IE83939B1 - Redirection of cellular immunity by receptor chimeras - Google Patents

Description

PATENT SPECIFICATION (11) 83939 (21) Application No. 1992/0716 (22) Date of Filing of Application: 05/03/1992 (30) Priority Data: (31) ()7/6()5,96l (32) 07/03/1991 (33) United States ofAmerica (US) (45) Specification Published: 13 July 2005 (51) Int. Cl.7 A61K 38/16 , C07K 17/00 , A61K 39/00 ,A6lP 31/00, 35/00, 37/00 //A61P 31/18 (54) Title: Redirection of cellular immunity by receptor chimeras (72) Inventor: CHARLES ROMEO BRIAN SEED (73) Patent Granted to: THE GENERAL HOSPITAL CORPORATION, a corporation organised under the laws of the State of Massachusetts, United States of America, Office of Technology Affairs, Thirteenth Street, Building 149, Suite 1101, Charlestown, Massachusetts 02129, United States of America © Copyright 2005, Government of Ireland PATENTS ACT, 1992 REDIRECTION OF CELLULAR IMMUNITY BY RECEPTOR CHIMERAS THE GENERAL HOSPITAL CORPORATION E LULAR IMMUN B RECEPTOR CHIMERAS Eield of the Invggtign The invention concerns functional T cell receptor I ECTION or Fc receptor chimeras which are capable of redirecting immune system function. More particularly, it concerns the regulation of lymphocytes, macrophages, natural killer cells or granulocytes by the expression in said cells of chimeras which cause the cells to respond to targets recognized by the chimeras. The invention also concerns functional T cell receptor or Fc receptor chimeras which are capable of directing therapeutic cells to specifically recognize and destroy either cells infected with a specific infective agent, the infective agent itself, a tumor cell, or an autoimmune—generated cell. production of T cell receptor or Fc receptor chimeras More particularly, the invention relates to the capable of directing cytotoxic T lymphocytes to specifically recognize and lyse cells expressing HIV envelope proteins. The invention therefore provides a therapy for diseases such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV virus. fiagggrougd of the Invention T cell recognition of antigen through the T cell receptor is the basis of a range of immunological The T cells direct what is called cell- This involves the destruction by phenomena. mediated immunity. cells of the immune system of foreign tissues or infected cells. A variety of T cells exist, including "helper" and "suppressor" cells, which modulate the immune response, and cytotoxic (or "killer") cells, which can kill abnormal cells directly.

A T cell that recognizes and binds a unique antigen displayed on the surface of another cell becomes activated; it can then multiply, and if it is a cytotoxic cell, it can kill the bound cell.

Autoimmune disease is characterized by production of either antibodies that react with host tissue or immune effector T cells that are autoreactive. In some instances, autoantibodies may arise by a normal T- and B- cell response activated by foreign substances or organisms that contain antigens that cross react with similar compounds in body tissues. Examples of clinically relevant autoantibodies are antibodies against acetylcholine receptors in myasthenia gravis; and anti- DNA, anti-erythrocyte, and anti-platelet antibodies in systemic lupus erythematosus.

HIV and Immunopathogenesis In 1984 HIV was shown to be the etiologic agent of AIDS. Since that time the definition of AIDS has been revised a number of times with regard to what criteria should be included in the diagnosis. However, despite the fluctuation in diagnostic parameters, the simple common denominator of AIDS is the infection with HIV and subsequent development of persistent constitutional symptoms and AIDS defining diseases such as a secondary infections, neoplasms, and neurologic disease.

Harrison's gginciplgs of Internal Medicine, 12th ed., McGraw Hill (1991).

HIV is a human retrovirus of the lentivirus group.

The four recognized human retroviruses belong to two distinct groups: the human T lymphotropic (or leukemia) retroviruses, HTLV-1 and HTLV-2, and the human immunodeficiency viruses, HIV-1 and HIV-2. The former are transforming viruses whereas the latter are cytopathic viruses.

HIV-1 has been identified as the most common cause of AIDS throughout the world. sequence homology between HIV has the usual retroviral genes (egg, ggg, and 991) as well as six extra genes involved in the replication and other biologic activities of the virus.

As stated previously, the common denominator of AIDS is a profound immunosuppression, predominantly of cell- mediated immunity. This immune suppression leads to a variety of opportunistic diseases, particularly certain infections and neoplasms.

The main cause of the immune'defect in AIDS, has been identified as a quantitative and qualitative deficiency in the subset of thymus—derived (T) y lymphocytes, the T4 population. This subset of cells is defined phenotypically by the presence of the CD4 surface molecule, which has been demonstrated to be the cellular receptor for HIV. Dalgleish et al., Nature, ;;;:763 (1984). Although the T4 cell is the major cell type infected with HIV, essentially any human cell that expresses the CD4 molecule on its surface is capable of binding to and being infected with HIV.

HIV binds specifically and with high affinity, via a stretch of amino acids in the viral envelope (gp120), to a portion of the V1 region of the CD4 molecule located near its N-terminus. Following binding, the virus fuses with the target cell membrane and is internalized. Once internalized it uses the enzyme reverse transcriptase to transcribe its genomic RNA to DNA, which is integrated into the cellular DNA where it exists for the life or the cell as a "provirus." The provirus may remain latent or be activated to transcribe mRNA and genomic RNA, leading to protein ' synthesis, assembly, new virion formation, and budding of virus from the cell surface. Although the precise mechanism by which the virus induces cell death has not been established, it is felt that the major mechanism is massive viral budding from the cell surface, leading to disruption of the plasma membrane and resulting osmotic disequilibrium.

During the course of the infection, the host organism develops antibodies against viral proteins, including the major envelope glycoproteins gp12o and gp41. progresses, resulting in a lethal immunosuppression Despite this humoral immunity, the disease characterized by multiple opportunistic infections, parasitemia, dementia and death. The failure of the host anti-viral antibodies to arrest the progression of the disease represents one of the most vexing and alarming aspects of the infection, and augurs poorly for vaccination efforts based upon conventional approaches.

Two factors may play a role in the efficacy of the humoral response to immunodeficiency viruses. First, like other RNA viruses (and like retroviruses in particular), the immunodeficiency viruses show a high mutation rate in response to host immune surveillance.

Second, the envelope glycoproteins themselves are heavily glycosylated molecules presenting few epitopes suitable for high affinity antibody binding. The poorly antigenic target which the viral envelope presents, allows the host -5.. little opportunity for restricting viral infection by specific antibody production.

Cells infected by the HIV virus express the gplzo glycoprotein on their surface. Gp120 mediates fusion events among CD4‘ cells via a reaction similar to that by which the virus enters the uninfected cells, leading to the formation of short-lived multinucleated giant cells.

Syncytium formation is dependent on a direct interaction of the gp120 envelope glycoprotein with the CD4 protein.

Dalgleish et al., su ra; Klatzman, D. et al., Nature, ;;;:763 (1984); Mcbougal, J.S. et al., ggigngg, ;;;:382 (1986); Sodroski, J. et al., Natu e, ;;;:470 (1986); Lifson, J.D. et al., Nature, ;;;:725 (1986); Sodroski, J. et al., Nature, ;;;:412 (1986).

Evidence that the CD4-gp120 binding is responsible for viral infection of cells bearing the CD4 antigen includes the finding that a specific complex is formed between gp12O and CD4. other investigators have shown that the cell lines, which were Mcbougal et al., supra. _ 5 - Zettlmeissl et al., pug Cell 3191. 9:347-353 (1990)).

T Cell and Fc Receptors receptor recognition of ligand (Weissman et al., EMBO J., ggigggg, g49:174-177 A 32kDa type I integral membrane homodimer, g unidentified function (Anderson et al., at 0 Recently it has been DC. ca that one of the murine low affinity Fc receptor isoforms Recently it has been shown In drawing parallels between the murine and human low affinity Fc receptor families, however, it has become clear that the human FcR1IIA and C isoforms have no _ 9 - murine counterpart. In part because of this, their function has yet to be defined.

Because humoral agents based on CD4 may have limited utility in vivo, the inventors began to explore the possibility of augmenting cellular immunity to HIV.

As a result they report the preparation of protein chimeras in which the extracellular domain of CD4 is. fused to the transmembrane and/or intracellular domains of T cell and IgG Fc receptor signal transducing elements. cytolytic T cells expressing the chimeras show potent MHC-independent destruction of cellular targets expressing HIV envelope proteins. An extremely important and novel component of this approach has been the identification of single T cell or Fc receptor chains whose aggregation suffices to initiate the cellular response. one particularly useful application of this approach has been the invention of chimeras between CD4 and g, n, or 1 that direct cytolytic T lymphocytes to recognize and kill cells expressing HIV gp120. fiummary Q: the Invention Although native T cell and Fc receptors are or can be highly complicated multimeric structures not lending themselves to convenient manipulation, the present invention demonstrates the feasibility of creating chimeras between the intracellular domain of any of a variety of molecules which are capable of fulfilling the task of target recognition. In particular, the formation of chimeras consisting of an extracellular portion which is capable of specifically recognizing and binding an agent or an cell wherein the extracellular portion comprises an HIV envelope—binding portion of CD4, or a functional HIV envelope—binding derivative thereof, joined to an intracellular or transmembrane portion which is capable of signalling a therapeutic cell to destroy a receptor—bound agent or a receptor—bound cell, wherein the intra—cellular portion or the transmembrane portion is a signal—transducing portion of CD3 delta or T3 gamma, Fggll, or a B cell receptor protein, or a functional derivative thereof allows the target recognition potential of an immune system cell to be specifically redirected to the antigen recognized by the extracellular antibody portion. - 10 _ Thus with an antibody portion capable of recognizing some determinant on the surface of a pathogen, immune system cells armed with the chimera would respond to the presence of the pathogen with the effector program appropriate to their lineage, e.g., helper T lymphocytes would respond by cytotoxic activity against the target, and B lymphocytes would be activated to synthesize antibody. their effector programs, including cytokine release, Macrophages and granulocytes would carry out phagocytosis, and reactive oxygen generation. Similarly, with an antibody portion capable of recognizing tumor cells, the immune system response to the tumor would be beneficially elevated. with an antibody capable of recognizing immune cells having an inappropriate reactivity with self determinants, the autoreactive cells Although these examples draw on the use of antibody chimeras as a could be selectively targeted for destruction. convenient expository tool, the invention is not limited in scope to antibody chimeras, and indeed, the use of specific nonantibody extracellular domains may have important advantages. For example with an extracellular portion that is the receptor for a virus, bacterium, or parasite, cells armed with the chimeras would specifically target cells expressing the viral, bacterial or parasitic determinants. The advantage of this approach over the use of antibodies is that the native receptor for pathogen may have uniquely high selectivity or affinity for the pathogen, allowing a greater degree of precision in the resulting immune response.

Similarly, to delete immune system cells which inappropriately react with a self antigen, it may suffice to join the antigen (either as an intact protein, in the case of B cell depletion therapies, or as MHC complex, in the case of T cell depletion therapies) to intracellular zeta, eta or gamma chains, and thereby affect the -11.- specific targeting of the cells inappropriately responding to self determinants.

Another use of the chimeras is the control of cell populations in vivo subsequent to other forms of genetic engineering. For example, the use of tumor infiltrating lymphocytes or natural killer cells to carry cytotoxic principles to the site of tumors has been proposed. The present invention provides a convenient means to regulate the numbers and activity of such lymphocytes and cells without removing them from the body of the patient for amplification in vitro. Thus, because the intracellular domains of the chimeric receptors mediate the proliferative responses of the cells, the coordination of the extracellular domains by a variety of aggregating stimuli specific for the extracellular domains (e.g., an antibody specific for the extracellular domain) will result in proliferation of the cells bearing the chimeras.

At present the most convenient method for delivery of the chimeras to immune system cells is through some form of genetic therapy. However reconstituting immune system cells with chimeric receptors by mixture of the cells with suitably solubilized purified chimeric protein would also result in the formation of an engineered cell population capable of responding to the targets recognized by the extracellular domain of the chimeras.

Similar approaches have been used, for example, to introduce the intact HIV receptor, CD4, into erythrocytes for therapeutic purposes. In this case the engineered cell population would not be capable of self renewal.

The present invention relates to functional simplified chimeras of CD3 delta or T3 gamma, EERVII, or a B cell receptor protein, or a functional derivative thereof which are capable of redirecting immune system function.

More particularly, it relates to the regulation of lymphocytes, macrophages, natural killer cells or granulocytes by the expression in said cells of chimeras which cause the cells to respond to targets recognized by the chimeras. The invention also relates to a method of directing cellular response to an infective agent, a tumor or cancerous cell, or an autoimmune generated cell. The method for directing the cellular response in a mammal comprises administering an effective amount of therapeutic cells to said mammal, said cells being capable of recognizing and destroying said infective agent, tumor, cancer cell or autoimmune generated cell.

In another embodiment, the method of directing cellular response to an infective agent comprises administering therapeutic cells capable of recognizing and destroying said agent, wherein the agent is a specific virus, bacteria, protozoa, or fungi. Even more specific- ally, the method is directed against agents such as HIV.

Specifically the invention provides for a method of directing cellular response to an HIV infected cell. The method comprises administering to a patient an effective amount of cytotoxic T lymphocytes, said lymphocytes being capable of specifically recognizing and lysing cells infected with HIV.

Thus, in one embodiment, there is provided according to the invention a method for directing cellular response to HIV infected cells, comprising administering to a patient an effective amount of cytotoxic T lymphocytes which are capable of specifically recognizing and lysing cells infected with HIV.

In yet another embodiment is provided the chimeric receptor proteins which direct the cytotoxic T lymphocytes to recognize and lyse the HIV infected cell.

Yet another embodiment of the invention comprises host cells transformed with a vector comprising the chimeric receptors.

In yet another embodiment, the present invention provides for an antibody against the chimeric receptors of the invention.

In order to obtain cytotoxic T lymphocytes which specifically bind and lyse cells infected with HIV, the present inventors therefore attempted, and herein receptor chimeras. These chimeric receptors are functionally active and possess the extraordinary ability of being able to specifically bind and lyse cells expressing gp120.

It is an object of the present invention, then, to provide for a method of treatment for individuals infected with HIV. The present invention thus provides a number of important advances in the therapy of AIDS.

These and other non-limiting embodiments of the present invention will be apparent to those of skill from the following detailed description of the invention.

In the following detailed description, reference will be made to various methodologies known to those of skill in the art of molecular biology and immunology.

Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J.D. et al., uolecular Biology of the Gene, Volumes I and II, the Benjamin/Cummings Publishing Company, Inc., ‘ publisher, Menlo Park, CA (1987); Darnell, J.E. et al., fiolegular Cell Biology, Scientific American Books, Inc., Publisher, New York, N.Y. (1986); Levin, B.M., Genes II, John Wiley & Sons, publishers, New York, N.Y. (1985); Old, R.W., et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2d edition, University of California Press, publisher, Berkeley, CA (1981); Maniatis, T., et al., figlggglg;_glgn;gg;__A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, NY (1989); and Current Protocols in Molecular Biology, Ausubel et al., Wiley Press, New York, NY (1989).

DEF N IONS By "cloning" is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to employ methods for generating DNA fragments for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.

By "cDNA" is meant complementary or copy DNA produced from an RNA template by the action of RNA- dependent DNA polymerase (reverse transcriptase).

"CDNA clone" means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.

Thus a _ 15 _ By "cDNA library" is meant a collection of recombinant DNA molecules containing CDNA inserts which comprise DNA copies of mRNA being expressed by the cell at the time the CDNA library was made. Such a CDNA library may be prepared by methods known to those of skill, and described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, su ra.

Generally, RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene. Preferred for the purpose of the present invention are mammalian, and particularly human, lymphocytic cell lines. A presently preferred vector for this purpose is the vaccinia virus WR strain.

By "vector" is meant a DNA molecule, derived, e.g., from a plasmid, bacteriophage, or mammalian or insect virus, into which fragments of DNA may be inserted or cloned. A vector will contain one or more unique restriction sites and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. Thus, by "DNA expression vector" is meant any autonomous element capable of directing the symthesis of a recombinant peptide. Such DNA expression vectors include bacterial plasmids and phages and mammalian and insect plasmids and viruses.

By "substantially pure" is meant a compound e.g., a protein, a polypeptide, or an antibody that is substantially free of the components that naturally accompany it.

Generally, a compound is substantially pure when at least 60%, more preferably at least 75%, and most preferably at least 90% of the total material in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. the context of a nucleic acid, "substantially pure" means _ 15 - a nucleic acid sequence, segment, or fragment that is free from the genes that flank it in its naturally- occurring state (e.g., free from the sequences that flank the nucleic acid in its native genomic position). By "functional derivative" is meant the "fragments," "variants," "analogues," or "chemical derivatives" of a molecule. A "fragment" of a molecule, such as any of the CDNA sequences of the present invention, is meant to refer to any nucleotide subset of the molecule. A "variant" of such molecule is meant to refer to a naturally occurring molecule substantially similar to An "analog" of a molecule is meant to refer to a non-natural either the entire molecule, or a fragment thereof. molecule substantially similar to either the entire molecule or a fragment thereof. A molecule is said to be "substantially similar" to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological activity.

Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional or fewer amino acid residues not found in the other, or if the sequence of amino acid residues is not identical. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th et., Mack Publishing Co., Easton, Penn. (1980). - 17 _ Similarly, a "functional derivative" of a gene of the T cell or Fc receptor chimera of the present invention is meant to include "fragments," "variants," or "analogues" of the gene, which may be "substantially similar" in nucleotide sequence, and which encode a molecule possessing similar activity to, for example, a T cell or Fc receptor chimera.

Thus, as used herein, a T cell or Fc receptor chimera protein is also meant to include any functional derivative, fragments, variants, analogues, or chemical derivatives which may be substantially similar to the "wild-type" chimera and which possess similar activity (i.e., most preferably, 90%, more preferably, 70%, preferably 40%, or at least 10% of the wild-type receptor chimera's activity). The activity of a functional chimeric receptor derivative includes specific binding (with its extracellular portion) to a targeted agent or cell and resultant destruction (directed by its intracellular or transmembrane portion) of that agent or cell; such activity may be tested, e.g., using any of the assays described herein.

A DNA sequence encoding the T cell and Fc receptor chimera of the present invention, or its functional derivatives, may be recombined with vector DNA in accordance with conventional techniques, including blunt- ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases.

Techniques for such manipulations are disclosed by Maniatis, T., et al., ggpgg, and are well known in the art.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a An operable linkage is a linkage way as to permit gene expression. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.

If desired, the non—coding region 3' to the gene sequence coding for the protein may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, Thus, by retaining the 3'-region naturally contiguous to the DNA such as termination and polyadenylation. sequence coding for the protein, the transcriptional Where the transcriptional termination signals are not termination signals may be provided. satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.

Two DNA sequences (such as a promoter region sequence and a T cell receptor or Fc receptor chimera encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the T cell receptor and Fc receptor chimera gene sequence, or (3) interfere with the ability of the T cell receptor and Fc receptor chimera gene sequence to be transcribed by the promoter region sequence. A promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.

Thus, to express the protein, transcriptional and translational signals recognized by an appropriate host are necessary.

The present invention encompasses the expression of a T cell receptor or Fc receptor chimera protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells, although eukaryotic (and, particularly, human lymphocyte) expression is preferred.

Antibodies according to the present invention may be prepared by any of a variety of methods. For example, cells expressing the T cell receptor or Fc receptor chimera protein, or a functional derivative thereof, can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding the chimera.

N.Y., pp. 563-684 (1981)). In general, such procedures involve immunizing an animal with the T cell receptor and present invention are monoclonal antibodies.

Fc receptor chimera antigen. The splenocytes of such animals are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention. After obtained through such a selection are then assayed to The hybridoma cells identify clones which secrete antibodies capable of binding the chimera.

Antibodies according to the present invention also may be polyclonal, or, preferably, region specific polyclonal antibodies.

Antibodies against the T cell receptor or Fc receptor chimera according to the present invention may be used to monitor the amount of chimeric receptor (or chimeric receptor-bearing cells) in a patient. Such antibodies are well suited for use in standard immunodiagnostic assay known in the art, including such immunometric or "sandwich" assays as the forward sandwich, reverse sandwich, and simultaneous sandwich assays. The antibodies may be used in any number of combinations as may be determined by those of skill without undue experimentation to effect immunoassays of acceptable specificity, sensitivity, and accuracy.

Standard reference works setting forth general principles of immunology include Roitt, I., sse gnnnnglggy, Sixth Ed., Blackwell Scientific Publications, Publisher, Oxford (1988); Kimball, J. W., Introdugtion tg Immunology, Second Ed., Macmillan Publishing Co., Publisher, New York (1986); Roitt, I., et al., Immunolggy, Gower Medical Publishing Ltd., Publisher, London, (1985); Campbell, A. , "Monoclonal Antibody Technology," in, Burdon, R., et al., eds., Laboratony Tegnnigngs in Bigcnenistrv and Molecular Bioloav, Volume 13, Elsevier, Publisher, Amsterdam (1984); Klein, J., Immunologvz The Science of Self-Nonself Discrimination, John Wiley 5 Sons, Publisher, New York (1982); and Kennett, R., et al., eds., Monoclonal Antibodies, b ' ° w Plenum Press, Publisher, New York (1980).

By "detecting" it is intended to include mension In '0 o ical Anal ses, determining the presence or absence of a substance or The term thus refers to the use of the materials, compositions, and quantifying the amount of a substance. methods of the present invention for qualitative and quantitative determinations.

For replication, the hybrid cells may be cultivated both in vitro and in vivo. High in vivo production makes this the presently preferred method of culture. Briefly, cells from the individual hybrid strains are injected intraperitoneally into pristane- primed BALB/c mice to produce ascites fluid containing high concentrations of the desired monoclonal antibodies.

Monoclonal antibodies of isotype IgM or IgG may be purified from cultured supernatants using column chromatography methods well known to those of skill in the art. _ 22 - Antibodies according to the present invention are particularly suited for use in immunoassays wherein they may be utilized in liquid phase or bound to a solid phase In addition, the antibodies in these immunoassays can be detectably labeled in various ways. carrier.

There are many different labels and methods of labeling known in the art. labels which can be used in the present invention Examples of the types of include, but are not limited to, enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, Those of ordinary skill in the art will know of other suitable bioluminescent compounds and metal chelates. labels for binding to antibodies, or will be able to ascertain the same by the use of routine experimentation.

Furthermore, the binding of these labels to antibodies can be accomplished using standard techniques commonly known to those of ordinary skill in the art. one of the ways in which antibodies according to the present invention can be detectably labeled is by linking the antibody to an enzyme. when later exposed to its substrate, will react with the This enzyme, in turn, substrate in such a manner as to produce a chemical moiety which can be detected as, for example, by spectrophotometric or fluorometric means. Examples of enzymes which can be used to detectably label antibodies include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha—glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, 3-galactosidase, ribonuclease, urease, catalase, glucose—VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.

The presence of detectably labeled antibodies also can be detected by labeling the antibodies with a _ 23 - radioactive isotope which then can be determined by such means as the use of a gamma counter or a scintillation Isotopes which are particularly useful for the 125 32 35 I: P: 5. counter. purpose of the present invention are 3H, 1‘C, 51Cr, 3‘Cl, 57Co, 5°Co, 59Fe and 75Se.

It is also possible to detect the binding of detectably labeled antibodies by labeling the antibodies with a fluorescent compound. when a fluorescently labeled antibody is exposed to light of the proper wavelength, its presence then can be detected due to the fluorescence of the dye. Among the most commonly used fluorescent labeling compounds are fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibodies of the invention also can be detectably labeled using fluorescent emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal chelating groups as diethyl- enteriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Antibodies also can be detectably labeled by coupling them to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of the chemical reaction.

Examples of particularly useful chemiluminescent labeling compounds are luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, and dioxetane.

Likewise, a bioluminescent compound may be used to label the antibodies according to the present invention.

Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The _ 24 - presence of a bioluminescent antibody is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling include luciferin, luciferase aequorin.

The antibodies and substantially purified antigen of the present invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the assay to be used.

The types of assays which can be incorporated in kit form are many, and include, for example, competitive and non-competitive assays. Typical examples of assays which can utilize the antibodies of the invention are radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), and immunometric, or sandwich, immunoassays.

By the term "immunometric assay" or "sandwich immunoassay," it is meant to include simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those skilled in the art. that antibodies according to the present invention will be useful in other variations and forms of assays which Those of skill will also appreciate are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.

In the preferred mode for performing the assays it is important that certain "blockers" be present in the incubation medium (usually added with the labeled soluble antibody). specific proteins, protease, or human antibodies to mouse The "blockers" are added to assure that non- immunoglobulins present in the experimental sample do not cross-link or destroy the antibodies on the solid phase support, or the radiolabeled indicator antibody, to yield false positive or false negative results. The selection of "blockers" therefore adds substantially to the specificity of the assays described in the present invention.

It has been found that a number of nonrelevant (i.e., nonspecific) antibodies of the same class or subclass (isotype) as those used in the assays (e.g., The concentration of the "blockers" (normally 1-100 pg/pl) is IgG1, IgG2., Igfl, etc.) can be used as "blockers." important, in order to maintain the proper sensitivity yet inhibit any unwanted interference by mutually addition, the buffer system containing the "blockers" occurring cross reactive proteins in human serum. needs to be optimized. Preferred buffers are those based on weak organic acids, such as imidazole, HEPPS, MOPS, TES, ADA, ACES, HEPES, PIPES, TRIS, and the like, at physiological pH ranges. Somewhat less preferred buffers are inorganic buffers such as phosphate, borate or carbonate. Finally, known protease inhibitors should be added (normally at 0.01-10 pg/ml) to the buffer which contains the "blockers." There are many solid phase immunoadsorbents which have been employed and which can be used in the present invention. Well known immunoadsorbents include glass, polystyrene, polypropylene, dextran, nylon and other materials, in the form of tubes, beads, and microtiter plates formed from or coated with such materials, and the like. The immobilized antibodies can be either covalently or physically bound to the solid phase immunoadsorbent, by techniques such as covalent bonding via an amide or ester linkage, or by absorption. Those skilled in the art will know many other suitable solid phase immunoadsorbents and methods for immobilizing antibodies thereon, or will be able to ascertain such, using no more than routine experimentation.

For ' according to the present invention either directly or by using an intermediary functional group. An intermediary group which is often used to bind radioisotopes which exist as metallic cations to antibodies is diethylenetriaminepentaacetic acid (DTPA). Typical examples of metallic cations which are bound in this 111m, 1311, 91Ru’ wen’ 67Ga and The antibodies of the invention can also be labeled manner are: 99‘Tc, 1231, "Ga. with non-radioactive isotopes for purposes of diagnosis.

Elements which are particularly useful in this manner are 151Gd' ssun’ 162Dy’ szcr and 56Fe_ The antigen of the invention may be isolated in substantially pure form employing antibodies according to Thus, present invention provides for substantially pure T cell the present invention. an embodiment of the receptor or Fc receptor chimera, said antigen characterized in that it is recognized by and binds to antibodies according to the present invention. In another embodiment, the present invention provides a method of isolating or purifying the T cell receptor or Fc receptor chimeric antigen, by forming a complex of said antigen with one or more antibodies directed against the T cell receptor or Fc receptor chimera.

The substantially pure T cell receptor or Fc receptor chimera antigens of the present invention may in turn be used to detect or measure antibody to the chimera in a sample, such as serum or urine. Thus, one embodiment of the present invention comprises a method of detecting the presence or amount of antibody to T cell receptor or Fc receptor chimera antigen in a sample, comprising contacting a sample containing an antibody to - 27 _ the chimeric antigen with detectably labeled T cell receptor or and Fe receptor chimera, and detecting said label. fractions and immunoreactive analogues of the chimera It will be appreciated that immunoreactive also may be used. By the term "immunoreactive fraction" is intended any portion of the chimeric antigen which demonstrates an equivalent immune response to an antibody directed against the receptor chimera. By the term "immunoreactive analogue" is intended a protein which differs from the receptor chimera protein by one or more animo acids, but which demonstrates an equivalent immunoresponse to an antibody of the invention.

By "specifically recognizes and binds" is meant an antibody which recognizes and binds a chimeric receptor polypeptide but which does not substantially recognize and bind other molecules in a sample, e.g., in a biological sample, which includes the receptor polypeptide.

By "autoimmune-generated cell" is meant cells producing antibodies that react with host tissue or immune effector T cells that are autoreactive; such cells include antibodies against acetylcholine receptors (leading, e.g., to myasthenia gravis) or anti-DNA, anti- erythrocyte, and anti-placelet autoantibodies (leading, e.g., to lupus erythematosus).

By "therapeutic cell" is meant a cell which has been transformed by a chimera of the invention so that it is capable of recognizing and destroying a specific infective agent, a cell infected by a specific agent, a tumor or cancerous cell, or an autoimmune-generated cell; preferably such therapeutic cells are cells of the hematopoietic system.

By "extracellular" is meant having at least a BY "intracellular" is meant having at least a portion of the portion of the molecule exposed at the cell surface. molecule exposed to the therapeutic cell's cytoplasm. }! "transmembrane" is meant having at least a portion of the An portion", an "intracellular portion" and a molecule spanning the plasma membrane. "extracellular "transmembrane portion", as used herein, may include flanking amino acid sequences which extend into adjoining cellular compartments.

By "oligomerize" is meant to complex with other proteins to form dimers, trimers, tetramers, or other higher order oligomers. Such oligomers may be homo- oligomers or hetero-oligomers. An "oligomerizing portion" is that region of a molecule which directs complex (i.e., oligomer) formation.

By "cytolytic" is meant to be capable of destroying a cell (e.g., a cell infected with a pathogen, a tumor or cancerous cell, or an autoimmune-generated) cell or to be capable of destroying an infective agent (e.g., a virus).

By "immunodeficiency virus" is meant a retrovirus that, in wild-type form, is capable of infecting T4 cells of a primate host and possesses a viral morphogenesis and morphology characteristic of the lentivirus subfamily.

The term includes, without limitation, all variants of HIV and SIV, including HIV-1, HIV-2, SIVmac, SIVagm, SIVmnd, SIVsmm, SIVman, SIVmand, and SIVcpz.

By "MC-independent" is meant that the cellular cytolytic response does not require the presence of an MHC class II antigen on the surface of the targeted cell.

By a "functional cytolytic signal-transducing derivative" is meant a functional derivative (as defined above) which is capable of directing at least 10%, preferably 40%, more preferably 70%, or most preferably at least 90% of the biological activity of the wild type molecule. As used herein, a "functional cytolytic signal-transducing derivative" may act by directly signaling the therapeutic cell to destroy a receptor- bound agent or cell (e.g., in the case of an intracellular chimeric receptor portion) or may act indirectly by promoting oligomerization with cytolytic signal transducing proteins of the therapeutic cell (e.g., in the case of a transmembrane domain). Such derivatives may be tested for efficacy, e.g., using the in gitgg assays described herein.

By a "functional HIV envelope-binding derivative" is meant a functional derivative (as defined above) which is capable of binding any HIV envelope protein.

Functional derivatives may be identified using, e.g., the in gitrg assays described herein.

THERAPEUTIC ADMINISTRATION The transformed cells of the present invention may be used for the therapy of a number of diseases. Current methods of administering such transformed cells involve adoptive immunotherapy or cell—transfer therapy. These methods allow the return of the transformed immune—system cells to the bloodstream. Rosenberg, S.A., gcientific American, 62 (May 1990); Rosenberg et al., The New Jou 'c' e, 323(9):570 (1990).

The pharmaceutical compositions of the invention may be administered to any animal which may experience the beneficial effects of the compounds of the invention.

Foremost among such animals are humans, although the invention is not intended to be so limited.

Detailed Description The drawings will first be described. The following drawings and examples shall explain the principle of the invention. The drawings and examples contain subject—matter described in the prior art which is not covered by the claims. _ 30 - grief Description of the Drawings FIG. 1 Characterization of CD4 chimeras. Fig. 1A presents the amino acid sequence about the site of fusion between CD4 (residues 1-369) and the different receptor chains. the amino acids encoded within the BamHI site used for infected with virus expressing CD4 chimeras or CD16PI, Cells were phycoerythrin-conjugated anti-CD4 MAb Leu3A. native CD4 expressed in CV1 cells. Lanes were run with reducing (R) or without reducing (NR) agent. Molecular mass standards in kD are shown at left.

CDIGTI following coinfection by viruses expressing CD16Tu The level of expression of the { chimeras was and coinfection of cells with viruses expressing CD16!" and g chimeras did not appreciably alter surface expression of the chimeras (data not shown).

FIG. 4 Increased intracellular free calcium ion follows crosslinking of mutant ( chimeras in a T cell line. Jurkat E6 cells (Weiss et al., J. Immunol., ;;;:123-128 (1984)) were infected with recombinant The results shown are for the gated CD4+ population, so that vaccinia viruses and analyzed by flow cytometry. crosslinking with goat antibody to mouse IgG.

Figs. 4C and 4D show Jurkat cells expressing cytolytic T lymphocytes (CTL) to kill targets expressing HIV-1 gp120/41. Fig. 5A: solid circles, CTL expressing -.32- Flow cytometric analysis of CD4 expression by To correct the target to chimera was determined by subtracting the scaled negative (uninfected) population by histogram superposition; for comparative purposes in this figure the uninfected cells were assigned an arbitrary threshold which gives roughly the same fraction positive for the other cell populations as would histogram subtraction.

FIG. 6 Specificity of the CD4-directed cytolysis.

Fig. 6A: with HeLa cells expressing CD16"; open circles, CTL incubated with Raji (MHC class II+) cells; open squares, The ordinate scale is expanded. fusion protein. The extracellular portion of the phosphatidylinositol-linked form of monomeric CD16 was joined to dimeric g just external to the transmembrane domain. The protein sequence at the fusion junction is shown at the bottom. analysis of calcium mobilization following crosslinking Fig. 7B shows a flow cytometric crosslinking in the REX33A TCR" mutant; open squares, the Solid squares, the open triangles, cytolysis mediated by cells expressing The peptide sequences Fig. 9B shows the cytolytic activity of monomeric chimera Solid triangles, ); open circles, CD16:5; solid circles, CD16:7. 9D shows calcium mobilization by mutant and tripartite chimeras in the TCR negative Jurkat JRT3.T3.5 mutant cell line. Open circles, response of cells expressing dimeric PIG. the activity of the 18 residue cytolytic signal- Contribution of individual amino acids to transducing motif. Figs. 10A and 10B show cytolytic activity and Fig. 10C shows calcium ion mobilization mediated by chimeras bearing point mutations near the Figs. 10A and 10B represent data collected on cells expressing low and high carboxyl terminal tyrosine (Y62). ); and open Figs. and cytolysis assays and are shown at right.

FIG. 11 Alignment of internal repeats of g and comparison of their ability to support cytolysis. Fig. 11A is a schematic diagram of chimeras formed by dividing the g intracellular domain into thirds and appending them The sequences of the intracellular domains are shown below, I16. 12 is a schematic diagram of the CD16:FcR7II Solid circles, cells expressing chimeras.

FIG. 13 Calcium mobilization following crosslinking of CD4:FcR1II and CD16:FcR7II chimeras.

Fig. 13A shows the ratio of violet to blue fluorescence emitted by cells loaded with the calcium sensitive fluorophore Indo-1 shown as a function of time following crosslinking of the CD16 extracellular domain with antibodies. Fig. 13B shows a similar analysis of the increase in ratio of violet to blue fluorescence of cells bearing CD4:FcR1II chimeras, following crosslinking with antibodies.

FIG. 14 Cytolysis assay of CD4:FcR1II and CD16:FcR1II chimeras. Fig. 14A shows the percent of 51Cr released from anti-CD16 hybridoma (target) cells when the cells are exposed to increasing numbers of cytotoxic T lymphocytes expressing CD16:FcR1II chimeras (effector cells). Fig. 14B shows a similar analysis of cytotoxicity mediated by CD4:FcRqII chimeras against target cells expressing HIV envelope glycoproteins.

FIG. 15 Identification of residues in the FcR1II A Fig. 15A is a Figs. 15B tail which are important for cytolysis. schematic diagram of the deletion constructs. _ 37 - and 15C shows calcium mobilization and cytolysis by carboxy1—terminal deletion variants of CD16:FcR7II A.

Figs. 15D and 15E show calcium mobilization and cytolysis by tripartite chimeras bearing progressively less of the amino terminus of the intracellular tail of CD16:FcR7II A.

FIG. 16 shows the amino acid sequence of CD3 delta; the boxed portion represents a preferred cytolytic signal transducing portion.

FIG. 17 shows the amino acid sequence of T3 gamma; the boxed portion represents a preferred cytolytic signal transducing portion.

FIG. 18 shows the amino acid sequence of mbl; the boxed portion represents a preferred cytolytio signal transducing portion.

FIG. 19 shows the amino acid sequence of B29; the boxed portion represents a preferred cytolytic signal transducing portion.

EXAMPLE I antibody sequences formed the extracellular portion.

Flow cytometry of COS cells transfected with a plasmid encoding the chimera showed high level expression of antibody determinants when an expression plasmid encoding a light chain cDNA was cotransfected, and modest expression of antibody determinants when the light chain expression plasmid was absent.

Similar chimeras including human IgG1 fused to n or 7 (see below), or any signal-transducing portion of a T cell receptor or Fc receptor protein may be constructed generally as described above using standard techniques of molecular biology.

To create a single transcription unit which would allow both heavy and light chains to be expressed from a single promoter, a plasmid encoding a bicistronic mRNA was created from heavy and light chain coding sequences, and the 5' untranslated portion of the mRNA encoding the 78kD glucose regulated protein, otherwise known as grp78, or BiP. grp78 sequences were obtained by PCR of human genomic DNA using primers having the sequences: CGC GGG CGG CCG CGA CGC CGG CCA AGA CAG CAC (SEQ ID NO: 9) and CGC GTT GAC GAG CAG CCA GTT GGG CAG CAG CAG (SEQ ID NO: 10) at the 5' and 3' ends respectively. Polymerase chain reactions with these oligos were performed in the presence of 10% dimethyl sulfoxide. The fragment obtained by PCR was digested with NotI and HincII and inserted between NotI and HpaI sites downstream from human IgG1 coding sequences. Sequences encoding a human IgG kappa light chain cDNA were then inserted downstream from the grp78 leader, using the HincII site and another site in the vector. The expression plasmid resulting from these manipulations consisted of the semisynthetic heavy chain gene, followed by the grp78 leader sequences, followed by the kappa light chain CDNA sequences, followed by polyadenylation signals derived from an SV40 Transfection of COS cells with the expression plasmid gave markedly improved expression of DNA fragment. heavy chain determinants, compared to transfection of plasmid encoding heavy chain determinants alone.

To create a bicistronic gene comprising a heavy chain/receptor chimera and a light chain, the upstream heavy chain sequences can be replaced by any chimeric heavy chain/ receptor gene described herein.

EXAMPLE II construction of CD4 Receptor chimeras from a murine thymocyte library. g, n and 1 cDNAs were To form the the sequence at the same approximate location (Fig. 1; SEQ ID NO: 2 and 5). into a vaccinia virus expression plasmid bearing the E; The gene fusions were introduced col; gp; gene as a selectable marker (M. Amiot and B.S., Flow cytometric analysis showed that The latter finding is consistent with a recent (1990)). vaccinia recombinants revealed that the fusion proteins Immunoprecipitation of cells infected with the of the fusion proteins are approximately consistent with The larger masses the greater length of the intracellular portion, which exceeds that of native CD4 by 75 (CD4:{) or 5 (CD4:1) residues.

EXAMPLE III cD4 chimeras cen Associate with other Receptor cheins Consistent with these reports, expression of the chimeras also allowed surface expression of CD16T") when delivered to the target cell either by cotransfection or by coinfection with recombinant vaccinia viruses (Fig. 2). more pronounced than promotion by 1 (Fig. 2) in the cell The promotion of (CD16Tu) surface expression by g was lines examined, whereas native CD4 (data not shown) did not enhance CD16T' surface expression.

EXAMPLE IV Asp g Mutants Do Not coassociate with re Receptor To create chimeras which would not associate with existing antigen or Fc receptors, mutant g fusion proteins which lacked either the intramembranous Asp or intramembranous Cys residue or both were prepared. Flow cytometry showed that the intensity of cell surface expression by the different mutant chimeras was not appreciably different from the unmutated precursor (data not shown) and immunoprecipitation experiments showed that total expression by the chimeras was similar (Fig. 3). transmembrane cysteine residue were found not to form disulfide linked dimers (Fig. 3). chimeras lacking Asp were incapable of supporting the As expected, the mutant chimeras lacking the The two mutant surface expression of CD16", whereas the monomeric chimeras lacking Cys but bearing Asp allowed CD16r' to be coexpressed, but at lower efficiency than the parental dimer (Fig. 3).

EXAMPLE V Mutant Receptors Retain the Ability to Initiate a calcium Response To determine whether crosslinking of the fusion proteins would allow the accumulation of free intracellular calcium in a manner similar to that known to occur with the T cell antigen receptor, cells of the human T cell leukemia line, Jurkat E6 (ATCC Accessior -42.. intracellular calcium levels.

EXAMPLE VI Expressing HIV gp12o/41 To determine whether the chimeric receptors would incubated with cells from a human allospecific (CD8+, CD4" mutant chimera. Fig. 5 shows that HeLa cells expressing cytometric analysis of CD4 expression by the CTL used in Fig. 5C shows a The CD4:7 fusion was less active, as was residues. However in both cases significant cytolysis was observed (Fig. 5). vaccinia-encoded surface proteins. In addition, CTLs expressing non-chimeric CD4 do not significantly lyse HeLa cells expressing gp120/41 (Fig. 6A).

EXAMPLE VII MHC class II-Bearing cells Are Not Targeted by the chineree .

Although a specific interaction EXAMPLE VIII sequence Requirements for Induction of cytolysis by the T Cell Antigen/Pc Receptor zeta chain Although chimeras between CD4 and g can arm cytotoxic T lymphocytes (CTL) to kill target cells expressing HIV gp120, an alternative to CD4 was sought in order to unambiguously compare the properties of zeta chimeras introduced into human T cell lines. Such lines _ 45 - T cell lines were infected with the vaccinia recombinants and the relative cytoplasmic free calcium ion concentration was measured following crosslinking of Both spectrofluorimetric (bulk population) and flow cytometric the extracellular domains with antibodies. (single cell) measurements were performed, with cells loaded with the dye Indo-1 (Grynkiewicz et al., J. Biol.

Chem. 260:3440 (1985); Rabinovitch et al., J. Immunol. l37:952 (1986)). collected from cells of the Jurkat human T cell leukemia Figure 7B shows an analysis of data (1984)); or a strong response to CD16 antibody when the chimera was expressed crosslinking was seen. Similar data have been collected on the REXZOA (Breitmeyer et al., su ra, 1987; Blumberg To evaluate the ability of the chimeras to redirect cell-mediated immunity, CTLs were infected with vaccinia recombinants expressing CD16 chimeras and used to specifically lyse hybridoma cells expressing membrane- bound anti-CD16 antibodies (see below). extension of a hybridoma cytotoxicity assay originally This assay is an Samples Nuclei were removed by The cytolytic activity of the mutant receptors was also tested. The mutated chimera deleted to residue 65 (CD16:{Cys11Gly/Asp15Gly/Asp66*) was, depending on the conditions of assay, two to eight fold less active in the cytolysis assay than the comparable unmutated chimera The reduction in activity of the mutant chimeras is similar structure (see above) and is most likely attributable to the lower efficiency of g monomers In contrast, the Asp", Cys" mutated chimera deleted to residue 59 had no cytolytic activity compared to dimers.

(Fig. 9B), supporting the hypothesis that association with other chains mediated by the transmembrane Cys and/or Asp residues was responsible for the weak persistence of cytolytic activity in deletions more amino terminal than residue 65.

Flow cytometric studies showed that the deletion mutants lacking transmembrane Asp and Cys residues could still promote an increase in free intracellular calcium ion in response to antibody crosslinking in a TCR" mutant Jurkat cell line (Fig. 9D). obtained for chimeras expressed in the parental Jurkat Similar results were line (not shown). In the case of CD16:gCys11Gly/Asp15Gly/Glu60*, these data demonstrate that the ability to mediate calcium responsiveness can be mutationally separated from the ability to support cytolysis. - 49 _ To examine the contributions of individual residues within the 18-residue motif, we prepared a number of mutant variants by site-directed mutagenesis, and evaluated their ability to mediate receptor-directed killing under conditions of low (Figs. 10A and 10D) or high (Figs. 10B and 10B) expression of chimeric receptor.

Fig. 10 shows that while a number of relatively conservative substitutions (i.e., replacing acidic residues with their cognate amides, or tyrosine with phenylalanine) which spanned residues 59 to 63 yielded moderate compromise of cytolytic efficacy, in general the variants retained the ability to mobilize calcium.

However collectively these residues comprise an important submotif inasmuch as their deletion eliminates cytolytic activity. Conversion of Tyr 62 to either Phe or Ser eliminated both the cytotoxic and calcium responses. At the amino terminus of the 18 residue segment, replacement of Tyr 51 with Phe abolished both calcium mobilization and cytolytic activity, while substitution of Leu with Ser at position 50 eliminated the calcium response while Without being bound to a particular hypothesis, it is suspected that the only partially impairing cytolysis. inability of the Leu50Ser mutant to mobilize calcium in short term flow cytometric assays does not fully reflect its ability to mediate a substantial increase in free intracellular calcium ion over the longer time span of the cytolysis assay. calcium-insensitive cytolytic activity has been reported for some cytolytic T cell lines, and the possibility that a similar phenomenon However, _ 50 - underlies the results described herein has not been ruled out. Replacement of Asn48 with Ser partially impaired cytotoxicity in some experiments while having little effect in others.

To investigate the potential role of redundant sequence elements, the intracellular domain of g was divided into three segments, spanning residues 33 to 65, 71 to 104, and 104 to 138. attached to a CD16:CD7 chimera by means of a MluI site Each of these segments was introduced just distal to the basic membrane anchoring sequences of the intracellular domain of CD7 (see below; 11A) . three elements showed they were essentially equipotent (Fig. 113). the second motif bears eleven residues between tyrosines, Fig. Comparison of the cytolytic efficacy of the Sequence comparison (Fig. 11A) shows that whereas the first and third motifs bear ten.

Although a precise accounting of the process of T cell activation has not been made, it is clear that aggregation of the antigen receptor, or of receptor chimeras which bear g intracellular sequences, triggers calcium mobilization, cytokine and granule release, and the appearance of cell surface markers of activation.

The active site of g, a short linear peptide sequence probably too small to have inherent enzymatic activity, likely interacts with one or at most a few proteins to mediate cellular activation. It is also clear that mobilization of free calcium is not by itself sufficient for cellular activation, as the ability to mediate cytolysis can be mutationally separated from the ability to mediate calcium accumulation.

As shown herein, addition of 18 residues from the intracellular domain of g to the transmembrane and intracellular domain of two unrelated proteins allows the resulting chimeras to redirect cytolytic activity against target cells which bind to the extracellular portion of the fusion proteins. Although chimeras bearing the 18 residue motif are approximately eight-fold less active than chimeras based on full length g, the reduced activity can be attributed to the loss of transmembrane interactions which normally allow wild-type g to form disulfide linked dimers. which have the same carboxyl terminus as the motif and That is, g deletion constructs lack transmembrane Cys and Asp residues typically show slightly less activity than chimeras bearing only the 18 residue motif.

The cytolytic competency element on which we have focused has two tyrosines and no serines or threonines, restricting the possible contributions of phosphorylation to activity. Mutation of either tyrosine destroys activity, however, and although preliminary experiments do not point to a substantial tyrosine phosphorylation following crosslinking of chimeric surface antigens bearing the 18 reside motif, the possible participation of such phosphorylation at a low level cannot be excluded. In addition to the effects noted at the two tyrosine residues, a number of amino acid replacements at the amino and carboxyl terminus of the motif weaken activity under conditions of low receptor density. - 52 _ redirected cytolysis (see above), n is not phosphorylated can be attributed to the first two motifs. in response to receptor stimulation (Bauer et al., supra, 1991). required for phosphorylation, or the third motif Thus either the presence of all three motifs is represents a favored substrate for an unidentified tyrosine kinase.

EXAMPLE I! cytolytic Signal Transduction by Human Fc Receptor corresponding to the transmembrane and cytoplasmic Specifically, CDNA sequences domains of the previously described FCRIIA, B1, and B2 isoforms were amplified from the preexisting clone PC23 or from a human tonsil CDNA library (Obtained bY Standard techniques) using oligonucleotide primers: CCC GGA TCC CAG CAT GGG CAG CTC TT (SEQ ID NO: 18; FCRIIA forward) CGC GGG GCG GCC GCT TTA GTT ATT ACT GTT GAC ATG GTC GTT (SEQ ID NO: 19; FCRIIA reverse); GCG GGG GGA TCC CAC TGT CCA AGC TCC CAG CTC TTC ACC G (SEQ ID NO: 20; FCRIIB1 and FcRIIB2 forward); and GCG GGG GCG GCC GCC TAA ATA CGG TTC TGG TC (SEQ ID NO: 21; FcRIIB1 and FcRIIB2 reverse). These primers contained cleavage sites for the enzymes BamHI and NotI respectively, indented 6 residues from the 5' end. The NotI site was immediately followed by an antisense stop codon, either CTA or TTA. more residues complementary to the 5' and 3' ends of the All primers contained 18 or desired fragments. The CDNA fragment corresponding to the FcRII1C cytoplasmic domain, which differs from the IIA isoform in only one amino acid residue (L for P at residue 268) was generated by site directed mutagenesis by overlap PCR using primers of sequence: TCA GAA AGA GAC AAC CTG AAG AAA CCA ACA A (SEQ ID NO: 22) and TTG TTG GTT TCT TCA GGT TGT GTC TTT CTG A (SEQ ID NO: 23). vaccinia virus expression vectors which contained the The PCR fragments were inserted into CD16 or CD4 extracellular domains respectively and subsequently inserted into wild type vaccinia by recombination at the thymidine kinase locus, using selection for cointegration of E ggli gpt to facilitate identification of the desired recombinants. The identities of all isoforms (shown in Fig. 12) were confirmed by dideoxy sequencing.

Production of the chimeric receptor proteins was further confirmed by immunoprecipitation studies.

Approximately 107 JRT3.T3.5 cells were infected for one hour in serum free IMDM medium with recombinant vaccinia Twelve hours post-infection, the cells were harvested and surface labeled with 0.5mCi 1251 per 107 cells using the The labeled cells were collected by centrifugation and lysed 1% NP- 40, 0.1mM Mgclz, 5mM KC1, 0.2M iodoacetamide and 1mM PMSF. Nuclei were removed by centrifugation, and CD16 at a multiplicity of infection of at least ten. lactoperoxidase/glucose oxidase method. fusion proteins immunoprecipitated with antibody 4G8 and anti-mouse IgG agarose. Samples were electrophoresed under reducing conditions. All immunoprecipitated chimeric receptor molecules were of the expected molecular masses.

To test the ability of the chimeric receptors to mediate an increase in cytoplasmic free calcium ion, the recombinant viruses were used to infect the TCR' mutant -54..

Jurkat cell line JRT3.T3.5 (as described herein) and cytoplasmic free calcium was measured in the cells (as described herein) following crosslinking of the receptor extracellular domains with monoclonal antibody 3G8 or Leu-3A (as described herein). that the intracellular domains of FcR1II A and C were These experiments revealed capable of mediating an increase in cytoplasmic free calcium ion after crosslinking of the extracellular domains, whereas the intracellular domains of FcR1II B1 and B2 were inactive under comparable conditions (Fig. 13 A and 13B). The CD4, CD5 and CD16 hybrids of FcR1II A shared essentially equal capacity to promote the calcium response (Fig. 13 and data not shown). other cell lines, from both monocytic and lymphocytic lineages, were capable of responding to the signal initiated by crosslinking of the extracellular domains (data not shown).

To explore the involvement of the different FcR7II intracellular domains in cytolysis, human cytotoxic T lymphocytes (CTL) were infected with vaccinia recombinants expressing CD16:FcR1II A, B1, B2 and C chimeras. The infected cells were then cocultured with 51Cr-loaded hybridoma cells (i.e., 3G8 10-2 cells) which expressed cell surface antibody to CD16. In this assay CTLs bearing the CD16 chimera killed the hybridoma target cells (allowing release of free 51Cr) if the CD16 extracellular domain of the chimera has been joined to an intracellular segment capable of activating the lymphocyte effector program; this cytolysis assay is described in detail below. Fig. 14A shows that CTL armed with CD16:FcR1IIA and C, but not FcR1II B1 or B2, are capable of lysing target cells expressing cell surface anti-CD16 antibody.

To eliminate the possibility that the specific cytolysis was in some way attributable to interaction with the CD16 moiety, cytolysis experiments were conducted in which the FcRII intracellular domains were attached to a CD4 extracellular domain. target cells were HeLa cells expressing HIV envelope In this case the gp12o/41 proteins (specifically, HeLa cells infected with the vaccinia vector vPEl6 (available from the National Institute of Allergy and Infections Disease AIDS As in the CD16 system, target cells expressing HIV envelope were susceptible to lysis Depository, Bethesda, MD). by T cells expressing the CD4:FcR1II A chimera, but not FcR1II B1 or B2 (Fig. 143).

Figs. 15B and 15C show that removal of the 14 carboxyl-terminal residues, including tyrosine 298, resulted in a complete loss of cytolytic capacity and a substantial reduction in calcium mobilization potential.

Further deletion to just before tyrosine 282 gave an identical phenotype (Figs. 15B and 15C). Deletion from the N-terminus of the intracellular domain to residue 268 had no substantial effect on either calcium profile or cytolytic potency, whereas deletion to residue 275 markedly impaired free calcium release but had little - 55 _ effect on cytolysis (Figs. 15D and 15E). Further deletion, to residue 282, gave FcR1II tails which lacked the ability to either mobilize calcium or trigger cytolysis (Figs. 15D and 15B). The 'active element‘ defined by these crude measures is relatively large (36 amino acids) and contains two tyrosines separated by 16 residues.

EXAMPLE X other intracellular and transmembrane signal transducing domains according to the invention are found in the CD3 delta, the T3 gamma, the mb1, and the B29 receptor proteins. The amino acid sequences of these proteins is shown in Fig. 16 (CD3 delta; SEQ ID NO: 24), Fig. 17 (T3 gamma; SEQ ID NO: 25), Fig. 13 (mbl; sag ID NO: 26) and Fig. 19 (B29; SEQ ID NO: 27). The portions of the sequences sufficient for cytolytic signal transduction (and therefore preferably included in a chimeric receptor of the invention) are shown in brackets.

EXAMPLE XI Experimental Methods Vaccinie Infection and Radioimmunoprecipitation Approximately 5 x 106 CV1 cells were infected for one hour in serum free DME medium with recombinant vaccinia at a multiplicity of infection (moi) of at least ten (titer measured on CV1 cells). The cells were placed in fresh medium after infection and labelled metabolically with 200uCi/ml 35$-methionine plus cysteine (Tran35S-label, ICN; Costo Mesa, CA) in methionine and cysteine free DMEM (Gibco; Grand Island, NY) for six hours. The labelled cells were detached with PBS containing 1mM EDTA, collected by centrifugation, and lysed in 1% NP-40, 0.1% SDS, 0.15 M Nacl, 0.05M Tris pH 8.0, 5mM EDTA, and 1mM PMSF. Nuclei were removed by centrifugation, and CD4 proteins immunoprecipitated with OKT4 antibody and anti-mouse IgG agarose (Cappel, Durham, NC). Samples were electrophoresed through 8% polyacrylamide/SDS gels under non-reducing (NR) and reducing (R) conditions. Gels containing 35$-labelled samples were impregnated with En3Hance (New England Nuclear, Boston, MA) prior to autoradiography.

Facilitated expression of the transmembrane form of CD16, CD16Tu, was measured by comparing its expression in CV1 cells singly infected with CD161" with expression in cells coinfected with viruses encoding CD16!" and g or 1 chimeras. After infection and incubation for six hours -57.. or more, cells were detached from plates with PBS, 1mM EDTA and the expression of CD16TM or the chimeras was measured by indirect immunofluorescence and flow cytometry. calcium rlux Assay Jurkat subline E6 (Weiss et al., Q. ;mmunol., l;1:123-128 (1984)) cells were infected with recombinant vaccinia viruses for one hour in serum free IMDM at an moi of 10 and incubated for three to nine hours in IMDM, % FBS. resuspended at 3 x 106 cells/ml in complete medium Cells were collected by centrifugation and Cells were analyzed for free calcium ion by fluorescence emission by flow cytometry (Rabinovitch et al., J. Immunol., 1;1:952-961 (1986)). calcium flux, either phycoerythrin (PE)-conjugated Leu-3A (anti-CD4) (Becton Dickinson, Lincoln Park, NJ) at 1 pg/ml was added to the cell suspension followed by long/ml of unconjugated goat anti-mouse IgG at time 0 or unconjugated 3G8 (anti-CD16) monoclonal antibody was added to the cell suspension at 1 pg/ml followed by 10 pg/ml of PE-conjugated Fab2' goat anti-monse IgG at time To initiate . collected from the PE positive (infected) cell population, which typically represented 40-80% of all cells. The T cell antigen receptor response in uninfected cells was triggered by antibody OKT3, without Histograms of the violet/blue emission ratio were crosslinking. For experiments involving CD16 chimeric receptors, samples showing baseline drift toward lower intracellular calcium (without antibody) were excluded from the analysis. Histogram data were subsequently analyzed by conversion of the binary data to ASCII using Write Hand Man (Cooper City, FL) software, followed by The violet/blue emission ratio prior to the addition of the analysis with a collection of FORTRAN programs. second antibody reagents was used to establish the normalized initial ratio, set equal to unity, and the resting threshold ratio, set so that 10% of the resting population would exceed threshold. cytolysis Assay Human T cell line WH3, a CD8+ CD4" HLA B44 restricted cytolytic line was maintained in IMDM, 10% human serum with 100 U/ml of IL-2 and was periodically stimulated either nonspecifically with irradiated (3000 rad) HLA-unmatched peripheral blood lymphocytes and lug/ml of phytohemagglutinin, or specifically, with irradiated B44-bearing mononuclear cells. After one day of nonspecific stimulation, the PHA was diluted to 0.5 pg/ml by addition of fresh medium, and after three days the medium was changed. Cells were grown for at least 10 days following stimulation before use in cytotoxicity assays. The cells were infected with recombinant vaccinia at a multiplicity of infection of at least 10 for one hour in serum free medium, followed by incubation in complete medium for three hours. Cells were harvested by centrifugation and resuspended at a density of 1 x 107 cells/ml. loopl were added to each well of a U-bottom microtiter plate containing 100 pl/well of complete medium. Cells were diluted in two-fold serial steps.

Two wells for each sample did not contain lymphocytes, to allow spontaneous chromium release and total chromium uptake to be measured. The target cells, from HeLa subline S3, were infected in 6.0 or 10.0 cm plates at an approximate moi of 10 for one hour in serum free medium, followed by incubation in complete medium for three In Vitro lutagenesis of the E sequence To create point mutations in amino acid residues and or 15 of the g sequence, synthetic oligonucleotide primers extending from the BamHI site upstream of the g transmembrane domain, and converting native ( residue 11 from Cys to Gly (C116) or residue 15 from Asp to Gly (D156) or both (C116/D156) were prepared and used in PCR reactions to generate mutated fragments which were reinserted into the wild type cD4:g constructs.

To prepare the construct CD16:(D66*, the 5 cDNA sequence corresponding to the transmembrane domain and the 17 following residues of the cytoplasmic domain was replaced by corresponding transmembrane and cytoplasmic domain obtained from the CD5 and CD7 cDNA. The CD5 and CD7 fragments were generated by a PCR reaction using forward oligcnucleotides including a BamHI restriction cleavage site and corresponding to the region just upstream of the transmembrane domain of CD5 and CD1 respectively and the following reverse oligcnucleotides overlapping the CD5 and CD7 sequences respectively and the g sequence which contained the sacI restriction Honomeric g cleavage site.

CD5:(': CGC GGG C‘I‘C.G'I'l‘ ATA GAG CTG G'1"1‘ CTG GCG CTG CT!‘ CT!‘ C'1‘G(SDQ ID NO:l2) synthetic oligonucleotide primers extending from the sacI site inside the { motif and converting native residue 48 from Asn to Ser (N488), residue 50 from Leu to Ser (L505) and residue 51 from Tyr to Phe (Y51P) were synthesized and used in a PCR reaction to generate fragments that were reintroduced into the wild type CD16:7:g(48-65) construct.

In Vitro nutagenesis of c-terninal Residues within the g cytolytie signal-rransducing xotif A CD7 transmembrane fragment bearing Klul and NotI sites at the junction between the transmembrane and intracellular domains was obtained by PCR using an oligonnucleotide with the following sequence: .CGC GGG GCG GCC ACG CGT CCT CGC CAG CAC RCA (SEQ ID NO:1A). The resulting PCR fragment was digested with BamHI and NotI and reinserted into the CD16:7:g(48-65) construct. : fragments encoding residues 33 to 65, 71 to 104, and 104 to 137 were obtained by PCR reaction using pairs of primers containing Hlul sites at the 5' end of the forward primers and stop codons followed by NotI sites at the 5' end of the reverse primers. In each case the restriction sites were indented six residues from the 5' terminus of the primer to insure restriction enzyme cleavage. { 104: CGC GGG ACG CGT ATT GGG ATG AAA GGC GAG CGC (SEQ ID NO:17L construction of rcnqzzn Deletion xutamts carboxyl terminal FcRIIA deletion mutants were constructed by PCR in the same fashion as for the full length constructs, converting the sequences encoding tyrosine at positions 282 and 298 into stop codons (TAA).

The N-terminal deletions were generated by amplifying fragments encoding successively less of the intracellular domain by PCR, using oligonucleotides which allowed the resulting fragments to be inserted between MluI and Natl restriction sites into a previously constructed expression plasmid encoding the CD16 extracellular domain fused to the CD7 transmembrane domain, the latter terminating in a Hlul site ant the juncition between the transmembrane and the intracellular domain. _ 53 - The examples described above demonstrate that aggregation of g, n, or 7 chimeras suffices to initiate the cytolytic effector cell response in T cells. The known range of expression of g, n, and 1, which includes T lymphocytes, natural killer cells, basophilic granulocytes, macrophages and mast cells, suggests that conserved sequence motifs may interact with a sensory apparatus common to cells of hematopoietic origin and that an important component of host defense in the immune system may be mediated by receptor aggregation events.

The potency of the cytolytic response and the absence of a response to target cells bearing MHC class II receptors demonstrates that chimeras based on g, n, or 1 form the basis for a genetic intervention for AIDS through adoptive immunotherapy. of endogenous g and 1 and evidence that Fc receptors The broad distribution - 54 _ intracellular site of action. _ 65 - -66.. extend the range and efficacy of current uses proposed for genetically engineered T cells.

U 0r 1 CDNA or genomic sequences encoding an extracellular domain of the receptor can be endowed with a restriction site introduced at a location just preceding the transmembrane domain of choice. The extracellular domain fragment terminating in the restriction site can then be joined to g, n, or 7 sequences. Typical extracellular domains may be derived from receptors which recognize complement, carbohydrates, viral proteins, bacteria, protozoan or metazoan parasites, or proteins induced by them. Similarly, ligands or receptors expressed by pathogens or tumor cells can be attached to g, n, or 1 sequences, to direct immune responses against cells bearing receptors recognizing those ligands.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover variations, uses, or adaptations of the invention and including such departures from the present disclosure as come within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (1) APPLICANTS: seed, Brien (ii) (111) (1') (3) (3) (C) (3) (3) (P) NUMBER OF SEQUENCES: Romeo, Charles TITLE OF INVENTION: Redirection of Cellular Immunity by Receptor Chimera: CORRESPONDENCE ADDRESS: ADDRESSES: Fish & Richardson STREET: 225 Franklin Street CITY: Boston ‘ STATE: MA COUNTRY: USA ZIP: 02110-2804 COMPUTER READABLE FORM: (A) NEDIUN TYPE: 3.5" Diskette, 1.44 Mb (3) (C) (3) (V1) (3) (3) (C) COMPUTER: IBM PS/2 Model 502 or sssx OPERATING SYSTEM: IBM P.c. DOS (Version 3.30) SOFTWARE: Wordperfect (Version 5.0) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (3) (3) (C) APPLICATION NUMBER: O7/665,961 FILING DATE: March 7, 1991 CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: clerk, Paul T. (3) (C) REGISTRATION NUMBER: 30,162 REFERENCE/DOCKET NUMBER: 00786/119002 (ix) TELECOMMUNICATION INFORMATION: (3) (3) (c) TELEX: TELEPHONE: (617) 542-5070 TELEFAX: (617) 542-8906 200154 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CEARACTERISTICS: (K) (3) (C) (D) LENGTH: 1728 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear . (11) uoLzcuLz rvpz: DNA (genomic) (xi) szguzncz DESCRIPTION: sag ID NO:1: ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC TGCAACTGGC 50 GCTCCTCCCA GCAGCCACTC AGGGAAACAA AGTGGTGCTG GGCAAAAAAG 100 GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA 150 TTCCACTGGA BAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC 200 CTTCTTLACT AAAGGTCCAT CCAAGCTGAA TGATCGCGCT GACTCAAGAA 250 GAAGCCTTTG GGACCAAGGA AACTTCCCCC TGATCATCAA GAATCTTAAG 300 ATAGAAGRCT CAGATACTTA CATCTGTGAA GTGGAGGACC AGAAGGBGGA 350 GGTGCAAITG CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC 400 TTCAGGGGCA GAGCCTGACC CTGACCTTGG AGAGCCCCCC TGGTAGTAGC 450 CCCTCAGTGC AATGTAGGAG TCCAAGGGGT ABAAACATAC AGGGGGGGAA 500 GACCCTCTCC GTGTCTCAGC TGGAGCTCCA GGATAGTGGC ACCTGGACAT 550 GCACTGTCTT GCAGAACCAG AAGAAGGTGG AGTTCAAAAT AGACATCGTG 600 GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTATAAGA AAGAGGGGGA 650 ACAGGTGGAG TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG 700 GCAGTGGCGA GCTGTGGTGG CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT 750 TGGATCACCT TTGBCCTGAA GAACAAGGAA GTGTCTGTAA AACGGGTTAC 800 CCAGGACCCT AAGCTCCAGA TGGGCAAGAA GCTCCCGCTC CACCTCACCC 850 TGCCCCAGGC CTTGCCTCAG TATGCTGGCT CTGGAAACCT CACCCTGGCC 900 CTTGAAGCGA AAACAGGAAA GTTGCATCAG GBAGTGAACC TGGTGGTGAT 950 GAGAGCCACT CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA 1000 CCTCCCCTAA GCTGATGCTG AGCTTGAAAC TGGBGAACAA GGAGGCAAAG 1050 GTCTCGAAGC GGGAGAAGCC GGTGTGGGTG CTGAACCCTG AGGCGGGGAT 1100 GTGGCAGTGT CTGCTGAGTG ACTCGGGACA GGTCCTGCTG GAATCCAACA 1150 TCAAGGTTCT GCCCACATGG TCCACCCCGG TGCACGCGGA TCCCAAACTC 1200 TGCTACTTGC TAGRTGGAAT CCTCTTCATC TACGGAGTCA TCATCACAGC 1250 CCTGTACCTG AGAGCAAAAT TCAGCAGGAG TGCAGAGACT GCTGCCAACC 1300 TOCAGGACCC GAATATGACO CAAACAGCAG AGAABGACAA AGGCGGAGAG AGCAGTOCAG GGACCCTTGG AGCCAATCCT GCCACCCBGT CAACCAGCTC TCTTGGAGBA BGGAGGAGGA GATGCCAGRA GCBAGGGGCA TTCGGGAACA GTTAAGAGCC GTGCCAGCGT AGCAGCTCCC TACAATGAGC GAAGCGGGCT ACCCCCAGGA cccracnarc cenrcaccrw GAAGAGAGAG ccccccnnna crrcnccarc AGCTCTAB (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: base pairs (3) TYPE: nucleic acid (c) srnannznnzss: double (D) TOPOLOGY: linear ATGAACCGGG GCTCCTCCCA GGGATACAGT TTCCACTOGA CTTCTTAACT GAAGCCTTTG ATAGAAGACT GGTGCAATTG TTCAGGGGCA CCCTCAGTGC GACCCTCTCC GCACTGTCTT GAGTCCCTTT GCAGCCACTC GGAACTGACC AAAACTCCAA AAAGGTCCAT GGACCAAGGA CAGATACTTA CTAGTGTTCG GAGCCTGACC AATGTAGGAG GTGTCTCAGC GCAGAACCAG (ii) MOLECULE TYPE: DNA (genomic) TAGGCACTTG AGGGAAACAA TGTACAGCTT CCAGATAAAG CCAAGCTGAA AACTTCCCCC CATCTGTGAA GATTGACTGC CTGACCTTGG TCCAAGGGGT TGGAGCTCCA AAGAAGGTGG rcnnrcrnac CGGGATCCAG AGGCGTATAC AGATCGGCAC TACCBGGACA AGAAGGTTCA ‘GTGAAAGCAC CCCACTCTGT (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CTTCTGGTGC AGTGGTGCTG CCCAGAAGAA ATTCTGGGAA TGATCGCGCT TGATCATCAA GTGGAGGACC CAACTCTGAC AGAGCCCCCC AAAAACATAC GGATAGTGGC AGTTCAAAAT GCGAAGAGAG AGATGGGAGG AATGCACTGC AAAAGGCGAG GCCACTTCCA GAACTCACAA CCAGCAGAGT GGAGTCCATG TGCAACTGGC GGCAAAAAAG GAGCATACAA BTCAGGGCTC GACTCAAGAA GAATCTTAAG AGAAGGAGGA ACCCACCTGC TGGTAGTAGC AGGGGGGGAA ACCTGGACAT AGACATCGTG GTGCTAGCTT ACAGGTOGAG GCAGTGGCGA TGGATCACCT CCAGGACCCT TGCCCCAGGC CTTGAAGCGA GAGAGCCACT CCTCCCCTAA GTCTCGAAGC GTGGCAOTGT TCAAGGTTCT TGCTATATCC GCTCTACTGT GTGAGAAATC ACATATGAGA TCCAGAAGGC TTCTCCTTCC GCTOTGGTGG TTGACCTGAA AAGCTCCAGA CTTGCCTCAG AAACAGGAAA CAGCTCCAGA GCTGATGCTG GGGAGAAGCC CTGCTGAGTG GCCCACATGG TGGATGCCAT CGACTCAAGA AGATGCTGTC CTCTGAAACA CTCCAGCATA CACTCGCCTT CAGGCGGAGA GAACAAGGAA TGGGCAAGAA TATGCTGGCT GTTGCATCAG AAAATTTGAC AGCTTGAAAC GGTGTGGGTG ACTCGGGACA TCCACCCCGG CCTGTTTTTG TCCAGGTCCG TACACGGGCC TGAGAAACCA (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: base pairs (B) TYPE: nucleic acid (c) SIRANDEDNESS: double (D) zroponocn linear (ii) MOLECULE TYPE: DNA (genomic) GTCTATAAGA TACAGTTGAA GGGCTTCCTC GTGTCTGTAA GCTCCCGCTC CTGGAAACCT GAAGTGAACC CTGTGAGGTG TGGAGAACAA CTGAACCCTG GGTCCTGCTO TGCACGCGGA TATGGTATTG AAAGGCAGAC TGAACACCCG CCCCAATAG (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AAGAGGGGGA AAGCTGACGG CTCCAAGTCT AACGGGTTAC CACCTCACCC CACCCTGGCC TGGTGGTGAT TGGGGACCCA GGAGGCAAAG AGGCGGGGAT GAATCCAACA TCCGCAGCTC TCCTTACCCT ATAGCCAGCC GAACCAGGAG ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC TGCAACTGGC GCTCCTCCCA GCAGCCACTC AGGGAAACAA AGTGGTGCTG GGCAAAAAAG GGGATACAGT GGAACTGACC TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA AAAACTCCAA CCAGATAAAG ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT CCAAGCTGAA TGATCGCGCT GACTCAAGAA GAAGCCTTTG ATAGALGACT GGTGCAATTG TTCAGGGGCA CCCTCAGTGC GACCCTCTCC GCACTGTCTT GTGCTAGCTT ACAGGTGGAG GCAGTGGCGA TGGATCACCT CCAGGACCCT TGCCCCAGGC CTTGAAGCGA GAGAGCCACT CCTCCCCTAA GTCTCGAAGC GTGGCAGTGT TCAAGGTTCT TGCTACCTGC CTTGTTCCTG BGCAGGGCCA GAGTACGATG AAAGCCGAGA AAGATAAGAT CGGAGGGGCA CAAGGACACC GGACCAAGGA CAGATBCTTA CTAGTGTTCG GAGCCTGACC AATGTAGGAG GTGTCTCAGC GCAGAACCAG TCCAGAAGGC TTCTCCTTCC GCTGTGGTGG TTGACCTGAB AAGCTCCAGA CTTGCCTCAG AAACAGGAAA CAGCTCCAGA GCTGATGCTG GGGAGAAGCC CTGCTGAGTG GCCCACATGG TGGATGGAAT AGAGTGAAGT GAACCAGCTC TTTTGGACAA AGGAAGAACC GGCGGAGGCC AGGGGCACGA TACGACGCCC AACTTCCCCC CATCTGTGAA GATTGACTGC CTGACCTTGG TCCAAOGGGT TGGAGCTCCA AAGAAGGTGG CTCCAGCATA CACTCGCCTT CAGGCGGAGA GAACAAGGAA TGGGCAAGAA TATGCTGGCT GTTGCATCAG AAAATTTGAC AGCTTGAAAC GGTGTGGGTG ACTCGGGACA TCCACCCCGG CCTCTTCATC TCAGCAGGAG TATAACGAGC GAGACGTGGC CTCAGGAAGG TACAGTGAGA TGGCCTTTAC TTCACATGCA (2) INFORMATION FOR SEQ ID N024: TGATCATCAA GTGGAGGACC CAACTCTGAC AGAGCCCCCC AAAAACATAC GGATAGTGGC AGTTCAAAAT GTCTATAAGA TACAGTTGAA GGGCTTCCTC GTGTCTGTAA GCTCCCGCTC CTGGAAACCT GAAGTGAACC CTGTGAGGTG TGGAGAACAA CTGAACCCTG GGTCCTGCTG TGCACGCGGA TATGGTGTCA CGCAGAGCCC TCAATCTAGG CGGGACCCTG CCTGTACAAT TTGGGATGAA CAGGGTCTCA GGCCCTGCCC GAATCTTAAG AGAAGGAGGA ACCCACCTGC TGGTAGTAGC AGGGGGGGAA ACCTGGACAT AGACATCGTG AAGAGGGGGA AAGCTGACGG CTCCAAGTCT BACGGGTTAC CACCTCACCC CACCCTGGCC TGGTGGTGAT TGGGGACCCA GGAGGCAAAG AGGCGGGGAT GAATCCAACA TCCCAAACTC TTCTCACTGC CCCGCGTACC ACGAAGAGAG AGATGGGGGG GAACTGCAGA AGGCGAGCGC GTACAGCCAC CCTCGCTAA 4oo 450 soo 550 eoo sso 7oo 15o eoo sso 9oo 95o ooo o5o oo oo oo soo Mot Ala Lys Ilo Gln 65 Asp Lys Asp Sor sor 145 Ly: Gln Val Sor Lou 225 Gln Ly: Gln Pro Thr 305 Gln (1) ssgunnca CHARACTERISTICS: (A) LENGTH: (ii) uoLacuLz (xi) szquzxcs Asn Lou Gly Gln 50 Gly Sor Asn Gln Asp 130 Pro Asn Asp Glu Ilo 210 Ala Ala Asn Mot Gln 290 Gly Lou Arg Lou Asp Pho Sor Arg Lou Lys 115 Thr Pro Ilo Sor Pho 195 Val Pho Glu Lyn Gly 275 Tyr Ly: Gln Glu 355 Gly Pro Thr His Pho Arg Lys 100 Glu Bis Gly Gln Gly 180 Lys Tyr Thr Arg Glu 260 Ly: Ala Lou Ly: Lou Lys Val Ala Val rrp Lou Sor 85 Ilo Gln Lou Sor Gly 165 Thr Ilo Ly: Val Ala 245 Val Asn 325 Sor amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear TYPE: protein DESCRIPTION: Pro Ala Glu Lyn Thr Lou Glu Val Lou Sor 150 Gly rrp Asp Lys Glu 230 Sor Sor Lou Sor Gln 310 Lou Pho Thr Lou Asn Ly: Trp Asp Gln Gln 135 Pro Lys Thr Ilo Glu 215 Lys Sor Val Pro Gly 295 Glu Thr Arg Gln Thr 40 Sor Gly Asp Sor Lou 120 Gly Sor Thr Cy: val 200 Gly Lou Sor Lys Lou 280 Asn Val Cys Val 360 sag ID Bis Gly Cys Asn Pro Gln Asp 105 Lou Gln Val Lou Thr 185 Val Glu Thr Lys Arg 265 His Lou Asn Gln Glu Lou Lou Asn Sor Gly 90 Thr Val Sor Gln Ser 170 Val Lou Gln Gly Sor 250 Val Lou Thr Lou Val 330 No:4: Lou Lys Ala Ilo Lys Asn Tyr Pho Lou Cys 155 Sor 235 rrp Thr Thr Lou Val 315 Trp Lys Lou Val Sor Lys Lou Pho Ilo Gly Thr 140 Arg Sor Gln Pho Glu 220 Gly Ilo Gln Lou Ala 300 Vol Gly Val Val Gln 45 Ilo Asn Pro Cys Lou 125 Lou Sor Gln Asn Gln 205 Pho Glu Thr Asp Pro 285 Lou Mot Pro Ala 365 Lou Lou Ly: Lou Asp Lou Glu 110 Thr Thr Pro Lou Gln 190 Ly: Sor Lou Pho Pro 270 Gln Gln Arg Thr Lys Gly Gly Lyn Gly Arg Ilo 95 Val Ala Lou Arg Glu 175 Lys Ala Pho rrp Asp 255 Lys Ala Ala Ala Sor 335 Lou Lys Sor Asn Ala 80 Ilo Glu Asn Glu Gly 160 Lou Lys S81‘ T1‘? 240 Lou Lou Lou Lys Thr 320 Pro Gln Lys 385 Cys Ala net 465 Asn Thr Asp Gly Gln Pro Met Ala Lys Ile Gln Asp Lys Asp ' Lou Len Ser 375 Ser Gln Val Leu Ala Cys Len 370 Asp Gly Len Pro Thr Val His Tyr Trp Pro Len Len Gly Ile Phe Ile Arg Tyr Asp Len are Len Len 420 Tyr Ala Lys Phe Ser Ala Tyr Gln 435 Gln Pro Asn Gln Len Asn Gln Len Gln Gln 450 Val Len 455 Tyr Asp Ala 460 Lys Lys Arg Gln Gln 470 Asp Gly Lys Pro 415 Arg Arg Asn Ala Len Gln Lys Arg Lyn Met Gln 490 G1y' Pro Tyr Gln 500 Phe Lys Gly Arg Arg Gly Lys Phe His 515 Gln Ser Gln Ala Val Gln Len Gly Asn Arg Len Thr Thr 535 Ser 530 Ser Ala 540 Ser Arg Gly Len Arg Gln Gln Ser 550 Gln Ala 565 SB!‘ Ser Cy: Thr Len Ser Ser Ser Pro Pro Tr? Tr? INFORMATION FOR SEQ ID N0:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 462 amino acids (3) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Asn Arg Gly Val Pro Phe Arg His Len Ala Ala Thr Gln Gly Asn Len Len Len Len Pro Val Thr Val Gln Th: 40 Gly Asp Len Thr Ala Ser Gln Phe His 50 Asn Gln Ile 55 Trp Lys Asn Lys Phe Thr Len 70 Ser Len Gly Pro Ser Lys Ser Gln Asn Phe 85 Ser Arg Arg Asp Gly Thr Asn Len Lys Gln Ser Ile Gln Asp Asp 105 Gln Gln Val Gln Len Val Phe Gly Lys 115 Thr Asp His Len Len Gln Gln Ser Len Thr Gln Asp Val Gln Asn 445 Arg Gln Ser Gly Arg 525 Arg Val Val Gln Ile Asn Pro Cys Len Len Ser Pro Ile Thr 430 Len Asp Gly Gln Len 510 Gln Pro Len Leu Lys Len Asp Len Gln 110 Ann Lys Ile 415 Ala Gly Pro Ile 495 Tyr Arg Lys Ser Len 575 Gly Lys Gly Arg Ile Val Ile Len 400 Thr Ala Arg Tyr 480 Gly Gln Gln Ile 560 Len Lys Ser Asn Ala Ile Gln Ser 145 Ly: Gln Val Ser Len 225 Gln Lys Gln Pro 305 Gln Lys Lys Gln Lys 385 Cys Leu Pro Ann Asp Gln Ila 210 Ala Ala Asn Met Gln 290 Gly Len Len Arg Cy: Val Tyr Len Arg Gln 450 Pro Ila Ser Pho 195 Val Pho Glu Ly! Gly 275 Tyr Lys Gln Met Gln 355 Len Leu Ile Tyr Glu Thr Gly Gln Gly 180 Lys Tyr Thr Arg Gln 260 Lys Ala Len Lys Leu 340 Lys Len Pro Len Cys 420 Lys Ser Gly 165 Thr 110 Lys Val Ala 245 Val Lys Gly His Asn 325 Ser Pro Ser Thr Asp 405 hrs !‘ INFORMATION FOR (A) LENGTH: Ser Pro 150 Gly Lys Trp Th: Asp Ile Glu 215 Lys Gln 230 Ser Ser Ser Val Len Pro Ser Gly 295 Gln Glu 310 Len Thr Len Lys Val Trp Ser 375 set up 390 Ala Ile Len Lys Asp Ala Thr Leu SEQ ID N026: Ser Thr Cys Val 200 Gly Len Ser Lys Len 280 Asn Val Cys Len Val 360 Gly Thr Leu Ila Val Lys Val Lou Thr 185 Val Gln Thr Ly: Arg 265 His Len Asn Glu Glu 345 Leu Gln Pro Gln 425 (i) SEQUENCE CHARACTERISTICS: 532 amino acids (3) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein Gln Ser 170 Val Len Gln Gly Ser 250 Val Len Thr Len Val 330 Asn Asn Val Val Len 410 Val Cys 155 Val Len Ala Val Ser 235 Trp Thr Thr Val 315 T1‘? Lys Pro Len His 395 Arg Gly Arg Ser Ser Gln Gln Asn Pha Gln 205 Glu Phe 220 Gly Gln Ile Thr Gln Asp Pro 285 Len Ala 300 Val Met Gly Pro Glu Ala Gln Ala 365 Len Gln 380 Ala Asp Gly Ile Lys Ala Len Asn 445 Pro Pro 460 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Pro Len Gln 190 Lys Ser Len Phe Pro 270 Gln Glu Arg Thr Lys 350 Gly Ser Pro Val Asp 430 Gln 462 Arg Glu 175 Lys Ala Phe TrP Asp 255 Lys Ala Ala Ala Ser 335 Val Met Asn Gln Len 415 Gly 160 Len Lys S81‘ was 240 Len Len Len Lys Thr 320 Ile Len 400 Thr Ala flat Asn Arg Gly Val Pro Phe Arg His Len Leu Len Val Len Gln Leu Ala Ly: I1: Gln A:p Ly: A:p Ser Ser 145 Ly: Gln Val Ser Lon 225 Gln Ly: Gln Pro 305 Gln Ly: Ly: Gln Ly: 385 Cy: Ala Tyr Len Gly Gln Gly Ser A:n Gln Asp 130 Pro Asn Asp Gln I10 2 10 Ala Ala Asn Met Gln 290 Gly Len Lon Arg Cy: Val Tyr Len Gln Gln 450 Len Asp P11: Ser Arg Len Ly: 1 15 Th: Pro Ile Ser Phe 195 Val Phe Gln Ly: Gly 27 5 Tyr Ly: Gln Mot Gln 3 55 Len Len Len Phe Gln Gln Thr El: Phe Arg Ly: 100 Gln Bi: Gly Gln Gly 180 Ly: Tyr 'l‘hr Arg Gln 260 Ly: Ala Len Ly: Len 340 Ly: Len Pro Len Len 420 'l‘yr Ala Val Tr? Len Ser I1: Gln Len Ser Gly 165 '.!.'hr Ile Ly: Val Ala 245 Val Ly: Gly Hi: Asn 325 Ser Pro Ser Thr Asp 405 Arg Ala Gln Ly: Thr Lou Gln Val Leu Ser 150 Gly Tr? Asp Ly: Gln 230 Ser Ser Len Ser Gln 3 10 Len Len Val Asp TIP 390 Gly Val Thr Len Asn Ly: Trp Asp Gln Gln 135 Pro Ly: Thr Ile Gln 215 Lys Ser Val Pro Gly 295 Gln Thr Lys Trp Ser 375 Ser Ile Lys Len 455 Gln Thr Ser Gly Asp Ser Len 120 Gly Ser Thr Cy: Val 200 Gly Len Ser Lys Len 280 Asn Val Cys Len Val 360 Gly Thr Len Phe Len Asp Cy: Asn Pro Gln Asp 105 Lou Gln val Leu Thr 185 Val Gln Thr Ly: Arg 265 Hi: Len Asn Gln Gln 345 Leu Gln Pro Phe Ser 425 Asn Thr Gln Ser Gly Thr Gln: Ser 170 Val Len Gln Gly Ser 250 Val Len Thr Len Val 330 Asn Asn Val Val Ile 410 Arg Ly: Ala Ile Lys Asn Tyr Phe Len Cy s 155 Val Len Ala Ser 235 TI’? Thr Len- Val 315 rrp Lys Pro Len Hi: 395 Tyr Ser Val Ser Lys Len Phe Ile Gly Thr 140 Arg Ser Gln Phe Gln 220 Gly Ile Gln Len Ala 300 Val Gly Gln Gln Len 380 Ala Gly Ala Gly 460 Val Gln Ile Asn Pro Cy: Len 125 Len Ser Gln Asn Gln 205 Phe Gln 'l‘hr Asp Pro 285 Len Met Pro Ala Ala 365 Gln Asp Val Gln Asn Arg Ly: Len Asp Len Gln 110 Thr Thr Pro Len Gln 190 Ly: Ser Len Phe Pro 270 Gln Gln Arg Thr Ly: 350 Gly Ser Pro Ile Pro 430 Gly Ly: Gly Arg Ile Val Ala Len Arg Gln 175 Ly: Ala Phe TIP Asp 255 Ly: Ala Ala Ala Ser 335 Val Met Asn Ly: Len 415 Pro Ly: Ser Asn Ala Ile Gln Asn Gln Gly 160 Len Ly: S61’ T1’? 240 Len Len Len Ly: Thr 320 Pro Ser T1-‘P Ile Leu 400 Thr Ala _ 75 - Net Gly Gly Ly: Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Ty: Asn 465 470 475 480 Glu Lou Gln Lyn Asp Lye Met Ala Glu Ala Tyr Set Glu Ile Gly net 485 490 495 Ly: Gly Glu Arg Arg Arg Gly Lys Gly Bis Asp Gly Leu Tyr Gln Gly 500 505 510 Leu Set Thr Ala Thr Ly: Asp Thr Ty: Asp Ala Leu Bis Met Gln Ala 515 520 525 Lou Pro Pro Arg 530 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 bases (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CGCGGGGTGA CCGTGCCCTC CAGCAGCTTG GGC 33 (2) INFORMATION FOR sag ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 bases (3) TYPE: nucleic acid (c) srxnunznnmss: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGCGGGGATC CGTCGTCCAG AGCCCGTCCA GCTCCCCGTC CTGGGCCTCA 50 (2) INFORMATION FOR SEQ ID NO:9: (1) szquzncs cnnnacrznrsrxcs: (A) LENGTH: 33 bases (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGCGGGCGGC CGCGACGCCG GCCAAGACAG CAC 33 (2) INFORMATION FOR seq ID NO:lO: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 bases (3) TYPE: nucleic acid (c) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CGCGTTGACG AGCAGCCAGT TGGGCAGCAG CAG 33 (2) INFORMATION FOR SEQ ID NO:l1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: COCGGGCGGC CGCTA 15 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 bases (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CGCGGGCTCG TTATAGAGCT GGTTCTGGCG CTGCTTCTTC TG 42 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 bases _ 78 _ (3) TYPE: nucleic acid (c) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l3: CGCGGGGAGC TCGTTATAGA GCTGGTTTGC CGCCGAATTC TTATCCCG 48 (2) INFORMATION ron 329 ID N0:l4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 bases (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CGCGGGGCGG CCACGCGTCC TCGCCAGCAC ACA 33 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 bases (8) TYPE: nucleic acid (c) srnnunznuzss: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CGCGGGACGC GTTTCAGCCG TCCTCGCCAG CACACA 36 (2) INFORMATION FOR SEQ ID NO:l6: (i) szqumxcn CHARACTERISTICS: (A) LENGTH: 33 bases (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CGCGGGACGC GTGACCCTGA GATGGGGGGA AAG 33 (2) INFORMATION FOR SEQ ID NO:l7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 bases (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CGCGGGACGC GTATTGGGAT GAAAGGCGAG CGC 33 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 bases (3) TYPE: nucleic acid (c) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CCCGGATCCC AGCATGGGCA GCTCTT 26 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 bases (3) TYPE: nucleic acid (c) srnaunznuzss: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9: CGCGGGGCGG CCGCTTTAGT TATTACTGTT GACATGGTCG TT 42 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GCGGGGGGAT CCCACTGTCC AAGCTCCCAG 30 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 bases (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCGGGGGCGG CCGCCTAAAT ACGGTTCTGG TC 32 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 bases (3) TYPE: nucleic acid (c) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TCAGAAAGAG ACAACCTGAA GAAACCAACA A 31 (2) INFORMATION FOR SEQ ID NO:23: (i) szquancz canxacwnnxsmxcs: (A) LENGTH: 31 bases (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: TTGTTGGTTT CTTCAGGTTG TGTCTTTCTG A Not So: Val Gly Lou 65 Asp Val Ala Glu Asn Set INFORMATION FOR SEQ ID N0:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 171 amino acids (3) TYPE: anino acid (c) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: amino acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Glu His Sor Thr Pho Lou Set Gly Leu Val Lou Ala Th: Pro Pho Lys Ile Pro Ile Lou Lou Gln Val So: Asp A811 Glu Glu Glu Leu Arg Pho Val Asn Thr Sor 40 Cy: Thr Val Glu Gly Trp Th: Val Thr 50 Ile 55 Lou Sor Asp Arg Leu Asp Leu Th: Lys Arg Ile Ile 70 Pro Arg Gly Arg Cys Asn Gly Ilo Arg Asp Ty: Lys 80 Glu Thr Cys 85 P130 Lys sor Gln Val His Tyr Met Gln Gly Set 95 Asp Glu Lou Ala Thr Val Ala Gly Asp Ilo 100 Ile Val Val 110 Ilo Thr Ala 115 Lou Ala Lou Val Ala Lou Pho Pho 125 Cys Gly His Thr 130 Lou sor Ala Thr Gln Asp Arg Gly 135 Asp Len Leu Arg Gln Val Gln 150 Tyr Lou Arg Asp Arg Ala Gln Lys Asp Tyr His Lou Gly Gly Ala Asn 170 Trp Arg INFORMATION FOR SEQ ID NO:25: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 182 anino acids (3) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: amino acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Mot Glu Gln Gly Lys Gly Leu Ala Val Lou Ilo Lou Ala Ile Ile Leu Lou Gln Gly Thr Lou Ala Gln Ser Ile Lys Gly Asn His Len Val Lys Val Glu Lau 65 Sar 145 Gln Mat Lau Lau 65 Gly Ala Cys Ila 145 Tyr Ala 50 Thr Arg Gln Thr Ala 130 Arg Asp Lys Glu Gly Val Ila 115 Val Ala Tyr Gln Glu Asn Ila Thr Asp Lys Lys Rat Gln Tyr 85 Tyr Tyr 100 Gly Pha Gly Val Tyr Sar Asp Lys Lys Asp Arg 165 Arg Arg Asn Asp Trp 55 Lys Cys Mat Lau Pha 135 Gly 40 Pha Trp Lys Cys Pha 120 Ila Lys Asn Gly Gln 105 Ala Ala INFORMATION FOR SEQ ID NO:26: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: (3) rypz: (c) STRANDEDNESS: single (n) TOPOLOGY: linear (ii) MOLECULE TYPE: amino acids Val Asp Lau Se: 90 Asn Glu Gly Gln 170 amino acids amino acid Lau Len Gly Lys 60 Gly Sat 75 Gln Asn Cys Ila Ila Val Gln Asp 140 Pro Asn 155 Tyr Sar (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Pro sar Gly Cys 50 Gln Thr Cys Gly Mat Lou Tyr Pro Glu Sar Thr Thr Thr 115 Gly Gly Lau Glu Ala Cys L80 Asn Asn Gly Thr 70 Asn Ila Gln 85 Gly Gly Gln 100 Tyr Lau Arg Glu Gly Thr Ala Pha Cys Ala Gly Thr Arg 55 Trp Pha Val Val Lys Val Len Pro Val 40 Asn Pro Pha Ila Arg 120 Arg Gly Asn Pro Pro Pro Glu 105 Asn Ala Cys Leu Asn Val Glu 90 Asn Pro Lau Pro Gln Ala Gly Glu Ila Thr 60 Pro Lau 75 Val Asn Asn Ila Val Pro Thr 140 Lau Thr 155 Thr 45 Hat Asn Lys Glu Sar 125 Gly Lau Lou Glu 45 TIP Gly Lys Lau Arg 125 Cys Ila Ala Sar Lau 110 Ila Val Lau Arg Ala Trp Pro Asn Lys 110 Pro Asp Gly Lys Lys 95 Asn Pha Arg Gln 175 Lau Val Arg Pha Gly Thr 95 Arg Pha Ala Pha Asp 80 Pro Ala Tyr 160 Gly Pha Glu Lau Sar Gln 80 Gly sar Leu Arg 160 Ly: Arg Trp Gln Asn Gln Lys Phe Gly Val Asp Met Pro Asp Asp Tyr 165 170 175 Gln Asp Gln Asn Len Tyr Gln Gly Len Asn Len Asp Asp Cys Ser Met 180 185 190 Tyr Gln Asp Ile Ser Arg Gly Len Gln Gly Thr Tyr Gln Asp Val Gly 195 200 205 Asn Len His Ile Gly Asp Ala Gln Len Gln Lys Pro 210 215 220 (2) INFORMATION FOR SEQ ID NO:27: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 228 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: amino acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Met Ala Thr Len Val Len Ser Ser Met Pro Cys His Trp Len Len Phe 10 15 Len Len Len Len Phe Ser Gly Gln Pro Val Pro Ala Met Thr Ser Ser 25 30 Asp Len Pro Len Asn Phe Gln Gly Ser Pro Cys Ser Gln Ile Trp Gln 40 45 His Pro Arg Phe Ala Ala Lys Lys Arg Ser Ser Met Val Lys Phe His 50 55 60 Cys Tyr Thr Asn His Ser Gly Ala Len Thr Trp Phe Arg Lye Arg Gly 65 70 75 80 Ser Gln Gln Pro Gln Gln Len Val Ser Gln Gln Gly Arg Ile Val Gln 85 90 95 Thr Gln Asn Gly Ser Va1 Tyr Thr Len Thr Ile Gln Asn Ile Gln Tyr 100 105 110 Gln Asp Asn Gly Ile Tyr Phe Cys Lys Gln Lys Cys Asp Ser Ala Asn 115 120 125 His Asn Val Thr Asp Ser Cys Gly Thr Gln Len Len Val Len Gly Phe 130 135 140 Ser Thr Len Asp Gln Len Lys Arg Arg Asn Thr Len Lys Asp Gly Ile 145 150 155 160 Ile Len Ile Gln Thr Len Len Ile Ile Len Phe Ile Ile Val Pro Ile 165 170 175 Phe Len Len Len Asp Lys Asp Asp Gly Lys Ala Gly Met Gln Gln Asp 180 185 190 His Thr Tyr Gln Gly Len Asn Ile Asp Gln Thr Ala Thr Tyr Gln Asp 195 200 205 Ile Val Thr Len Arg Thr Gly Gln Val Lys Trp Ser Val Gly Gln His 210 215 220 Pro Gly Gln Gln 225

Claims (1)

1.Claims: A method for manufacturing a medicament for the therapeutic application in an infection, a tumor disease or an autoimmune disease, characterized in that therapeutic cells directing a cellular response to an infective agent, to a cell infected with said agent, to a tumor or cancerous cell, or to an autoimmune- generated cell in a mammal are used, said therapeutical cells being capable of specifically recognizing and destroying said agent or said cell; wherein said therapeutic cells are selected from the group consisting of: (a) T lymphocytes; (b) cytotoxic T lymphocytes; (c) neutral killer cells; (d) neutrophils; (e) granulocytes; (t) macrophages; (g) mast cells; (h) HeLa cells; wherein said therapeutic cells express a membrane-bound, proteinaceous chimeric receptor, comprising (a) an extracellular portion which is capable of specifically recognizing and binding said agent or said cell wherein said extracellular portion comprises an HIV envelope-binding portion of CD4, or a functional HIV envelope~binding derivative thereof; and (b) an intracellular or transmembrane portion which is capable of signalling said therapeutic cell to destroy a receptor-bound agent or a receptor-bound cell, wherein said intracellular portion or said transmembrane portion is a signal~transducing portion of CD3 delta or T3 gamma, FCRYH, or a B cell receptor protein, or a functional derivative thereof. The method of claim 1, wherein said cellular response is MHC—independent. The method of claim 2, wherein said portion of FCRYII is FCRYIIA or FcRyllC;or wherein said portion of a B cell receptor protein is mbl or B29. 10. 85 The method of claim 3, wherein said chimeric receptor comprises: (a) amino acids YMTLNPRAPTDDDKNIY; (b amino acids 132-171 of