AU723073B2 - CDR-grafted type III anti-CEA humanized mouse monoclonal antibodies - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): IMMUNOMEDICS, INC.

Invention Title: CDR-GRAFTED TYPE III ANTI-CEA HUMANIZED MOUSE MONOCLONAL ANTIBODIES 0 0.0.

0* to.

a The following statement is a full description of this invention, including the best method of performing it known to me/us: 1A- CDR-GRAFTED TYPE III ANTI-CEA HUMANIZED MOUSE MONOCLONAL ANTIBODIES BACKGROUND OF THE INVENTION The invention relates to immunological reagents for diagnostic and-_heieapeutic use in colon and other cancers. In particular, the invention relates to humanized anti-carcinoembryonic antigen ("CEA") monoclonal antibodies ("mAbs") that have the binding affinity characteristics of corresponding mouse anti-CEA 10 mAb (MN14) and the antigenic and effector properties of a human antibody. Further, the invention relates to humanized mAbs in which the complementarity determining regions ("CDRs") of an anti-CEA murine mAb is grafted into the framework regions of a human antibody, to DNAs that encode such CDR-grafted antibodies, to vectors and transformed hosts for propagating and expressing the DNAs, and to conjugates of the antibodies useful in diagnostic and therapeutic applications.

A promising approach to cancer diagnosis and therapy 20 involves the use of targeting antibodies to deliver diagnostic and therapeutic agents directly to the site of a malignancy. Over the past decade, a wide variety of tumor-specific antibodies and antibody fragments have been developed, as have methods to conjugate the antibodies to drugs, toxins, radionuclides or other agents, and to administer the conjugates to patients.

These efforts have produced great progress, but a variety of largely unanticipated problems have limited the diagnostic and therapeutic utility of some of the reagents thus far developed.

Among the most intractable problems is that which is caused by the human immune system itself, which may respond to the targeting conjugate as a foreign antigen.

For instance, patients treated with drugs or 2 radionuclides complexed with murine monoclonal antibodies (which have been the most commonly used targeting antibodies for human) develop circulating human antimouse antibodies (HAMAs) and a generalized immediate type-III hypersensitivity reaction to the antibody moiety of the conjugate. Furthermore, even when adverse side effects are minimal (for example, as in a single administration) circulating HAMAs decrease the effective __concentration of the targeting agent in the patient and therefore limiting the diagnostic or therapeutic agent from reaching the target site.

Several approaches have been developed to overcome or avoid this problem, with only limited success. One strategy has been to chemically modify the targeting antibody to suppress its antigenicity. For example, .conjugation of polyethylene glycol to the targeting antibody (PEGylation) is reported to reduce antigenicity of antibodies. Another approach has been to characterize the situs of antigenicity in an antibody and then remove it. In this vein, Fab', F(ab) 2 and other antibody fragments have been used in place of whole IgG. In addition, attempts have been made to reduce the adverse effects of HAMA by plasmaphoretically removing HAMA from blood. Immunosuppressive techniques also have been used to ameliorate the adverse effect of the foreign antibody sufficiently to permit multiple treatments with the targeting agent.

None of these approaches has proven altogether satisfactory. An important need persists for a means to reduce or eliminate the adverse immune response to targeting antibody and antibody conjugates in order to gain the full benefit of these diagnostic and therapeutic agents.

This goal has been achieved with the CDR-graftedhumanized murine anti-human CEA mAbs that are described below.

3 SUMMARY OF THE INVENTION It is an object of the present invention to provide a humanized Class III anti-CEA mAb in which the CDRs of a murine Class III anti-CEA mAb (MN14) are functionally engrafted to the amino acid sequence of a human antibody or antibody fragment to provide an immunological reagent with the anti-CEA binding properties of the murine Class III, anti-CEA mAb and the immunogenic properties of a human mAb in a human patient.

It is another object of the present invention to provide DNA constructs encoding such antibodies.

Particular objects in this regard are substrate DNAs that facilitate genetic manipulation to produce improved antibodies and DNAs encoding the antibodies with 15 advantageous properties in cell culture and antibody production.

Yet another object of the invention is to provide vectors for propagating the DNA and for expressing the antibody. A related object of the invention is to provide cells containing a vector for the purposes of storage, propagation, antibody production and therapeutic applications.

Still another object of the invention is to provide compositions comprising the antibodies for use in diagnosis and therapy. In this regard it is an object of the invention to provide conjugates comprising the antibodies complexed with imaging agents and therapeutic agents for ex vivo and in vivo imaging, diagnosis, prognosis and therapy, among others.

In accomplishing the foregoing objects, there has been provided, in accordance, with one aspect of the present invention, a humanized mouse mAb, comprising the CDRs of a murine Class III, anti-CEA mAb (MN-14) engrafted to the framework regions of a heterologous-- (human) antibody, wherein the thus humanized mAb antibody retains the Class III, anti-CEA binding specificity of the murine mAb but in the patient is less immunogenic than is the parent MN-14 murine monoclonal antibody.

In a highly preferred embodiment, the light chain variable regions of the humanized antibody are characterized by the formula: FRLI CDRL FR CDR FR CDR FR wherein each FR is separately a framework region of a human antibody, and each CDR is separately in a complementarity-determining region of the light chains of -N-14, and the subscripts refer to light chain regions. The heavy chain variable regions are characterized by the formula: FRI -CDR -FR CDR -FR -CDR -FRH 4 wherein FR and CDR have the same meanings as above, and wherein the subscripts refer to heavy chain regions.

In one embodiment, CDR u has the amino acid sequence KASQD VGTSVA (SEQ. ID NO. 20); CDRU has the amino acid sequence WTSTR HT (SEQ. ID NO. 21); CDR u has the amino acid sequence QQYSL YRS (SEQ. ID NO. 22); CDRH has the amino acid sequence TYWMS(SEQ. ID. NO. 23); CDR, has the amino acid sequence EIHP DSSTI NYAPS LKD (SEQ. ID NO.

24); and, CDRm has the amino acid sequence LYFGF PWFAY (SEQ. ID NO. In another embodiment, FRLI has the amino acid sequence DIQLT QSPSS LSASV-'GDRVT ITC' (SEQ. ID NO. 26); FRU has the amino acid sequence WYQQK PGKAP KLLIY (SEQ.

ID NO. 27); FRL 3 has the amino acid sequence GVP(S or D) RF SGS(G or V)S GTDFT FTISS LQPED IATYY C SEQ. ID NO. 28); FR, has the amino acid sequence FGQGT KVEIK (SEQ. ID NO.

29); FR, has the amino acid sequence EVQLV ESGGG WQPG RSLRL SCSSS GFDFT (SEQ. ID NO. 30), EVQLV ESGGG WQPG RSLRL SCSAS GFDFT (SEQ. ID NO. 31), or QVQLQ ESGPG LVRPS QTLSL TCTSS GFDFT (SEQ. ID NO. 32); FR, has the amino acid sequence WVRQA PGKGL EWVA (SEQ. ID NO. 33), WVRQA PGKGL EWIA (SEQ. ID NO. 34), or WVRQP PGRGL EWIA (SEQ. ID NO. 35); FR, has the amino acid sequence RFTIS RDNSK 35 NTLFL QMDSL RPEDT GVYFC AS (SEQ. ID NO. 36), RFTIS RDNAK NTLFL QMDSL RPEDT GVYFC AS (SEQ. ID NO. 37), or RVTML RDTSK NGSFL RLSSV TAADT AVYYC AS (SEQ. ID NO. 38); and *a o 5

FRH

4 has the amino acid sequence WGQGT PVTVS S (SEQ. ID NO. 39), or WGQGT TVTVS S (SEQ. ID NO. 40); and wherein C may be in the sulfhydryl or disulfide form.

Another preferred embodiment comprises a diagnostic or therapeutic agent complexed to Class III, anti-CEA humanized mAb in which the CDRs of the antibody are derived from those of the MN-14 murine mAb and the FRs are derived from those of the heterologous (human) antibody, wherein thecon.-jgate retains the Class III, anti-CEA binding specificity of MN-14, but is in humans less immunogenic than is murine MN-14. In one such embodiment the light chain and heavy chain variable regions are characterized as shown above and have amino acid sequences also as described above.

a In yet another preferred embodiment, a method for diagnosing or treating a patient comprises the step of administering in an appropriate regimen the conjugate of "the previous preferred embodiment.

Another preferred embodiment comprises an isolated, purified DNA that encodes the light chain, the heavy a chain or both chains of the humanized antibody described above.

Another preferred embodiment comprises the DNA sequence of the CDRs and FRs described above.

S. 25 Other objects, features and advantages of the present invention will become apparent from the following detailed description and appended claims.

ILLUSTRATIVE GLOSSARY The following terms or abbreviations are used in the present application. The meanings set out in this glossary are for illustrative purposes only. The full meaning of the terms will be apparent to those of skill in the art.

"CDR" is used as an abbreviation for Complementarity Determining Region. These are the regions within the variable regions of an antibody that are primarily, but 6 not exclusively, responsible for antigen-antibody binding.

"FR" is an abbreviation for Framework Region.

Broadly speaking, these are the portions of the variable regions of an antibody which lie adjacent to or flank the CDRs. In general, these regions have more of a structural function that affects the conformation of the variable region and are less directly responsible for the -specific binding of antigen to antibody, although, nonetheless, the framework regions can affect the interaction.

"Chimeric" refers to an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from an antibody from a S 15 heterologous (other) species.

"Humanized" refers to a chimeric antibody as defined above, but one in which the FR variable regions are derived from a human antibody.

"HAMA" refers to human antibodies directed to a mouse 20 antibody, that are produced when a mouse antibody is administered to a human subject.

"HAHA" refers to human antibodies directed to a humanized mouse antibody.

"CEA" refers to carcinoembryonic antigen, a 180 kDa 25 glycoprotein that is expressed in most adenocarcinomas of endodermally-derived digestive system epithelia and in some other cancers such as breast cancer and non-small cell lung cancer.

The letter as a prefix means "humanized".

Other abbreviations are used in accordance with Roitt et al., IMMUNOLOGY, 3rd ed. Mosby Year Book Europe Ltd.

(1993), the entirety of which is herein incorporated by reference.

These and other terms used in the present disclosureare used in the same sense as ordinarily they are employed in the arts to which this invention pertains.

BRIEF DESCRIPTION OF THE FIGURES FIGURES 1A& B(SEQ. ID. NOS. 1 and 2) shows the consensus DNA sequence of murine NEWM N-14 variable region hea-/ chain and its protein translation product. The CDRs are enclosed in boxes.

FIGURES 2A&2B(SEQ. ID. NOS. 3 and 4) shows the consensus DNA sequence of murine MN-14 variable region light chain and its protein translation product. The CDRs are enclosed in boxes.

FIGURE 3 shows a vector for the expression of chimeric or humanized MN-14 heavy chain gene. The schematic diagram shows both the chimeric and a reshaped heavy chain immunoglobulin (Ig) gene and the pSVgpt expression vector. The diagram at top, labeled "CHIMERIC, is a map showing DNA encoding the MN-14 mouse VH region joined to DNA encoding a human IgGI constant region. In the MN-14 VH region the three CDRs are indicated by the horizontally-lined areas. The FRs are indicated by the four diagonally-lined areas. The middle diagram, labeled 20 "RESHAPED" shows the humanization of the MN-14 VH region in which the mouse FRs have been replaced by human FRs, indicated by the four clear areas in the "Human"

VH

region. The circular.map of the expression vector pSVgpt at bottom shows the HindIII/BamHI insertion site for the 25 reshaped MN-14 antibody gene just downstream from an Igh enhancer element. The map also indicates some important functional domains in the vector, including the replication origins for propagation in E. coli (colEl ori) and in mammalian cells (SV40 promoter region), and genes encoding selective markers for culturing bacterial (Apr) and mammalian (gpt) cells transformed with the vector. Expression of the antibody gene in this case is mediated by the Ig promoter indicated by the solid circles near the HindIII site in the maps of the antibody genes.

A i FIGURE 4 shows a vector for the expression of chimeric or humanized MN-14 kappa chain gene. The 8 diagram shows a chimeric and a reshaped MN-14 kappa ligh: chain gene and the pSVhyg expression vector. The diagram at top, labelled "CHIMERIC, is a map showing DNA encoding the MN-14 mouse kappa light chain variable region joined to DNA encoding a human kappa constant region. In the mouse VK region the three CDRs are indicated by the horizontally-lined areas and the FRs are indicated by the four diagonally-lined areas. The middle diagram, labelled "RESHAPED" shows the humanization of the M-14 VK region in which the mouse FRs have been replaced by human FRs, which are indicated by the four clear areas in the "Human"' VK region. The circular mao of the expression vector pSVhyg at bottom shows the HindlII/BamH1 insertion site for the reshaped MN-14 antibody gene just downstream from an Igh enhancer element. The map indicates some important functional domains in the vector, including the replication origins for E. coli (col El ori) and mammalian propagation promoter region), and genes encoding selective markers 20 for culturing bacterial and mammalian (gpt) cells transformed with the vector. Expression of the antibody gene in this 'case is mediated by the Ig promoter schematized by the solid circles near the HindIII site in the maps of the antibody genes.

25 FIGURES 5A 5B show the alignments of the murine MN-14 variable regions (SEQ. ID. NOS. 2 and 4) with the human variable regions NEWM VH (SEQ. ID NO. 5) and REI VK (SEQ.

ID NO. 6) (Figure 5A). and with the human KOL VH region

S*

(SEQ. ID NO. 7, Figure 5B) CDRs are boxed, and the S" 30 murine VH FRs, which are incorporated into the humanized VH, are marked with their positions according to the numbering system of Kabat et al. SEQUENCES OF PROTEINS

OF

IMMUNOLOGICAL INTEREST, U.S. Government Printing Office, Washington, 1987. Murine residues outside the CDRs that were included in the KLHuVH are indicated by a filled circle.

FIGURES 6A, 6B 6C show a comparison of the amino acid sequence between murine (SEQ. ID NO. 2) and humanized (SEQ. ID NOS. 57, 8-11, 58 and 12-15). MN-14 VH framework residues Only human FR residues different from the mouse are shown. CDAs for NEWM and KOL are also not shown.

Position of the substitution is indicated according to the Kabat et al. numbering system. The 3 CDRs are boxed.

Figure 7 shows the DNA sequence and corresponding amino acid sequence (SEQ. ID. NOS. 16 and 17) of the MN-14 HuVH region (KLHuVh) CDRs are boxed.

FIGURE 8 shows the DNA sequence and corresponding amino acid sequence (SEQ. ID NOS. 18 and 19) of the MN- 14HuVK region. CDRs are boxed.

FIGURE 9 is a graph of 14 blocking competition) assays comparing relative binding affinities of KLHuVH variants, including: KLHuVH/HuVK; KLHuV~AIG/HuVK; KLHuVHAIGAY/HuVK; KLHuVkAIGA/uVK; chimeric control; and, 0 murine control.

FIGURE 10 shows MN-14 blocking assays comparing hMN- 14 with 2'MN-14-NVT (glycosylated in FR 1 region) FIGURE 11 is a radioautogram of the abdomen of a colon cancer patient following administration of I 31 ilabelled hMN14IgG (left panel) or mMN14IgG (right panel) 25 in the same patient.

DETAILED DESCRIPTION OF THE INVENTION Notwithstanding past failures to develop an effective non-kAMA-inducing anti-CEA antibody having, the CEAbinding characteristics of MNV-14, it has been discovered that the CDRs of the MN-14 mAb can be grafted onto the FRs of a human antibody to provide antibodies and antibody-derived reagents that have the antigen binding properties of the MN-14 anti-CEA mAb, while also exhibiting reduced induction of KAMA and augmented effector activities.

A The murine anti-CEA IgGI monoclonal antibody MN-14, and its production, have been described previously.

10 Hansen et al., Cancer, 71 3478 (1993); Primus et al., U.S. Patent No. 4,818,709. MN-14 meets all of the criteria of a Class III, anti-CEA monoclonal antibody, being unreactive with meconium antigen by EIA and not reacting with normal tissues.

Blocking studies are carried out according to Hansen et al. 1993, above, Losman et al., Int. Cancer, 56 580 (1994); Hansen et al., Clin. Chem., 35: 146 (1989).

Usinggte._- same conditions as described in those references for quantification of CEA, binding of humanized MN-14 may be assessed relative to a labeled MN- 14 probe. A typical probe is MN-14 conjugated to horse radish peroxidase (HRP). Both labeled and unlabeled MN- 14 are added to a CEA sample fixed to a solid support 15 such as microtitre plate wells. The degree of "blocking" S- of labeled MN-14 binding to CEA is a direct reflection of unlabeled MN-14 activity. Using standard MN-14, the relative activity of an unknown sample of humanized MN-14 or derivatives thereof can be determined. Typically, the 20 reactions are performed in the wells of a microtitre plate where wells are charged directly with CEA at a level of, for example, 25 pg/well or indirectly where the wells are precharged with an antibody reactive with CEA but to an epitope different than that to which MN-14 interacts; such an antibody may be the MN-15 mAb. CEA can thus be indirectly fixed to the well. A competitive binding EIA assay can then be performed with such a charged plate.

Alternate to the aforementioned HRP-labeled mAbs, antibodies can be.radioiodinated conventionally with, for example, 131I by the chloramine-T method to a specific activity of about 10 mCi/Ag, and free radioisotopes removed by chromatography on acrylamide gel columns (see Hansen et al., 1993, above).

Molecular biological techniques suitable to carrying out the invention as herein described also are known to those skilled in 'the art. Suitable teachings are described in numerous manuals and primary publications, 11 including inter alia, Sambrook et al., MOLECULAR

CLONING:

A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1987, 1988, 1989), which are herein incorporated by reference in their entirety including supplements.

MN-14 light and heavy chain CDRs disclosed herein, and modified MN-14 CDRs can be integrated into other antibodies using well-known recombinant techniques, such as those described in the above references.

Specific methods suitable to this end are shown below in the examples. Based on the amino acid sequences set forth herein, oligonucleotides encoding MN-14 CDRs can be synthesized. Oligonucleotides that encode modified CDRs may be made, as well as those that encode exactly the amino acid sequences herein set forth. Also, the oligonucleotides may contain nucleotides in addition to those of an MN-14 CDRs, to facilitate cloning, for instance. Oligonucleotide synthesis techniques are well known, and can be carried out on automated equipment available from a number of manufacturers. Moreover, oligonucleotides of any specified sequence can be 25 obtained commercially.

Oligonucleotides encoding the MN-14 CDRs and/or specific FR residues, or representing the complementary strand thereof, may be used to introduce codons for these residues into VH or VK DNA by site-directed mutagenesis provided that the ends of the oligonucleotides, generally 12 nucleotides, are designed to anneal perfectly to the template DNA. The template DNAs are typically singlestranded DNAs representing M13 vectors that carry a variable region DNA encoding the required FRs. In one method, the mutagenic oligonucleotides are phosphorylated at their 5' ends and, together with an oligonucleotide priming 5' to variable region DNA, are annealedd to the SssDNA template. The oligonucleotides are extended using 12 T7 polymerase and the fragments linked together by T4 DNA ligase to give a complete mutant strand covering the whole variable region. Using the mutant strand as a template, multiple copies of its complementary strand can be synthesized from a suitable primer using Taq DNA polymerase in a thermal cycling reaction. Once the mutant strand has been preferentially amplified in this manner, the DNA can be amplified by conventional PCR for cloning, sequencing and expression.

Suitable antibody-encoding DNAs are illustrated by the disclosure herein, but include practically any such DNA. A variety of human antibody genes are available in the form of publically accessible deposits. Many sequences of antibodies and antibody-encoding genes have 15 been published and suitable antibody genes can be synthesized from these sequences much as described above.

The scope of this invention encompasses all alleles, variants and mutations of the DNA sequences described herein.

20 CDR grafting in accordance with the present e: disclosure may be carried out using established -techniques. Antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a 25 variety of laboratory manuals and primary publications.

For instance, techniques suitable for use in the invention as described below are described in CURRENT PROTOCOLS IN IMMUNOLOGY, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.

RNA may be isolated from the original hybridoma cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed bycentrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligodT cellulose. Techniques suitable to these purposes are well known in the art as described in the foregoing references.

cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known methods. It may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences.

PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.

The necessary techniques are well known to those of skill in the art, are set forth in the foregoing Sambrook and Ausubel references and are illustrated by the examples set forth below.

cDNAs 'that encode the light and the heavy chain of an antibody can be propagated in any suitable vector in 20 any suitable host prior to isolation of the CDR. Often the clones will most conveniently be propagated for this purpose in E. coli as illustrated in the examples below.

However, a variety of other vectors and host cells well knowN to those of skill profitably may be employed in 25 this aspect of the invention. A variety of such vectors are described in the foregoing references.

DNA, typically plasmid DNA, may be isolated from the cells, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail 30 in the foregoing references relating to recombinant DNA techniques.

DNAs encoding antibody heavy and light chains and fragments thereof in accordance with the vector are used to construct chimeric and CDR-grafted humanized MN-14 antibodies.

The CDRs of the MN-14 anti-CEA mAb are herein identified and described, and illustrated in Figures 1A and 1B RAZ, and 2A and 2B (SEQ.ID NOS.2 and 4, respectively). Using these A$l 11 14 sequences, CDRs of the MN-14 heavy and light chain can be synthesized for use in the present invention. It is not necessary to redone MN-14 CDRs from a natural source.

The DNA and amino acid sequences are set forth herein.

Oligonucleotide synthesis techniques suitable to this aspect of the invention are well known to the skilled artisan and may be carried out using any of several commercially available automated synthesizers. In addition,-DNAs encoding the CDRs set forth herein can be obtained through the services of commercial DNA synthesis vendors.

Polynucleotides synthesized in accordance with this aspect of the invention may include those not derived from an MN-14 CDR as well as those that make up the CDR.

15 The additional bases may be included to facilitate joining the CDR to the FRs from a heterologous source.

They may comprise restriction sites or overlapping complementary regions for this purpose. The synthesis of longer, double-stranded DNAs from shorter, overlapping, 20 single-stranded DNAs is well known to those of skill in the art. Likewise, well known is the end-to-end joining of DNAs, including blunt-ended DNAs and those with at least partially overlapping complementary termini. These techniques are illustrated in the foregoing references on recombinant DNA techniques, for instance.

The CDRs of the MN-14 heavy and light chains may also be modified particularly after incorporation into a chimeric or humanized antibody using well-known recombinant DNA techniques for deleting, inserting and altering bases in a cloned or synthetic DNA or RNA.

Site-specific mutagenesis techniques suitable to this end are well known to those of skill in the art, and are illustrated in the foregoing references on recombinant DNA techniques. Also illustrated are deletional andinsertional techniques. These methods can be used to introduce practically any desired alteration into polynucleotides that encode the MN-14 CDRs or into other regions of a closed heavy or light chain gene.

15 MN-14 CDRs and modified MN-14 CDRs can be introduced into practically any set of FRs in accordance with the present invention. It will be appreciated by those of skill in the art that a variety of well known techniques for cloning and manipulating -polynucleotides may be effectively employed in this regard. Such techniques are illustrated by the methods set forth in the foregoing recombinant DNA-related references.

i- In a particularly preferred embodiment of -the-P4.asent invention, MN-14 CDRs are grafted into a human antibody.

It will be understood that human antibody in this context refers to any antibody that occurs in a human or an engineered antibody that has been designed, in some respect, to be compatible with the human immune system.

15 Particularly preferred for this purpose are antibodies that, broadly, do not engender an adverse immune response in a patient. More particularly, the expression "human antibody" is intended to mean an antibody encoded by a gene actually occurring in a human, or an allele, variant or mutant thereof.

Once DNA encoding an MN-14-derived CDR-grafted antibody has been assembled from MN-14 VH and VK region DNAs and the variable regions thus formed combined with their respective light and heavy chains of human constant domains, it may be inserted into a vector for propagation and expression by conventional techniques. In this manner desired amounts of the antibody may be' obtained.

The MN-14 CDR-grafted human antibody can be used in imaging applications by administrating to a subject the humanized antibody or Fab' thereof conjugated with an imaging compound or isotope.

The antibody is conjugated to a label for imaging using conventional methods. Such conventional methods include, but are not restricted to: 1) directradioiodination of the antibody protein or fragments thereof or 2) direct attachment to the antibody or fragments thereof of metallic nuclides (see, Hansen et al.,Cancer, 73: 761 (1994)). The use of bifunctional 16 chelates that can be used to bind various diagnostic or therepeutic metals to the antibody or fragment thereof is also within the scope of the present invention (see, Antibodies in Radiodiagnosis and Therapy, ed. M.R.

Zalutsky, 1989, CRC Press, Boca Raton, FL, and Cancer Therapy with Radiolabeled Antibodies, ed. D.M.

Goldenberg, 1994, CRC Press, Boca Raton, FL). Following the conjugation procedure and characterization of the product, satisfactorily labelled-conjugates are purified to homogeneity under conditions that conform to Good Manufacturing Procedures appropriate to the production of diagnostic compositions for use in human patients.

The reaction of serum antibody with the MN-14 CDR- 15 grafted antibody and imaging agent portions of the conjugate can be determined over the course of the diagnostic procedures, including the reaction of control sera obtained prior to administration of conjugate.

Similar determinations are made in other patients treated 20 with similar conjugates of MN-14 itself. The sera ee° antibody reactive with CDR-grafted MN-14 human antibodies detected by these tests is much less than the antibody reactive with antibody portion of the conjugate in patients treated with the murine MN-14-containing conjugates.

Humanized MN-14 antibodies conjugated to aminodextran and to boron may be used for diagnostic purposes. MN-14 and CDR-grafted MN-14 antibodies can be prepared as set forth above for conjugation to an aminodextran-boron adduct. Amino-dextran-boron adducts can be prepared by reaction of a suitable boron cage compound a 12boron carborane suitably derivatized with an aminodectran functional group). In a preferred embodiment, the amino-dextran is reacted with an excess of ahaloacetyl acid ester or anhydride (such as iodoacetic anhydride), thereby producing an amino-dextran with a desired number of haloacetyl groups, usually ranging from 10-1000 groups, depending on the reaction conditions and 17 the size of the amino-dextran. A suitable boron derivative such as mercaptocarborane-B12 is reacted, in a desired molar excess, with the haloacetyl-amino-dextran via an alkylation reaction. In a preferred embodiment, a number of haloacetyl groups on the boronated haloacetyl amino-dextran remain unreacted, and can be used as a "handle" to attach the adduct to protein thiol groups.

MN-14 CDR-grafted humanized antibodies and their derivatives,-because of their reduced immunogenicity, are useful in therapy, for passive immunization without negative immune reactions such as serum sickness or anaphylactic shock, for localization and in vivo imaging of tumors as described above, for specific treatment of disease cells, site directed delivery of 15 cytotoxins, immunomodulators or other pharmaceutically active molecules where local concentration of the active agent is an important factor, or the like, thereby establishing the practical utility of these humanized antibodies. As described above, for in vivo imaging, the 20 humanized, CDR-grafted MN-14 monoclonal antibody is radiolabeled or conjugated with a metal chelator complexed with a radionuclide, iodine, ytrium, technetium, or the like, and radio-scanning techniques may be used to detect primary and metastatic CEA tumors.

To that end, the radioactive antibody is injected, e.g., intravenously, and the patient scanned with a gamma imager at regular intervals. Tumors expressing CEA will take up more radioantibodies than other tissues and will be easily recognized by the imaging camera.

Preferentially, monoclonal antibodies labelled with 1 31I are used in amounts of 3 to 10 Ag representing 15 to pCi per kg body weight. For therapy with cytotoxic agents, the antibodies are conjugated to any of a variety of known therapeutic agents such as doxorubicin;methotrexate, taxol, ricin A, radioactive atoms, cytoxic agents, and the like, formulating such conjugate in a pharmaceutically acceptable sterile vehicle, and administering the formulation by conventional means. The 18 therapeutic dosages can be readily determined conventionally by the user of average skills in these arts. The therapeutic dose for mammals is between about 1 mg and 5 mg per kg body weight for the monoclonal antibodies themselves, and between 0.1 mg and 5 mg per kg body weight for conjugates with cytotoxic drugs, depending on the status of the patient and the mode of administration. Alternately, the humanized antibodies can be used in combination with components of the host'simmune system, complement or cell mediated responses, in order to remove from the subject CEApresenting cancer cells. The immune responses of patients may be monitored in accordance with the foregoing procedures. For additional procedures for 15 radioimaging and therapy, see EP 0 323,806, Hansen et .i al., Cancer 71: 3478-85 (1993), and U.S. Patent No.

4,818,709 and references contained therein, all of which are incorporated by reference.

Preferred are pharmaceutical preparations for 20 parenteral administration, such as are described in Remington's Pharmaceutical Sciences, Mack Publishing Co. Easton, PA, 1989. The final preparations contain from 0.01% to 50% of active ingredients. Methods for the production of such conjugates and their use in diagnostics and therapeutics are provided in, for example, Shih et al., U.S. Patent No. 5,057,313; Shih et al., Int. J. Cancer 41:832 (1988); copending, commonly owned USSN 08/162,912; and, McKearn et al., U.S. Patent No. 5,156,840, the contents of which are incorporated by reference.

As noted above, for purposes of therapy, a humanized antibody conjugate and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. A combination of aconjugate and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is "physiologically significant" 19 if its presence results in a detectable change in the physiology of a recipient patient. A targeted therapeutic agent is "therapeutically effective" if it delivers a higher proportion of the administered dose to the intended target than accretes at the target upon systemic administration of the equivalent untargeted agent.

To be therapeutically effective the conjugate and carrier may need to be administered in combination with other therapeutic agents or as part of a broader treatment regimen. Physicians are currently of the opinion that the effectiveness of targeted therapeutics can often be greatly increased when used in a combination therapy approach. For example, high-dose 15 radioimmunotherapy for B-cell lymphomas, which causes severe hematologic toxicity when used alone, has been

S.

shown to be highly effective when used in combination with autologous bone marrow reinfusion. Press et al., "Treatment of Relapsed B Cell Lymphomas with High Dose S. 20 Radioimmunotherapy and Bone Marrow Transplantation" in CANCER THERAPY WITH RADIOLABELED ANTIBODIES, Goldenberg, ed. (CRC Press, Boca Raton, 1994) ch. 17. In another example a five-fold enhancement of tumor uptake of a radiolabeled antibody is observed when the tumor is preirradiated. Leichner et al., Int. J.-Radiat. Oncol.

Biol. Phys. 14:1033 (1987). Mechanisms which have been shown to have the potential for improving the clinical efficacy of radioimmunotherapy are also discussed in DeNardo et al., "Overview of Obstacles and Opportunities for Radioimmunotherapy of Cancer" in CANCER THERAPY WITH RADIOLABELED ANTIBODIES, Goldenberg, Ed. (CRC Press, Boca Raton, 1994) ch. 11. Methods of developing such combination protocols, as well as to investigate doselimiting side effects and to potentiate and amplify targeting, uptake, and beneficial side effects, are well known to skilled clinical artisans in this field and would not require undue experimentation to develop.

20 In vivo experiments using conjugates of the humanized MN-14 with diagnostic and therapeutic agents have been carried out with animal models and with human patients (see Example 11 below). The CDR-grafted humanized antibody conjugate exhibited a better therapeutic profile and could be used in longer treatment regimens than the parental MN-14 antibody conjugate. The CDR-grafted antibody conjugate provided a better therapeutic effect and fewer deleterious side effects- than the control murine antibody conjugates.

For example, the antibody was covalently complexed to aminodextran-conjugated methotrexate using the method described by Shih et al., above using carbohydrate hydroxyl groups for derivatization purposes. In order to S 15 determine the contribution of antibody carbohydrate groups on immunoactivity, a mutation can be introduced at position 18-20 in the VK FR1 region of hMN-14 (the prefix is intended to mean "humanized") so as to introduce a glycosylation site, NVT, prior to expression of the 20 blocking gene in mammalian cells. Comparison of hMN-14 antibody with mutated hMN-14-NVT in a blocking cell binding assay (Figure 8) has demonstrated that the carbohydrate moiety at position 18 is without influence on immunoreactivity of this humanized antibody.

Aminodextran, average molecular mass 40 kDa, is oxidized by NaIO 4 to form aldehydes (by the oxidation of hydroxyl groups). About 50 to 150 moles of aldehydic groups are introduced per mole of aminodextran by careful control of the reaction conditions and timing. The aldehydes then are reacted with an excess of 1,3-diamino- 2-hydroxypropane to form Schiff bases with virtually all of the aldehydes. The Schiff bases are then reduced by treatment with excess NaBH4. The amine-derivatized dextran then is purified by gel-exclusion chromatography...

The cytotoxic drug methotrexate (MTX) is activated by treatment with dicyclocarbodiamide, followed by reaction with N-hydroxysuccinimide, both in dimethylformamide. Activated MTX is mixed in a 50:1 21 ratio with the amino derivatized dextran in aqueous solution. The product provides, after purification, MTXderivatized dextran having about 35 MTX moles per mole.

The MTX adduct thus obtained is conjugated to a MN-14 CDR-grafted antibody using methods described in Shih, et al., supra. For example, the antibody carbohydrates are oxidized and the resultant aldehydes are reacted with the remaining amines on the dextran in the adduct. The Schiff-base product obtained thereby is reduced by treatment with sodium cyanoborohydride in 10-fold molar excess over antibody. The reduced antibody-dextran-MTX product is thoroughly purified prior to assay, and formulated for administration to patients.

Parental MN-14 antibody is conjugated to dextran-MTX 15 in the same way, as a control.

The purified CDR-grafted antibody conjugate can be administered to patients with a CEA-producing cancer (see above). The response to therapy is monitored, including adverse side effects, particularly those which are 20 mediated by the patient's immune systems. Patients treated with the CDR-grafted antibody conjugate show improved therapeutic results, decreased immune response to the agent and notably decreased immune-mediated adverse effects of therapy. Therapy with the CDR-grafted antibody conjugate can be carried out at higher dosages and for longer periods of time then with the parental murine MN-14 antibody, allowing more aggressive therapies and improved responses.

The present invention is further described by reference to the following, illustrative examples. It will be appreciated that the techniques related to isolating DNA clones encoding MN-14 light and heavy chain genes are illustrated by cloning techniques useful to isolate light and heavy chain genes of any antibody from.

producing cells. There is no necessity, given the disclosed sequence, to reisolate MN-14 heavy'and light chain genes to carry out the invention.

22 It should be understood that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the following illustrative description.

EXAMPLE 1 Culturing Antibody Producer Cells A mouse/mouse hybridoma cell line producing Class III, anti-CEA monoclonal antibodies was established according to Hansen et al. (1993) above and Primus et al.

(1983) above.

Cells were selected for secretion of kappa IgGi by 15 testing conditioned medium using standard isotyping techniques. A variety of kits for this purpose are commercially available. Such cells were screened for production of antibody by testing conditioned medium using a standard blocking assay described above. Stocks 20 of producer cells that proved out in the assay were expanded and frozen in liquid nitrogen.

EXAMPLE 2 Isolating RNA From Producing Cell Lines MN-14-producing cells were expanded in culture, collected by centrifugation and washed. Total RNA was isolated from the cells in the pellet according to Favaloro et al., Methods in Enzymology 65: 718 (1980) and Orlandi et al., Proc. Nat'l Acad. Sci., USA 86: 3833 (1989), which are incorporated by reference.

EXAMPLE 3 cDNA Synthesis And Amplification Of The Heavy Chain Variable Region mRNA from MN-14 producing cells was used to synthesize cDNA using standard techniques of cDNA synthesis and DNA amplification by PCR, as described below. In general, the primers used for PCR included a restriction endonuclease cleavage site at their 5' ends to facilitate cloning of the amplification product. An 23 oligonucleotide complementary to the end of the sense strand of the DNA encoding the first constant region domain of the murine IgG, heavy chain ("CH1i") was used to prime f irst strand cDNA synthesis by reverse transcriptase. The sequence of this primer, CG1FOR, is shown in Table 1. Table I below provides other oligonucleotide sequences used herein.

TABLE 1 QLIGONCLEOTIDE

SEQUENCES

0 SEQ.

ID

NO.

S

S. S

S.

S.

S

*S

*SS*

S S S.

S.

*S*e

S

'S

Sr.. S S *5 CG1FOR VHlFOR 15 VI{1BACK SH1BACK SH2BACK CK2FOR VK1FOR VK3FOR VK1BACK VK213ACK VK3 BACK VK41BACK 25 VK5BACK VK61BACK VK7BACK VK8 BACK GGAAGCTTAGACAGATGGGGGTGTCGTTTTG 3' TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG 3' AGGTSMARCTGCAGSAGTCWGG 3' TGGAATTCATGGPATGGAGCTGGRTCWT2BHTCTT 3' TGATCTRCTDGTACKRT 3' GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3' GTTAGATCTCCAGCTTGGTCCC 3' GTTAGATCTCCAGTTTGGTGCCT 3' GACATTCAGCTGACCCAGTCTCCA 3' GACRTTCAGCTGACCCAGGMTG{A 3' GACATTCAGCTGACCCA 3' GACATTGAGCTCACCCAGTCTCCA 3' TTGAATTCGGTGCCAGAJCCWSAHATYGTkATG 3' TTGAATTCGGTGCCAGAKCWSAHATYGTKCTC 3' TTGATTCGGAGCTGATGGGAACA.TTGTAATG 3' CWGAGAAATTCAGCTGACCCAGTCTC 3' sites incorporated in primers t( Restriction facilitate cloning are underlined.

The variable region of the heavy chain cDNA- -then was amplified by the POR using the same primer, CG1FOR, and a primer based on the consensus sequence of the 5' end of VII genes (VH1BACK) as described in Orlandi et al. (1989) cited above. The PCR product of this 24 reaction was analyzed by agarose gel electrophoresis, which, upon ethidium bromide staining and fluorescence illumination, revealed one major band of about 400 bp, as.

expected.

For confirmatory sequences from a second cDNA preparation, signal sequence primers were used in the PCR to allow determination of the authentic amino acids of the N-terminus. SH1BACK and SH2BACK, degenerate oligonucleotides based on heavy chain signal sequence coding regions, were used in separate reactions in concert with CG1FOR. A diffuse product band was obtained from CG1FOR, SH1BACK amplification.

In order to increase the VH content of the product it was excised from low melting point agarose and 15 amplified using SH1BACK and an oligonucleotide complementary to a fourth framework region consensus sequence, VH1FOR. This product of this reaction was a discrete band when analyzed by agarose gel electrophoresis.

20 EXAMPLE 4 Cloning And Sequencing DNA Encoding The MN-14 Heavy Chain Variable Region Obtained By PCR The amplification product obtained using the CG1FOR, .o VH1BACK primer pair was digested with HindIII and PstI separately. The cleavage sites of these enzymes are included in the PCR primers. It was preferable to determine whether there were also sites internal to the VH. Agarose gel analysis of the restriction fragments indicated the presence of an internal PstI site close to one end of the DNA. The PCR product was digested with HindIII and PstI, cloned into M13mpl8 and 19 and the DNA sequence of the inserts of representative clones determined. The majority of the clones contained inserts of the same VH DNA.

The sequencing confirmed the presence of this additional, unexpected PstI site, which was close to the 3' end of the sequence of the CG1FOR primer partially=.encoding the final two amino acids of the VH. Although several full length VH clones were obtained by this method, further PCR product DNA was cloned as PstI-PsgI fragments. These clones thus contained full VH sequences but none of the constant region given by CG1FOR. A total of 16 full-length clones were obtained from the VK1BACK, CG1FOR product. In these experiments about 25% of the clones that were analyzed contained inserts unrelated to the VH region.

In order to confirm the VH sequence from a second cDNA preparation and, at the same time, to obtain the authentic, rather than primer-dictated, DNA sequence corresponding to the N-terminus of the VH, the PCR product from VH1FOR and SH1BACK primers was cloned.

These primers contain BstEII and EcoR1 restriction sites, rather than the PstI and HindIII sites of CG1FOR and VH1BACK described above. The PCR product of this reaction was cloned by digesting with BstEII, filling in the BstEII ends, digesting with EcoRI, and ligating the EcoRI and blunt ends to the vector, which had been 20 digested with EcoRI and HindII. The sequences of the cloned fragments were determined. The yield of VH fragments was relatively low, perhaps reflecting a lack of specificity in the PCR caused by degeneracy of SH1BACK. However, four of the 18 clones that were 25 sequenced contained DNA encoding the MN-14 VH region as previously sequenced. The other inserts did not derive from VH-encoding DNA.

In all, 20 full-length MN-14 VH clones were obtained.

i Five transition mutations were observed amongst the 30 sequences in the MN-14 VH region clones. These mutations are likely to have been introduced during amplification as a result of misincorporation by Taq polymerase.

EXAMPLE Analysis Of The Amino Acid Sequence Of The Heavy Chain Variable Region Of MN-14 The amino acid sequence of murine MN-14 heavy chain variable region, translated from the VIj DNA sequence, is T shown in Figures 1A and 1B(SEQ.ID.NOS.1 and Comparison of this sequence with sequences representing the murine Vsubgroups indicated that the variable heavy region of MN- 14 belongs to subgroup IIIB (see, Kabat et al. SEQUENCES OF PROTEINS OF IiMMUNOLOGICAL INTEREST, U.S. Government Printing Office, 1987).

The MN-14 CDR sequences are different from any of those reported by Kabat et al. (1987), supra.

Furthermore, the amino acids at four positions in the MN- 14 heavy chain VH framework regions are different from those in the framework regions of other subgroup IIIB VE sequences. These four substitutions (Ser 14, Thr 30, Ser 98 and Pro 108) have been observed in other murine VH regions outside the IIIB VH subgroup, however, with the proline in Kabat position 108 being the most unusual.

Any unusual residues in the VH or VK may represent somatic mutations which proved advantageous to the binding of murine MN-14.

EXAMPLE 6 ScDNA Synthesis And Amplification Of DNA Encoding The MN-14 VK c DNA encoding the kappa light chain of MN-14 was cloned in much the same fashion as the cDNA encoding the variable region of the heavy chain, as described above.

Several primers were used to prime reverse transcriptase 25 for synthesis of the first strand of the kappa chain S° o cDNA. The sequence of one primer, CK2FOR, was derived from the sequence of the 5' end of the constant region of kappa light chain genes The sequences of two other primers, VKIFOR and VK3FOR, were based on the 30 sequence of the 3' end of the variable region of kappa light chain genes The first strand DNA product was amplified by PCR using a number of primer pairs. Synthesis in one direction was primed by the primers used to make the first strand. Polymerization in the other, "backward," direction initiated from a series of kappa light chainspecific primers.which had sequences based on either the sequence at the 5' end of the VK region, VK1BACK, 27 VK2BACK, VK3BACK, VK4BACK and VK8BACK, or the sequence encoding the last four amino acids of the signal peptide and the first four amino acids of the variable region, VK6BACK and VK7BACK. In addition, cDNA primed by CK2FOR also was amplified using VK1FOR and VK8BACK.

The amplification products were analyzed by gel electrophoresis in the manner described in the Examples above. The products from the reactions primed by VK1BAC-IjVK3BACK, VK5BACK, VK7BACK and VK8BACK gave rise to the expected 350 bp band.

EXAMPLE 7 Cloning and Sequencing the MN-14 Kappa Light Chain Variable Region Obtained by PCR Selected PCR products were cloned into M13mpl8 and 15 19 using the restriction sites included in the amplification primers in a manner similar to that described for VH in Example 4 above. Nucleotide sequencing revealed that most inserts were not VKrelated. This is not uncommon when attempting to clone S 20 VK cDNAs and it appears to be more difficult to design VK-specific primers than VH specific primers.

From the VK1FOR/VK8BACK combination, a VK cDNA insert was obtained, but this did not yield a functional VK due to a frameshift within the cDNA encoding CDRu and absence 25 of the invariant Cys at position 23. This VK cDNA has 0. been isolated from other hybridoma cells and it is derived from the Sp2/0 fusion partner. CK2FOR/VK1BACK product yielded a further four different aberrant VK cDNA inserts, in this case lacking the conserved residues of framework 4. A fifth VK insert obtained using this primer pair was that of a functional VK with the exception of a frameshift at the 3'end of VK1BACK, a phenomenon apparently due to mismatch-induced slippage of the primer. This problem may be avoided by the use of-- VK8BACK which does not extend as far into the VK gene.

However, analysis of further clones from VK1FOR/VK8BACK product did not yield the.desired insert.

28 In order to amplify preferentially the putative VK, VK3FOR was designed from its genuine fourth framework sequence, and synthesized as an alternative to VK1FOR.

This strategy proved successful when amplification of VK3FOR-primed cDNA with VK3FOR and VK8BACK yielded 4 clones containing the desired VK.

The DNA and amino acid sequence of the murine MN-14 kappa light chain variable region is set out in Figs.2A&2B (SEQUENCE ID. NOS. 3 and This MN-14 VK can be placed in Kabat's VK subgroup V. Only 3 residues in the MN-14 VK framework regions (Met 10, Val 66 and Thr 76) do not appear in other members of this subgroup. Met 10 and Val 66 are the most unusual of the three. They are not found in any murine VK listed in Kabat. None of the MN-14 VK CDRs are previosuly-reported sequences in Kabat.

EXAMPLE 8 Graftina of the MN-14 VH and VK CDRs into Variable Regions of Human Antibody 8.1. Construction of chomeric anitbody expression vectors In the production of a chimeric antibody consisting of murine variable regions and human constant regions, testing alongside the parent mouse antibody served to check that the correct variable region cDNAs have been isolated. A successful chimeric antibody also acts as a useful control when assessing the binding of humanized versions. The scheme used in cloning the variable regions for expression is described by Orlandi et al.

(1989) supra, and is illustrated in Figures 3 and 4.

30 VH DNA was amplified from the M13 clone MNVH41 using the PCR with oligonucleotides VH1BACK and VH1FOR. The PstI and BstEII restriction sites in the primers allowed the VH to be inserted into M13VHPCR1 in the correct context for expression. At this point, the internal BamHI restriction site of the VH was removed by site directed mutagenesis. The reaction product, which encompassed the entire HindIII-BamHI fragment of 29 M13VHPCR1 was cloned into pSVgpt and the VH sequence confirmed.

The human IgG 1 constant region gene, published by Takahashi et al., Cell, 29: 671-679 (1962) above then was added to the construct as a BamHI fragment, which yielded the vector referred to as pSVgptMN14MuVHHuIgG1.

VK DNA was similarly obtained from the M13 clone MNVK154 by PCR amplification with the primers VKSBACK and VK3FOR and the PvuII, BglII-digested-product cloned into 0 M13VKPCR, whereupon the sequence of the variable region was checked. The HindIII-BamHI fragment was excised from RF DNA and transferred to the plasmid pSVhyg. The Sconstruct already contains a human kappa constant region gene as described in Hieter et al., Cell, 22: 197-207 (1980). The final vector thus obtained was designated pSVhygMN14MuVKHuCK.

IE' 8.2 Expression and testin of the hbrid antibod *The HindIII-BamHI fragment of M13KLHuVHAIGA was 20 inserted into a plasmid pSVgpt to yield the expression vector pSVgptKLHuAIGAHuIgG1. Similarly, the HindIII- SBamHI fragment of Ml3HuVK was inserted into the plasmid p.Vhyg to yield the expression vector pSVhygMN14REIHuVKHuCK. About 5g pSVgptMN14MuVHHuIgG 1 and 10g pSVhygMN14MuVKHuCK DNAs were linearized with 25 Pvul and transferred into about 107 subconfluent myeloma cells by electroporation conventionally using a BioRad Model 165BRI160 Gene Pulser Electroporator with a single pulse of 170 V, 960 gF. Cells were selected for the expression of the gpt gene in 2 4-well plates by addition of mycophenolic acid and xanthine to the DMEM FCS growth medium.

Wells which contained colonies of surviving cells were identified. The supernatant medium was removed from these wells and assayed for human antibody. Colonies that secreted antibodies were expanded to give 0.5 L of conditioned medium for isolation of larger amounts of antibody.

Antibody was purified conventionally from the medium by protein-A agarose affinity chromatography, initially.

The purified antibody was characterized further with reference to native MN-14 antibody, human antibodies and other controls.

The antibody was also characterized by its reaction profile in a MN-14 blocking assay, which provided an informative comparison of CEA binding affinities of the hybrid antihod-ies with CEA binding by the MN-14 murine antibody which served as a positive control.

8.3 Humanization of the MN-14 antibody The human NEWM VH, KOL VH and REI VK frameworks were chosen as the basis for reshaping the antibody, as they are likely to be tolerated in humans. Alignments of the MN-14 VH (SEQ. ID NO. 2) and VK (SEQ. ID NO. 4) with these human variable regions are shown in Figures 5A and (SEQ. ID. NOS.5-7).

A. NEWM based humanization.

The starting points for the introduction of MN-14 20 CDRs are DNAs encoding the required FRs and irrelevant CDRs. These template variable regions are in a form compatable with' the expression vectors used, that is, within HindIII-BamHI fragments, that also include promotor regions, signal peptide and intron DNA (Figures 25 3 and For the NEWM VH version, the template is M13VHPCR1 (Orlandi et al, above, and section 8.3 below).

A derivative of this template, containing KOL FRs and irrelevant CDRs, was used to generate the KOL coding region. A derivative of M13VKPCR1 (Orlandi etal. above) .i 30 was used in the creation of the HuVK vector. The resulting vectors were termed M13NMHuVH, M13KLHuVH and M13HuVK. The HindIII-BamHI fragments containing the humanized MN-14 variable region DNAs were transferred from these M13 vectors to the expression vectors essentially as described for the construction of the chimeric MN-14 expression vectors in Example 8.1.

The NEWM FR is described in Poljak et al.

Biochemistry 16: 3412-20 (1977). Construction of a hMN- 14 with an affinity for CEA comparable to that of its murine counterpart was achieved in a stepwise approach.

Production of the chimeric antibody provided a useful control when assessing the binding of the humanized versions. The human NEWM VH and REI VK frameworks were initially chosen as the basis for reshaping the antibody as they are known to be tolerated in man. MN-14 residues Phe27, Asp28 and Thr30 were retained because, although not part of the Kaba-t-s hypervariable region, CDR1 residues 31-35, those amino acids are part of the CDR1 structural loop (Chothia et al., J. Mol. Biol. 176: 901- 917 (1987)). In addition, the following residues were also selected for incorporation into the humanized VH for the following reasons: Ala24, this residue contacts CDR1; Arg71, the side chain of this residue pokes through the center of the domain to interact with CDRs 1 and 2; substitution of the smaller Val may alter the conformation of these CDR loops; and, Ser94, the majority II: of antibodies have Arg in this position where it is thought to interact with an Asp residue on CDR3, and the inclusion of the Arg of NEWM could create an unwanted interaction with the murine CDR. Other changes were made to this version (NMHuVH) in three areas corresponding to regions which had been proved important in other reshaped 25 molecules. These changes were: Gln77Phe78Ser79 to ThrLeuTyr (NMHuVhHTLY,

SEQ.

ID NO. 9) (ii) Ser82Thr83Ala84Ala85 to LysArgSerGlu (NMHuVhHKRSE, SEQ. ID NO. 30 (iii) Arg66Val67Thr68Met69Leu70 to LysPhelleValSer (NMHuVhKFIVS, SEQ. ID NO. 11) The alignment of the different versions of the NEWM VH frameworks (SEQ. ID NOS. 5 and 8-11) with the MN-14 VH (SEQ. ID NO. 2) is shown inFigures 6A,6B&6C. Each of these versions has been paired with the same HuVK. The inclusion of either the TLY or KFIVS motifs gave about a two-fold improvement.

32 o *0 There are 2 differences from the NEWN framework sequences given in Kabat et. al (1987) above: S107 to T and L108 to T. Kabat lists residue 1 as PCA and residues and 6 as E or Q.

B. KOL based humanization.

In parallel to the use of NEWM VH, we have also reshaped the human KOL VH. KOL VH is described in Schmidt et al., Z. physiol. Chem, 364: 713-747 (1987). The FR used-for humanizing the antibody is as given in Kabat.

The original version of the KOL-based VH contained the MN-14 CDRs, as defined in terms-of residue variability (Kabat et al., above) and three additional murine residues. As with the NEWM VH, two of these substitutions were made because the actual peptide CDR1 structural loop, which extends from the 3-sheet framework, consists of residues 25 to 32 (Chothia et al.

1987, above). Changes at positions 28 and 30 allowed this loop to be transplanted as a whole from the murine antibody. MN-14 residue 94 was included because the Arg residue of the KOL VH is involved in a salt bridge with AspllO and, like with the NEWM VH, it was felt that retention of Arg 94 might perturb the MN-14 CDR3 structure. The side-chain of residue 94 may also interact with residues of CDR1. Other changes made to this basis MN-14 KOLHuVH (SEQ. ID NO. 12) were as follows: Ser24 to Ala24 and Val48Ala49 to IleGly (KLHuVhAIG, SEQ. ID NO. 13).

(ii) Ser24 to Ala24, Val48Ala49 to IleGly and Ser74 to Ala (KLHuVhAIGA, SEQ. ID NO. 14).

(iii) Ser 24 to Ala24, VA148Ala49 to IleGly, Ser74 to Ala and Phe79 to Tyr (KLHuVhAIGAY, SEQ. ID NO.

Mutation rationale Ala24 The loop of CDR1 is anchored by the penetration of the side chain of residue 29 into the framework. Residue 24 is one of those with which it interacts (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)) Ile48Gly49 Although both these residues are adjacent to the CDR2 hypervariable region they are far removed from the actual structural loop. Both residues are completely buried (Padlan, Mol. Inmunolog. 28: 489- 498, 1991) and it was considered possible that these would effect binding via their packing interaction.

Ala74 This residue is part of the fourthloopf.ound at the VH antigen-binding surface and its side chain is almost completely exposed to solvent (Padlan, 1991 above). Direct interaction of this residue with antigen could be envisaged.

Tyr79 Like residue 74, this residue is close to the antigen-binding site and could effect binding.

The alignment of the different versions of KOL VH frameworks (SEQ. ID NOS. 7 and 12-15) with NEWM based versions (SEQ. ID NOS. 5 and 8-11) and the murine MN14 (SEQ. ID NO. 2) is shown in Figures 6A, 6B and 6C.

S: 20 The DNA sequences and translation products of MN14HuVH and MN14HuVL are shown in Figures 7 and 8, Orespectively (SEQ. ID NOS. 16-19, respectively).

.The humanized KLHuVH ,yarients, .such as antibody KLHuVHAIGA/HuVK, were purified and tested in a blocking assay carried out as follows. Antibodies were added at 9the indicated concentrations together with HRP-labeled S" MN-14 to a final volume of 0.1 ml. Following 30 mins. of incubation at 37C, and washing to remove unbound antibodies, the relative affinities of the antibodies 30 were determined from the remaining bound peroxidase activity. A shown by the assay data of Figure 9, the activity of the "reshaped" CDR-engrafted on FR) humanized antibody was similar to that of chimeric and murine positive controls.

Results obtained using supernatant fluids from cells secreting KLHuVHAIG/HuVK and KLHuVHAIGAY/HuVK antibodies suggests that these have blocking activities that are RA/ similar to that of the KLHuVHAIGA/HuVK antibody.

34 C. REI based humanization REI VL is described in Epp et al., Eur. J. Biochem., 513-24 (1974). No change4s were made in REI framework to improve binding. The changes from the sequences given in Kabat et al. (1987) are: M4 to L; T39 to K: Y71 to F; L104 to V; Q105 to E: and T107 to K.

These changes preexisted in the template used when grafting the MN-14 CDRs and were not made specifically to improve binding. The M4 to L__change incorporates a restriction site but the remaining differences eliminate unusual residues in the REI framework. A similar framework has been referred to as a consensus of huma kappa subgroup I by Foote et al., J. Mol. Biol. 224: 487- (1992) 15 EXAMPLE 9 S: Expression of MN-14 CDR-grafted Humanized Antibodies Cells that were stably transformed for expression of the MN-14 CDR-grafted human antibodies were selected in the manner described above and cloned out to establish individual producer lines. Each of the lines was assayed Sto determine production of the correct antibody and to assess the efficiency of production. The antibody class was determined and the anti-CEA binding affinity assessed.

25 The best producers were further characterized for the overall amount of the antibody produced and, for the best of these, sequences were obtained from the mRNA to insure that the mutation has not occurred in the antibody genes during transfection, integration, propagation or selection.

EXAMPLE Purification of MN-14 CDR-grafted Humanized Antibodies Expressed in Cell Culture The best producer lines of the MN-14 CDR-grafted human antibody were cultured, the growth medium collected and filtered through a 0.2 micron membrane. The antibody was then purified by protein A chromatography followed by other conventional purification steps such as ion 35 exchange and size exclusion chromotography. The cells were pelleted and from the supernatant by conventional centrifugation. The antibody was purified from the supernatant fluid as described above.

EXAMPLE 11 USES OF HUMANIZED MN-14 MONOCLONAL ANTIBODY IN

DIAGNOSES

A. Animal studies The biodistribution of labeled humanized MN-14 IgG in nude mice bearing human colon cancer was determined.

For radiolocalization studies, at 4-5 weeks female athymic mice (nu/nu, Harlan, Indianapolis, IN) were given S: s.c. 0.2 ml of a 10% suspension of LS174T human colon adenocarcinoma prepared from a xenograft serially 15 propagated in an athymic mouse (Sharkey et al., Cancer Res., '50: 828-34 (1990)). After waiting 2 weeks for tumor development, the mice were injected i.v. with 20 jiCi (about 2 4g) of 31 -labelled humanized MN-14 monoclonal antibodies. Groups of 4-5 mice were sacrificed at intervals thereafter, and radioactivity localized in tissues according to Sharkey et al., above. The data of Table 2 show the injected dose/g tissue and tumor:nontumor ratios.

The results show excellent tumor accretion of the 25 antibody, with maximum accretion occuring within 2 days.

Blood clearance of the hMN-14 antibody was more rapid than the parental mMN-14 antibody. In addition, there was higher uptake of hMN-14 by the spleen than there was of mMN-14, reflecting the fact that the former antibody is "foreign" to the mouse. Tumor;nontumor ratios were excellent. These results demonstrate that the inventive hMN-14 mAb is capable of targeting CEA-producing tumors.

*i.

S

Si. S S S S S S S S S. S S S S S CS S S S 5*5 55 TABLE 2 1 Percent Injected Dose Per Gram Tissue MN 4 to 5 animals) I Time Post-injection 1 3 1 -hMN-14 IgG Tissue 4 hour 1 iday 2 days 5 days 7 days 14 ldays LS 174T 11.8 ±2.9 J 18.1 14.9 J 32.6 17.2 j 30.2 ±13.4 10.6 15.2 11.6 ±5.6 weight J 0.31 ±0.07 J 03±0.2 J 0.27 0.08 0.31 ±0.9 J 0.3 0.2 f 0.40± 0.7 Liver 10.8 ±2.1 6.0 3.3 -2.8 0.5 0.8 0.4 0.5 0.7 0.08 0.05 Spleen 17.0 ±4.8 10.5 ±8.8 4.9 0.4 1.3 0.6 0.6 0.8 0.14 0.09 Kidney 7.0 0.8 3.1 ±0.7 2.3 0.9 0.9 0.4 0.4 0.6 0.07 0.04 Lungs 8.7 0.4 3.7 ±1.4 3.7 1.4 1.4 0.6 0.6 1.0 0.11 0.06 Blood 15.4 6.6 5.5 ±4.7 6.8 4.1 2.3 1.2 0.9 1.8 0.14 0.13 Tumor (LS1474T)INontumor Ratios (N 4 to 5 animals) Time Post-Injection 131 1-hMN- 14 lgG Tissue 4 hour 1 day 2 days 5 days 7 days 14 days Liver 1.1 0.3 5.0 5.0 11. 1 ±4.7 39.5 -t 7.5 24.6 ±1 4.8 160 :1 28 Spleen 0.7 0.3 4.2 4.6 6.6 ±3.4 24.7 ±7.8 15.8 4.4 91 21 Kidney 1.7 0.5 6.3 ±3.5 13.3 ±3.6 36.1 4.0 39.4 ±12.9 173 41 Lungs 1.4 0.4 4.2 ±2.2 8.2 ±2.1 22.3 2.1 25.4 ±7.3 112 Blood 0.9 0.5 3.7 ±0.9 5.4 ±1.4 14.2 2.9 26.0 ±12.9 1111 37 B. Clinical Studies with 31 I-labeled Humanized MN-14 IgG Patients were entered into an Institutional Review Board-approved protocol at the Center for Molecular Medicine and Immunology, Newark, NJ for a pilot investigation of the targeting and pharmacokinetic behavior of the humanized MN-14 IgG. In the case the results of which are shown in Figure 12, the male patient had colorectal cancer that had metastasized to the liver.

He was injected i.v. with "'I-hMN-14 IgG (8 mCi, 0.6 mg antibody) and images were taken over a six day period.

The patient was subsequently injected with an identical dose of mMN-14 IgG. The images shown in Figure 12 shaw the anterior abdominal view about 140 h after each o 1 injection. The images are adjusted to exactly the same 15 intensity so that they are directly comparable. The results indicate that the humanized antibody is taken up by the CEA-producing tumor as well as the parental murine antibody. These experiments establish the practical utility of diagnosing human CEA-producing colon cancers with the inventive humanized MN-14 mAb.

This application is divided from our copending application 37196/95 (Serial No.689331) and the entire disclosure in the complete specification and claims of that application is by this cross-reference incorporated into the present specification.

SEQUENCE LXSTING GE2NEPAL IXTF0RNATI0N.: Wi APPLICANVT: )(ANsEN, Hans J.

ARMouR, Kathryn L.

(ii) T17LE OF INVENTION: CDR-GRAFTED TYPE III ANTZ-CEA H{tmANIZ;: MOUSE MONOCLONAL

ANTIBODIES

(iii) NUMBER OF SEQUENCES: 58 (jv) COR.RESPONDENCE

ADDRESS:

ADDRESSEE: Foley LaZ-dner STREET; 3000 K Street, Suite Soo CITY: Washington STATE: D.C.

COU14TRY: USA ZIP: 20007-53.0.

COMUTR READABLE FORM:- ME~DIUM TYPE: Floppy disk COMPUTERl: IBM PC compatible OPERATlNG SYSTEM:

PC-DOS/MS-DOS

SOMARwE. Patentln Release 41.0, Version #*1.30 (vi) CURRM- APPLICATION

DATA:

APPLICATION NUMBER: US 08/3l8,LS7 So:* FILING DATE: OS-OCT-1-994 CLASSIFICATION: 424 (viii)ATTORNEYAGNT

INFORMATION:

NAME: SAXE, Bernhard fl.

REGISTRATION NUMBER: 28,66S REFERENCE/DOCKET NUMBER: 18733/464 5(ix) TELCONIv=ICATION NOP4TIN S TrELEPHONE: (202)672-5300 TELEFAX: (202)672-5399 TELEX: 904136 INFORMATIONr FOR SEQ ID No:j: SEQUNCE CgARACTERISTICS.

L-EGTH: 357 base pairs TYPE: nucleic acid 0(Cc) STRANlErNqESS: double TOPOLOGY: linear (ii) MOE- TYPE: DNA (genomic) (ix) FEATURE: NAMEY:

CDS

LOCATION: 1. .357 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: GAG GTG AAG CTT CTC GAG TCT GGA GGT GOC CTC GTO3 CAC; TCT GGA GGA 48 Glu val Lys Le" Leu Glu Ser Gly Gly Gly Leu Val Gizi Ser Gly Gly 1. 5 10 1S TCC CTG AAA CTC TCC TGT GCA GCC TCA GGA TTC GAT =T ACT ACA TAT Ser Leu Lys Leu Ser Cys Ala Ala S.r Gly Phe Asp ?he Thr Thz- Tyr 25 30 TGG ATG ACT TGG CTC CGG CAC GCT CCA COG AAA GGC CTrA GAA T-G ArT Trp Met Ser Trp Val. Arg Gin Ala Pro Gly Lys Gly Leu Giu Trp Ile 40 45 GGA GAA AT? CAT CCA GAT AGC AGT ACG ATT MAC TAT GCG CCG TCT CTA Giy Gu Ile His Pro Asp Ser Ser Thr Ile Asri Tyr Ala Pro Ser Leu 55 60 AAG, GAT AAA TTC ATC GTC TCC AGA CAC AAC GCC AAA AAT ACG CTG TAC Lys Asp Lys Phie Ile Val Ser Arg Asp Asn Ala Lys Asn Thr Lau Tyr 70 7S

SO

CTG CMA ATC AGC AAA GTG AGA TCT GAG GAC ACA GCC CTT TAT TAC TGT Leu Gin Met Ser Lys Val Axg Ser Giu Asp Thr Ala Lau Tyr Tyr Cys as 90 GCA AGC CT? TAC TTC CCC TTC CCC TGG TT? OCT TAT TGG GOqC CAA GG Ala Ser Lau Tyr Phe Gly Phe Pro Trp Phe Ala Ty-r Trp Gly Gin Gly 100 105 110 ACT CCG GTC ACT OTC TCT GCA Thr Pro Val Thr Val Ser Ala 115 96 144 192 240 288 336 357 INFORMATION FOR SEQ ID NO:2: SEQUENCE

CRARCA=R~ISTICS:

LENGTH: 119 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECUiLE TYPp,: protein (xi) SEQUTENCE DESCRIPTION: SEQ 11) Glu Val Lys Leu Leu Giu Ser Gly Gly Gly I 1 S 10 Ser Leu Lys Leu Ser Cys Ala Ala Ser ClyP 25 Trp, Met Ser Trp Val Arg Gin Ala Pro Gly L 35 40 Gly Giu Ile His Pro Asp Ser Ser Thr Ile A 55 Lys Asp Lys Phe Ile Val Se: Arg Asp Asn A 70 Leu Gin Met Ser Lys Val Arg Ser Giu Asp TI 90 Ala Ser Leu Tyr ?he Gly Phe Pro Trp Phe A 100 l0s Thr Pro Val Thr Val Se: Ala INFORMATION FOR SEQ, ID NO:3: Wi SEQUENCE

CHARACTERISTICS:

40: 2: ~et Val he Amp ya Giy an Tyr la Lya 75 ir Ala la Tyr Gn Se: ly Gly is Phe Thr Thr Tyr Lu Gu Trp Ile 4S Ala Pro Sr Leu Aeqn Thz Leu Tyr Leu Tyr Tyr Cys Trp Gly Gn ly 12.0 LENGTH: 31.8 base pairs TYPE: nuclaic acid STRANflEDNESS: double TOPOLOGY: linear (iMOLECULZ TYPE: DNjA (genomic) (iX) FEATURE: ZqAME/Kvy:

CDS

LOCATIONq: 1. .31.8 (xi) SEQUZNCZ DESCRIPTION: SEQ ID NO;:: GAA ATT CAG CTG ACC CAG TCT CAC AA.A ATC ATO TCC ACA TCA GTO GGA 4 Glu Ile Gin Leu Thr Gin Ser His Lys Mec Met Ser Th Ser Val Gly 1.20 125 130 135 CGAC AGG GTC ACC ATC ACC TOC AAO GCc AGT CAG CAT GTG GGTr AcT TCT 96 Asp Arg Val Ser Ie Thr Cys Lys Ala Ser Gin Asp Val Gly Th~r Ser 140 145

ISO

GTA 0CC TGG TAT CAA cAG AGA CCA GGA CAA TCT CCT AAA CTA CTG ATT 144 Val Ala Trp Tyr cin ain Arg Pro Gly Gin Ser Pro Lys Lau Leu Ile Iss 160 165 TAC TOG ACA TCC ACC CG43 CAC ACT OA GTC CCT CAT CGC TTC ACA GGC 192 Tyr Tzp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 2.70 175 18O AGT GTG TCT GOO ACA GAT TTC ACT CTC ACC Arr ACC AAT GTG CAG TCT 240 -Se: Val Ser Gly Thr Asp Pixe Thr Leu Thr le Thr Ann Val Gin Ser Las 190 195 .*GAA GAC TTG OCA OAT TAT TTC TGT CAG CAA TAT AGC CTC TAT CGG TCG, 288 Glu Asp Leu Ala Asp Tyr Phe Cys Gin Gin Tyr Ser Leu Tyr PArg Se: *.*200 20S 21.021 TT1C G GAGOC ACC AAA, CTG GAG ATC AAA 319 Phe Gly Gly Gly Thr Lys Leu 01u Ile Ly's 220 22S IN~oRmxTioN FOR sEQ rr o4 .*SEQUENCE

CHARACTERISTICS:

LENGTH; 106 amino acids TYPE: amino acid TOPOOGY linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Giu le Gin Leu Thr Gin Ser His Lys Met Mt~e Ser Thr Ser Val Gly 1 5 10 i Asp Arg Val Ser le Thr Cys Lys Ala Ser Gin Asp Val Gly Thr Ser 25 Val Ala Trp Tyr Gin Gin Arg Pro Gly Gin Ser Pro Lys Leu Lau le 3S 40 Tyr Trp Th~r Se: Thr Arg His Th: Gly Val Pro Asp Arg Phe Thr Gly so S 41 Ser Vri Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gin Ser 70 75 so Clu Asp Leu Ala Asp Tyr Phe Cys Gin Gin Tyr Ser Leu Tyr Arg Ser 90 Phe Gly Giy Gly Thr Lys Leu Ciu Ile Lys 100 lOs INFORMATIoN FOR SEQ ID NOS: SEQUENCE

CHARACTERISTICS:

LENGTH: 115 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Gin Val Gin Leu Gin Glu Ser Gily Pro Gly Leu Val Arg Pro Ser Gin 1 S 10 iS Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Th Phe Ser Asn Asp 25 Tyr Tyr Thr Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Val Phe Tyr His Gly Tfri Ser Asp Asp Thr Thr Pro Ser Leu SS 60 Arg Ser Arq Val Thr Met Leu Val Asp Thr Ser Lys Asn Gin Phe Ser 70 75 Lau Arg Leu Ser Ser Val Thr Ala Ala Asp Thz Ala Val Tyr Tyr Cys 90 Ala Arg Asn Leu Ile Ala Gly Cys Trp Ile Asp Val Trp Gly Gin Gly 100 105 130 Thr Thr Val Thr Vai Ser Ser 115 INFORAIITION FOR SEQ inD NO:6: (W E SEENCE CARACTERISTICS: LENGTH: 107 amino acids TYPE; amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Asp lie Gin Leu Thr Gix Ser Pro Ser Ser Leu Ser Aa Ser Vai Giy 1 5 10 ASP Arg Val Thr le Thr Cya Gir Leu A-sn Trp Tyr Gin Gin Lys Prc 40 TYr Glu Ala Ser Asn Le~u Gin Ala 55 Ser Gly Ser Giy Thr Asp Phe Thr 70' Glu ASP Ile Ala Thr Tyr Tyr Cys 8S Thr Phe Gly Gin Gly Thr Lys Val 100 INFORM'ATIONJ FOR SEQ ID NO:7: Wi SEQUNC

CHARACTERISTICS:

LENGTH: 1.26 amino acids TYPE: amino acid

STRANDEDNESS.

TOPOLOGY: linear (ii) MOLECULE~ TYPE: protein LAla Ser Giln Asp Ile le Lys 25 Gly Lys Ala Pro Lys Leu. Leu Zie Cly Val Pro Ser Arg Phe Ser Gly Phe Thr le Ser Ser Leu. Gin Pz-o 75 so Gin Gin Tyr Gin Ser Leu Pro r 90 Giu Ile Lys 1.05 (Xi) SEQUIENCE DESCRIPTION: SEQ ID NO:7: Giu Val Gin Leu Val Glu Ser Gly Gly Gly Val 1. S 2.0 Ser Leu Arg Leu Ser Cys Ser Ser Ser Gly Phe 20 25 Ala Mec Tyr Trp Val Arg Gin Ala Pro Gly Lys 35 40 Ala Ile Ile Trp Asp Asp Gly Ser Asp din His so 55 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asm Ser 65 70 75 Leu Gin Met Asp Ser Leiu Arg Pro dlii Asp Thr 85 90 Ala Arg Asp Gly Gly Hijs Gly Phe Cys Ser ger 100 10s Pro Asp Tyr Trp Gly Gin Gly Thr Pro Val Thr 2.15 2.20 rN-FORWATIN FOR SEQ ID NC:a: Wi SEQUENCE

CRARACTERISTICS:

LENGTH: 11.9 amino acids TYPE: arnino acid

STRANDEDNES.

TOPOLOGY: linear (ii) MOLECULE TYPE- protein Val Ile Gly Tyr Lys Ala Val l PoGly Aru Ple Ser er Tyr Leu Gu TZp Val Ala Asp Ser Val Asn Thr Leu Phe so Val Tyr Phe Cys Ser Cys Phe Gly Ila.

Ser Ser 125 43 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Gin Val Gin Leu Gin Giu Ser Gly Pro GI? Leu Val Arg Pro Ser Gin 1 5 10 is Thr Leu Ser Leu Thr Cys Thr Ala Ser cly Pbe Asp Phe Th-r Thr Tyr 25 Trp Met Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp Ile 40 Gly Glu lie His Pro Asp Ser Ser Thr Ile A-sn Tyr Ala Pro Ser Leu so 55 Lys Asp Arg Val Thr Met Leu Azg Asp Thr Ser Lys Ann Gin Phe Ser 70 75 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 90 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gin Gly 100 lOS 110 Thr Thr Val Thr Val Ser Ser 115 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear *ii MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gin s 10 1 oI Thz Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asp Phe Thr Thr Tyr 25 Trp Met Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Glu Trp Ile 40 Gly Glu le His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 55 Lye Asp Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Thr Leu Tyr 70 75 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Th Ala Val Tyr Tyr Cys 0 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Giy Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 44 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ 1D Gin Val Gin Leu Gin Giu Ser Gly Pro Gly Leu Val. Azg Pro Ser Gin 1 S 10 is Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asp Phe Tht Thr Tyr 25 Trp Met Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Lau Giu Trp Ile 3S40 Giy Giu Ile His Pro Asp Ser Ser Thr Ilie Asn Tyr Ala Pro Ser Leu so 55 so Lys Asp Arg Val. Tbhr Met Leu Ag Asp Thr Se= Lys Asn Gin. ?he Ser 6S70 75 8 Leu Arg Leu Ser Lys Val Arg Ser Glu Asp Thx Ala Val Tyr Tyr Cys as 90 9S a..*Ala Ser Leu Tyr Phe dly Phe Pro Trp Phe Ala Tyr Trp Giy Gin Gly :100 105 110 Thr Thr Val Thr Vali Ser Ser INFORMATION FOR SEQ ID NO;il: Wi SEQUENCE CHARACTERISTICS: LRNGTH: 119 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION; SEQ ID NO:il: Gin Val Gin. Leu Gin Gil± Ser Gly Pro Gly Leu. Val Arg Pro Ser Gin 10 is Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asp Phe Thr Thr Tyr Is Trp Mez Ser Trp Val Arg Gin Pro Pro Gly Arg Gly Leu Giu Trp Ile 40 Gly Giu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu so 55 Lys Asp Lys Phe Tie Val Ser Arg Asp Thr Ser Lys Asn Gin Phe Ser 70 75 so Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys as 90 Ala Ser Leu Tyr Pbe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gin Gly 100 10s 110 Thr Thr Val Thr Val Ser Ser 115 INFORMATION FOR SEQ ID NO:12: i) SEQUENCE CHA.RACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid

STP.ANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRPTION: SEQ ID NO:12: Glu Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg S 10 iS Ser Leu Azg Leu Ser Cys Ser Ser Ser Gly Phe Asp Phe Thz Thr Tyr 75 Trp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 3S 40 Ala Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu fh Lys Asp AIl he Thr le Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 Leu Gin Met Asp Ser Leu Arg Pro Giu Asp Thr Gly Val Tyr Phe Cys 90 Ala Ser Leu Tyr Phe Gly Phe Pro Tzp Phe Ala Tyr Trp Gly Gin Gly 100 105 110 Thr Pro Val Thr Val Ser Ser 115 *feel: INFORMATION FOR SZQ ID NO:13: SEQUENCE CHARACTERISTICS: 0.009: LENGTH: 12.9 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: Linear (ii) MOLECULE TYPE: protein *999 .9 9* 9 .9 9 99 99 9 9 9..

9* 9* 9 99*999 9 9. 9 9 9* (Xi) SEQUENCE DESCRIPIOaJ. SEQ ID NZO:13: Glu Val Gin Leu Val. Glu Ser Gly Gly GlY Val. Val Gin Pro Cly Arg 1 5 10 is Ser Leti Arq Lau Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr'Thr ryr 2S Trp met Ser:

T

rp Val A-g Gin Ala Pro Gly Lys GyLuG~ r i 40 45 euGuTr l GlyfGlu tIle His Pro Asp Ser Ser 'Thr Ile Asn Tyr A1a Pro Ser Leu SS go Lys AS A PeTrLeSrAgApAnSrLsAs h e h 7 0 75 so Leu Gin Met Asp 5cr Leu Arg Pro Glu Asp Thr GlY Val Tyr ?he Cys as50 Ala Ser ILau Tyr Phe Gly Phe Pro Tzm Phe Ala TrTrp Giy Gin Gly 100 105 If. 110 Thr Pro Val Thr Val Ser Ser 3.15 INFORZ-IATZON FOP- SEQ ID NO-.14: i)SEQUENCE

CHARACTERISTICS:

LENGTH: 119 amino acids TYPE: amino acid STRANflEDNESS: TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GiU Val Gin Leu Val Giu Ser Gly Gly Gly Val Val. Gin Pro Gly Arg 1I 0i Ser Leu Arg Leu Scr Cys Ser A .la Sar Gly Phe Asp Phe Thrryhr Tyr 20 25 Trp Met Se ]Try Val Arg Gin Ala Pro cGly LYS Gly Leu Giu Trp Ile 40 G Clu le His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu s Lys Asp7Arg Phe Th Ile Ser Arg Asp Aaen Ala Lys Asin Thr Leu. Phe, 5 70 75 so Leu Gin Met Asp Ser Leu Arg Pro Giu Asp Thr Gly Val Tvyr Phe Cys 90 Ala Serr eu Tyr Phe Gly Phe Pro Trp Phe Ala T Trp Gly Gin Gly IL 100 105 I 110 Thr Pro Val Thr Val Ser Ser ils INFORMATION FOR SEQ ID 47 ()SEQUENCE

CHARACTERISTICS;

LENGTH: 119 amino acids TYPE: amino acid

-STRAIDEDNESS:

TOPOLOCY: linear- (iMOLSCULE TYPE: protein -x)SEQUENCE DESCRIPTION: sEQ Inl No:1S_: Giu Val Gln Leau Val Giu Ser Gly Gly Cly Val Val Gln Pro Gly Arg 1 5 1.0 is Ser Lau Axrg Leu Ser Cys 5cr Ala Ser Gly Phe Asap Phe Thr Thr 77r 25 Trp Met Ser Trp Val Axg Gln Ala Pro Cly Lyn GlY LOU Giu Trp Ile 40 Giy Glu Ile His Pro Asp Ser 5cr Tbr Ile Agn Tyr Ala Pro Ser Leu so 55 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lyn Asn Thr Leu Tyr 6S 70 75 so Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Tbz Gly Val Tyr Phe Cys as 90 Ala Ser Leu Tyr Phe Gl Phe Pro Trp Phe Ala Tryr Try, Gly Gin Gly 100 10as 110 *Thr Pro Val Thr Val Ser Ser INFORM4ATION' FOR SEQ 11) NO: 16: 0, 0: i) SEQUENjCE CHARACTERpISTICS: LENGTH: 357 base pairs (B T,'YPE:ncli acid 0 STRANDEDNESS. double 0 TOPOLOGY: linear- (ii) MOLECU2 TYPE: DNA (genomic)

FEATURE:

NAME/lcEY:

CDS

.0 0(B] LOCATZON.: 1.-357 0 (xi) SEQUENCE DESCRIPTION't SEQ ID NO:iE: GAG GTCCA CMG GTG GAG AGC GGT GGA CC? CT? GTG CAA CCT GGC CGG 4 0 0 0 0Glu Val Gin Leu Val Glu Ser Gly Gly Cly Val Val Gin Pro Gly Arg 110 115 12(.

*TCC CTG COc CTO TCC TGC TCC TICG TCT GGC TIC CAT TTC ACC ACA TAT 96 Ser Leu Arg Leu Ser Cys Scr Ser Scr dly Phe As9p Phe Thr rhr Tyr 1.25 130 13S TCG ATG ACT TGG GTO AGA CAG OCA CC? GGA AAA GGT C'-r GAG TGG GTT 144 Trp I-et Ser Trp Val Axg Gin Ala Pro Cly Lys Gly Leu Giu Trp Val 140 1.45 150 48 GCA GAA ATT CAT CCA GAT AGC AGT AC; ATT AAC TAT GCG CCG TCT CTA A.a Glu II. His Pro Asp Ser Ser Thr lie Asn Tyr Ala Pro Ser Leu 155 160 165 170 AAG GAT AGA TTT ACA ATA TCG CGA GAC AAC AGC AAG AAC ACA TTG TTC 240 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 175 ISO 185 CTG CAA ATG GAC AGC CTG AGA CCC GAA GAC ACC GOG CTC TAT TT TGT 288 Leu Gin Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 190 195 200 GCA AGC CTT TAC TTC GGC TTC CCC TGG TTT GCT TAT TGG GGC CAA GGG 336 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gn Gly 205 210 215 ACC CCG GTC ACC GTC TCC TCA 37 Thr Pro Val Thr Val Sar Ser 220 225 INFORMATION FOR SEQ ID NO:17: SEQOM;CE

CHARACTERISTICS:

LENGTH: l1 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQ ME DESCRIPTION: SEQ ID NO:17: Gi Val Gn Leu Val Gu Ser Gly Gly Gly Val Val Gin Pro Gly Arg 0 1 5 1.0 SSer Leu Arg Lu Ser Cys Ser Ser Ser Gly Phe Asp Phe Thr Thr Tyr 0*020 25 *0 0. Trp Met Se: Trp Val Ag Gn Ala Pro Gly Lys Gly Leu Gu Trp Val 0 35 40 45 Ala Glu ie Hisi Pro Asp Ser Se: Thr Ile Asn Tyr Ala Pro Ser Leu o 55 Lys Asp Arg Phe Thr Ile Se: Arg Asp Asn Ser Iys Asn Thr Leu Phe 70 75 Leu Gin Met Asp Ser Leu Arg Pro Gu Asp Thr Gly Val Tyr Phe Cys 985 0 Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gin Gly 0 a 100 105 110 Thr Pro Val Thr Val Ser Se: 0 115 INFORMATIoN FOR SEQ ID NO:l8: SEQUENCE

CHARACTERISTICS.

LENGTH: 318 base pairs TYPE: nucleic acid STXNDEDNESS: double TOPOLOGY: linear (ii) MOLECLE TPE: DA (genomic) (ix) FEATURE: NAJIE/KEY: CD)S LOCATION: 1. .318 (Xi) SEQUENCE DSCRIPTION. SEQ ID NO:iB: GAC ATC CAC CTG ACC CAG AGC CCA AGC AGC CTG AGC GCC AGC GTG OGT 48 Asp Ile Gin Leu Thr Gin Ser Pro Ser Ser Leu Set Ala Set Val Gly 120 125 130 2-3S GAC AGA GTG ACC ATc ACC TCT AMG OCC AGT CAG GAT GTG GGT ACT TCT 56 Asp Arq Val Thr Ile Thr Cys Lys Ala Ser Gin As p Val Gly Thr Ser 140 1-45

ISO

GTA GCT TOO TAC CAG CAG AAG CCA GGT AAG GCT CCA AAG CTG CTG ATC 144 Val. Ala Trp Tyr G3.n Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu le 155 1.60 165 TAC TGO ACA TCC ACC COO CAC ACT GGOT GTG CCA AGC AGA TTC AGC GGT 192 Tyr Trp Thr Set Thr Arg His T~ir Gly Val Pro Ser Arg Phe Ser Gly 170 175 is0 AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC ATC AGC AGC CTC CAG CCA 240 Set Gly Set Gly Thr Asp Phe Thr Phe Th Ile Ser Ser Lau Gin Pro Bss 190 195 GAG GAC ATC GCC ACC TAC TAC TOC CAG CAA TAT AGC CTC TAT COG TCG 288 Glu Asp Ile Ala Thr Tyr Tyr Cys Gin Gin Tyr Ser Leu Tyr Arg Sez 200 20S 210 215 TT~C GGC CAA GGG ACC AAG GTG GXM ATC AAA 318 o.*Phe Gly Gin Gly Thr Lys Val C1u Ile Lys *220 22S feet so** INFORMATION FOR SEQ 1D NO:19.: 0 *0 SEQUENCE

CHARACTERISTICS:

LENGTH: 1.06 amino acids TYPE: amino aicid S TOPOLOGY: linear (ii) MOLECULE~ TYPE: prtein (xi) SEQUENCE DESCRIPTION: SEQ ID SAsp Ile Gin Leu Tb~r Gla Ser Pro Set Set Leu Ser Aia Set Val Gly l5 10 15 *fe*:Asp Arg Val. Thr Ile Thr Cys\ Lys Ala Ser Gin Asp V/al Gly Thr Ser **20 25 Val Ala ITrP Tyr Gin Gin Lys; Pro Gly Lys Aia Pro Lys Leu Leu Ile So:Tyr (Trp Thr SrT Arg HsTh11rjlGly Val Pro Ser Arg Phe Set Gly 0 *-50 55 60 Set Gly Ser Gly Thr Asp Phe Th-r Phe Thr Ile Ser Ser Leu Gin Pro 70 75 s Glu Asp Ile Ala Thr Tyr Tyr CysGln Gln Tyr- Set Leti Tyr Arg Set 90 Phe Gly Cin Gly Thr Lys Val Glu Ile Lys i00 105 (21 INFORMATION FOR SEQ ID NO. C)SEQUNC

CHARACTERISTICS:

LENGTH: 11 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLE CUL E TYPE: r~otejn (xi) SEQUENCE DESCRIPTION: SEQ ID LYS Ala Ser 01n Asp Val Gly Thr Ser Vfal Ala .1 5 INFORMATION FOR SEQ ID NO:21: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 7 amino acids TYPE: amino acid STRANDEDNzS.9s TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENrCE DESCRIPTION: SEQ ID NO:21: Trp Thr Ser Thr Arg His Thx 1 INOMTO FOR SEQ ID NO.22: W. SEUNE..R=RSI LENGTH: 8 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (iMOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ In No.:22.

Gin Gin Tyr Ser Leu Tyr Arg Ser INFORMATION FOR SEQ ID NO:23: (W SEQUECE

CHARACTERISTICS.

LENGTH: S amino acids TYPE: amino acid STRANDEDNEss: TOPOLOGY; linear (ii) MOLECUL~E TYPE: protein 51 SEQUENCE DESCRIPTION: SEQ ID XO:23: Thr Tyr Trp met Ser 1 INFORMATION FOR SEQ ID NO:24: W1 SEQUtJNC CHARACTrERISTICS: LENGTH: 1.7 amino acids TYPE: amnino acid

STANDEDNESS:

TO'POLOGY: linear (ii) MOLECUL~E TYPE: protein (xi) Glu 1 Asp SEQUENCE DESCRIPTION: SEQ ID NO:24: Ile IF±s Pro A-sp Ser Ser Thr Ile Asri Tyr Al~a Pro Ser Leu Lys 5 10 INFORMATION pOR SEQ ID X0:25: ()SEQUENCE~

CHARACTERISTICS:

LENGTH: 10 amino acids TYPE: amino acid STRAN4DEDNESS.

TOPOLOGY: linear (ii) M1OLECULE TYPE: protein (xi) 58QUENCE DESCRIPTION. SEQ ID lTOz2Sz Leu Tyr Phe Oly Phe Pro Trp Phe Ala Tyr 1. S INFORMATION FOR SEQ ID NO:26: Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 23 amino acids TYPE: amino acid STRAEDNEsS: TOPOLOGY: linear (ii) MOLECUfLE TYPE: protein (xi) Asp

I

SEQUENCE DESCRIPTION. SEQ ID NO:2g: Ile Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 5 10 is INFORMATION FOP. SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 4 OTHER INFORMATION: /note= "At site 4, Xaa Ser or Asp. (ix) FEATURE: NAME/KEY: Modified-site LOCATION: OTHER INFORMATION: /note= "At site 10, Xaa Gly or Val." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Gly Val Pro Xaa Arg Phe Ser Gly Ser Xaa Ser Gly Thr Asp Phe Thr 1 5 10 Phe Thr Ile Ser Ser Leu Gin Pro Glu Asp Ile Ala Thr Tyr Tyr Cys 20 25 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Phe Gly Gin Gly Thr Lys Val Glu lie Lys S* 1 5 INFORMATION FOR SEQ ID (i2SQUWCE CHARACER ISTIcS LENCT.H: 30 amino acids TYPE: armino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) M-OLECUjLE TYPE: protein SEQECE DESCRIPTION: SEQ II) N0:30: Glu Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg 1 5 10 1 Ser Leu Arg Leu Ser Cys Ser Ser Ser Gly ?he Asp Phe Thr 2s INFORM.ATIN FOR SEQ ID NO!31L: SEQtrMNCH CRARACTERIS TICS: LENGTH: 30 amino acids TYPE: amino acid

SRANDEVNESS:

TOPOLOGY: linear (iMOLECULE TYPE: protein (xi) SEQUENKCS DESCRIPTION: SEQ ID NO:3i: ~~Glu Val Gin Leu Val Glu Ser Gly Gly Gly ValVaGiPr lAz 000. Va 15Po Gl r Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr 25 INFORMATION FOR SEQ ID NO:32: ()SEQUENCE

CHARACTERISTICS:

LENGTH: 30 amino acids TYPE; amino acid

STR.ANP)EDNESS.-

TOPOLjOGY: linear *Goes:(ii) MOLECUL~E TYPE: protein ses:(xi) SEUNEDSRPIN SEQ ID NO:32: **Gi-n Val GIn Leu Gin Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gin 1 S 10 is Thr Leu Ser Leu Thr Cys Thr Ser Ser Gly Phe Asp Phe Thr 25 INFORMATION FOR SEQ ID NO:33: SEQUENCE

CHACTERISTICS:

LENGTH: 14 amino acids TYPE: amino acid t/ C) STRANDEDNESS: TOPOLOGY: linear (ii) MOLZCULE TYPE. protein (xi) SEQUENCE DESCR.IPTION; SEQ :D NO: 3 1 Trp Val Gln Ala Pro Cly Lys Gly Leu Glu Trp Val Ala 1 5 IrN'ORM-zoN FOR SEQ ID NO:34: iiSEQUENCE C8ARACTERISTICS:- LENGTH; 14 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQtXENR DESCRIPTION: SEQ ID NO:34: Trp Val Arg Gin, Ala Pro Cly Lys Gly Leu Ulu Trp Ile Ala 1 5 INFORKAT.ION FOR SEQ ID V.09 i) SEQUENCE

CHARACTERISTICS:

.9 LENGTH: 14 armino acids V. TYPE: amino acid .00

STRANDEDNES.

TOPOLOGY: linear (ii) MOZ.ECULE TYPE: protein Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:3S: Trp Val Axg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Ala 1 5 INFORMA7ION FOR SEO ID N'O:36: Ci SEQUENCE

CHARACTERISTICS:

LENGTH: 32 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOL.ECULE TYPE: protein Cxi) SEQ.UENCE DESCRIPTION: SEQ ID N0:36: Arg Phe Thr Ile Ser Arg Asp Asn Ser LyS Asa Thr Leu Phe Leu Gin 1 5 10 is Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Ser 2S INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid

STRANEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Arg Phe Tbir Ile Ser Arg Asp Asn Ala Lys Asn Tihr Lau Phe Leu Gin 1 5 10 is Met Asp Ser Leu Arg Pro Glu. Asp Thr GIly Val. Tyr ?he Cys Ala Ser 25 INFORMATION FOR SEQ ID zNo:38; Wi SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid

STRANDFMNESS:

TOPOLOGY: linear (ii) MOEUETYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO;.38: Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gly 5cr Phe Leu Arg 1 5 10 is **.Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Ser 25 INFORMATION FOR SEQ ID NO:39: Wi SEQUENC CHARACTE ISTICS: LENGTH: 11. amino acids TYPE: amino acid STRANflEDN'ESS: TOPOLOGY: linear (ii) MOLECULE TYPE:. protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: TrP GlY Gin Gly Thr Pro Val Thr Va, e Ser I. s INFORMATION FOR SEQ rD SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUTENCE DESCRIPTION: SEQ ID NOA:0 Trp Gly Gin Gly Thx Th-r Val Thr Val. Ser Ser I. 5 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENTH: 31. base pairs TYPE: nucleic acid STANDEDNESS; single TOPOLOGY: linear (ii) MOLECULE TYPE: D14A (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID) NO: 41: GGAAGCTTAG ACAGATGGO GTGTCGMrr G 31 INFORMlATIONI FOR SEQ ID NO:42: Wi SEQUENCE CHARACTERISTICS: LaNGTH: 34 bass pairs TYPE: nucleic acid STP.ANDEDNESS: single TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: TGAGGAGACG GTGACCGTGG, T CCCTTGGCC CCAG 34 INFORMATION FOR SEQ ID NO:43.

Wi. SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iMOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: AGGTSMARCT OCAGSAGTCW GG 22 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: 'LENGTH: 34 base pairs TypE:. nitcleic acid STRANDEDNESS: Single TOPOLOGY: linear (ii4) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: TGGAATTCAT GGRATGGAGC TGQRTCWTBH TCTT 34 INFORMATION FOR SEQ 10 NO:4S: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS- Single TOPOLOGY: linear (ii) MOLECULE TYPE. DNA (genomic) (xi) SEQUENCE DESCRIP'TION: SEQ ID TGGAATTCAT GUACTTCDGG YTCAACTKCPR TTT 33 INFORMATION FOR SEQ ID NO:46: C)SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: mucleic acid OV C) STRANDEDNESS: single TOPOLOGY: linear (iMOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46G: :GGAAGCTTGA AGATGGATAC AGTrGGTGCA GC 32 INFORMATION FOR SEQ ID 140:47: CW SEQUENCE CHARACTERXSTICS: LENGTH: 22 base pairs TYPE: nucleic acid STR.ANEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: GTTAGATCTC CAGCTTGGTC CC 22 INFORMATIONT FOR SEQ ID NO:48: SEQUENCE CHAACTRISTICS: LENOTH: 23 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (gancinic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:- GTTAGATCTC CAG5TTrGGTG CCT 23 :NFORMATION FOR SEQ I= NO;49: Wi SEQUENCE CHARACTERISTICS: LENGTHI: 24 bame pairs TYPE: nucleic acid STRANDEDNESS; single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genamic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO.-49- *GACAT"-TCAQC TGACCCACTC TCCA 24 INFOP.MATION FOR SEQ ID NO:SO: SEQUENCE CBRACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRI~PTION: SEQ iD No:SO: :GACRTTCAGC TGACCCAGGM TGMA 2 IINFORMATION FOR SEQ ID NO:52.: Wi SEQUENCE CHARATERISTICS: LENGTH- 17 base pairs TYPE: nucleic acid STRANDEDNESSz single TOPOLOGY: linear (1i) MOLECULE TYPE: DNA (genomic) (xNi). SEQUENCE DESCRIPTION: SEQ ID NO:51: GACATTCAGC TGACCCA 1.7 INFORMATION FOR SEQ ID NO:52: Wi SEQUMNCE CHARACTERISTICS: LENGTH; 24 b~ass pairs TYPE: naucleic acid STRAM~EDNESSt single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genlotic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: OACATTGAGC TCACCCAGTC TCCA 24 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pair3 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYE: DNA (genontic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: TTGAATTCGG Tc3CCAGAKtCW SAHATYGTHA TG 32 INFORMATION FOR SEQ ID NO:54: Ci) SEQUENCE CHARACTERISTICS: LENGTH.- 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: a a 'TGAATTCGG TGCCAGAXCW SARATYGTKC TC 32 INFORMATION FOR SEQ IDl Wi SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANIDEDNESS. single TOPOLOGY: linear (ii) MOLECULE TYPE: D11A (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:SS: TTGAATTCGG AGCTGATGGG AACATTGTAA TG 32 INFORMATION FOR SEQ ID NO:S6: SEQUENCE CHARACTERISTICS: (W LENGTH: 26 base pairs TYPE: mucleic acid STPANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNAL (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:.

CWGAGAAA'rT CAGCTGACCC AGTCTC INFORM.ATION FOR SEQ ID NO:S7: Wi SEQUENCE CHARACTERISTICS: LENGTH. 119 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: Gin Val Gin Leu GIn Glu Ser Gly Pro Gly Lau Val. Arg Pro Ser Gin.

1 10 is :**Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Thbr Phe Ser Thr Tyr 25 Trp Met Ser Trp Val Arg Gin Pro Pro Gly Axrg G3y Lau Giu Trp Ile 3S 40 Gly Giu Ie His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu Lys Asp Arg Val Thr Met Lau Val Asp Thz Ser Lys Asn Ginx Phe Ser 70 75 Lau Arg Leu Ser Ser Val Thr Al a Ala Asp Thr Ala Val Tyr Tyr Cys as 950 **:Ala Arg Leu Tyr ?he Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gin Gly 100 105 110 Thr Thr Val Thr Val. Ser Ser INFORMATION FOR SEQ 1rD NO:SB: SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid STRANDEDNESS; single TOPOLOGY: linear 61 (xi) SEQUENCE DESCRIPTIONi SEQ ID NO:SB: GLL Val Gin Lau Val Giu Ser cfly Gly Gly Val Val. Gin Pro Gly Arg 1 5 IQ is Ser Lou Axrg lau Ser Cys Ser Ser Ser Gly Phie Ile Phe Ser Tbhr Tyr 7025 Trp Met Ser .rp Val Ari9 Gin Ala Pro Gly Lys Gly L.eu Giu TZ-p Val 40 Ala Glu Ile Pro Asp Ser 3cr Thr Ile Asn Tyr Ala Pro Ser Leo so 55 6 Lys Azp Arg ?he Thr Ile Ser Arg Asp A.Bn Ser Lys Asn Th~r Leu Phe 70 75 s0 Leu. Gin Met Asp Ser Leu Arg Pro Lys Asp Thxr Gly Val. Tyr Phe Cys as go Ala Arg Leu '-yr Phe Gly Phe Pro Trp Phe Ala Tyrr Trp Gly Gin Gly -a0 105. 110 Thr Pro Val .I=h Val. Ser 2cr 115 .so GO 6 so 00 Co so so

Claims (29)

1. A method for diagnosing or treating a patient comprising administering a conjugate to said patient in an effective amount for diagnosis and treatment, wherein said conjugate comprises: a diagnostic or therapeutic agent bound to a humanized Class III, anti-CEA, monoclonal antibody (mAb), wherein the complementarity- determining regions (CDRs) of said humanized mAb @000 are CDRs from a parental murine Class III, anti- CEA mAb, and each of the framework regions (FRs) is from a human antibody, wherein said humanized I mAb retains the binding specificity of said parental murine Class III, anti-CEA mAb but is less immunogenic in a patient than is said •parental murine Class III, anti-CEA mAb.

2. The method of claim 1, wherein said murine Class III, anti-CEA mAb is the MN-14 mAb.

3. The method of claim 2, wherein: 0* the light chain variable regions are characterized by the formula: FRL -CDRLi -FRL 2 -CDRL 2 FRL3-CDRL 3 -FRL4, wherein each FR is a different framework region of a human antibody, and each CDR is a different CDR of the light chain of the MN-14 mAb; and, the heavy chain variable regions are characterized by the formula: FRH -CDRH -FRH 2 CDRH 2 -FRH3-CDRH 3 FRH4, wherein each FR is a different framework region of a human antibody and each CDR is a different CDR of the heavy chain of MN-14 mAb.

4. The method of claim 3, wherein the amino acid sequence of: CDRl is KASQD VGTSV A (SEQ. ID. NO. CDRL 2 is WTSTR HT (SEQ. ID. NO. 21); SCDRL 3 is QQYSL YRS (SEQ. ID. NO. 22); H: \PC1arke\KeeP\specis\73209-98 imrnnomedics cm.doc 3/03/99 63 CDRH1 is TYWMS (SEQ. ID. NO. 23); CDRH 2 is EIHPD SSTIN YAPSL KD (SEQ. ID NO. 24); and, CDRH 3 is LYFGF PWFAY (SEQ. ID NO.

5. 0 S 0* 0 S S. S 00 US SO U pr, *0 *O The method of claim 3, wherein: FRLI comprises a region of about that occurs naturally in the FRL1 of a human antibody; FRL 2 comprises a region of about that occurs naturally in the FRL22 of a human antibody; FRL 3 comprises a region of about that occurs naturally in the FRL33 of a human antibody; FRL4 comprises a region of about that occurs naturally in the FRL 4 of a human antibody; FRH1 comprises a region of 28-32 occurs naturally in the FRH1 of a human antibody; FRH2 comprises a region of 12-16 occurs naturally in the FRH2 of a human antibody; FRH3 comprises a region of 30-34 23 amino acids 15 amino acids 32 amino acids 10 amino acids amino acids that amino acids that amino acids that occurs naturally in the FRH3 of a human antibody; and, FRH4 comprises a region of 9-13 amino acids that occurs naturally in the FRH 4 of a human antibody.

The method of claim 5, wherein FRLI is DIQLT QSPSS LSASV GDRVT ITC (SEQ. ID NO. 26); FRL 2 is WYQQK PGKAP KLLIY (SEQ. ID NO. 27); FRL3 is GVP(S or D)R FSGS(G or V) SGTDF TFTIS SLQPE DIATY YC (SEQ. ID NO. 28); FRL 4 is FGQGT KVEIK (SEQ. ID NO. 29); FRH1 is EVQLV ESGGG VVQPG RSLRL SCSSS GFDFT (SEQ. ID NO. 30), or H:\PClarke\Keep\specis\73209-98 immunomedics cm.doc 3/03/99 64 EVQLV ESGGG VVQPG RSLRL SCSAS GFDFT (SEQ. ID NO. 31); FRH2 is WVRQA PGKGL EWVA (SEQ. ID NO. 33); FRH 3 is RFTIS RDNSK NTLFL QMDSL RPEDT GVYFC AS (SEQ. ID NO. 36), or RFTIS RDNAK NTLFL QMDSL RPEDT GVYFC AS (SEQ. ID NO. 37); and FRH 4 is WGQGT PVTVS S (SEQ. ID NO. 39); and wherein C may be in the sulfhydryl or disulfide form.

7. The method of claim 6, wherein the amino acid ,sequence of: 00. CDRLI is KASQD VGTSV A (SEQ. ID. NO. CDRL 2 is WTSTR HT (SEQ. ID. NO. 21); 0* 2 CDRL 3 is QQYSL YRS (SEQ. ID. NO. 22); S. 15 CDRH1 is TYWMS (SEQ. ID. NO. 23); CDRH 2 is EIHPD SSTIN YAPSL KD (SEQ. ID NO. 24); and, CDRH 3 is LYFGF PWFAY (SEQ. ID NO.

8. The method of claim 1, wherein said humanized mAb heavy and light chain variable regions are KLHuVHAIG/HuVK (SEQ. ID NO. 13/SEQ. ID NO. 19), KLHuVHAIGA/HuVK (SEQ. ID NO. 14/SEQ. ID NO. 19) or KLHuVHAIGAY/HuVK (SEQ. ID NO. ID NO. 19).

9. The method of claim 1, wherein said humanized mAb heavy and light chain variable regions is KLHuVHAIGA/HuVK (SEQ. ID NO. 14/SEQ. ID NO. 19).

10. The method of claim 1, wherein said therapeutic agent is a cytotoxic agent.

11. The method of claim 10, wherein said cytotoxic agent is doxorubicin, methotrexate, taxol, ricin A, or a radionuclide.

12. The method of claim 11, wherein said radionuclide is 131I.

13. The method of claim 1, wherein said therapeutic agent is a cytokine.

14. The method of claim 13, wherein said cytokine is interferon, interleukin or tumor necrosis factor.

H:\PCarke\Keep\pecis\73209-98 immunomedics cm.doc 3/03/99 65 The method of claim 14, wherein said cytokine is interferon-g or interleukin-2.

16. The method of claim 1, wherein said diagnostic reagent comprises an imaging agent.

17. The method of claim 16, wherein said imaging agent is a radionuclide.

18. A method for treating a patient comprising administering a humanized Class III, anti-CEA, monoclonal antibody (mAb) to said patient in an effective amount for treatment, wherein said mAb comprises: a humanized Class III, anti-CEA, monoclonal .*00 antibody (mAb), wherein the complementarity- determining regions (CDRs) of said humanized mAb are CDRs from a parental murine Class III, anti- 15 CEA mAb, and each of the framework regions (FRs) is from a human antibody, wherein said humanized mAb retains the binding specificity of said parental murine Class III, anti-CEA mAb but is less immunogenic in a patient than is said 20 parental murine Class III, anti-CEA mAb.

19. The method of claim 18, wherein said murine Class III, anti-CEA mAb is the MN-14 mAb.

20. The method of claim 19, wherein: 0 the light chain variable regions are characterized by the formula: FRLI- CDRLI- FRL 2 -CDRL 2 -FRL 3 -CDRL3-FRL 4 &see wherein each FR is a different framework region of a human antibody, and each CDR is a different CDR of the light chain of the MN-14 mAb; and, the heavy chain variable regions are characterized by the formula: FRH -CDRH1 -FRH2-CDRH 2 -FRH 3 -CDRH 3 -FRH 4 wherein each FR is a different framework region of a human antibody and each CDR is a different CDR of the heavy chain of MN-14 mAb.

21. The method of claim 20, wherein the amino acid sequence of: H:\PCiarke\Keep\speci9\73209-98 immunomedics cm.doc 3/03/99 66

22. OS e S S. 0 S OSOS S. S S. S. *e S S S es 5 S.. *505 0e 55 S 500e S. S@ S S CDRLI is KASQD VGTSV A (SEQ. ID. NO. CDRL 2 is WTSTR HT (SEQ. ID. NO. 21); CDRL 3 is QQYSL YRS (SEQ. ID. NO. 22); CDRH1 is TYWMS (SEQ. ID. NO. 23); CDRH 2 is EIHPD SSTIN YAPSL KD (SEQ. ID NO. 24); and, CDRH 3 is LYFGF PWFAY (SEQ. ID NO. The method of claim 20, wherein: FRL comprises a region of about 23 amino acids that occurs naturally in the FRL, of a human antibody; FRL 2 comprises a region of about 15 amino acids that occurs naturally in the FRL 2 2 of a human antibody; FRL3 comprises a region of about 32 amino acids that occurs naturally in the FRL33 of a human antibody; FRL 4 comprises a region of about 10 amino acids that occurs naturally in the FRL 4 of a human antibody; FRH1 comprises a region of 28-32 amino acids that occurs naturally in the FRH1 of a human antibody; FRH 2 comprises a region of 12-16 amino acids that occurs naturally in the FRH2 of a human antibody; FRH3 comprises a region of 30-34 amino acids that occurs naturally in the FRH3 of a human antibody; and, FRH4 comprises a region of 9-13 amino acids that occurs naturally in the FRH 4 of a human antibody. The method of claim 22, wherein FRL1 is DIQLT QSPSS LSASV GDRVT ITC (SEQ. ID NO. 26); FRL 2 is WYQQK PGKAP KLLIY (SEQ. ID NO. 27); FRL3 is GVP(S or D)R FSGS(G or V) SGTDF TFTIS SLQPE DIATY YC (SEQ. ID NO. 28); o.oo *0 *S.

23. H:\PCarke\Keep\specis\73209-98 imnunomedics cm.doc 3/03/99 67 FRL 4 is FGQGT KVEIK (SEQ. ID NO. 29); FRHI is EVQLV ESGGG VVQPG RSLRL SCSSS GFDFT (SEQ. ID NO. 30), or EVQLV ESGGG WQPG RSLRL SCSAS GFDFT (SEQ. ID NO. 31); FRH2 is WVRQA PGKGL EWVA (SEQ. ID NO. 33); FRH3 is RFTIS RDNSK NTLFL QMDSL RPEDT GVYFC AS (SEQ. ID NO. 36), or RFTIS RDNAK NTLFL QMDSL RPEDT GVYFC AS (SEQ. ID NO. 37); and FRH 4 is WGQGT PVTVS S (SEQ. ID NO. 39); and wherein C may be in the sulfhydryl or disulfide form.

24. The method of claim 23, wherein the amino acid sequence of: 15 CDRL1 is KASQD VGTSV A (SEQ. ID. NO. CDRL2 is WTSTR HT (SEQ. ID. NO. 21); CDRL 3 is QQYSL YRS (SEQ. ID. NO. 22); CDRH1 is TYWMS (SEQ. ID. NO. 23); CDRH 2 is EIHPD SSTIN YAPSL KD (SEQ. ID NO. 24); 20 and, CDRH 3 is LYFGF PWFAY (SEQ. ID NO.

25. The method of claim 18, wherein said humanized ,6 mAb heavy and light chain variable regions are KLHuVHAIG/HuVK (SEQ. ID NO. 13/SEQ. ID NO. 19), KLHuVHAIGA/HuVK (SEQ. ID NO. 14/SEQ. ID NO. 19) or KLHuVHAIGAY/HuVK (SEQ. ID NO. 15/SEQ. ID NO. 19).

26. The method of claim 18, wherein said humanized mAb heavy and light chain variable regions is KLHuVHAIGA/HuVK (SEQ. ID NO. 14/SEQ. ID NO. 19).

27. The method of claim 18, further comprising administering a therapeutic agent to said patient.

28. The method of claim 27, wherein said therapeutic agent is doxorubicin, methotrexate, taxol, ricin A, a radionuclide or a cytokine.

29. The method of claim 28, wherein said radionuclide is 131 The method of claim 28, wherein said therapeutic H:\PClarke\Keep\specis\ 7

3209-98 immunomedics cm.doc 3/03/99 68 agent is a cytokine. 31. The method of claim 30, wherein said cytokine is interferon, interleukin or tumor necrosis factor. 32 The method of claim 31, wherein said cytokine is interferon-g or interleukin-2. Dated this 3rd day of March 1999 IMMUNOMEDICS, INC. :By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia *0 00 0 H:\PClarke\Keep\specis\73209-98 iinzunomedics cn,.doc 3/03/99