AU631545B2 - Il-2 receptor-specific chimeric antibodies - Google Patents

Description

WO 89/09622 cPCT/US89/01578 IL-2 RECEPTOR-SPECIFIC CHIMERIC ANTIBODIES Field of the Invention The present invention relates generally to the S combination of recombinant DNA and monoclonal antibody technologies for developing novel therapeutic and other agents and, more particularly, to the production of chimeric antibodies specific for the human interleukin-2 receptor and the use of such chimeric antibodies in treating T-cell mediated human disorders.

Background of the Invention In mammals, the immune response is mediated by two types of cells that interact specifically with foreign material, antigens. One of these cell types, B-cells, are responsible for the production of antibodies. The second cell class, T-cells, include a wide variety of cellular subsets controlling the in vivo function of both B-cells and a wide variety of other hematopoietic cells, including Tcells.

One way in which T-cells exert this control is through the production of a lymphokine known as interleukin-2 i originally named T-cell growth factor. IL-2's prime function appears to be the stimulation and maintenance of Tcells. Indeed, some immunologists believe that IL-2 may be at the center of the entire immune response (see, Farrar, J., et al., Immunol. Rev. 63:129-166 (1982), which is incorporated herein.by reference).

To exert its biological effects, IL-2 interacts with a specific high-affinity membrane receptor (Greene, W., et al., Progress in Hematology XIV, E. Brown, ed., Grune and Statton, New York (1986), at pgs. 283 ff). The human IL-2 receptor is a complex glycoprotein, with one chain 55kD in size (Leonard, W. et al., J. Biol. Chem. 260:1872 (1985)). A gene encoding this protein has been isolated, and predicts a 272 amino acid peptide, including a 21 amino acid signal i peptide, (see, Leonard, W. et al., Nature 311: 626 (1984)). i 7 WO 89/09622 PCT/US89/01578 2 Much of the elucidation of the human IL-2 receptor's structure and function is due to the development of specifically reactive monoclonal antibodies. In particular, one monoclonal antibody, known as anti-Tac, (Uchiyama et al., J. Immunol. 126:1393 (1981)) has shown that IL-2 receptors can be detected on T-cells, but also on cells -of the monocyte-macrophage family, Kupffer cells of the liver, Langerhans' cells of the skin and, of course, activated T-cells. Importantly, resting T-cells, B-cells or circulating machrophages typically do not display the IL-2 receptor (Herrmann, et al., J. Exp. Med. 162:1111 (1985)).

The anti-Tac monoclonal antibody has also been used to define lymphocyte functions that require IL-2 interaction, and has been shown to inhibit various T-cell functions, including the generation of cytotoxic and suppressor T lymphocytes in cell culture. Also, based on studies with anti-Tac, a variety of disorders are now associated with improper IL-2 receptor expression by T-cells, in particular adult T-cell leukemia.

More recently, the IL-2 receptor has been shown to be an ideal target for novel therapeutic approaches to T-cell mediated diseases. The anti-Tac monoclonal antibody can be used either alone or as an immunoconjugate with Ricin A, isotopes and the like) to effectively remove cells bearing the IL-2 receptor. These agents can, for example, theoretically eliminate IL-2 receptor-expressing leukemic cells, certain B-cells, or activated T-cells involved in a disease state, yet allow the retention of mature normal Tcells and their precursors to ensure the capability of mounting a normal T-cell immune response as needed. In general, other T-cell specific agents can destroy essentially all peripheral T-cells, which limits therapeutic efficacy.

Overall, the use of monoclonal antibodies specific for the IL-2 receptor can be expected to have therapeutic utility in autoimmune diseases, organ transplantation and any unwanted response by activated T-cells. Indeed, clinical trials have been initiated (see, generally, Waldman, Science 232:727- 732 (1986), which is incorporated herein by reference).

!a WO 89/09622 PC/US89/01578 WO 89/09622 3 Unfortunately, the use of the anti-Tac monoclonal antibody has certain drawbacks, particularly in repeated therapeutic regimens. As a mouse monoclonal, it does not fix human complement well, whereas a human equivalent may be more efficient.

More importantly, however, anti-Tac monoclonal antibody contains substantial murine amino acid sequences that will be antigenic when injected into a human patient.

Numerous studies have shown that the immune response elicited by a patient against the nonbinding portion of an injected mouse monoclonal antibody can be quite strong, essentially eliminating the antibody's therapeutic utility after an initial treatment. As increasing numbers of different mouse monoclonal antibodies can be expected to be developed to treat various diseases, after the first and second treatments with different mouse monoclonal antibodies, subsequent treatments can be dangerous in themselves.

Thus, there is a need for improved forms of the anti-Tac monoclonal antibody that are substantially less antigenic, yet easily and economically produced in a manner suitable for therapeutic formulation. The present invention fulfills these needs.

Summary of the Invention The present invention provides novel compositions useful in the treatment of T-cell mediated human disorders, the compositions containing a chimeric antibody specifically capable of binding to human IL-2 receptors, such as at the epitope bound by the anti-Tac monoclonal antibody. The IL-2 chimeric antibody can have two pairs of light chain/heavy chain complexes, wherein at least one pair has chains comprising mouse variable regions joined with human constant region segments, with or without naturally-associated J and D segments.

The chimeric antibodies, or binding fragments thereof, of the present invention may be produced by a variety of recombinant DNA techniques, with -ultimate expression in transfected cells, preferably immortalized WO 89/09622 PCT/US89/01578 fII

N-

eukaryotic cells, such as myeloma or hybridoma cells.

Polynucleotides comprising a first sequence coding for a human immunoglobulin constant region and a second sequence coding for the desired mouse immunoglobulin variable or hypervariable region can be produced synthetically or by 5 combining appropriate cDNA and genomic DNA segments.

When the chimeric antibodies are complexed with a cytotoxic agent, such as a radionuclide, a ribosomal inhibiting protein or a cytotoxic agent active at cell surfaces, the compounds will be particularly useful in treating T-cell mediated disorders. These compounds can be provided in a pharmaceutically accepted dosage form, which will vary depending on the mode of administration.

1 j

C

WO 89/09622 PC/US89/01578 BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts the DNA coding sequence and putative amino acid sequence of the V and J regions of the anti-Tac light chain.

Figure 2 depicts the DNA coding sequence and putative amino acid sequence of the V and J regions of the anti-Tac heavy chain.

Figures 3-10 are schematic diagrams of the plasmids utilized to demonstrate the present invention.

Figure 11 represents an overview of a preferred strategy for preparation of V and J regions for insertion into plasmids.

WO 89/09622 PCT/US89/01578 6 DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, DNA sequences encoding mouse variable/human constant region chimeric antibodies capable of binding epitopes on human IL-2 receptors are provided. When placed in expression vectors and suitable hosts, large quantities of chimeric antibodies can be produced. Preferably, the chimeric antibodies will have substantially the same binding profile or characteristics as be cross-reactive or capable of blocking) the binding of the anti-Tac monoclonal antibody, such as antibodies produced by the myeloma cell line deposited with the A.T.C.C. and designated accession number CRL 9688. These chimeric antibodies find use, for example, i in the treatment of T-cell mediated disorders in human patients.

The basic immunoglobin structural unit is known to comprise a tetramer. Each tetramer is composed of two i identical pairs of polypeptide chains, each pair having one "light" (about 25kD) and one "heavy" chain (about 50-70kD).

The NH -terminus of each chain begins a variable region of S 20 about 100 to 110 or more amino acids primarily responsible for antigen recognition. The C00H terminus of each chain defines a constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains are classified (and subclassified) as gamma, mu, alpha, delta, or epsilon, and define the immunoglobulin's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a region of about 12 or more amino acids, with the heavy chain also including a region of about 12 more amino acids. (See, generally, Fundamental Immunology, Ed. Paul, W. Chapter 7, pgs. 131-166, Raven Press, N.Y. (1984), which is incorporated 'herein by reference.) Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin gene segments belonging to

_J

i IX WO 89/09622 PCT/US89/01578 7 different species. For example, the variable segments of the genes for a mouse monoclonal antibody may be joined to human constant segments, such as 1 and A preferred therapeutic chimeric antibody is thus a hybrid protein consisting of the V or antigen-binding domain from a mouse antibody and the C or effector domain from a human antibody, although other mammalian species may be used.

Human chimeric antibodies have at least three potential advantages over mouse antibodies for use in human therapy: 1) Because the effector portion is human, it may interact better with the other parts of the human immune system destroy the target cells more efficiently by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity

(ADCC))

2) The human immune system should not recognize the C region of the chimeric antibody as -foreign, and therefore the antibody response against an injected chimeric antibody should be less than against a totally foreign mouse antibody.

3) Injected mouse antibodies have been reported to have a half-life in the human circulation much 'i shorter than the half-life of normal antibodies (Shaw, D. et al., J. Immunol. 138:4534-4538 (1987)). It is possible that injected chimeric antibodies will have a half-life more like that of human antibodies, allowing smaller and less frequent doses to be given.

In one aspect, the present invention is directed to recombinant DNA segments encoding the heavy and/or light chain variable or hypervariable regions from the anti-Tac monoclonal antibody. The DNA segments encoding these regions will typically be joined to DNA segments encoding appropriate constant regions, such as human gamma heavy chain regions or human kappa light chain regions. The preferred variable region DNA sequences, which on expression code for the I C- W89/09622 PCT/US89/01578 SWO 89/09622 polypeptide chains comprising the anti-Tac light and heavy chain variable regions (with naturally-associated J regions), are shown in Figures 1 and 2, respectively. Due to codon d.generacy and non-critical amino-acid substitutions, other DNA sequences can be readily substituted for those sequences, as detailed below.

The DNA segments will typically further include an expression control DNA sequence operably linked to the chimeric antibody coding sequences, including naturallyassociated or heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the light chains, heavy chains, light/heavy chain dimers or intact chimeric antibodies may follow.

It is well known that native forms of "mature" immunoglobulins will vary somewhat in terms of length by deletions, substitutions, insertions or additions of one or more amino acids in the sequences. Thus, both the variable b and constant regions are subject to substantial natural modification, yet are "substantially identical" and still capable of retaining their respective activities. Human constant region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells, but V preferably immortalized B-cells. Suitable source cells for Lthe DNA sequences and host cells for expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection ("Catalogue of Cell Lines and Hybridomas," Fifth edition (1985) Rockville, Maryland, which is incorporated herein by reference).

In addition to these naturally-occurring forms of immunoglobulin chains, "substantially identical" modified heavy and light chains can be readily designed and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art. For example, the IWO 89/09622 PCT/US89/01578 9 chains can vary from the naturally-occurring sequence at the primary structure level by several amino acid substitutions, terminal and intermediate additions and deletions, and the like. Alternatively, polypeptide fragments comprising only a portion (usually at least about 60-80%, typically 90-95%) of the primary structure may be produced, which fragments possess one or more immunoglobulin activities complement fixation activity), while exhibiting lower immunogenicity. In particular, it is noted that like many genes, the immunoglobulin-related genes contain separate functional regions, each having one or more distinct biological activities. These may be fused to functional regions from other genes enzymes, see, commonly assigned U.S.S.N. 132,387, filed Dec. 15, 1987, which is incorporated herein by reference) to produce fusion proteins immunotoxins) having novel properties. In general, modifications of the genes may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see, Gillman and Smith, Gene 8:81-97 (1979); Roberts, S. et al, Nature 328:731-734 (1987); and U.S. Patent no. 4,703,008, all of which are incorporated herein by reference). Preferred DNA segments encoding variable regions of the present invention will typica'ly be substantially homologous to the sequences of Figures 1 and 2 capable of hybridizing to the sequences under stringent conditions of low salt and high temperature), most preferrably at least about 90-95% homologous or more.

The nucleic acid sequences of the present invention capable of ultimately expressing the desired chimeric antibodies can be formed from a variety of different y 30 polynucleotides (genomic or cDNA, RNA, etc.) and components V, J, D, and C regions), as well as by a variety of different techniques. Joining appropriate genomic sequences is presently the most common method of production, but cDNA sequences may also be utilized (see, European Patent Application Nos. 85102655.8, 85305604.2, 84302368.0 and 85115311.4, as well as PCT Application Nos. GB85/00392 and US86/02269, all of which are incorporated herein by i* I i PCT/US89/01578 WO 89/09622 reference).

As stated previously, the DNA sequences of the present invention (typcially at least about 30 contiguous nucleotides encoding 10 amino acids from the sequences in Figures 1 and 2) will be expressed in hosts after the sequences have been operably linked to positioned to ensure the functioning of) an expression control sequence.

These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, U.S. Patent 4,704,362, which is incorporated herein by reference).

E. coli is one prokaryotic host useful particularly for cloning the DNA sequences of the present invention.

Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell an origin of replication). In addition, any number of a variety of wellknown promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.

Other microbes, such as yeast may also be used for expression. Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.

In addition to microorganisms, mammalian tissue cell culture may also be used to produce the polypeptides of WO 89/09622 PCT/US89/01578 11 the present invention (see, Winnacker, "From Genes to Clones," VCH Publishers, N.Y. (1987), which is incorporated herein by reference). Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, etc, but preferably transformed B-cells or hybridomas. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen, C. et al., Immunol. Rev. 89:49-68 (1986), which is incorporated herein by reference), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, Adenovirus, Bovine Papilloma Virus, and the like.

The vectors containing the DNA segments of interest the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment may be used for other cellular hosts. (See, generally, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, (1982), which is incorporated herein by reference.) Once expressed, the whole chimeric antibodies, their dimers, or individual light and heavy chains of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fraction column chromatography, gel electrophoresis and the like. (See, generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982).) Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like. (See, generally, Immunological WO 89/09622 PCT/US89/01578 12 Methods, Vols. I and II, Eds. Lefkovits and Pernis, Academic Press, New York, N.Y. (1979 and 1981).) The chimeric antibodies of the present invention will typically find use individually in treating a T-cell mediated disease state. Generally, where the cell linked to a disease has been identified az IL-2 receptor bearing, then the chimeric antibodies ar:. suitable (see, U.S.S.N. 7- 085,707, entitled "Treating Human Malignancies and Disorders," which is incorporated herein by reference).

For example, typical disease states suitable for treatment graft versus host disease and most patients undergoing an organ transplant, such as heart, lungs, kidneys, liver, etc. Other diseases include Type I diabetes, multiple sclerosis, rheumatoid arthritis, Lupus erythematosus, and Myasthenia Gravis.

The antibodies of the present invention may also be used in combination with other antibodies, particularly human chimeric antibodies or human monoclonal antibodies reactive with other markers on cells responsible for the disease. For example, suitable T-cell markers can include those grouped into the so-called "Clusters of Differentiation," as named by the First International Leukocyte Differentiation Workshop, Leukocyte Typing, Eds. Bernard, et al., Springer-Verlag, N.Y.

(1984), which is incorporated herein by reference.

The chimeric antibodies can also be used as separately administered compositions given in conjunction with chemotherapeutic or immunosuppressive agents.

Typically, the agents will include a cephalosporin or a purine analog methotrexate, 6-mercaptopurine, or the Slike), but numerous additional agents cyclophosphamide, sulfa drugs, etc.) well-known to those skilled in the art may also be utilized.

A preferred pharmaceutical composition of the present invention comprises the use of the subject chimeric antibodies in immunotoxins. Immunotoxins are characterized by two components and are particularly useful for killing selected cells in vitro or in vivo. One component is a cytotoxic agent which is usually fatal to a cell when Jr~llsl WO 89/09622 PCT/US89/01578 13 attached or absorbed. The second component, known as the "delivery vehicle," provides a means for delivering the toxic agent to a particular cell type, such as cells comprising a carcinoma. The two components are commonly chemically bonded together by any of a variety of well-known chemical procedures. For example, when the cytotoxic agent is a protein and the second component is an intact immunoglobulin, such as a chimeric antibody, the linkage may be by way of heterobifunctional cross-linkers, SPDP, carbodiimide, glutaraldehyde, or the like. Production of various immunotoxins is well-known with the art, and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al, Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982), which is incorporated herein by reference.

A variety of cytotoxic agents are suitable for use in immunotoxins. Cytotoxic agents can include radionuclides, such as Iodine-131, Yttrium-90, Rhenium-188, and Bismuth-212; a number of chemotherapeutic drugs, such as vindesine, methotrexate, adriamycin, and cisplatinum; and cytotoxic proteins such as ribosomal inhibiting proteins, pokeweed antiviral protein, abrin and ricin (or their Achains, diphtheria toxin A-chains, Pseudomonas exotoxin A, etc.) or an agent active at the cell surface, such as the phospholipase enzymes phospholipase (See generally, "Chimeric Toxins," Olsnes and Phil, Pharmac.

Ther., 25:355-381 (1982), and "Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Byers, pp.

159-179, 224-266, Academic Press (1985), both of which are incorporated herein by reference.) The delivery component of the immunotoxin will include the chimeric antibodies of the present invention.

Intact chimeric immunoglobulins or their binding fragments, such as Fab, F(ab 2 etc., are preferably used. Typically, the chimeric antibodies in the immunotoxins will be of the human IgM or IgG isotype, but other mammalian constant regions may be utilized as desired.

The chimeric antibodies and pharmaceutical i i; ,xic a ided ming 1

I

i i i ij use 89/09622 PCT/US89/01578 compositions thereof of this invention are particularly useful for parenteral administration, subcutaneously, intramuscularly or intravenously. The compositions for parenteral administration will commonly comprise a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody in these formulations can vary widely, from less than about usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile buffered water, and 50 mg of antibody. A typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of antibody. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th Ed., Mack Publishing Company, Easton, Pennsylvania (1980), which is incorporated herein by reference.

The antibodies of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immune globulins and art-known lyophilization and reconstitution techniques can be employed. It will be j i1

I

a 1i f WO 89/09622 apprec and re activi antibo antibo 5 compen chimeri for the therape 10 patient partial amount "therap use wil 15 general general dose, w commonl this in 20 states, threate minimiz probabi achieve 25 vention treatin these a contain 30 adminis enhance to be a precise health 3 from 0.: patient l

.I

l WO 89/09622 PCT/US89/01578 WO 89/09622 27 gamma 3 heavy chain plasmid pGTAC3 described before) and the non-producing line Sp2/0 itself, were plated in 1 ml nf nMT WO 89/09622 heavy show ti WO 89/09622 PC/US89/O1578 appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss with conventional immune globulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted to compensate.

The compositions containing the present human chimeric antibodies or a cocktail thereof can be administered for the prophylactic and/or therapeutic treatments. In therapeutic application, compositions are administered to a patient already, in an amount sufficient to cure or at least partially arrest the infection and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the infection and the general state of the patient's own immune system, but generally range from about 1 to about 200 mg of antibody per dose, with dosages of from 5 to 25 mg per patient being more commonly used. It must be kept in mind that the materials of this invention may generally be employed in serious disease states, that is life-threatening or potentially lifethreatening situations. In such cases, in view of the minimization of extraneous substances and the lower probability of "foreign substance" rejections which are I achieved by the present human chimeric antibodies of this invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these antibodies.

In prophylactc applications, compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactically effective dose." In this use, the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per patient.

I frm Ol to25 g pe doe, epecally0.5to 25 m p2 g WO 89/09622 PCT/US89/( 16 )1578 it ii :j 1 i' Vi Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of the antibody(ies) of this invention sufficient to effectively treat the patient.

Chimeric antibodies of the present invention can further find a wide variety of utilities in vitro. By way of example, the chimeric antibodies can be utilized for T-cell typing, for isolating specific IL-2 receptor bearing cells or fragments of the receptor, for vaccine preparation, or the like.

For diagnostic purposes, the chimeric antibodies may either be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the chimeric antibody, such as antibodies specific for human immunoglobulin constant regions. Alternatively, the chimeric antibodies can be directly labeled. A wide variety of labels may be employed, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), etc. Numerous types of immunoassays are available and are well known to those skilled in the art.

Kits can also be supplied for use with the subject antibodies in the protection against or detection of a cellular activity or for the presence of a selected antigen.

Thus, the subject antibody composition of the present invention may be provided, usually in a lyophilized form in a container, either alone or in conjunction with additional antibodies specific for the desired cell type. The antibodies, which may be conjugated to a label or toxin, or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, serum albumin, or the like, and a set of instructions for use. Generally, these materials will be present in less than about 5% wt. based on the amount of active antibody, and usually present in total amount of at least about 0.001% wt. based again on the antibody t- I WO 89/09622 PCT/US89/01578 17 concentration. Frequently, it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% wt. of the total composition. Where a second antibody capable of binding to the chimeric antibody is employed in an assay, this will usually be present in a separate vial. The second antibody is typically conjugated to a label and formulated in an analogous manner with the antibody formulations described above.

The following examples are offered by way of illustration, not by limitation.

II -r

A

WO 89/09622 Const 1.

S

(I

I

15 S 20 ruction of PCT/US89/01578 18

EXPERIMENTAL

Plasmids Construction began with the plasmid pKcatH described in the literature (Garcia, J.V. et al., Nature 332:383-385 (1986); Fig. This plasmid has the following parts; clockwise: a. A 700 bp fragment containing the mouse immunoglobulin heavy chain enhancer (EH in the Fig.

3).

b. An 1100 base pair (bp) fragment from the mouse immunoglobulin light chain kappa gene MOPC 41, containing its promoter. There is a Bgl II site bp after the transcription startsite of this promoter.

c. The bacterial CAT gene (800 bp).

d. A splice and polyadenylation signal from the animal virus SV40 (850 bp).

e. Another part of SV40 containing its origin of replication (700 bp); f. Part of the plasmid pBR322, extending from the Sph I site to the Eco R1 site of the plasmid pML1 (Lusky, M and Botchan, M. Nature 293:79-81; 2300 bp) including the Amp gene and origin of replication.

2. pKcatH was cut with Bgl II, and the ends filled in with Klenow polymerase. An Xba I linker, having the sequence GCTCTAGAGC, was inserted at the filled-in Bgl II site.

The resulting plasmid is called pKcatH Xba.

3. pKcatH Xba was cut partially with Xho II, run on an agarose gel, and full-length linear plasmid plasmid cut exactly once with Xho II) was isolated. The DNA was cut with Bam HI, run on a gel and the 5600 bp fragment isolated. Because of its size, this fragment had to extend from the Bam site to the Xho II site at the end of the CAT gene (Fig. The fragment was WO 89/09622 PCT/US89/01578 19 ligated by itself. The resulting plasmid is called pKcatH Xba BX. It is similar to pKcatH but has an Xba I site in place of the Bgl II site and is missing the Xho II Bam HI SV40 fragment (Fig. 4).

4. pKcatH Xba BX was cut partially with Eco RI, run on an agarose gel, and full-length linear plasmid was isolated. This DNA was cut with Bam HI, and the 2600 bp fragment isolated. This fragment extended from the Eco R1 site before E H to the Bam HI site. The plasmid pSV2gpt, described in the literature (Mulligan, R.C. and Berg, Proc. Nat. Acad. Sci. USA 78:2072-2976 (1980)), was cut with Eco R1 and Bam HI and the large Eco RI Bam HI fragment ligated to the Eco RI Bam HI fragment from pKcatH Xba BX. The resulting plasmid is called pSV2gpt E, K, (Fig. The plasmid pSV2neo (Southern, P.J. and Berg, P. J. Mol.

App. Genet. 1:327-341 (1982)), was cut with Eco RI and Bam HI, and the large Eco RI Bam HI fragment ligated to the same Eco RI Bam HI fragment from pKcatH Xba BX used in The resulting plasmid is called pSV2neo EH c, which is like pSV2gpt EH x, but has the Neo gene in place of the Gpt gene.

6. pSV2gpt E, was cut with Xba I and Bam HI and the ends filled in with Klenow polymerase. A fragment of the cloned human K constant segment gene (Hieter, P.A.

et al., Cell 22:197-207 (1980)) was purified, extending from a Hind III site 336 bp before the coding region, to an Xba I site about 800 bp beyond the coding region, and the ends filled in. The two fragments were ligated together, and a plasmid selected in which the Hind III site of the second fragment was joined to the Xba I site and the Xba I site of the second fragment to the Bam HI site. Because of the sequences of these sites, this recreated an Xba I site and a Bam HI site. The new plasmid is called pVcl (Fig. 6).

WO 89/09622 PCT/US89/01578 WO 89/09622 7. An Xba I fragment containing the VJ region of the cloned anti-Tac light chain gene was prepared by in vitro mutagenesis (see, below). pVxl was cut with Xba I, treated with phosphatase, and ligated with the Xba I fragment. A plasmid was selected in which the VJ region had the same orientation as the following C region, and called pLTAC2 (Fig. 7).

8. pSV2neo E x was cut with Xba I and Bam HI and the ends filled in with Klenow polymerase. A 2800 bp fragment containing the human Cyl gene was purified from the phage HG3A (Ellison, J.W. et al., Nucleic Acids Res.

10:4071-4079 (1982)), extending from a Hind III site 210 bp before the CH1 exon to a Pvu II site about 1100 bp after the CH3 exon, and the ends filled in. The two fragments were ligated together, and a plasmid selected in which the Hind III site of the second fragment was joined to the Xb;i I site and the Pvu II site of the second fragment to the Barn HI site. Because of the sequences of these sites, this recreated an Xba I site and a Bam HI site. The new plasmid is called pVl1neo (Fig. 8).

9. pSV2neo EH K was cut with Xba I and Bam HI and the ends filled in with Klenow polymerase. A 3600 bp fragment containing the human C-3 gene (Takahashi, N. et al., Cell 29:671-679 (1982)) was purified extending from a Hind III site 210 bp before the CH1 exon to a Pvu II site about 1100 bp after the CH3 exon, and the ends filled in. The two fragments were ligated together, and S 30 a plasmid selected in which the Hind III site of the second fragment was joined to the Xba I site and the Pvu II site of the second fragment to the Bam HI site.

Because of the sequences of these sites, this recreated an Xba I site and a Bam HI site. The new plasmid is called pV-3neo and is identical to pVylneo except it has the C-3 gene instead of C71.

r WO 89/09622 PCT/US89/01578 21 An Xba I fragment containing the VJ region of the cloned anti-Tac heavy china gene was prepared by in vitro mutagenesis (see, below). pVylneo was cut with Xba I, treated with phosphatase, and ligated with the Xba I fragment. A plasmid was selected in which the VJ region had the same orientation as the following C region, and called pHTAC.

11. Three additional plasmids were prepared respectively from pVylneo, pV73neo and pHTAC, called respectively pVyl, pVy3 and pGTAC1. In each case, the original plasmid was cut with Hind III and Bam HI, and the large Hind III Bam HI fragment purified. The plasmid pXBohph containing the Hyg gene (Blochlinger, K. and Diggelmann, H. Mol. Cell. Biol. 4:2929-2931 (1984)) was cut with Hind III and Bam HI and a 1600 bp fragment containing the Hyg gene purified. The fragments from the original plasmids were each ligated to the pXbohph fragment. pVyl is shown in Fig. 9 and pGTAC1 in Fig.

12. pHTAC was cut with Xba I and the small Xba I fragment, containing the heavy VJ region, purified. pVy3 was cut with Xba I, treated with phosphatase, and ligated with the small Xba I fragment. A plasmid was selected in which the VJ region had the same orientation as the following C region, and called pGTAC3. It is similar to pGTAC1, but has the Cy3 region instead of the Cyl region.

c rl

-JI

-22- Cloninq of Light and Heavy Chain cDNA mRNA was extracted from approximately 10-8 ascites cells of the anti-Tac hybridoma by the guanidium isothiocyanate method followed with poly A selection on Hybond-mAP paper (Amersham). Anti-Tac hybridoma may be obtained by the public from the authors of the Uchiyama et al (1981) paper cited at page 2. cDNA was prepared by the method of Gubler and Hoffman (Gubler, U. and Hoffman, B.J.

Gene 25:263-269 (1983)), treated with Eco Ri methylase, ligated to Eco RI linkers, cloned into IgtlO arms (Promega Biocec), packaged (Strategene packaging extract) and plated on C600Hfl cells. Oligonucleotides respectively 43 and 37 nucleotides long that hybridizes to the 5' ends of the mouse CK and Cy2 segments were synthesized (Applied Biosystems Model 380B DNA synthesizer). The oligonucleotides were 5' end-labelled and used to screen the plaques (Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, (1982), which is incorporated herein by reference).

Approximately 10,000 plaques were screened with the oligonucleotide probes. About 100 plaques were positive for the K probe and 40 for the y probe.

The cDNA inserts from 4 K positive and 4 y2a positive phage were subcloned into the Eco R1 sites of 25 pUC19 and M13mpl9. Partial sequencing by the dideoxy method (Sanger, R. et al.,Proc. Nat. Acad. Sci. USA 74:5463-5467 (1977)) showed that two of the K isolates had one sequence, and the other two had another sequence. In one pair, a K V gene segment was joined to the JK2 segment 30 out of its reading frame. In addition, the conserved Cys at position 23 was absent in this V segment, and the sequences of the two isolates differed slightly.

Presumably, these clones were the result of an aberrant joining event in one K allele, which continued to undergo somatic mutation after the formation of the hybridoma.

S920827,GSRLET084,35445.SUB,22

J

I 2 -22A- The VJ seynients of the other pair of K clones were sequences completely and were identical (Fig. 1).

This light chain uses the JK5 segment and contains all the amino acid residues conserved in mouse K light chains (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, particularly page. 45 et seq, all of which is incorporated 920827,GSRLETD84,35445.SUB,23 II _I _I WO 89/09622 PCT/US89/01578 herein by reference).

Partial sequencing of the four 72a clones showed they were from the same gene. The VJ segments of two were sequenced completely and had no differences. This heavy chain (Fig. 2) uses the JH2 segment and is of subgroup II (Kabat, E.A. et al., supra, at pgs. 121-127). The sequence is somewhat unusual because it contains no detectable D segment and has an N region containing 10 Gs. As both alleles of the x light chain were accounted for and only one heavy chain sequence was detected, we tentatively assigned these sequences to the anti-Tac antibody genes.

Preparation of the Chimeric Genes Plasmid vectors were prepared for the construction and expression of chimeric light and heavy chain genes as described above. The -lasmid pVcl (Fig. 6) contains the human c genomic C segment, including 336 bp of the preceding intron and the poly A signal. It also contains a strong promoter sequence from the MOPC 41 K gene, and the heavy chain enhancer sequence. There is a unique Xba I site between the promoter and the C intron. The plasmid also contains the gpt gene for selection. Two other very similar plasmids were prepared by using the human 71 and 73 C regions in place of the c C region. In each case, the region inserted between the Xba I and Bam HI sites extended form about 210 bp 5' of the CH1 exon to about 1100 bp 3' of the CH3 exon. In addition, the gpt gene was replaced with the hyg gene to confer resistance to hygromycin.

.Our strategy was to insert the VJ region from the anti-Tac x cDNA, followed by a splice donor signal, at the Xba I site of pVcl. Doing so would create a chimeric K gene, with a synthetic intron between the mouse VJ and human Cn segments. For this purpose, we devised a form of primerdirected mutagenesis (Fig. 11). The n Eco R1 cDNA clone was moved into a variant of M13mpll, in which the Xba 1 and Eco R1 sites in the polylinker were abuting. An oligonucleotide was synthesized, of which the first 22 residues hybridized to the last 22 bp of the JK5 segment. The next 16 nucleotides WO 89/09622 PCT/US89/01578 WO 89/09622 24 were the same as the sequence that follows JK5 in mouse genomic DNA, and therefore included a splice donor signal.

The final nucleotides of the oligo consisted of an Xba I site followed by a short irrelevant sequence.

This oligonucleotide was hybridized to the M13 phage DNA containing the x cDNA and extended with Klenow polymerase (Fig. The DNA was denatured and hybridized to I a "reverse primer," which represents M13 DNA 5' to the cDNA insert. The reverse primer was extended, and the DNA cut with Xba I. The Xba I fragment consisting of the extended VJ segment of the cDNA was purified, and cloned in the correction orientation into the Xba I site of pVcl to obtain the plasmid pLTAC2 (Fig. Hence, the final chimeric K gene has a VJ-C intron, of which the first 14 bp and last 209 bp are respectively the same as in mouse and human .genomic DNA, as verified by direct sequencing of the construct. The gene is transcribed from a n promoter, stimulated by the heavy chain enhancer. Based on results obtained with deleted introns, we expected that the intron would be correctly spliced from the transcribed RNA.

In an analogous manner, the VJ region from the Santi-Tac -2a heavy chain cDNA, followed by a splice donor signal, was inserted into the Xba I site of pV7-neo. The resulting plasmid pHTAC contains a chimeric heavy chain gene, with a synthetic intron between the mouse VJ and human C1l segments. The additional plasmids pGTAC1 and pGTAC3, containing the Hyg gene and chimeric heavy genes with the human Cyl Cy3 constant regions respectively, were constructed as described above (Fig. i _I WO 89/09622 PCT/US89/01578 Details of Insertion of VJ cDNA Regions Into Plasmids RF DNA of the phage M13mpll was cut with Eco R1 and Xba I, the ends were filled-in, and the DNA ligated was ligated and transformed into JM101 cells. A plaque was picked and the DNA was sequenced to verify that the ends of the DNA had joined correctly, recreating the Eco R1 and Xba I sites with the intervening DNA segment deleted. This phage is designated M13mpllD. Eco R1 fragments containing the anti-Tac light and heavy chain cDNAs were separately cloned into the Eco R1 site of M13mpll, so that their 5' ends abuted the Xba I site. The resulting phage are respectively denoted M13mpllL and M13mpllH.

The following 48-nucleotide long primer was synthesized and gel-purified: CCAGAATTCTAGAAAAGTGTACTTACGTTTCAGCTCCAGCTTGGTCCC. From the 3' end, the first 22 residues of the primer are the same as the last 22 bp of the JK5 segment (non-coding strand). The next 16 nuc'.eotides are the same as the sequence that follows in mouse genomic DNA and therefore includes a splice donor signal (abbreviated SD in Fig. 11). The final nucleotides of the oligo consist of an Xba I site followed by a short irrelevant sequence. Approximately 1 ug of singlestranded M13mpllL DNA was mixed with 50 ng of primer in 35 ul of 10 mM Tris, pH 7.4, 60 mM NaCl, 10 mM MgC1 2 and incubated for 15 min at 50'C and then 15 min at 23'C. 4 ul of a solution of 200 uM each dNTP was added, together with Klenow polymerase. The solution was incubated for 30 minutes at 37'C. 50 ng of the "reverse primer" AACAGCTATGACCATG (New England Biolabs), which can hybridize to the newly synthesized strand upstream of the Xba I site (Fig. 11), was added. The solution was incubated at 95'C for 3 min and put on ice. An additional 4 ul of 200 uM each dNTP and 5u Klenow polymerase was added, and the solution incubated for minutes at 37'C. The solution was extracted with phenolchloroform, precipitated with ethanol, resuspended, and digested with 20u Xba I. The digested DNA was run on a 4% polyacrylamide gel and visualized with ethidium bromide. In addition to high molecular weight DNA, an approximately 400 I_ I_ WO 89/09622 PCT/US89/01578 26 bp fragment was visible, corresponding to the VJ region of the light chain cDNA with a "tail" (Fig. 11). The fragment was cloned directly into the Xba I site of pVl in the correct orientation.

The following 50-nucleotide long primer was synthesized and gel-purified: CCAGAATTCTAGAGGTTTTAAGGACTCACCTGAGGAGACTGTGAGAGTGG. From the 3' end, the first. 21 residues of the primer are the same as the last 21 Yp of the JH2 segment (non-coding strand). The next 19 nucleotides are the same as the sequence that follows JH2 in mouse genomic DNA and therefore includes a splice .donor signal. The final nucleotides of the oligo consist of an Xba I site followed by a short irrelevant sequence. This primer was hybridized to M13mpllH DNA following the same protocol as above, in order to synthesize a fragment containing the VJ region of the heavy chain cDNA. The fragment was cloned directly into the Xba I site of pVylneo in the correct orientation.

Chimeric Antibody Specificity CR2-2 and CEM cells are human T cell lines that are respectively positive and negative for surface expression of the IL2 receptor (IL2R). Whole CR2-2 cells were used to demonstrate binding specificity of the chimeric antibody in an ELISA assay, and CEM cells were used as a negative control.

Antibody to be tested was prepared in several ways.

Anti-Tac monoclonal antibody, supplied by T. Waldmann, was purified from mouse ascites by passage through a DEAE-dextran column. The cell line L40H4, created by transfecting cells with the chimeric light and gamma 1 heavy chain plasmids pLTAC2 and pGTAC1 (described above), was injected into mice to form an ascites. From 3 mls of ascites fluid, 650 gg of chimeric gamma 1 antibody was purified by passage through a column of Baker Abx and a size exclusion column.

Also, to detect antibody production in tissue culture, 106 cells each of L40H4 and the cell lines 51.3 and L40H2 (which had been created by transfecting SP2/0 with pLTAC2 and the I WO 89/09622 PCT/US89/01578 27 gamma 3 heavy chain plasmid pGTAC3 described before) and the non-producing line Sp2/0 itself, were plated in 1 ml of DME medium each. The media supernatent, containing any secreted chimeric antibodies, was harvested after 24 hr. and used directly in the assay, Cultured cells of each type were washed in FACS buffer BSA, .01% sodium azide in Dulbecco's phosphate buffered saline). 2 x 106 cells were mixed with either purified antibody in 20 pl FACS buffer or 20 1l media supernatent from the transfected cell lines, and incubated on ice for 2 hr. The cells were washed 3 times with 1 ul of PACS buffer (being collected by brief centrifugation after each wash). The cells that had been incubated with anti-Tac antibody (or no antibody as a control) were mixed with 0.5 p1 peroxidase-conjugated goat anti-mouse antibody (Fab)' fragment (Tago Immunologicals, Burlingame, California) in .l FACS buffer, and the cells that had been incubated with chimeric antibody were mixed with 0.5 pl peroxidaseconjugated goat anti-human gamma chain (Fab)' 2 fragment (Tago) in 20 p1 FACS buffer. The cells were incubated for 30 min on ice and then washed 3 times with FACS buffer. They were then mixed with 100 gi peroxidase development solution and incubated for 5 min at room temperature. The cells were spun out and the si:ternatents were transferred to a 96-well plate and the OD's determined in an ELISA reader. The OD's at 414 nm for the cells treated with each antibody are given in Table 1.

As expected the anti-Tac antibody itself bound to the IL2R+ CR2-2 cells but not to the IL2R- CEM cells. An equal amount of purified gamma 1 chimeric antibody gave an equivalent amount of binding to the CR2-2 cells as the anti- Tac, and also failed to bind to the CEM cells. All the supernatants from both the chimeric antibody producing cells bound to the CR2-2 cells. As additional negative controls, the 51.3 supernatant did not bind to the CEM cells, and S supernatant from the parental SP2/0 cells did not bind the CR2-2 cells.

In combination with the derivation of the light and I i 1 J i _u i-l l ii. li- i i i-iil- iii~ r~i WO 89/09622 PCT/US89/01578 28 heavy chain genes from the anti-Tac hybridoma, these results show that the chimeric antibodies retained specificity for the human IL2R.

7 PCT/US89/01 578 WO 89/09622 29 TABLE 1 Whole-Cell ELISA Assay of Chimeric Antibodies CR2-2 cells (IL2R CEM cells (IL2R Mouse Gamma 2a LO none .037 anti-Tac (100ng) .679 .024 Human Gamma 1 none .084 purified ascites (100ng) .641 .044 L40H4 (20 4l sup.) .481 Human Gamma 3 51.3 (20 g1 sup.) .381 .038 L40H2 (20 Al sup.) .932 -i WO 89/09622 PCT/US89/01578 To quantitate the rate of secretion of the chimeric antibodies, an ELISA assay was performed, using standard methods. The results are presented in Table 2. About 106 cells each of the chimeric gamma 1 secreting line L40HO and the chimeric gamma 3 secreting line 51.3 were plated in 1 ml of DME medium with 5% fetal calf serum, and the supernatents collected after 24 hr. A 96-well plate was coated with goat anti-human antibodies (Tago Immunologicals). Different wells were incubated with increasing known amounts of gamma 1 chimeric antibody (purified from ascites) and with 1 gl of the chimeric antibody supernatents. Briefly, the plate was washed, incubated with peroxidase-conjugated goat anti-human gamma chain antibody, washed again, incubated with peroxidase developing solution and the OD's at 414 nm determined in an ELISA reader. By comparison with the standard curve from the purified antibody, the media supernatants contained respectively about 8 and 7 ng per that is the cells secreted 8 and 7 gg antibody per 106 cells per 24 hr.

o!* WO 89/09622 PTU8/17 PCF/US89/01578 31 TABLE 2 Rate of Chimneric Antibody Secretion OD414 Purified gamma 1 chimeric 3 ng 4 ng 6 ng 8 ng .426 .535 .734 1. 004 1.014 .845 L40H4 (1 Al1 sup.) 51.3 (1 4l sup.) r r WO 89/09622 PCT/US89/01578 32 The mixed lymphocyte reaction is a model for transplant rejection and was used to analyze the effectiveness of the chimeric anti-Tac antibodies. The experiments shown in Table 3 below were performed by standard methods (see, Strong, D.M. et al., In Vitro Stimulation of Murine Spleen Cells Using a Microculture System and a Multiple Automated Sample Harvester, J. Immunol. Meth., 2:279 (1973)). Briefly, human peripheral blood lymphocytes from two unrelated human donors were purified. The lymphocytes from the two donors were incubated in wells of a 96-well plate, either separately, mixed together, or mixed together with 1 gg/ml of the indicated antibody added each day. When mixed together, the T-cells recognize each other as foreign, as T cells recognize an organ transplant as foreign. They therefore proliferate, as measured by uptake of 3H-labeled thymidine after three days. The numbers in Table 3 are the average thymidine uptake in cpm from triplicate wells the standard deviation. Addition of either the original anti-Tac antibody or either of the chimeric antibodies strongly inhibits proliferation of the cells (percent inhibition shown in parentheses), by binding to the IL2R. This prevents IL2 from binding to the IL2R, which is required for T-cell proliferation. These experiments show that the chimeric antibody can be used to reduce the response of T-cells to foreign cells and may therefore have medical applications in preventing transplant rejection.

WO 89/096 115 22 PCr/US89/O1 578 22 33 TABLE 3 Anti-IL2R Antibody Inhibition of Mixed Lymphocyte Reaction Exp. 1 Donor A 510 292 Donor B 901 38 A B 11740 1593 A B anti-Tac 4262 346 (73%) A B Human Gamma 1 4126 370 (74%) A B+Human Gamma 3- ExrP. 2 151 712 29 19806 2034 7311 461 (66%) 6721 981 (69%) 6423 423 (71%)

I

"Li Li WO 89/09622 PCT/US89/01578 34 From the foregoing, it will be appreciated that the chimeric antibodies of the present invention offer numerous advantages of other human IL-2 receptor-specific antibodies. In comparison to anti-Tac mouse monoclonal antibodies, the present human chimeric antibodies can be more economically produced and contain substantially less foreign amino acid sequences. This reduced likelihood of antigenicity after injection into a human patient represents a significant therapeutic improvement.

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that II certain changes and modifications may be practiced within the scope of the appended claims.

is

Claims (29)

1. A method of treating T-cell mediated disorders in a human patient, said method comprising administering to said patient a therapeutically effective dose of a mouse variable/human constant region chimeric antibody composition specifically reactive with IL-2 receptors on human T-cells.

2. A method according to Claim 1, wherein the mouse variable region comprises the entire variable region from heavy and light chains of an anti-Tac monoclonal antibody as produced by the cell line designated A.T.C.C. Accession No. CRL 9688.

3. A method according to Claim 1, wherein at least one of the mouse variable regions is joined with a naturally-associated mouse J segment.

4. A method according to Claim 1, wherein the human light chain constant region comprises a i chain constant region.

A method according to Claim 1, wherein the human light chain constant region comprises a 7 or 7 chain constant region.

6. A method according to Claim 1, wherein the I composition comprises the chimeric antibody complexed with Sa cytotoxic agent.

7. A method according to Claimk/, wherein the cytotoxic agent is a ribosomal inhibiting protein, a radionuclide or cytotoxic agent active at cell surfaces. 1 WO 89/09622 PCT/US89/01578 WO 89/09622 36

8. A chimeric antibody capable of binding to a human IL-2 receptor epitope, said antibody having two pairs of light chain/heavy chain complexes, wherein at least one pair has chains comprising mouse variable region and human constant region segments.

9. A chimeric antibody according to Claim 8, wherein the mouse variable region is adjacent to a naturally associated J segment.

10. A chimeric antibody according to Claim 8, which inhibits the binding of monoclonal antibodies secreted by the cell line designated A.T.C.C. Accession No. CRL 9688.

11. A chimeric antibody according to Claim wherein at least one variable region comprises the amino I acid sequence of a variable region of the anti-Tac Imonoclonal antibody.

12. A chimeric antibody according to Claim 8, which was produced in a myeloma or hybridoma cell. V

13. A chimeric antibody according to Claim 8, which was expressed in a myeloma cell from a transfected DNA sequence comprising a mouse cDNA segment joined to a human genomic DNA segment.

14. A chimeric antibody according to Claim 8 complexed with a cytotoxic agent or signal agent.

15. A heavy immunoglobulin chain comprising a human heavy chain constant region and a variable chain region which is substantially identical to a monoclonal antibody heavy chain variable region secrsted by the cell line designated A.T.C.C. Accession No. CRL 9688. :I ,rr r Ir Cr'.Of, /.nl r'0 WO 89/09622 Yi/ U oY/ui /o 37

16. A heavy immunoglobulin chain according to Claim 15, wherein the variable and constant region are joined by a mouse J segment.

17. A light immunoglobulin chain comprising a human light chain constant region and a variable chain region which is substantially identical to a monoclonal antibody light chain variable region secreted by the cell line designated A.T.C.C. Accession No. CRL 9688.

18. A polynucleotide molecule comprising a first sequence coding for a human immunoglobulin constant region and a second sequence coding for a mouse immunoglobulin variable region, wherein said second sequence encodes substantially for one of the amino acid sequences of Figures 1 or 2.

19. A cell line transfected with a polynucleotide of Claim 18.

20. The cell line designated A.T.C.C. Accession No. CRL 9688.

21. A DNA segment encoding a portion of an immunoglobulin light chain, said segment comprising at least about 30 contiguous nucleotides from the sequence of Figure 1.

22. A DNA segment according to Claim 21, wherein the segment encodes a full length light chain variable region substantially homologous to the sequence in Figure 1.

23. A DNA segment encoding a portion of an immunoglobulin heavy chain, said segment comprising at least about 30 contiguous nucleotides from the sequence of Figure 2. WO 89/09622 PCT/US89/01578 38

24. A DNA segment according to Claim 23, wherein the segment encodes a full length heavy chain variable region substantially homologous to the sequence in Figure 2.

25. An expression vector comprising a heterologous promoter operably linked to a DNA segment according to Claim 21 or 23.

26. An immortalized cell line transformed with an expression vector according to Claim

27. A protein composition comprising at least about ten contiguous amino acids from the protein sequence of Figure 1 or Figure 2 fused to a heterologous polypeptide.

28. A protein composition according to Claim 27, wherein the heterologous polypeptide is an immunoglobulin constant region.

29. A protein composition according to Claim 27, wherein the protein is glycosylated. A recombinantly produced antibody exhibiting substantially the same antigen binding profile as an immunoglobulin secreted by a cell line designated A.T.C.C. Accession No. CRL 9688. WO 89/09622 PCr/US89/O 1578 1/6 ~20 30 40 50 AAATTGAAAGA CA AA AT GATTTTCAA GTGCGAG ATTTT CAGOTT OCT GCTAATCGAG TGCC Met H-isPheG InVa IGln 11ePheSerPheLeu LeullIe SerA Ia 80 90 100 110 120 TOAGTCATAATG TCCAGAGGAGAAA TTGTTCGTCACCGAGTCTC CAGOAA TCATG TC TGCA SerVa 11IeMet SerArqGIyG InI IeVal LeuThrGI nSer ProAi alle~et SerAlo 130 140 150 160 170 180 TCOTC CAG GGGAGAAG GT CACCATAACGCTGCAG TGCCGAGGTCAA GTA TAAGTTAC AT GCAC Se r Pr oG ly GIlu Lys Va IT h r IIeT hr Gys S erA Ia Se rSe rS e r IIe S er Ty rMe t H is 190 200 210 220 230 240 TGGTTC CAGCAGAAG CCAGGCACTTGCCAAAC TGTGGATT TATA CCACAT CAA CCTG Tr pPhe G I nGI n LysP roGlIyTh rS erP ro LysL eu TrplI I eTy rT hrT hrS erAsn Le u 250 260 270 280 290 300 GCTTCTGGAGTCGCTGCT GGCTTCAGTGGGAGTGGATCTGGGACCTGTTACTCTCTGAA AlIoaSerG Iy Va IProA Ia Arg Ph eSer G Iy~er GlySer G IyTh rSe rTy rSe rLe uTh r 310 320 330 340 350 360 AT CAGGGGAAT GGA GGCT GAA GAT GCTGOCACTTATTACTGCCGATCAAA GGAGTACTTAC I Ie S erAr gMe t G I uA I a G Iu Asp A Ia A I Th rTy rTy r CysH is G I n Ar gSe rThr Ty r 370 380 390 400 COACT CAC GTT GG TT CTGG GAGCAA G TG GAGCCTG AA AC ProLe uTh rPh eGlySe r Gly Thr Lys Lou GIu LouLys FIG..J. .1 o~ 89/0' )9622 CGATO CTOC C TT C CTGT' u Ph e L e uP ITUAACTIGG aG I uLeuA TA OTAG CT eThrSerT 2 ATAT ATTA yTyrII eA 3 ATTGACTG rLeuThrA 3 GGA TOT G uAspSerA 4 AACCACTC yThrThrL p. Ig Promoter p BR32 2 FIG.au. SUEZ71TUTE SNP-27- PCT/US89/O 1578 WO089/09622 2/6 20 30 40 50 GOAT CGCTC CAGAG A CACT GAAA ACTCOTGACT CA CAATG GAAAGG CA GTGG ATOTT TOT Met GlIuArgH isT rpIIeP he Le 80 90 100 110 120 CTTCCTGTTTTCAGTAACTGCAGGTGTGCACTCCAGGTCCAGCTTCAGCAGTCTGGGGC u Ph eL e uPh eSe r VI Th rA Ia GlIyVaINi s S er GInV alIG In L euG In GlIn 5e rGIy A I 130 140 150 160 170 180 TGAACTGGGAAAAGCTGGGGCOTCOAGTGAAGATGTCGTGGAAG GCTTCTGGCTAOACCTT oGlu LeuAloLysProGlyAloSerVolLyst~etSerOysLysAloSerGlyTyrThrPh 190 200 210 220 230 240 TAOTAGCTACAGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGG eThrSerTyrArg~etH i sTrp Vol LysGlInArgProG IyG In G-1yLeuG.I uTrp II eG I 250 260 270 280 290 300 ATATATTAAT CCTAGCACTGGGTATACTGAATACAATCAGAAGTTCAAGGACAAGGCCAC yTy rIlIeA s nPr o 5erT h rG IyTyrT hr G IuTy rAs nG In Lys P heLysA s pLys A IaTh 310 320 330 340 350 360 ATTGACTGGAGACAAATCCTGCAG CACAGCCTACATGGAACTGAGCA GCCTGAOATTTGA rLeuThrAl aAsp LysSe rSerSerTh rAlIaTyr~etGlInL euSerSe rLeu ThrPh eGl 370 380 390 400 410 420 GGAC TCTGCAGTCTATTACTGTGCAAGAGGGGGGGGGGTCTTTGACTACTGGGGCCAAGG uAs p SerAl o VatI TyrTyr CysAla ArgGI yG ly G y Va I Ph eAsp Ty rTrpG IlyG I G I 430 440 AACGACTCTCACAGTOTCTCTAG ylhrThrLeuThrV I SerSer FIG..2. EH Ig Promoter pBR322 FIG..A SUBSTITUTE SHEET I WO 89/09622 PCT/US89/O 1578 p R3 22 Promoter )Got Ig Promoter pBR322 FIG.J. SUSSTITUT- r -E- -1 WO 89/09622 PCr/US89/01578 Ig Promoter pBR322 Ft pBR322 fg Promoter FIG...8. SUESTCT1JTt SHEZT I1 PCI', US89/01578 WO 89/09622 Ig Pro mater pBR322 FIG..9 1g Promoter pBR322 SY41 VHJ ct 1 SUBTITUTE SHEET WO 89/09622 PCr/US89/01578 primer Xba I V Extend -x0 Denature. Hybridize rev prim er 0 1 Extend and Cutj Xb I V J FIG.. 11. SUBTTLITE S11E:ET i 1I.---P6CI-II-^- INTERNATIONAL SEARCH REPORT ?blwll C/U S89/oi578 I. CLASSIFICATION OF JECT MATTER (it several ctass ctetSiolics aQri, inoic8e all) 7 According to International Patent Classirication ilPCt or !o ootn National Cassification ano [PC IPC(4): A6LK 39/395; C2N 15/00; C12N 5/00; C07K 13/00; C07K 15/04. 424/89-21, 42/858: 530/387: 435/6R ,4-19/172.2- 4 5/240- 27- -3/27 II. FIELDS SEARCHED Minimum Documento.tion Sarcnea Classifiction System Clssifiction Symools U.S. 424/85.8; 424/85-91; 530/387; 530/388; 530/391; 530/808; 530/821; 530/825; 435/68; 43570; 435/172.2; 435/172.3; 435/240.27; 435/240.2; 536/27; 935/32; 935/15. Documentation Searched other than Minimum Documentation to the Etent that such Documents are tncluded in the Fields Searched 8 rrputer database searches on APS, Biosis. Ill. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category Citation ot Document, 11 with indication, wnere aporooriate, ot the relevant passages 12 Relevant to Claim No. 1 Y US, A, 4,664,911 (UHR et al), 12 May 1987 (12.05.87), 6,7,14 see columns 2 to 6. Y EP, A, 0125023 (GQNT& i et al), 14 November 1984 11-30 (14.11.84), see pages 16-25, 28, 29, and 31 to 43. Y EP,A, 0173494 (THE BOARD OF TRUSTEES OF THE LELAND 1-30 STANFORD JUNIOR UNIVERSITY), 05 March 1986 (05.03.86), see pages 4-6 and 18-20. Y EP,A,0184187 (TEIJIN LIMITED), 11 June 1986 (11.06.861 1-30 see pages 8 to 23. Y JWO,A, 86/01533 (.JLLTECi LIMITED), 13 March 1986 1-30 (13.03.86), see pages 3-8 and 19-22. Y The Journal of Immunology, Vol.126, No.4, 1-30 issued April 1981 (USA), UCHIYAMA et al, "A Monoclonal Antibody (ANTI-Tac) Reactive with Activated and Functionally Mature Human T Cells," see pages 1393 to 1397. Special categories of cited documents:0 later document published alter the international filing dale or priority date and not in conflict With the aoolication but document defining the general state of the art Nnich is not cited to understand the principle or theory unoerlying the considered to be ot particular relevance invention earlier document but published on or alter the international document of particular relevance: the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claimts) or involve an inventive step which is cited to establish the publication date of another document of Particular relevance: the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, eshibiion or document is comoined with one or more other such docu- other means ments, such combination being Obvious to a person skilled document published Prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family V. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report July 1989 27JUL 1989 I! i Li Intern ations ISA/US if Searching Authority Signature of Authorized Ofyri Jeff P. Kushan klevll i rrrl Fom PCTWlA,10 (ucom ~lrc (Pav. 1 i7