandlmerierani. A Practical Approach. Qt 19, p. 345, modified as follows. Purified human tonsillar B Mb are stimulated for 3 days with ILA and immobilized aati-IgM. Proliferation is measured by the incorporation of Ή thymidine.

3B9 (murine) inhibited *H-thymidine mcotporetion by human peripheral blood T lymphocytes stimulated with 133 pM QA and human tonsillar B lymphocytes stimulated by 167 pM ILA. ZL2-stimulated T lymphocytes were not affected. The 1C» for inhibition of T cell proliferation was 30 pM, and fo^ B cell

V 30 proliferation US pM. The corresponding values for the Fab fragment of 3B9 (marine) were 106 and 393 pM.

D. Inhibition of CD23 Induction

0523 is the low affinity receptor for IgE (FcERH) and is induced on the membrane of resting B lymphocytes by low concentranocs of TLA as a necessary prerequisite for lg£ production. Purified human tonsillar B cells are stimulated for 2 days with QA. The percentage of cells expressing the CD23 receptor are determined by flow cytometry (Defiance et aL I. Exp. Med.. 1459-1467

AP/P! 9 6 / 0 0 7 8 2

-33C19S7)]· 3B9 (murine) inhibited CD23 expression on human tonsil B lymphocytes stimulated with 8.3 pM ILA with an IC^o value of 136 pM.

£ Inhibition of IgE Secretion

Unlike ether assays where £4 was added ai EC* concentrations [Pern « al. 5 Proc. Natl. Acad. Sci.. 35:6880-6884 (1988)], IgE secretion was investigated in the presence of concentrations of HA giving maximal secretion in order to reduce the variability inherent in thia system. T ceil proliferation was measured as follows. Human peripheral blood lymphocytes are incubated with IL4 for between 10-18, preferably 12, days. The concentration of IgE in the culture supernatant is determined by ELISA.

IgE secretion was inhibited by 3B9 (marine), and the Fab fragment of 3B9, ia the presence ci 1.7 nM HA giving IC* values of 1.9 and 5.0 nM respectively. The experiment was repeated using a lower concentration of ILA, 667 pM, which reduced the IC* value to 0.65 uM for 3B9 (murine). The molar ratio of antibody (IgG) to ILA remained unchanged (1:1) over the concenuation ranges examined.

F. Summary and Interpretation of Data

The molar ratios of ILA to various MAbs required for 50% inhibition of function in bioassays it given in Table 2.

AP/P/ 9 6 / 0 0 7 8 2

-34A’-*' - - - ο b ό

j Table 2	Com para., vc activity of mAhs 3B9, 6Λ1 and Humanized 3B9 [IgGl and IgG4 variants] in IL-4 dependent bioassays
i Assay	IC5(	) (pM) (range) n
	Marine 3B9	Murine 3B9 (Fab)	| RattiAl	Humanized 3B9
				ItGl	IgG4·
RBA	63{17-lWh	93	>50000
TeeD	30(10-40)4	108	«7	44(30-56)3	40
BeeU	103 [79-120)3	393	187	47(10-80)3	79
CD23 induction	136 [53-27214	216	80	333
IgB synthesis	658 (370-1070^	1170	623 [412-833)2	54 [35-83)3	406

a MHtar of aapani an orM ml me IgGl red 1|G« retires we reaped at afferent tinea

In nil assays, except lg£ secretion. ILA was added at approximate EDjo 3 concentrations. The molar ratios of antibody »ILA required for 50% idlsbtaoB wen similar for humanized 3B9, murine 3B9, and 6A1 in the two lymphocyte proliferation assays, bat higher for humanized 3B9 in the CD23 induction assay. The latter is a particularly sensitive assay apparently requiring very low ¢. 5%) \ receptor occupancy (Kruse et aL, EMBQJ. 123121 1993) and. as is βνώββί from the nmtia obtained with murine 3B9.sob)ect to inter assay vsriaticn.

A comparison of the activities of nt 6A1 and murine 3B9 demonstrated a similar profile of fueeoonal effects, but an unexpected failure of 6A1 to fully inhibit the binding of radioiodinated ILA to its receptor. The radioiodinated ILA used in the receptor binding assay is thought to be iodinated at die accessible ryresine. residue 124. When the ability of 6A1 to inhibit CD23 expression induced by either

AP/P/ 9 6 / 0 0 7 8 2

-35i - r urJabelled or lodinamd IL4 compared, it was found that inhibition was less efficient against iodinated ligand. These results indicate that 6A1 binds to IL4 in the region of, but not specifically to, tyrosine 124.

Thus on current data, 6A1 is a neutralizing antibody of high affinity, binding to a very different region of IL4 than 3B9.

Examnle 9 - Pharmacoldnetia

The pharmam kinetics of humanized 3B9 was investigated in the male 10 Sprague Dawley rat Humanized 3B9 was administered to four animals as an iv bolus dose at 1 mg/kg. Wood sampling was continued for 5 weeks post doting. Plasma humanized 3B9 concentrations were determined using an IL-4/anti-haman IgG sandwich ELISA designed to confirm not only the presence of circulating human IgG but also its ability to bind to recombinant human IL-4.

Results from this study are summarized in Table 3.

Table 3

Pharmacokinetics of Humanized 3B9 in male Sprague-Dawley Rate (dose: 1 mg/kg iv bolus)

Op (rnL/h/ka)
Rati	0.442
Rat 2	0.635
Rat 3	0.555
Rat 4	0.447
Mean	0.525
SD	0.101

Abbreviation of the phmeokiaetic penexter is as follow Clp, apparent plana

Z 8 I 0 0 / 9 6 IdldV cl

Data indicated that inter-animal variability was relatively email and 25 disappearance of humanized 3B9 from plasma appeared to be biphase. The apparent plasma clearance was low (0.5 mUh/kg). The half-life appeared to be 11 days. Thus, the pharmacokinetic characteristics of the CHO cell-derived humanized 3B9 are consistent with other hemanized monoclonal antibodies in rats. The long circulating half life of humanized 3B9 in the rat also suggests that when

- 36administered io man. humanized - 59 is likely to be effective over m extended period of time.

Numerous modifications and variations of the present invention are included 5 in the above-identified specification and are expected to be obvious to one of skill in the art. For example, human framework regions or modifications thereof, other than the exemplary antibodies described above, may be used in the construction of humanized antibodies. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

AP/P/ 96/00782

-37AP .

(I! GENERAL INFORMATION:

(1) APPLICANT: Heines, Stephen D.

Gross, Mitchell S.

Sylvester, Denlel R.

(li, TITLE or INVENTION: Recoebinant XL4 Antibodies Useful ir. Treatment ot 114 Mediated Disorders (iii) NUMBER cr SEQUENCES: SB (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: SniCAKiine Beeches Corporation (I) STREET: Corporate Intellectual Property, UW2220 - 709

Swedeland Rd.

(C, CITY: Χϊης of Prussia CD, STATE: PA (E) COUNTRY: USA (FJ SXP: 19406-2799 tv, eowonn nbadabuc form:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC coepatibla (C) OPERATOR STSTEM: PC-DOS/MS-DOS (D, KmORE: Patent In Release »1.0, Version >1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/117,366 () FILING DATE: 07-SEP-1993 (C, CLASSIFICATION: , (A) APPLICATION NUMBER: US 08/136,783 (B) FILMC DATE: 14-OCT-1993 (C, CLASSIFICATION;

(Till) ATTORNEY/AGENT INFORMATION;

(A) ΧΑΜΕ: Suttor., Jeffrey A.

AP/P/ 9 6 / 0 0 7 8 2

AP . ^Λ *? 5 9 3

IB) REGISTRATION NWBEK. 34,028 (Cl RXFERENCE/COCKET NUMBER: P5C18S-2 (ix; TELECOMMUNICATION INTCRMATION: iA) TELEPHONE: (2lS> 270-5024 (B) TELEFAX. (2151 270-509C (2! INFORMATION FOR SEC ID NO:1:

<±> SEQUENCE CHARACTERISTICS: UI LENGTH: 396 base pairs (9} TYPE: nucleic acid (C) STRANDEDNESS: Oouble (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: c&KA (ix! FEATURE:

(A) NAME/KEY: CDS (9) LOCATION: 1..396 (Xi) SEQUENCE DESCRIPTION: S2Q ID NO:!· /p; 9 6 / 0 0 7 8 2

ATC Met 1

GAG Gi'J

ACA Thr

GAC Asp

ACA ATC CTG CTA

TGG GTG CTG CTG CTC TGG GTT CCA

49

Thr Tie S

Leu Leu

Trp

Val 10

leu

leu Leu

Trp

val IS

Pm .

GGC

TCC

ACT

GGT

GAC

ATT

GTG

CTC

ACC

CAR

TCT

CCA

GCT

TCT

TTG

GCT

96

Gly

Ser

Thr

Gly

A«P

Ile

Val

Leu

Thr

Gin

Ser

Pro

Ala

Ser

Leu

Ala

20

* « » *

30

GTS

TC?

CTA

GGG

CAS

AGG

GCC

ACC

ATC

TCC

TGC

AAG

CCC

AGC

CAA

AGT

144

Val

Scs

Leu

Gly

Gin

Aru

Ala

Thr

lift

Ser

Cya

Lys

Ala

Ser

Gin

Ser

CL <

A F’

J t

- 45

co:

GAT

TAT

GAT

GGT

GAT

AGT

TAT

ATG

AAC

TCC

TAG

CAA

CAG

AAA

CCA

192

7a 1

Asp

Tyr

Asp

Gly

Asp

Ser

Ty;

net

Asn

Ty;

Gin

Gin

Lys

Pro

50

55

6C

GGA

CAG

CCA

CCC

AAA

CTC

CTC

ATC

TAT

GCT

3CA

TCC

AAT

CTA

GAA

TCT

240

siy

Gin

Pro

Pro

Lys

Leu

Leu

Ile

Tyr

Ala

Ala

Ser

Asn

Leu

Glu

Ser

63

70

75

ao

ATC

CCA

AGG

TTT

AGT

GGC

AGT

GGG

TCT

GGC

ACA

GAC

TTC

ACC

288

Sly

lie

Pro

Ala

Arg

Phe

Ser

Gly

Ser

Gly

Ser

Gly

Thr

Asp

Phe

Thr

S3

90

95

CTC

AAC

ATC

CAT

CCT

GTG

GAG

GAG

GAG

GAT

GCT

GCA

ACC

TAT

TAC

TGT

336

Leu

Asc

ile

His

Pro

val

Glu

Glu

Glu

Asp

Ala

Ala

Thr

Tyr

Tyr

cys

100

103

110

CAG

CAA

AGT

AAT

GAG

GAT

CCT

CCG

ACG

TTC

GGT

GGA

GGC

ACC

AAG

CTG

314

Sin

Gin

Ser

Asn

Glu

Asp

Pro

Pre

Thr

Phe

Gly

Gly

Gly

Th;

LyS

Leu

115

120

125

SAA

ATC

AAA

CGG

396

βία Xie Lys Arg 133 (2) INFORMATION ΓΟΑ SEQ ID HO:2:

ti) SEQUENCE CHARACTERISTICS:

(A) LENGTH; 132 aaino acids (3) TYPE: Mine acid

O) TOPOLOGY: linear _k (ii) MOLECULE TTK: protein txi) SEQOENCE DESCRIPTION: SEQ 10 N0.2:

Met Glu Th; As? Thr lie Leu Leu Trp Val Le. Leu La. Irp v»i Pro 15 10 13

AP/P/ 9 6 / 0 0 7 8 2

Ml*

8 3

Gly

Ser

Thr

Gly

A3F

lie

Val

Leu

Thr

Cln

Ser

Pro

Ala

Ser

Leu

Ala

20

25

30

Val

Ser

Lau

Gly

Gin

Arg

Ala

Thr

lie

Ser

Cys

lys

Ala

Ser

cm

Ser

35

40

45

Val

lap

Tyr

Asp

Gly

Asp

Ser

Tyr

Het

Asn

Trp

Tyx

Gin

Gin

Lys

Pro

50

55

60

Gly

Gin

?xo

Pro

lys

Leu

Leu

Zle

Tyr

Ala

Ala

Set

Asn

Leu

Glu

Ser

65

70

75

80

Gly

lie

Pro

Ala

Arg

Phe

Ser

Gly

Ser

Gly

Ser

Gly

Thr

Asp

Phe

ThX

65

90

9S

leu

Asn

lie

Mis

Pro

Val

Glu

Glu

Glu

Asp

Ala

Ala

Thr

Tyr

Tyr

cy*

100

105

110

Gin

Glc

Sex

Kin

Glu

Asp

Pro

Pro

Thr

Phe

Gly

Gly

Gly

Thr

Lys

Leu

115 120 125 din He Lys Arg 130 (2) IKTOWailOB FOX no XD NO:):

(1) SXQVTKCX CHARACTtRISTXCS:

CA) LtNSTX: 463 Ms· pel»

CB) TIPS: nucleic acid (C) STJtANDS&HKSS: OOufcle (OI TOPOLOGY: unknown (ii) MOLSCBLS TTPS: cOMA ti*) rSArintE;

(Al ΗΑΚ5/ΚΕΪ: COS (8) LOCATION: 6(..433

AP/P/ 9 6 / 0 0 7 8 2

AP . ,ί υ 5 8 3 (xi: SEQUENCE CESCSIPTiON: SEQ TO K0:3:

GAATTCK3G CCCCTATCCA GG5ACAATCA GCAGCAGCAA TGAGGAAGTA AGCCTGTGCA 60

GAT

ATS

AAC

AGG

ctt

ACT

τα

TCA

TTC

CTG

CIS

CTG

ATT

3TC

CCT

GCA

101

Het

Asn

Arg

Leu

Thi

Ser

Ser

Leu

Leu

Leu

leu

ile

val

Pro

Ala

1

a

10

IS

TA?

GTC

CTS

TCC

CAG

GTT

ACT

CTG

AAA

GAS

>tv**

GGC

CCT

GGG

AT A

TTC

156

Tyr

Val

Ser

Sir.

Val

Thr

Leu

Lys

Glu

Ser

Gly

Pro

Gly

lie

Leu

20

21

30

CAC

ccc

ICC

CAS

ACC

CTC

ACT

CTG

ACT

TGI

TCT

TTC

TCT

GGG

TIT

TCA

204

Gin

Pro

Se:

Sin

Thr

Leu

Se:

Leu

Thr

Cys

Ser

Phe

Ser

Gly

Phe

5er

AS

40

45

CTG

AGC

ACT

TCT

SGT

ATG

GGT

GTG

AGC

TGG

ATT

CGT

CAG

CCT

TCA

GGA

252

Leu

Ser

**··.

Ser

Sly

Met

Gly

Val

Ser

Trp

Tie

Arg

Sic

Pro

Ser

Gly

so

55

60

AAG

GGT

CI3

gag

IGG

CTG

OCA

CAC

AST

TAC

tea

GAT

GAT

CAC

AAG

CSC

300

tya

siy

IM

βία

trp

leu

Ala

Bla

lie

Tyr

Tap

Aap

Aap

Aap

i-y·

Arg

es

TO

75

TAT

AAC

CCA

ICC

AAG

AGC

CSS

CTC

ACA

ATC

TCC

AAG

SAI

ACC

1«

34»

Tyr

Am

Pro

Ser

Leu

Lys

Ser

Arg

Leu

Thr

Xie

Ser

Lys

Asp

Thr

Ser

to

BS

»0

95

AGC

AAC

GAS

5TA

TTC

CTC

AAG

ATC

ACC

AST

GTG

CAC

ACT

OCA

GAT

ACT

396

Ser

Am

Gia

Val

Phe

Leu

Ly»

Xie

Thr

Set:

val

Asp

Thr

Ala

A«p

Thr

100

iOS

110

AP/P/ 9 6 / 0 0 7 8 2

CCC ACA TAC TAC Ala Thr Tyr Tyr

TOT CCT CGA

AGA GAG ACT GIG TTC TAC TGG TAC TTC

444

Cys Ala

Arg

Arg Glu Thr 120

val Phe Tyr

Trp 125

Tyr Phe

IIS

GAT

GTC

TGG

GGG

GGA

GGG

ACC

ACG

GTC

ACC

GTC

TCC

TCA

483

ASp

val

Trp Sly

Ala

Gly

Thr

Thr

Val

Thr

Val

Ser

Se-

AP . '? 0 5 8 3

130 :35 145

C2) INFOWaTTQW FOA SS2 1C KO: 4:

(1) SZOUENOt CHARACTERISTICS:

(A) U3f9TB: 140 asu.no acids (Bt TYPE: aud.no acid (D! TOPOLOGY: linear

ii) :

KOXXl

TITLE

ttp:

P

rote

in

txi)

SEQUENCE

DESCRIP

TICK

: $X

Q ID

NO:

4:

Kat

Asn

Arg

lou

Thr

Set

ser

Leu

Lou

leu

Lou

He

Val

Pre

Ala

Tyr

1

5

10

15

Val

Lou

Sox

Gin

Val

Thr

Leo

Lye

Glu

Set

Gly

Pro

Gly

Xie

Lou

Gia

20

25

30

Pro

Sor

Gin

HU

Lou

Sot

toil

Thr

Cys

Se;

Phe

Ser

Gly

Phe

Ser

Leu

35

40

45

Ser

Thr

Sor

Gly

Kat

Gly

Val

ser

Ttp

tie

Are

Gia

Pre

Ser

Gly

Lys

50

53

eo

Sly

Lou

Glu

Trp

xeo

Ala

Bis

Xie

Tyt

Trp

Asp

Asp

Asp

Lys

Are

»y*

CS

TO

35

to

Asn

Pro

Sor

Lou

Lya

Sor

Arp

Leu

Thr

lie

Ser

Lys

Asp

Thr

Ser

Ser

•5

90

95

Asn

Gin

val

Ph·

Lou

lys

Xie

tut

Ser

Val

Asp

Thr

Ala

Asp

Thr

1, Ale

100

105

110

Thr

Tys

tyr

cys

Ala

Axg

Are

Glu

Thr

Val

Pha

Tyr

Trp

Tyr

Phe

Atp

115

120

125

v*i

Tt?

Gly

Ala

Cly

Thr

Tr.r

Val

Thr

Val

ser

Ser

135

135

140

CM r-*.

<o o>

CL

Ql <

(21 INFORMATION FOR SSQ ID NO:5:

(ii SEQUENCE CHARACTERISTICS: (Al LENGTH: ¢0 base parrs (Bl TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown iii) MOLECULE TYRE: cDNA (ix! FEATURE;

(A) NAME7KEY: CDS (B> LOCATION: 1..60 (xi! SEQUENCE DESCRIPTION: SEQ ID NO:S:

ATG GTG TTG CAG ACC CAG GTC TIC ATT TCT CTG TTG CTC TGG ATC TCT 48

Het Val Leu Gin Thr Gin Val Phe lie Ser Leu Leu Leu Trp Ile Ser

IS 10 13

GGT SCC TAC GGG «0

Gly Ala Tyr Gly

(2) INFORMATION FOR SEQ ID 140:6:

(il SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 aaino acids (B> TYPE: amino add '

ID) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ι Xii SEQUENCE DESCRIPTION: ESQ ID NO:6.

Met Val Leu Sin Thr Gin val Phe Ile Ser Leu Leu Leu Trp lie Sec

AP/P/ 9 6 / 0 0 7 8 2

AH . G ’j 5 8 ύ

10 IS

Gly Ala Tyr Gly 20 (2) INFORMATION FOR SEQ ID HO:7:

(1) SEQCZNCE CHARACTERISTICS:

(λ) LENGTH: 57 base pairs IB) TYPE: nucleic icld

CC) STRANDEDNESS: double

CD) TOPOLOGY: unknown

Cii) MOUCULE TTPS: cDHA (ia) FEATURE:

(A) SAME/KEY: CDS (Bl LOCATION: 1..57 (ai) SEQGENCE DESCRIPTION: SEQ ID MO:7:

ATG <WA TM AGC T6T ATC ATC CTC TTC TTC GTA GCA ACA GCT ACA GGT 41

Met Sly Trp Sec Cya Xie llo Leo Fbe Leu Val Ala Thr Ale Thr Gly

10 IS

GTC CAC TCC 37 vai His ser

(2) INFORMATION FOR SEQ IS MO;8:

Ci) SSOOBCS CHARACTERISTICS:

CA) LENGTH: 19 «aion acids Cl) TYPE: aatino acid CD! TOPOLOGY: linear (11) MOLECULE TYPE: pretext;

AP/P/ 9 6 / 0 0 7 8 2 (xi: sequence cesc=e?t;cn: sec id :.q.ί.

Met Gly Trp Ser Cys lie He Leu Pre Leu Val A.j Thr Ala Thr Gly

5 CO 15

Val His Sex (2) INFORMATION FOR SEQ ID NO:9:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 base pairs O) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (il) MOLECULE TYPE: COMA (ix, FEATURE:

(A, NAME/KEY: CDS (B) LOCATION: 1..423 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

ATG GTG TTO CM ACC CAG CTC TTC ATT TCT CTG TTG CTC TGG ATC TCT
Met val Leu Gia Thr Sin Val Phe lie Ser	Leu	Leu	Leu	Trp	lie 13	Ser
1	5	10
GOT GCC	TAC GGG CAG GTT	ACC CTG AAA GAG* TCT	GGC	CCT	GGG	ATA	TTG
Cly Ala	Tyr Gly Gia Val 20	Thr Leu Lys Glu 23	Ser	ciy	Pro	Gly 30	lie	Leu
CAG CCC	TCC CAG ACC CTC	AGT CTG ACT TGT	TCT	TTC	TCT	GGG	TTT	?CA
filn Pro	Sex Gin Thr Leu 33	Sez Leu Thr Cys 40	Sez	Phe	sex 43	Gly	Phe	Sez
CTG AGC	ACT TCT GGT ATG	GCT GTG AGC TGG	ATT	CGT	CAG	CCT	TCA	GGA
Leu Se: 53	T3r Ser Sly Met	Giv Val Ser Trp 55	lie	A.-T s:	Glr.	Fro	Ser	Gly

144

AP/P! 9 6 / 0 0 7 8 2

192

AA3 Lys «5

QZZ CTG 3aG

TGG Trp

CTG GCA Leu Ala 10

CAC His

ATT lie

TAC TGG

GA? ASP

*5AT CAT AAC C3C

240

Gly

Leu

Glu

Tyr

Trp 75

Asp

AS?

lys

Arg 80

TAT

AAC

CCA

TCC

CTG

AAG

AGC

CGG

CTC

ACA

ATC

TCC

AAG

SAT

ACC

TCC

286

Tyr

Asn

Pro

Sec

Leu

Lya

Ser

Arg

Leu

Thr

lie

Ser

Lys

Asp

Thr

Ser

85

90

95

AGC

AAC

CAfl

GT*

TTC

cue

AAG

ATC

ACC

AGT

GTG

GAC

ACT

GCA

Gat

ACT

336

Sec

Asr.

Gin

Val

Phe

Leu

Lya

lie

Thr

Ser

Val

Asp

Thr

Ala

Asp

Thr

100

105

110

CCC

ACA

TAC

TAC

TGT

GCT

CGA

AGA

GAG

ACT

GTG

TTC

TAC

TGG

TAC

TTC

384

Ala

Tnr

Tyr Tyr Cys

Ala

Arg

Arg

Glu

Thr

Val

Phe

Tyr

Trp

Tyr

Phe

115

120

125

SAT

GTC

TGG

GGC

GCA

GGG

ACC

ACG

GTC

ACC

CTC

TCC

TCA

423

Asp

val

Trp Gly

Ala

Gly

unr

Thr

val

Thr

Val

Ser

Ser

130

135

140

CM

OO

(2) ΥΝΤΟΜΜΑΠΟΜ FOR SEQ ID )10:10:

til SEQUENCE CHARACTERISTICS·.

(A) length: 141 aaino acids (B) TYPE: aad.no acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein [Xi) SEQUENCE DESCRIPTION: SEQ W NO:10:

Met Val Lea 61a Thr 61a Val Ph* lie Ser Lea Lea Lea Trp lie Ser . I 5 10 15

AP/P/ 9 6/00

Sly AJ.4

Tyr Gly

Gin Val Thr Leu ,ys Glu Ser Gly Pro Gly lie Leu 25 30

* > r

? ro

Ser

Glr.

Thr

Lev

Ser

Leu

Thr

Cys

Ser Phe Ser

Gly

Phe

Ser

35

40

45

Le·-

Ser

Thr

Ser

Gly

Her

Gly

Val

Ser

Tr?

•—e *

Pro

Ser

G.y

SO

55

60

Lys

Gly

Lei

Glu

Trp

Leu

Ala

His

lie

Tyr

Trp Asp Asp

ASP

ty»

Arg

<5

70

75

80

Tyr

Aon

Pre

Ser

Leu

Lys

Ser

Arg

Leu

Thr

lie Ser Lya

Asp

Thr

Ser

85

90

95

Sec

Asn

Sis

Val

Pfie

Leu

Lys

Ila

Thr

Ser

Val Aap Thr

Ala

Asp

Thr

100

105

110

Aii

Thr

Tyr

Tyr

Cys

Ala

Arg

Arg

Glu

The

Val Phe Tyr

Trp

Tyr

Phe

115

120

125

AS?

val

Tr?

Gly

Ala

Gly

Thr

Thr

Val

Thr

val Ser Ser

120 . 135 140

CM

OO

(2) IXTOAXAIXON TO* no ID »0:11:

Ci) SEQUENCE CHARACTERISTICS:

CA) LENGTHt 423 Me· pairs (Η) TYPE: nucleic acid (C) STRANDEDNESS: dOUDlt CD) TOPOLOGY: unknown (ii) KOLECULE ΤΥΡΙ: cDNA (ix) PIATOBE:

(A) ΝΜβ/XSt: CDS (8) LOCATION: 1..423 (xu.) SEQUINCS DESCRIPTION; SSQ ID NO;1

AP/P/ 9 6 / 0 0 7

ATG GTG TTG CAS ACC CAC GTC TTC ATT TCT CTC TTG CTC TGG ATC TCT 48 ν* i j ) . j-j;

*· <. vjj'jo

K«t

Val

Leu Gin

Thr

Gin

val

Phe

lie

Ser

' Leu

t Leu Leu Trp 21«

ι Ser

1

5

10

15

^GC

TAG GGG

CAG

GTT

ACC

CTG

GAA

TCC

; GGT

CCG GCA

. CTA

GTT

96

Gly

All

Tyr Gly

Gin

Val

Thr

Leu

Arg

Glu

Ser

Gly

Pro Ala

Leu

vai

20

:5

30

AAA

CCG

ACC CAG

ACC

CTG

ACG

TTA

ACC

TGC

ACC

TTC

TCC GGT

TTC

TCC

144

Lys

Pre

Thr Gin

Thr

Leu

Thr

Leu

Thr

Cys

Thr

Pfce

Ser Gly

Phe

Ser

35

40

45

CTG

TCG

ACC TCC

GGT

ATG

GGT

GTT

TCC

TGG

ATC

CGT

CAG CCG

CCG

GGT

192

Leu

Ser

Thr Sex

ciy

Met

Gly

Val

Ser

Trp

lie

Arg

Gin Pro

Pro

Gly

50

55

65

ΑΛΑ

GGT

CTA GAA

TG5

CTG

GCT

CAC

ATC

TAC

TGG

GAC

GAC GAC

AAA

CGT

240

Lyj

Gly

Leu Gi-

Trp

Leu

Ala

His

lie

Tyr

Trp'

Asp

Aap Asp

lys

Arg

65

70

75

80

TAC

AAC

CCG AGC

CTG

AAA

TCC

CGT

CTG

ACG

ATA

TCC

AAA GAC

ACC

TCC

288

Tyr

Asn

Pro Ser

Lea

lye

Ser

Arg

lee

Thr

lie

Ser

Lys Asp

Thr

Ser

es

90

95

CCT

AAC

CAG GTT

CTT

CTG

ACC

ATG

ACT

AAC

ATG

GAC

CCG GTT

GAC

ACC

336

*rg

Aje.

Gin Val

Val

Leu

Thr

Met

Thr

Asn

Het

Asp

Pro Val

Asp

Thr

100

105

110

CCT

ACC

TAC TAC

TGC

CCT

CGA

CGC

GAA

ACC

GTT

TTC

TAC TGG

TAC

TTC

384

All

Thr

Tyr Tyr

cy»

Ala

Arg

Arg

Glu

Thr

Val

Phe

Tyr Trp

Tyr i

Phe

115

120

125

GAC

GTT

TGC GGT

cgt iat

ACC

CCA

CTT

ACC

GTG

AGC

TCA

423

Mp

Val

Trp Gly Arg Oly

Thr

Pre

Val

Thr

val

Ser

Ser

130

135

140

CM

OO r* <o o>

CL £

<

(2) sntormaticn for sic

NO :12:

A-s ί * ,)

		ii;		CHARACTERISTICS:
			(Al LENGTH: 141 aeiiao aoiis
			IS) TYPE: ardco acii
			(D) TOPOLOGY: linear
	ί	111	molecule	TYPE: proceir.
	(xl)	SEQUENCE	DESCRIPTION: SEQ D NO:	12:
Met	Val	Leu	Gin Thr	Gia Val Phe lie Ser Leu	Leu	Leu	Trp	Tie	Ser
1			5	13				15
Gly	Ala	Tyr	flly Gin	Val Thr Leu Arg Glu Ser	Gly	Pro	Ala	Leu	Val
			20	25			30
Ly*	Pro	Thr	Gl.i Thr	Leu Thr Leu Thr Cys Thr	?he	Ser	Gly	Phe	Ser
		35		40		45
Leu	Ser	Thr	Ser Gly	net Gly Val Ser Trp lie	Arg	Gin	Pro	Pro	Gly
	50			55	60
Lys	Gly	Leu	Gio Trp	Leu Ala Sis lie Tyr Trp	Asp	Asp	Asp	Lys	Arg
es				70 75					so
Tyr	Asa	Pro	Sec Leu	Lys Ser Arg Leu Thr Xie	Ser	Lys	Asp	Thr	Ser
			15	90				95
Arg	Asn	Gin	Val Val	Leu Thr Met Thr Asn Met	Asp	Pro	val	Asp	Thr
			100	105			lio
Ai·	Thr	Tyr	Tyr Cys	Ala Arg Arg Glc Thr Val	Phe	Tyr	Trp	Tyr	Phe
		US		120		125
Asp	val	Trp Gly Arg	Gly Thr Pro Val Thr Val	Ser	Ser
	130			135	140
(2:	INFORMATION fcr	SEQ IO NO :13:

C\J

OD o

(O

CL

Ql <

(Ιί SEQUENCE CHARACTERISTICS:

(A) LENGTH: 393 base pairs {>) TYPE: cucleic acid (C) STRANDSONSSS: double !□) TOPOLOGY; unxr.ovn (ii) MQUC3LE TYPE: eDNA (Lx) ΓΕΑΤΟΜ:

(A) SAME/KSY: CDS (B) LOCATION: 1..393 (xi) SEGUENCE DESCRIPTION: SEQ ID NO:13:

ATS SGA Met. Gly 1	T3G AGC TGT ATC AtC	CTC TTC TTC CTA GCA ACA GC? ACA GST	43
Trp Sec Cys 3	lie Xie	Leu Phe	Leu 10	Val	Ala Thr	Ala Thr Gly IS
STC CAC	TCC GAT ATC	GTS ATS	ACC CAG	TCT	CCA	GAC TC3	CTA GCT GTG	96
Val Kis	Ser Asp lie ao	Val Met	Thr Gin 25	Ser	Pro	Asp Ser	Leu Ala Val ' 30
TCT CTG	GGC GIG AGO	GCC ACC	ATC AAC	TGC	AAG	GCC TOC CAA AGT GTT	144
Sec Leu	Gly Glu Arg 35	Ala Thr	He Asa 40	Cys	Lys	Ala Ser 45	Gin Ser Val
GAT TAT	GAT GOT GAT AGT TAT	ATG AAC	res	TAT	CAG CAG	AAA CCC GGG	192
Aap Tyr 50	Asp Gly Asp	Sec Tyr Si	Mat Asa	Trp	Tyr	Gia Gia SO	Lys Pro Gly
CAG CCT	CCT AAG TTS	CTC ATT	TAG GCT	GCA	TCC	AAT CTA	GAA TCT GGG	240
Gin Pro ¢5	Pro Lye Leu	Leu lie 70	Tyr Ala	Ala	Ser 75	Asa Liu	Glu Ser-*Giy SO
GTA CCT	GAC CGA TTC	ACT GGC	AGC GGG	TCT	GGG	ACA GAT	TTC ACT CTC	288
Val Pro	Asp Arg pb· 65	Sec Gly	Ser Gly	Ser 9C	Gly	Thr Asp	Phe Thr Leu 95
ACC ATC	AGC AGC CTG	CAG GCT	GAA GAT	GTG	GCA	GTA TAC	TAC TGT CAG	33S
Thr Tie	Sec Ser Leu	Gin Ala	GlU Asp	Val	Ala	val Tyr	Tyr Cys Gin

CM

OD co

GU &

<

100

105

HO

CAA

A3T

AAT

GAG

gat

CCT

CCG

AGG

T TC GG\.

GGA

GCC

ACC

AAG

CTG

GAG

384

Gin

Ser

Asn

SI-j

AS?

?ro

?£S

Arg

Phe Gly

Gly

Gly

The

lys

Val

Glu

115

120

12«

ATC AAA CGT 393

Xie Lya Arg

130 (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH; 131 aalno acids (9) TYPE: aalno acid (□) TOPOLOGY: linear (ll) MOLECULE TYPE: protein

C\l

CO

Jzi) SEQUENCE DESCRIPTION

: SEQ W

NO:

14:

Met 1

Gly Trp Ser Cys 5

lie

Ila

Leu

Phe

Leu 10

vai

Ala

Thr

Ala

Thr Gly 15

Val

Hli Sec Asp lie 20

vai

Met

Thr

Gin 25

Sec

Pre

Asp

Ser

Uu 30

Ala

Val

Ser

Leu Gly Glu Arg 35

Ala

Thr

lie 40

Asn

Cys

Lys

Ala

Ser 45

Gin

Ser

Val >

Asp Tyr Asp Gly Asp 53

Mr

Tyr 55

Met

Aan

Trp Tyr

Gin €0

Gia

Lya

Pro

Gly

Gin 65

Pre Pr; lys Leu

Leu 70

lie

Tyr

Ali

Ala

Ser 75

Asn

Leu

Glu

Ser

Gly 80

co

Ch

CL flL <

AP . G ύ 5 8 3

val

Pro

Aap

Arg

Phe

Ser

Gly

Ser

Giy

Ser

Gly

IB

Ao?

Phe

Thr

Leu

85

90

95

Thr

Xie

Ser

Ser

Leu

Glr.

Ala

Glu

Asp

Val

Ala

val

Tyr

Ty:

Cyj

Gin

ICO

105

110

Gin

ser

Α»

Glu

Asp

Pro

Pro

Arg

Phe

Gly

Gly

Gly

Thr

Lya

Val

Glu

115 120 125

Xie lys Arg 130 (2) INFORMATION TOR SEQ ID NO:15:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pair» (S) TUI; nucleic acid to STRANDEDNESS: double (0) TOPOLOGY: unknown (ii) MO1ECEI4E TYPE: eDNA (lx) PEAJOTE:

(A) HAMS/KEY: CDS (S) LOCATION: 1..45 (Hi) SEQUENCE DESCRIPTION: SEQ ZD MO:15:

AAG GCC ACC CAA AGT GTT GAT TAI GAT GOT GAT AGT TAT ATG AAC Lys Ala Sec tin Ser Val A*p Tyr Asp Gly Asp Se: tyr mt Asn

AP/P/ 96/00782

(2i INFORMATION FOR SIQ XO NO: 16:

(1) SEQUENCE CHARACTERISTICS:

fi τ'* (λ) LENGTH: IS »m:rs acids (B) TYPE; asir.o acid (0) topology·, linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ IO NO:16:

Lys Ala Ser Gin Ser val Asp Tyr Asp Gly Asp ser Tyr Met Asn 15 10 15 (2) INTORMATIQN FOR SEQ ED NO :17:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 Base pairs (B) TYPE: nucleic acid (C) STRANBEDNESS: double (3) TOPOLOGY: unknown (ii) MOLECULE TYPE: cfiKA (xx) FEATURE:

(A) ΚΑΜΕ/KEY: COS (B) LOCATION: 1..21 (Xi) SEQUENCE DESCRIPTION: SEQ ID HO:IT:

GCT GCA TCC AAT CTA GAA TCT Ala Ala Ser Asn Leu Glu Ser

5 e

(2) INFORMATION FOB SEQ ID »0:18:

(i) SEQUENCE CHARACTERISTICS:

;a: LENGTH: 7 aAins acids <S> TYPE: anine acid (D) TOPOLOGY; linear

AP/P/ 9 6 / 0 0 7 8 2

ΜΗ

Iii) MOLECULE TYPE: protein (xl) SEQUENCE DESCRIPTION: SE3 :□ NC:18:

Ala Ala Sat Asn Leu Glu S«r 1 s (2) INFORMATION FOR SEQ ID NO :19:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNXSS: double (0) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ia) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1..27 (ai) SEQUENCE DESCRIPTION: SEQ IS NO:19:

CAG CAA AST AAT SAG OAT CCT CCS ACS Gin Sin Sat Asn Glu AspPro Pro Thr

S

AP/P/ 96/00782

(2) INFORMATION TOR SEQ IS NO :20:

(1) SagOENCE CHARACTERISTICS:

(A) LENGTH: 9 aaino acids (B> TYPE: anino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: pretext

Ϊ) .S-'Jk/iN'C- iiJZP-XrT.w 5—^ ID .’’J.22.

Gin Gin Sar Asn Glu A$= Pro Pro Thr 1 5 ¢2) INFORMATION FOR SBC ID NC:2i:

(i) SEQUENCE CBARACTERISTXCS:

(A) LE8GT8: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double <D) TOPOLOGY: unknown

Iii) MOLECULE TYPE: cDNA (iJt) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1..21 !xi) SEQUENCE DESCRIPTION: SEQ 12 NO:21:

ACT TCT GGT ATG GGT GTG AGC

Thr Sar Gly Mat Gly Val Sax 1 9

(2) INFORMATION FOR SEQ ID NO:22:

<1) SBQUtNCt CSASACTERISTICS:

(A) LENGTH: 1 amino acids (B) TYPE: amine acid (0) TOPOLOGY: linear (ii) MOLECULE TYPE: protein <Xi! SEQUENCE DESCRIPTION: SEQ ID KO:22:

Thr Ser Gly Met Gly Vii Ser 1 5

AP/P/ 96/00782 (2) INFORMATION FOR SEQ ID NO :22:

Ci! SEQUENCE CHARACTERISTICS:

(A! LENGTH: 43 base pairs (Bl TYPE; nucleic acid (C) STRANDEDNIS3: double CD) TOPOLOGY: unknown

Cii) MOLECULE TYPE: cDNA (ix) FEATURE:

(A) NAME/KEY: COS (3) LOCATION: 1..48

Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

CAC ATT TAC TGG 6AT GAT GAC AAG CSC TAT AAC CCA TCC CTO AAG ACC

His Lie Tyr Trp Asp Asp Asp Lys Arg Tyr Asn pro Ser Leu Lye sec

5 10 15 (2) INFORMATION FOR SEQ ID NO:24:

ci; SEQOENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids.

CB) TYPE: amino acid φ) TOPOLOGY: linear (ii) MOLECULE TYPE: protein )

(xi) SEQUENCE DESCRIPTION: SSQ IO 110:24:

His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 15 10 15

AP/P/ 96/00782 (2) INFCRMATICN FOR SEQ ID NC:25:

;x: SEQUENCE CHARACTERISTICS:

(A) LENGTH; 33 base pairs (fi, TYPE: nucleic acid (C! STWUCEDNESS: double (DI TOPOLOGY: ur.Known (11) MOLECULE TYPE: CDMA (iX, FEATURE:

(A) NAME/KEY: CDS (>; LOCATION: 1..33 txi) SEQUENCE DESCRIPTION: SEC ID NO:25:

AGA GAG ACT GTG TTC TAC TGG TAC TTC GAT GTC Arg Gia Thr val pae Tyr Trp Tyr Pae Asp val

5 10

(2, INFORMATION TOR SEQ ID NO:26:

(1) SEQUENCE CHARACTERISTICS:

(A, LENGTH: 11 amino acids (B) TYRE: aaino acid CD) TOPOLOGY: linear (11) MOLECULE TTPS: protein (Xi) SEQUENCE DESCRIPTION; SEQ TD NO :25:

Arg gi-j Tit Val Phe lyr Trp Tyr ?ne Asp Val 1 5 10

AP/P/ 96/00782 (2) INFORMATION FOR SEQ XD NO :27:

(i) SSQUENCE CHARACTERISTICS:

(A) LENGT8: Z7 ba 3« pairs (8) TYPE: nucleic add CO STRANDEDNESS: double CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: C3NA (lx) FEATURE:

(A) NAME/KEY: CDS (1) LOCATION: 1..27 (Xi) SEQUENCE DESCRIPTION: SEQ 12 NC:27:

CAG CAA ACT AAT SAG GAT CCT CCG AGG Gin 51c 5«; Asn Glu Asp Pre Pro teg

5

(2) INFORMATION FOR SEQ ID 80:28:

<1! SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 aalno acids (8) type: anino acid (D) TOPOLOGY: linear <11) MOLECULE TYPE: protein (xl) SEQUENCE DESCRIPTION: SEQ ID 80:28:

Gin Gia Ser Asn Glu Asp Pro Pre teg 1 9

Z 8 L 0 0 / 9 6 Zd/dV (2) INFORMATION FOR SEQ 10 NO:25:

(i; sequence character:s?res:

(A) LENGTH: 36 base pales (Β) TYPE: nucleus acid (C> STRANDEDNESS: single 101 TOPOLOGY; unknown (11! MOLECULE TYPE; DNA (genomic) (xl! SEQUENCE DESCRIPTION: SEQ S> NC;29:

CTAACACTCA TTCCT3TTGA A3CTCTTGAC AATGGS 36

12) INFORMATION FOR SEQ ID NO:30:

(Ai SECCENCE CHARACTERISTICS:

(A) DENOTE: 29 base pairs (8) TYPE; nucleic acid (C) STRANDEDNESS: ·ingle (D) TOPOLOGY: unknown (11) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

CYACATATGC AAGGCTTACA ACCACAATC ₂₉

28/00/96 /d/dV (2) INFORMATION FOR SEQ ID »0:31:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 117 base pales (9) TYPE: nucleic acid iC) STSANDEDNSSS: single (D) topology; unknown iii) MOLECULE TYPE: DNA (genomic)

AP. ο μ :83 (Xi· SEQUENCE DESCRIPTION: SEQ ID NO :31:

GGTTACCCTG CGTGAATCC5 GTCCGGCACT AGTTAAACCG MZZZ&CACZZ TGAC3TTAAC

C73CACC7TC TCCGGXTTCT CCCT3TCGAC CTCCGGTATS GGT5TTTCCT GGATCCG (2) DiTORMATTON FOR SEQ ID KO:32:

(ii SEQUENCE CHARACTERISTICS:

(A) LENGTH: 120 base pairs |B) TYPE: nucleic acid (CJ STRANDEDNESS: Single (D) TOPOLOGY: unknown (il! MOLECULE TYRE: DNA (genomic) (xi! SEQUENCE DESCRIPTION: SEQ ID NO:32:

TCAGCCGCTC GGTAAASGTC 7ACAATCGCT GGCTCACATC 7ACTGGGACS ACRAfiAAATS

TTACAACCOG A9CCTGAAAT CCCGTCTGAC CATATCCAAA GACACCTCCC GTAACCASGT s:

111 so

120

AP/P/ 9 6 / 0 0 7 8 2 (2) XMFOMOT1Q· FOR SEQ XX) MOi32:

(X) SEQUENCE CHARACTERISTICS: (A) LENGTH: 120 base pairs (Ei ty?S: nucleic acid (C) STRANDEDNESS: single (0) TOPOLOGY: unknown

AH .

iii) MOLECULE TYPE; DNA (geno.-iic;

!xi! SEQUENCE DESCRIPTION: SEQ ID NO:33:

TCCTCCGACC ATGGACCCGG TTGACACCCC TACCTACTAI TSCGCTC3TC GCGAAACCGT 6C

TTTCTACTGG TACTTCGACG TTCGGCCTCG TGGTACCCCA GTTACCCTGA GCICCCAACC 120 (2) INFORMATION FOR SEQ ID NO:34;

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (Cl TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (geneaie) (xl) SEQUENCE DESCRIPTION: SEQ Ο HO:34:

ACCCGGCGGC TGACGGATOC AGGAA (21 INFORMATION FOR SEQ ID MO:33:

<i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs (8} TYPE: nucleic acid (C) STRANDEDNESS: single >

(DJ TOPOLOGY: unknown (ill MOLECULE TYPt: DNA (genomic) ;xi) SEQUENCE DESCRIPTION: SEQ ID NO:IS:

AIGGTCAGAA CAACCTGGTT ACGG 24

8 / 0 0 / 9 6 /d/dV (2) TNFORHATXCN TOR SEC XD NO: 36:

Ci) SEQUENCE CHARACTERISTICS:

CA) LENGTH: 25 base pairs .

(Bj type: nucleic acid (C) STRANDEDNESS: single

CD) TOPOLOGY: unknown

CU) MOLECULE TYPE: DNA (genomic)

Cxi) SEQUENCE DESCRIPTION: SBQ XD NO:36:

CTCGGGTTAC CCTGCGTGAA TCCGG 25

C2) INFORMATION FOR SEQ XD NC:37:

HI SEQUENCE CHARACTERISTICS:

CA) LENGTH: 21 base pairs (B> type: nucleic acid CC> «HANDEDNESS: single CD) TOPOLOGY.· unknown (ii) MOLECULE TYPE: DNA igencaic) (Xi) SEQUENCE DESCRIPTION: SBQ ID NO:37:

CCAAOCCTCG AGTGCCATTG A 21

8 L 0 0 / 9 6 /d/dV (2) INFORMATION FOR SEQ IO NO :38:

Ii) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 43 base pairs (B) TYPE: nucleic acid (C; STAV.-DSCNSSS: single CD) TOPOLOGY: unknown (IX) MGL£2LT_£ ΙΥ7Ξ. 3.\'Λ (genorair.

(xl) SSOUENCS DiSCRIPCION: sea id NO:38:

CIAGCTGTG7 CTCIGOGCQA GA3GGCCACC ATCAAQTGCA AGG 93 (2) INFORMATION FOR SEC W HO:39:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs (Β) TYPE: nucleic acid (C) STRANSESNESS: single

CD) TOPOLOGY; unknown (ii) MCLECCLE TYPE: DNA (geneaie) (xi! SEQUENCE DESCRIPTION: SEO ID NC:39:

CCTTGCASTI 2ATGGTGGCC CTCTC3CCCA GAGACACAG 39

(2) INFORMATION FOR 8SQ ZD NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: SI base pairs (B) TYPE: nucleic (rid (C) STRANDED» SS: single (0) TOPOLOGY: unknown (ii) MOLECULE TYPE: DMA (genoaic) (xi) SEQUENCE DESCRIPTION: SEQ'ID NO:40:

TCSASAGGCC TCCCAAAGTG TTGATTATGA TG5TGATAGT TATATGAACT GGTATCACCA «0

IAAACCC £*

AP/P/ 9 6 / 0 0 7 8 2

Ar . ·. ·.· οδό (2) INFORMATION FOR S£J JO NO:41:

ti) SEOUINCI CHARACTERISTICS:

(A) LENGTH: ¢3 base pairs (B! TTPS: nucleic acid (C> STRANDEDNESS: single (91 TOPOLOGY: unknown (11) MOLECULE TYPE: ONA (gecoalc)

Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

GGGTTTCTGC TGATACCAGT TCATATAACT ATCACCATCA TAATCAACAC TTTGGGAGGC CO (2) INFORMATION TOR SEQ a NO :42:

(1) SEQUENCE CHARACTERISTICS:

IA) LENGTH: 51 Base pairs (8) TYRE: nucleic acid (C> mANOnmSS: single (D) TOPOLOGY: unknown (11) KOXXCVU TYRE: DNA (genomic) <xl) SEQUENCE DESCRIPTION: SEQ ID NO:42:

ATACTACTGT CAGCAAAGTA ATGAGGATCC TCCGAGGTTC CGCGGAGGGA C 51

AP/P/ 9 6 / 0 0 7 8 2 (2, INFORMATION POE «Ο ZD 80:43:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 53 bas« pales IS; type·, nucleic acid IC) STRANDEDNESS: single (0) TOPOLOGY: unknown (it: MOLECULE TYPE: DNA ;c».-.3ir_C (xi) SEQUENCE DESCRIPTION: SEQ 10 NO:43:

CTTGGTCCCT CCOCCGAACC TCGGAGGATC CTCATTACTT TGCTSACAGT AGT 53 (2) INFORMATION FOR SCO ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (gensaie) (xi) SXQQSNCE DESCRIPTION: SEQ IC NC:44:

GGGCAGCCTC CJAASTTGCT CATTTACOCT GCATCCAATC TAOAATOTGG GGTAC 55 (2) INFORMATION FOR SEQ ID NO:45:

(i) SSOCSICB CHARACTERISTICS:

(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANOEDHBSS: Single (D) TOPOLOGY: unknown (ill MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

CCCAGATTCT AGATTGGATG CAOCGTAAAT OAGCAKCTTA GGA08CTGCC C 51

AP/P/ 9 6 / 0 0 7 8 2 (2) INFORMATION “OR SEQ ID NO :46·

MP {;; SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 base pales CB) TYPE: nucleic acid (T) STRANDEDNESS: single CD) TOPOLOGY: unknown (ii) MOLECULE TYPE: DBA (genomic, (xi, SEQUENCE DESCRIPTION: SEQ ID NO:46:

AATTCSAGGA C3CCAGCAAC AT3GTGTTGC AGACCCAGGT CfTCATTTCT CTGTTGCTCT «0

GGAICTCTGG TGCCTACGGG CAG 83 (2) INFORMATION FOR SEQ ID NO:47:

(i, SEQUENCE CHARACTERISTICS:

(A) LENGTH: 64 base pairs (B) TYPE: nucleic acic (C) STRANDEDNESS: «ingle CD) TOPOLOGY: unknown

Cii) MOLECULE TYPE: DMA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO :47.· fftAACCTGCC CGTAGCCACC AGACATCCAG AOCAACASAG AAATGAASAC CTSGGTCTGC 60

AACACCATGT TGCTSGCSTC CTCG 84

AP/P/ 96/00782 (2) INFORMATION FOR SEQ ID NO:48:

Ci) SBQOSNCS CHARACTERISTICS:

(A) LENGTH: 20 base paisa (B) TYPE; nucleic acid (C, STRANDEDNESS: single CD) TOPOLOGY: uaknown

(ii) MCLSCCLZ TYPE: DNA (gencsic' (xi) SEQUENCE DESCRIPTION: SEQ IE NC:48:

CAGGTTACCO TGAAAGAGTC (2) INFORMATION TOR SEQ ID NO:49:

(il SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs (3) TYPE: nucleic acid (C) STRANDEDKESS: single (D) TOPOLOGY: unknown (il) MOLECULE TYPE: ONA (geneaiu) (xi! SEQUENCE DESCRIPTION: SEQ ID NO:49:

gaagtagtcc TTGACCAG IB (2) INFORMATION FOR SEQ ID NO;50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pais· (*} TYPE: nucleic acid (0) STRASDECNESS: single (0) TOPOLOGY: unknown (11) MOLECULE TYPE: DNA (genaaie) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:SO:

I

GTCACCGTCT CCTCACCTAG CACCAAGGGG C 31

79/00/96 fd/dV (2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs

3 (5) TYPE; nucleic acid (CS STRANDEONESS: single (D) TOPOLOGY: unknown (US MOLECULE TYPE: DNA (gencnic) txi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

CTTG6T6CTA CCTSAGGAQA CG 22 (2) INFORMATION FOR SEQ ID NO:52:

<i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs (1) TYPE: nucleic acia (C) STRANDEONESS: single (0) TOPOLOGY: unknown

Cii) MOLECULE TYPE: DNA (gencaiic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

CATCTAGAie GCQCOGCCAC M»ACGTTTG ATCTCCAGCT TGGTCCC 47 (2} INFORMATION FOR SEQ ZD NO :53:

SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs (!) Type*, nucleic acid IC) ETRAHDEDNCSA: double (0) TOPOLOGY: unknown

MOLECULE TYPE: CCNA

SEQUENCE DESCRIPTION: SEQ IO NO:53:

iii (xi)

AP/P/ 9 6 / 0 0 7 8 2 (ii d S

AAGGCCTCCC AAAGCSTTGA TTATQATCOT GAIAGTTATA TOAA’ (2) INFORMATION FOR SEQ IO NC:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2: base pairs Ο) TYPE: nucleic acid !C; STPANUSDNESS: double (0) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA (Xi) SEQOENCE DESCRIPTION: SEQ ID NO;54:

ACCTCCGGTA TGGGTGTTTT C 21 (2) INFORMATION FCR SEQ ID NO :55:

(il SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 baa· pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION; SEQ ID N0;S5:

CACATCTACT gggacgacga CAAACGTTAC AACCCGAGCC TGAAATCC 4 a

V-S (2) INFORMATION FOR SEQ ID NO :55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pair»

AP/P/ 96/00782

AP.00585

(3! ZYSZ; aucl^ib acid (C) STRANDEDNESS: double (D! TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: S2Q 2D $0:5$:

CGCGAAACCG TTTTCTACTG GTACTTCGAC GTT (2) INFORMATION FOR SEQ ID NO:57;

(1} SEQUENCE CHARACTERISTICS:

(A) LENGTH: 393 baa· paisa (B, TYPE: nucleic acid (Ci STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: CDMA (ix) FEATURE;

(A) ΚΑΜΕ/KEY: CDS (·) LOCATION: 1..393 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

ATG GGA TCC AGC TCT ATC ATC CTC TTC TTG GTA GCA ACA GCT ACA GGT

Mat 1

Gly Trp Sac

Cya 5

Ila na

Leu

Phe Las Val Ala Thr Ala Thr Gly

10

15

GTC

CAC

TCC

GAT

ATC

GTG

ATG

ACC

CAG

tct

CCA

GAC

tcc

CTA

GCT

GTG

Val

Bis

Ser

up

21·

V*1

Mae

TRr

Gia

Ser

Pre

up

Ser

Leu

Ala

val

20

25

30

TCT

CTG

GGC

GAS

AGG

GCC

ACC

ATC

AAC

TGC

AA3

GCC

TCC

CAA

AST

GTT

ta *

Law

Gly

Glu

Ar3

Ala

Τ.-.Σ

lie

Asn

cys

Lys

Ala

ser

Gin

Ser

Val

35

40

4<

CM

CO r*.

o>

Gl £

<

144 “1

AP

0 5 8 3

GAT

TAT

jAT

G GT

GAT

AGT TAT ATG

AAC

TGG

TAT CAls

CAG

AAA

ccc

GGG

192

Arp

Tyr

Asp

Gly

Asp

Ser Tyr Met

Asn

Tcp

Tyr sjlr.

Gin

Lys

Pro

Gly

53

55

60

CAG

CCT

AAG

TTG

CTC ATT TAG

GCT

GCA

TCC AAT

CTA

GAA

TCT

SG*3

240

Gin

Pre

Pre

Lys

Leu

L«u lie Tyr

Ale

Ala

Sar Asa

Leu

Glu

Ser

Ciy

65

73

75

83

GTA

CCT

SAC

CCA

TTC

AGT GGC AGC

aoe

TCT

GGG ACA

GAT

TTC

ACT

CTC

288

val

Pre

AS?

Arg

Phe

Ser Gly ser

Gly

Sar

Gly Thr

Asp

Phe

Thr

Leu

85

90

95

ACC

ATC

AG_

CTG

CAG GCT GAA

GAT

GTG

GCA GTA

TAC

TAC

TGT

CAG

336

Thr

lie

Ser

Ser

Leu

Sin Ala Glu

Asp

Val

Ala val

Tyr

Tyr

Cy*

Gin

ICO

105

110

CAA

AGT

AAT

GAG

GAT

CCT CCG ACG

TTC

GGC

GGA GGG

ACC

AAA

GTG

GAG

384

Gin

Ser

Ass

Glu

Asp

Pre Pr; T.-.r

Phe

Gly

Gly Gly

Thr

Lys

Val

Glu

ILS

120

125

ATC

AAA

CGT

393

Ila Lyt Arg 130 (2) INTOWaTIOK rOR SEQ id no:58:

(11 SEQUENCE CHARACTERISTICS:

(A) LENGTH: 131 aaino acids (Bi TYPE: aaino add (Pl TOPOLOGY: linear (ii) MOLECULE TYPE: protein ©

(Xi) S2QCENCE DESCRIPTION: SEQ ID NO:58:

Ma; Gly Trp Ser Cys lie lie Leu ?r.e Leu Val Ala Thr Aia Thr Gly 1 5 10 15

AP/P/ 9 6 / 0 0 7 8 2

AP.00583

val

His

Ser

Asp

lie

val

Met

Thr

Gin

Ser

Pro

ASP

Ser

Leu

Ala

Val

20

25

30

Ser

Leu

Gly

Glu

Arg

Ala

Thr

lie

Asn

Cys

Lys

Ala

Ser

GIT.

Ser

val

35

40

45

Asp

Tyr

Asp

eiy

Asp

Ser

Tyr

Met

Asn

Trp

Tyr

Gia

file

Lys

Pro

Gly

50

35

SO

Sir.

Pro

Lys

Leu

Leu

lie

Tyr

Ala

Ala

Ser

Asn

Leu

Glu

Ser

Gly

SS

70

75

SO

Val

Pro

Asp

Arg

Phe

Ser

Gly

Ser

Gly

Ser

Gly

Thr

Asp

Phe

Thr

Leu

05

90

95

Ths

xie

Ser

Ser

Leu

Gio

Ala

Slu

Asp

val

Ala

val

Tyr

Tyr

Cys

Gin

100

105

110

Gin

Ser

Asa

Glu

ASp

Pre

Pro

Thr

Phe

Gly

Gly

Gly

Thr

Lys

Val

Glu

115 120 125

He Lys Arg 130

8 / 0 0 / 96 /d/dV

V

Applicants cr agents file refrrenc; number

P50186-2 intemaaonil Application No^3PW/· /in?rt_e indications relating to a deposited microorganism (PCT Rai* UUi)

Λ. The indicadoos made below relax co the nncnxxginism referred to in the description on page 32,

Boa 14.

B. IDENTmCArrONOF »;π<·λΊ ftrtw depone η» identified oa aaaddtonnal abaci □

European Collection of Animal Cdl Cultures (ECACC1

Addnsi cf depo&ary initiation QptdmisBj postal eode and connory)

Pane· Dow*

W0bdnr«,SP4OJC Uaitad Eaganai

Date of Deposit 06 October 1993	Areesaoo Number 9310M20
C. ADDITIONAL INDICATIONS 0er* Mmfc if χ φρίΐοΜ·) This infonnuioB is coocuuwd OP in iddidoftii cboct Q

Ia respect of those designations ia which a European or Australian Pateoi is sought or in any other states having equivalent provisoes, a sample of the deposited micro-organism wQl be made available until the publication of the meattou of die grant of the patent or until the dam on which the application has been refused or withdrawn, only by the issue of such a sample to aa expert nominated by the person «questing the sample.

D. MSOWAIED STATES KM WffiCH INWCATTONS ΛΒ& MAS£«ra

AB

E. SEPARATE FURNISHING OF INDICATIONS 0-· «* t * ι of the tadteadbea, eg, ’Acccsion timber of Depoau*)

AP/P/ 9 6 / 0 0 7 8 2

	ror wawni a wag ise anv
Authorized officre	Authorized officer —— - -

Claims (36)

1. A fusion protein having binding specificity for human interleukin-4 (E4) which comprises complementarity determining regions (CDRs) denved from i non-human neutralizing monoclonal antibody characterized by a dissociation constant equal to or less than

2 χ IO^-10 M for human 1L4, and a first fusion partner.

1 The fosioo protein according to claim 1 which is operatively baked to a second fusion partner.

3. The fusion protein according to claim 1 wherein said non-human neutralising moooelooal antibody is selected from the group consisting of 3B9 and 6A1.

4. The fusion protein according to claim 2 wherein said second fusion partner comprises all or pan of an immunoglobulin constant heavy chain or immunoglobulin constant light chain, or both.

5. The fusion protein according to claim 1 wherein said first fusion partner sequence is the heavy chain sequence of: amino adds 21-50,56-71,88-119, and 131441 of SEQ ID NO:li

6. The fusion protein according reclaim 1 wherein said first fosioo partner sequence is the light chain sequence of: amino acids 20-42,58-72,80-111, and 121431 of SEQ ID NO: 14.

7. The fiisioa protein according to claim 1 wherein said amino add sequences of die complementarity determining regions for the heavy chain are (a) ThrSeCly MetGJy ValSer. SEQ ID NO-.22, (b) HislkTyrTrpAspAapAspLysArxTyrAsnPro.

SetLeuLyxSer SEQ ID NOs24, or (c) ArgGbThrVaffheTyrTrpPheAspVal: SEQ ID N026.

8. The fusion protein according to claim 1 wherein said amino acid sequences of the complementarity determining regions for the light chain arc;

(a) LeuAlaSetGlnSerValAspTyrAspGlyAspSerTyrMetAan: SEQ ID

NO: 16,

AP/P/ 9 6 / 0 0 7 8 2 ο

fb; AlaALaSe-’AsnLeuGluSer: SEQ ID NO:IS, or (c) GlnG'.r-SerAsnGluAspPrcProArg: SEQ ID \’O:2S

9. The fusion protein according to claim 1 wherein said amino acid 5 sequences of the complementarity determining regions for the light chain are:

(a) LysAlaSerGlnSerValAspTyrAspGlyAspSetTyrMetAsn: SEQ ID

NChl6, (b) AlaAlaSerAsaLeuGluSer. SEQ ID NO: 18, or (c) doGlnSerAanGluAipProProThr SEQ ID NO:20.

10. An immunoglobulin heavy chain complementarity determining region (CDR), the imino add sequence of which is selected from the group consisting of:

(a) TbrScrGlyMctGly ValSer SEQ ID NO22,

15 (b) HisDeTytTrpAspAspAspLysArgTyrAsnProSerLeuLysSer: SEQ ID NOr24, and f (c) ArgGluThrValPheTyxTrpPheAspVal: SEQ ID NO26.

11. An immunoglobulin light chain complementarity determining region (CDR), the amino acid sequence of which is selected from the group consisting of:

(a) LeuAlaSoGlnSerValAspTyrAspGlyAspSeftyiMetAsa; SEQ ID

NO:16.

(b) AlaAlaScrAsaLeuGluSer SEQ ID NO: 18, (c) GlnGlnSerAsnGluAspProProArg. SEQ ID NO:2S; and (d) GlnGtoSerAsaGhiAspProPToThr. SEQ ID NO-JO.

11 A nucleic acid molecule encoding an immunoglobulin heavy chain complementarity determining region (CDR), the sequence of which is selected from the group consisting of:

30 (a) ACT TCT GGT ATC GGT GTC ACC: SEQ ID NO Jl, (b) CAC ATT TAC TGG GAT GAT GAC AAG CGC TAT © AACCCATCCCTOAAGAOC: SEQ ID NO-23, (e) AGA GAG ACT GTC TTC TAC TGG TAC TTC GAT

GTC: SEQ ID NQ25.

35 (d) ACC TCC GGT ATG GGT GTT TCC: SEQ ID NO: 54.

(e) CAC ATC TAC TGG GAC GAC GAC AAA CGT TAC AAC CCG AGC CTG AAA TCC: SEQ ID NO 55, and

AP/P/ 9 6 / 0 0 7 8 2

AP . «? π 5 θ 3 (fi CGC GAA ACC Gl x 3'. C TAC TGG TAC TTC GAC GTT.· SEQ

ID NO: 56.

13 A nucleic acid molecule encoding an immunoglobulin light chain

5 complementarity determining region (CDR), the sequence of which is selected from the group consisting of:

(a) AAG GCC AGC CAA ACT GTT GAT TAT GAT GGT CAT AGT TAT ATG AAC SEQID NO:15, (b) AAG GOC TCC CAA AGT GTT GAT TAT GAT GGT GAT AGT

10 TAT ATG AAC SEQ Π> NO: 53.

(c) GCT GCA TCC AAT CTA GAA TCT: SEQ ID NO. 17, (d)

CAG CAA AGT AAT GAG GAT CCT CCG ACC SEQ ID NCH19, and (e) CAG CAA AGT AAT GAG GAT CCT CCG AGG: SEQ ID NO27.

15 14. A hemanized antibody comprisiflg.a heavy chain and a tight chain, said antibody characterized by a dissociation constant equal to or less than about 2 x IO'¹* M for human IL4, wherein the framework regions of said heavy and light chains an derived from at least one selected human antibody and the amino add sequences of the complementarity determining regions of each said chain are

20 derived from a noa*hnman neutralizing monoclonal antibody specific for human ILA characterized by a dissociation cocsant equal to or less than aboot 2 x 10r“Mfor human ILA

15. The antibody according to claim 14 wherein said antibody is

25 optionally linked to an effoeut agent selected from the group consisting af a nooprocein earner molecule, polystyrene, and plastic beads.

16. A chimeric antibody comprising a heavy chain aula light chain, said antibody characterized by a dissociation constant equal to or less than about 2 a Kt¹* o 30 M for human ILA, wherein the amino add sequence* of the «yi*————ty φ tir e j mining regions of said heavy chain ad said tight chaia are derived from a aonbuman neutralizing monodoul antibody specific for human IL4 chancaerized by a dissociation constant equal to or less than about 2 x 10*’* M for human 114.

AP/P/ 9 6 / 0 0 7 8 2

35

17. A pharmaceutical cotuposjaoa comprising the fusion protein of claim

1 and a pharmaceutically acceptable carrier.

Μ Η . ό ύ 3 δ 0

Ο 30 e

18. A method of treating allergies and other conditions associated with excess IgE production ia a human comprising the Step of administering to said human in need thereof an effective amount of the fusion protein of claim 1.

19. An isolated nucleic acid sequence which is selected from the group consisting of:

(a) a nucleic acid sequence encoding the fusion protein of claim 1;

(b) a nucleic acid sequence complementary to (a);

(c) a nucleic acid sequence of 18 or more nucleotides capable of hybridizing to (a) or (b) under stringent conditions; and (d) a fragment or analog of (a), (b), or (c) which encodes a protein characterized by having specificity for human interleuldo-4;

wherein said sequence optionally contains a restriction site.

20. The isolated nucleic acid sequence according to claim 19. wherein the sequence encoding the fusion protein comprises the nucleic acid sequence of Fig. 5. SEQ ID NO: 13.

21. The isolated nucleic acid sequence according to claim 19, wherein the sequence encoding the fusiou protein comprises die nucleic acid sequence of Fig. 4, SEQID NO:11.

22. Au isolated nucleic acid sequence which is selected from the group consisting of:

(a) a nucleic acid sequence encoding a complementarity determining region (CDR) wherein said CDR is obtained from a neutralizing murine monoclonal antibody specific for human inrerieukin-4 sad having a dissociation constant equal to or less than about 2 x 10** M;

(b) a nucleic acid sequence complementary to (a);

(c) a nndeic acid sequence of 18 or more nucleotides capable of hybridizing under stringment conditions to (a) or fb>: and (d) a fragment or analog of (a), (b) or (c) which encodes a protein characterized by having specificity for human intrr'.eukin-4.

AP/P/ 9 6 / 0 0 7 8 2 r-\ !

23. The isolaiid nucleic acid scquv.ce according to ciaim 22, wherein said sequence is selected iron the group of heavy chain complementarity demrminiflg region-encoding sequences consisting of:

(a) ACT TCT GGT ATG GGT GTG AGC SEQ ID NC21.

5 (b) CAC ATT TAC TGG GAT GAT CAC AAG CGC TAT

AAC CCA TCC CTC AAG AGC: SEQ ID NO23, (c) AGA GAG ACT GTG TTC TAC TGG TAC TTC GAT

GTCSEQIDNO25.

(<n ACC TCC GGT ATG GGT GTT TCC SEQ ID NV. 54,

10 (e) CAC ATC TAC TGG GAC GAC GAC AAA CGT TAC AAC CCG

AGC CTC AAA TCC SEQ ID NO: 55, and (f) CGCGAA ACC GTT TTC TAC TGG TAC TTC GAC GTT;

SEQ ID NO: 56.

15

24. The isolated nucleic add sequence according to claim 22, wherein said sequence is selected from the group of light chain complementarity determining region-encoding sequences consisting of:

(a) AAG GCC ACC CAA AGT GTT GAT TAT GAT GGT

GAT AGT TAT ATC AAC SEQ ID NO:15,

20 (b) AAG GCCTCC CAA AGT GTT GAT TAT GAT GGT GAT AGT

TAT ATG AAC SEQ ID NO: 53, (c) OCT GCA TCC AAT CTA GAA TCT: SEQ ID NO; 17, (d)

CAG CAA AGT AAT GAO GAT CCT CCG ACG: SEQ ID NO: 19. and CAG CAA AGT AAT GAG GAT CCT CCG AGG: SEQ ID NO:27.

A recombinant plasmid comprising the nucleic add sequence of (e)

25. claim 19.

26.

30 claim 22.

27.

28.

29.

A recombinant plasmid comprising the nucleic acid sequence of

AP/P/ 9 6 / 0 0 7 8 2

A'host ceO mnsfoned with the recombinant plasmid of claim 25.

A host cell transfected with the recombinant piasmic of claim 26.

A process for producing a humanized antibody specific for human tnterleukin-4 comprising culturing & cell Hne transfected with the recombinant ύ

plasmid of claim 25 under the control cf selectee regulators· sequences capable of directing the expression thereof in said cells

30. A method for diagnosing allergies and other conditions associated 5 with excess immunoglobulin E production ic a human which comprises contacting a sample of biological Quid with a high titer monoclonal antibody for human ILA and assaying for the occurrence of binding between said monoclonal antibody and human interleukin 4.

10

31 A method for screening monoclonal antibodies which have ahigh titer for human interleukin 4 which comprises:

a) preparing a hybridoma cell line characterized by secretioo of a monoclonal antibody to human interleukin 4; and

b) screening said hybridoma cell line with aldehyde-coupled 12 human interleukin-4 or biotinylated human interleukin-4.

32. A neutralizing monoclonal antibody having a high titer for human interleukin-4, a Fab fragment or a F(ab% fragment thereof, produced by screening a library of hydridotua products with aldehyde-coupled human interieukin-4 or

20 biotinylated human mterienkin-4.

33. A rodent aeaoalizing monoclonal antibody for human interleukin-4 and having a binding affinity characterized by a dissociation constant equal to or less than about 2 χ ΙΟ^-1® M

34. The monoclonal antibody according to claim 33 wherein said rodent is a mouse.

~_;

35. The monoclonal antibody according to claim 34, which comprises the

30 light chain amino add sequence of SEQ ID KO: 2, and the heavy chain amino acid θ sequence of SEQ ID NO; 4.

36. The monoclonal antibody according to claim 33. w herein said rodent is a rat.

37. The monoclonal antibody according to claim 36 having the identifying characteristics of 6AI.

AP/P/ 9 6 / 0 0 7 8 2

AH . 0 0 5 8 3

38. A hybricoma having the identifying characteristics of cell line 3426A11C1B9.