RU2165703C2 - Method of suppression of hiv-infected mammalian cells and protein recombinant receptor for its realization - Google Patents

Abstract

FIELD: genetic engineering, medicine. SUBSTANCE: invention proposes method of administration to mammal of effective amount of therapeutic cells expressing membrane-bound recombinant receptor. Receptor has exocellular part that involves CD4 fragment showing ability to recognize specifically and bind HIV-infected cell but which does not mediate HIV-infection. Also, invention describes the protein recombinant receptor that is able also to recognize specifically and bind HIV-infected cell but it does not mediate HIV-infection. Invention ensures to operate cellular immune response directed against HIV-infected mammalian cell. EFFECT: improved method of suppression. 11 cl, 26 dwg

Description

 The invention relates to receptors that include CD4 fragments that bind HIV but do not make host cells susceptible to HIV infection. Thus, the present invention has created a new and effective therapeutic agent against HIV.

 The recognition of antigen by T-cells using a T-cell receptor is the basis of numerous immunological phenomena. T cells control what is called cell-mediated immunity. It includes a mechanism for the destruction of foreign tissue or infected cells by cells of the immune system. There are varieties of T cells, including helper and suppressor cells that modulate the immune response, and cytotoxic (or "killer") cells that can directly destroy abnormal cells.

 A T cell that recognizes and binds the corresponding antigen found on the surface of another cell becomes activated; it can then multiply and if it is a cytotoxic cell, then it will destroy the attached cell.

HIV and immunopathogenesis
In 1984, it was found that HIV is the etiological factor of AIDS. Since then, the definition of AIDS has been repeatedly changed many times in accordance with the applicable diagnostic criteria. However, despite fluctuations in the diagnostic parameters, an obvious common beginning is HIV infection, followed by the development of persistent constitutional symptoms and AIDS-determining diseases, such as secondary infections, neoplasms, and neurological diseases.

 Harrison Basics of Therapy, 12th ed. McGraw Hill (1991).

 HIV is a human retrovirus from the lentivirus group. Four recognizable human retroviruses belong to two different groups: T-lymphotropic (or leukemic) human retroviruses. HTLV-1 and HTLV-2; and human immunodeficiency viruses, HIV-1 and HIV-2. The first group is represented by transforming viruses, while the second group is represented by cytopathic viruses.

 HIV-4 has been identified as the most common cause of AIDS in the world. The sequence homology between HIV-2 and HIV-1 is about 40% of HIV-2, which is closer to the group of immunodeficiency viruses of apes (SIV). Curran et al., Nature 324: 572-575 (1986).

 HIV-2 is represented by the usual retroviral genes (env, gag and pol), as well as six other genes involved in the replication and other biological functions of the virus. As indicated above, the common denominator of AIDS is deep immunosuppression, leading to the appearance of a variety of diseases caused by opportunistic microorganisms, in particular infectious and tumorous.

 The main cause of immunity disorders in AIDS is considered as a quantitative and qualitative insufficiency of the population of T4 cells, lymphocytes produced by the thymus (T). This group of cells is phenotypically detected by the presence of surface CD4 molecules, which are cellular receptors for HIV. Dalgleich et al., Nature 312: 763 (1984). Although a T4 cell is the main type of HIV-infected cell, virtually any human cell that expresses a CD4 molecule on its surface is able to bind and become infected with HIV.

 Traditionally, CD4 T cell molecules have been assigned the role of helpers / inducers indicating their function in providing an activated signal for T cells or inducing the formation of T lymphocytes carrying a similar CD8 marker for cytotoxic / suppressor cells. Reinherz and Schlossman, Cell 19: 821-827 (1980); Goldstein et al., Immunol, rev. 68: 5-42 (1982).

 HIV specifically binds with high affinity, as a result of the amino acid extension of the viral envelope (gp120), with a region of region VI of the CD4 molecule located near its N-terminus. After binding, the virus fuses with the membrane of the target cell and is internalized. Immediately after internalization, he uses the enzyme reverse transcriptase to transcribe his genomic RNA from DNA, which is integrated into cellular DNA and where it exists throughout the life of the cell as a “provirus”.

 Provirus can remain latent or activate by transcribing mRNA or genomic RNA, leading to protein synthesis, assembly, the formation of new viral particles and the separation of the virus from the cell surface. Although the exact mechanism by which the virus causes cell death has not been established, it is obvious that the main mechanism is the massive separation of viral particles from the cell surface, leading to the destruction of the cytoplasmic membrane and impaired osmoregulation.

 During infection, the host organism produces antibodies against viral proteins, including the main envelope glycoproteins gp120 and gp41. Despite this humoral immunity, the disease progresses, leading to lethal immunosuppression, characterized by multiple opportunistic infections, parasitemia, dementia and death. The lack of host antiviral antibodies that impede the development of the disease is one of the most painful and disturbing aspects of infection, and portends the low effectiveness of vaccination efforts by traditional methods.

 Two factors can affect the effectiveness of the humoral response in viral immunodeficiency. First, like other RNA viruses (and in particular, like retroviruses), immunodeficiency viruses exhibit a high mutational rate in response to host immune surveillance. Secondly, envelope glycoproteins themselves are highly glycosylated molecules, representing several epitopes with high affinity for binding of antibodies. The insufficient antigenic target that the viral envelope represents leaves the host little opportunity to limit viral infection by the formation of specific antibodies.

 HIV infected cells express gp120 glycoprotein on their surface, gp120 mediates fusion events between CD4 cells by a reaction similar to that by which the virus invades an uninfected cell, resulting in the formation of short-lived multinucleated giant cells. The formation of syncytium depends on the direct interaction of the envelope glycoprotein gp120 with protein CD4. Dalgleish et al., Supra; Klatzman et al., Nature 312: 763 (1984); McDougal et al., Science 231: 382 (1986); Sodroski et al., Nature 322: 470 (1986); Lifson et al., Nature 323: 725 (1986); Sodroski et al., Nature 321: 412 (1986).

 Evidence that CD4-gp120 binding is responsible for viral infection of cells carrying the CD4 antigen includes the detection of a specific complex that forms between gp120 and CD4 (McDougal et al., Supra). Other researchers have shown that cell lines not infected with HIV turned into infected cell lines after transfection and expression of the CD4 cDNA gene in humans. Maddon et al., Cell 46: 333-348 (1986).

 Soluble CD4-based therapeutic programs as a passive agent for suppressing viral adsorption and syncytia-mediated intercellular transmission have been proposed and successfully demonstrated in vitro by a number of groups (Deen et al., Nature 331: 82-84 (1988); Fisher et al., Nature 331: 76-78 (1988); Hussey et al., Nature 331: 78-81 (1988); Smith et al., Science 238: 1704-1707 (1987); Traunecker et al., Nature 331: 84-86 (1988); a subsequently created fusion CD4-immunoglobulin proteins with extended half-life and moderate biological activity (Capon et al., Nature 337: 525-531 (1989); Traunecker et al., Nature 339: 68 -70 (1989); Byrn et al., Nature 344: 667-670 (1990); Zettlmeissl et al., DNA Cell Biol. 9: 347-353 (1990). Although conjugates, CD4 immunotoxin or fusion proteins show strong cytotoxicity to infected cells in vitro (Chaudhary et al., Nature 335: 369-372 (1988); Till et al., Science 242: 1166-1168 (1988), the latent state of immunodeficiency syndrome makes it unlikely that any single therapy will be effective in eliminating viral damage, and the antigenicity of foreign fusion proteins is likely to limit their tolerability in repeated dose treatments. Tests performed on monkeys affected by SIV have shown that soluble CD4 when administered to animals without distinct CD4 cytopenia can lower the SIV titer and improve in vitro myeloid potential (Watanabe et al., Nature 337: 267-270 (1989). induced repeated viral manifestation was observed after interrupted treatment, indicating that lifelong administration of CD4 seems to be necessary to prevent progressive depletion of the immune system.

T cells and Fc receptors
The cell surface expresses the most abundant form of the antigenic T cell receptor (TCR), requiring coexpression of at least 6 different polypeptide chains (Weiss et al., J. Exp. Med. 160: 1284-1299 (1984); Orloffhashi et al., Nature 516: 606-609 (1985); Berkhout et al., J. Biol. Chem. 263: 8528-8556 (1988); Sussman et al., Cell 52: 85-95 (1988), α / β antigen, binding chain, three CD3 complex polypeptides, and s. If no chain is present, stable expression of the remaining members of the complex does not occur.s is a limiting polypeptide for surface expression of the full complex (Sussman et al., Cell 52: 85-95 ( 1988)) presumably mediates the fraction that triggers cell activation programs with a ligand-recognizing receptor (Weissman et al., EMBO J. 8: 3651-5656 (1989); Frank et al. Science 249: 174-177 (1990)). 1 with a molar mass of 32 kDa, s (zeta), fixed in the membrane, has 9 extracellular residues of a domain that has no sites for N-attached incremental glycan, and 112 residues (mouse) or 113 residues (human) of the intracellular domain (Weissmaim et al., Science 238: 1018-1020 (1988); Weissman et al., Proc. Natl. Acad. Sci. UCA 85: 9709-9713 (1988)). Isoform s, named η (eta) (Baniyash et al., J. Biol. Chem. 263: 9874-9878 (1988); Orloff et al., J. Biol. Chem. 264: 14812-14817 (1989), which emerged from an alternative mRNA splicing pathway (Jin et al., Proc. Natl. Acad. Sci. UCA 87: 3319-3255 (1990)), present in reduced amounts in cells expressing the antigen receptor. It is believed that s-η heterodimers mediate the formation of inositol phosphates, as well as the programmed cell death of the cell called apoptosis (Mercep et al., Science 242: 571-574 (1988); Mercep et al., Science 246: 1162-1165 (1989).

Like the s and η, the γ (gamma) chain associated with the Fc receptor expresses complexes with additional polypeptides on the cell surface, some of them mediate ligand recognition, while others have an indefinite function, the γ chain supports homodimeric structure and general organization, very similar to the one formed by the s-chain and is a component of the high affinity mast / basophilic IgE receptor, Fc RI, which consists, as a minimum, of three different polypeptide chains (Blank et al., Nature 337: 187-189 (1989) ; Ra et al., Nature 241: 752-754 (1989), and one of the low affinity IgE receptor, represented in mice FcyRII α (Ra et al, J. Biol Chem 264:... 15323-15527 (1989) and in man - CD17-subtype expressed by macrophages and natural killer cells, CD16 _TM ( transmembrane molecules CD16) (Lanier et al., Nature 342: 803-805 (1989); Anderson et al., Proc. Natl. Acad. Sci. UCA 87: 2274-2278 (1990) and an unknown function polypeptide (Anderson et al., Proc. Natl. Acad Sci. UCA 87: 2274-2278 (1990)). A recent publication reported that the γ chain is expressed by the mouse T cell line, CTL, in which it forms homodimers, as well as the γ-s and γ-η heterodimers (Orloff et al., Nature 347: 189-191 (1990) .

 Fc receptors mediate phagocytosis with immune complexes, transcytosis, and antibody-dependent cell cytotoxicity (ADCC) (Ravetch and Kinet et al., Annu. Rev. lmmunol. 9: 547-492 (1991); Unkeless et al., Annu. Rev. Immunol. 6: 251-281 (1988); and Mellman, Curr. Opin. Immunol. 1: 16-25 (1988)). Recently, it has been shown that one of the murine low-affinity isoforms of the Fc receptor, FcRylllBI, mediates the internalization of Ig coated targets in the clathrin-coated pits, and the other low-affinity receptor, FcRylllA, mediates ADCC through association with one or more members of a small start-up family molecules "(Miettinen et al., Cell. 58: 317-327 (1989); and Hunziber and Melllman, J. Cell Biol. 109: 3291-3302 (1989)). These trigger molecules, the T cell receptor s chain (TCR), the TCR η chain, and the Fc receptor γ chain interact with a ligand that recognizes receptor domains of different immune systems and can autonomously initiate cell effector programs, including cytolysis with subsequent aggregation (Samelson et al., Cell 43: 223-231 (1985); Weissman et al., Science 239: 1018-1020 (1988); Jin et al., Proc. Natl. Acad. UCA 87: 3319-3323 (1990); Blank et al., Nature 337: 187-189 (1989); Lanier et al., Nature 342: 803-805 (1989); Kurosaki and Pavetch, Nature 343: 805-807 (1989); Hibbs et al., Science 246: 1608-1611 (1989); Anderson et al., Proc. Natl. Acad. Sci. UCA 87: 2274-2278 (1990); and Irving and Weiss, Cell 64: 891-901 (1991) .

 However, when parallels are drawn between the low affinity mouse and human Fc receptor families, it becomes apparent that human FcRyllA and C isoforms do not duplicate mouse. This is partly because their function has not yet been determined.

 Since humoral factors based solely on CD4 may have limited in vivo use, a preliminary study probed the possibility of enhancing cellular immunity to HIV. Chimeric protein preparations were identified (in which the extracellular domain of CD4 was fused to the transmembrane and / or intracellular domains of the T-cell receptor, the IgG Fc receptor, or to B-cell signal converting elements (USSN 07/847566 and 07/665961 included in the present description by reference) Cytolytic T cells expressing chimeras that include the extracellular domain of CD4 exhibit a strong, MHC independent ability to destroy cell targets expressing HIV envelope proteins. A very important and new component of this This method was the identification of a single T-cell receptor, Fc receptor, and B-cell receptor chains that are sufficiently aggregated to initiate a cellular response.One of the most valuable achievements of this method is to create chimeras between CD4 and s, η, or γ, which directly destroy T-lymphocytes upon recognition and destroy cells expressing HIV gp120 glycoprotein (USSN 07/847566 and 07/665961, incorporated herein by reference).

Summary of the invention
In accordance with the main objective of the present invention, the most important place is given to a method for controlling a cellular immune response directed against an HIV-infected mammalian cell. This method involves administering to the mammal an effective amount of therapeutic cells, namely, therapeutic cells expressing a membrane-bound protein chimeric receptor comprising (a) an extracellular portion that includes a CD4 fragment that is capable of specifically recognizing and binding HIV an infected cell, but which does not mediate HIV infection, and (b) the intracellular part, which has the ability to signal the therapeutic cell about the need to destroy l receptor-linked HIV-infected cell.

 In accordance with an objective related to this, the present invention relates to a cell that expresses a membrane-bound protein chimeric receptor, which includes (a) the extracellular part, which includes a CD4 fragment that has the ability to specifically recognize and bind HIV-infected a cell, but which does not mediate HIV infection and (b) the intracellular part, which has the ability to signal to the therapeutic cell the need to destroy the HIV-infected receptor th cage.

 In accordance with a second objective, the present invention relates to a method for treating HIV in a mammal, comprising administering to the mammal an effective amount of therapeutic cells, namely, therapeutic cells expressing a membrane-bound protein chimeric receptor comprising an extracellular portion that includes a fragment CD4, which has the ability to specifically recognize and bind an HIV-infected cell, but which does not mediate HIV infection.

 In accordance with an objective related to this, the present invention relates to a cell that expresses a membrane-bound protein chimeric receptor comprising an extracellular portion that includes a CD4 fragment that has the ability to specifically recognize and bind an HIV-infected cell, but which does not mediate HIV infection.

 In preferred embodiments, the primary and second objectives, the CD4 fragment is a fragment of the amino acid sequence 1-394 of the CD4 molecule or a fragment of the amino acid sequence 1-200 of the CD4 molecule; this CD4 fragment was separated from the intracellular part using the CD7 transmembrane domain shown in FIG. 26 or using the hinge, CH2 and CH3 domains of the human IgGI molecule shown in FIG. 25: a CD4 fragment was separated from the therapeutic cell by a distance of at least 48 angstroms (and preferably by a distance of at least 72 angstroms). In preferred embodiments of the first objective, the intracellular portion is a portion that converts a signal of a T-cell receptor protein (e.g., s), a B-cell receptor protein, or an Fc receptor protein; and therapeutic cells are selected from the group consisting of: (a) T lymphocytes; (b) cytotoxic T lymphocytes; (c) natural killer cells; (d) neutrophils; (e) granulocytes; (f) macrophages; (g) mast cells; and (i) embryonic stem cells (ES).

 In accordance with other purposes related to this, the present invention relates to DNA encoding a chimeric receptor of the present invention; and a vector including the DNA of this chimeric receptor.

 Although a specific embodiment of the present invention is a chimera between CD4 and zeta molecules, any receptor chains having a function similar to those molecules, for example, in granulocytes or B-lymphocytes, could be used for the purposes disclosed here. Distinctive features of the necessary triggering molecule of an immune cell include the ability to express autonomously (i.e., as a single chain), the ability to fuse with the extracellular part of the CD domain so that the resulting chimera is present on the surface of the therapeutic cell, and the ability to initiate cell effector programs for secondary aggregation when meeting a target ligand.

 Currently, most traditional methods of delivering chimeras to cells of the immune system are carried out using some form of genetic therapy. However, reproduction of cells of the immune system with chimeric receptors by mixing cells with the corresponding dissolved purified chimeric protein will also lead to the formation of a population of bioengineered cells capable of responding to HIV-infected targets. For therapeutic purposes, a similar method was used, for example, to introduce a CD4 molecule into red blood cells. In this case, the population of bioengineered cells did not possess the ability to reproduce itself.

 The present invention relates to functional and simplified chimeras between CD-4 and fragments and the T-cell receptor, B-cell receptor and Fc receptor subunits, which have the ability to control immune cells to recognize and lyse HIV-infected cells. This method for managing the cellular response in mammals involves the introduction of an effective amount of therapeutic cells (e.g., cytotoxic T-lymphocytes) into the body of a mammal, since the cells are able to recognize and destroy an HIV-infected cell.

 The present invention includes chimeric receptor proteins that direct cytotoxic T lymphocytes to HIV-infected cells for recognition and lysis, host cells transformed with a vector including chimeric receptors, and antibodies to chimeric receptors.

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

 In the following detailed description, references are made to various techniques known to those skilled in the art of molecular biology and immunology. Publications and other recited materials regarding such well-known referenced techniques are incorporated herein by reference in full.

 Standard working references setting forth recombinant DNA technology are described herein by Watson et al., Molecular Biology of a Gene, Vol. I and II, the Benjamin / Cummings Publishing Company, Inc., publisher, Menlo Park, CA (1987); DarnelI et al., Molecular Cell Biology. Scientific American Books, Inc., publisher, New York, N.Y. (1986); Lewin, Genes II, John Wiley & Sons, publishers. Not York, N.Y. (1985); Old et al., Principles for Working with Genes: Introduction to Genetic Engineering, 2nd ed., University of California Press, publisher, Berkely, CA (1981); Maniatis et al., Molecular Cloning: Laboratory Manual, 2nd ed. Cold Spring Harbol Laboratory, publisher, Cold Spring Harbor, NY (1989); Ausubel et al., Modern Methods in Molecular Biology, Wiley Press, New York :, NY (1989).

TERMINOLOGY
By "cloning" is meant the use of in vitro recombination methods to introduce a single gene or other DNA sequence into a vector molecule.

 By "cDNA" is meant complementary or copy DNA obtained from an RNA template resulting from the activity of an RNA-dependent DNA polymerase (reverse transcriptase). Therefore, "cDNA clone" means a double-stranded DNA sequence complementary to the corresponding RNA molecule represented in the cloning vector.

 By "cDNA library" is meant a collection of recombinant DNA molecules containing cDNA inserts that include DNA copies of mRNA expressed by the cell during the creation of the cDNA library. Such cDNA can be prepared using methods known to those skilled in the art, as described for example by Ausubel et al. (see above) and in Maniatis et al. (see above). In general, RNA is first isolated from the cells of the body, from the genome of which they intend to clone a single gene. For the purpose of the present invention, mammalian, in particular human, lymphocyte cell lines are preferred. Currently, the preferred vector for this purpose is the vaccinia virus, strain WR.

 By "vector" is meant a DNA molecule isolated, for example, from a plasmid, bacteriophage, or from a mammalian or insect virus into which DNA fragments can be inserted or cloned. The vector will contain one or more unique restriction sites and may have the ability to autonomously replicate in the organism of a particular host or in such an intermediary organism in which the cloned sequence can be reproduced. Therefore, by “DNA expression vector” is meant any autonomous element capable of driving the synthesis of a recombinant peptide. Such DNA expression vectors include bacterial plasmids and phages, as well as mammalian and insect plasmids and viruses.

 By “substantially pure” is meant a compound, for example, a protein, polypeptide or antibody, which is substantially free of the components naturally accompanying it. In general, a compound is substantially pure when at least 60%, more preferably at least 75%, and most preferably at least 90% of the total volume in the sample is the corresponding compound. The degree of purification can be measured by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. In the context of a nucleic acid, the term “substantially pure” means a nucleic acid sequence, segment or fragment that is not directly in contact with (i.e., not covalently linked to) both coding sequences with which it is directly in contact (i.e., one in 5 '-end, and the other at the 3'-end) in the naturally occurring genome of the body from which the DNA of the present invention was isolated.

 A “fragment” of a molecule, such as, for example, any of the cDNA sequences of the present invention, means that it refers to any subclass of nucleotide contig molecules. The “analogue" of a molecule means that it refers to a non-natural molecule that is practically similar to any original molecule or its fragment. The indicated molecule is “practically similar” to another molecule if the amino acid sequence of both molecules is practically the same. In particular, a “substantially similar” amino acid sequence is one that exhibits at least 50%, preferably 85%, and most preferably 95% identity of the amino acid sequence with the natural or reference amino acid sequence and / or which differs from the natural or reference sequence only in conserved amino acid replacements. Virtually similar amino acid molecules have similar biological activity. As used herein, said molecule is a “chemically derivative” of another molecule when it contains chemically different constituents of a single molecule. Such constituents can improve the solubility, absorption, biological half-life of the molecule, etc. The constituents can alternatively reduce the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Components capable of mediating such effects are disclosed, for example, in the pharmaceutical sciences of Remington, 16th ed., Mack Publishihg Co., Easton, P.A. (1980).

 A “functional derivative” of a chimeric receptor gene of the present invention means the inclusion of “fragments” or “analogs” of a gene that are “substantially similar” in nucleotide sequence. A “substantially similar” nucleic acid encodes substantially similar amino acid sequences (as described above) and may also include any nucleic acid sequence capable of hybridizing to a natural or test sequence under appropriate hybridization conditions (see, for example, Ausubel et al., Modern methods in molecular biology, Wiley Press, New York, NY (1989) for the corresponding stringent hybridization conditions).

 A “substantially similar” chimeric receptor has similar activity to a wild-type T-cell, B-cell or Fc-chimeric receptor. Most preferably, when the derivative has 90%, more preferably 70% and preferably 40%, of the activity of the wild-type chimeric receptor. The activity of a derivative of a functional chimeric receptor includes specific binding (with regard to its extracellular CD4 part) to an HIV-infected cell and, ultimately, the destruction of this cell; in addition, the chimeric receptor does not make the cell carrying the receptor susceptible to HIV infection. Chimeric receptor activity can be analyzed using, for example, any of the methods presented here.

 The DNA sequence encoding the chimeric CD4 receptor of the present invention, or its functional derivatives, can be recombined with vector DNA in accordance with traditional methods, including blunting or extension of the ends for ligation, digestion with restriction enzyme to obtain the corresponding ends, building up the sticky ends properly, processing alkaline phosphatase to avoid undesired compounds, and ligation using suitable ligases. Techniques for such manipulations are disclosed in Maniatis et al. (see above) and are well known to specialists in this field.

 A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide, if it contains nucleotide sequences containing transcriptional and translational regulation information, then such sequences are "operably linked" to nucleotide sequences that encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the expressed DNA sequence are connected so as to express the gene. The exact nature of the regulatory regions necessary for gene expression can vary from organism to organism, but it always includes the promoter region, which in prokaryotes contains both the promoter sequence (which controls the initiation of RNA transcription) and DNA sequences whose transcription from RNA is protein synthesis initiation signal. Such regions typically include 5 ′ non-coding sequences involved in transcription and translation initiation, and are represented by a TATA box, a capping sequence, a CAAT sequence, and the like.

 If necessary, using the above methods, you can get the connection 3'-non-coding region with the sequence of the gene encoding the protein. This region can be used for regulatory sequences terminating its transcription with sequences regulating transcription termination for its termination and polyadenylation. Thus, using the 3 ′ region naturally in contact with the DNA sequence encoding the protein, signals can be created that terminate transcription. When transcriptional termination signals do not function satisfactorily in the expressing host cell, then the functional 3 ′ region of the host cell can be replaced.

 Two DNA sequences (such as the sequence of the promoter region and the sequence encoding the chimeric CD4 receptor), as indicated, are operably linked if the nature of the linkage between the two sequences 1) does not lead to the introduction of a mutation in the reading frame, 2) does not inhibit the ability of the region sequence a promoter to control transcription of a chimeric receptor gene sequence or 3) does not inhibit the ability of a chimeric receptor gene sequence to be transcribed using promoter region sequences. The promoter region will be operably linked to the DNA sequence if the promoter is able to enhance transcription of this DNA sequence. Thus, when expressing a protein, it is necessary for the appropriate host to recognize transcription and translation signals.

 The present invention encompasses the expression of a chimeric CD4 receptor protein (or a functional derivative thereof) in both prokaryotic and eukaryotic cells, but preferably relates to the expression of eukaryotic cells (and especially human lymphocytes).

 In accordance with the present invention, antibodies can be prepared by any available method. For example, cells expressing a protein of a chimeric CD4 receptor, or a functional derivative thereof, can be administered to an animal to cause the formation of serum containing polyclonal antibodies that are capable of binding to the chimera.

 In a preferred embodiment of the method, the antibodies of the present invention are monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256: 495 (1975); Kohler et al., Eur. J. Immunol. 6: 511 (1976); Kohler et al., Eur. J. Immunol. 6: 292 (1976); Hammerling et al., B: Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY pp. 565-684 (1981)). Basically, such techniques involve the immunization of an animal with a chimeric CD4 receptor antigen. Splenocytes obtained from such animals are extracted and fused to a suitable myeloma cell line. Any myeloma cell line may be used in accordance with the present invention. After fusion, the resulting hybridoma cells are selectively maintained on HAT medium and then cloned by limiting dilution as described by Wands et al. (Castroenterology 80: 225-232 (1981)). The resulting hybridoma cells are then analyzed to identify clones that secrete antibodies capable of binding to the chimera.

 In accordance with the present invention, the antibodies can also be polyclonal or, preferably, region-specific polyclonal antibodies.

 In accordance with the present invention, antibodies to a chimeric CD4 receptor can be used to control the amount of a chimeric receptor (or cells carrying a chimeric receptor) in a patient. Such antibodies are well suited for use in standard immunodiagnostic methods known in the art, including such immunometric or “sandwich” methods in the form of direct sandwich analysis, reverse sandwich analysis, and combinations thereof. These antibodies can be used in any combination selected by those skilled in the art to avoid undue experimentation to perform immunoassays of acceptable specificity, sensitivity and accuracy.

 References to key works formulating the main principles of immunology include Roitt, Fundamentals of Immunology, 6th ed., Blackwell Scientific. Publications, publisher, Oxford (1988); Kimbal, Introduction to Immunology, 2nd ed., Macmillan Publishing Co. , publisher, New York (1986); Roitt et al., Immunology, Gower Medical Publishing Ltd., publisher, London, (1985); Campbell, Monoclonal Antibody Technique, by Burdon et al., Eds., Laboratory Technique in Biochemistry and Molecular Biology, Volume 13, Elsevier, publisher, Amsterdam (1984); Klein, Immunology: The science of Self-Nonself Discimination, John Wiley & Sons, publisher. New York (1982) and Kennet et al. Monoclonal antibodies. Hybridoma: New Approaches in Biological Research, Plenum Press, publisher, New York (1980).

 By "detection" is meant the presence or absence of a substance or a detectable amount of a substance. Therefore, this term refers to the materials, preparations and methods of the present invention relating to qualitative and quantitative determinations.

 The antibodies and the substantially pure antigen of the present invention are ideal for kit development. Such a kit may include a portable module for packaging closely spaced one or more containers, for example in the form of bottles, tubes, etc., each of these containers includes individual elements used in the analysis.

 The types of analyzes that can be arranged as a set are numerous and are presented, for example, by competitive and non-competitive analysis. Typical examples of assays in which the antibodies of the present invention can be used are: radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELISA) and immunometric or "sandwich" immunoassays.

 By the term “immunometric assay” or “sandwich” immunoassay is meant a combination of a sandwich method, a direct sandwich method and an inverse sandwich method. These terms are well understood by specialists in this field. It will also be appreciated by those skilled in the art that, in accordance with the present invention, antibodies are suitable for other variants and assays currently known or that may be improved in the future. These terms are intended to be used within the scope of the present invention.

 The term “specifically recognizes and binds” means that the antibody recognizes and binds the polypeptides of the chimeric receptor, but practically does not recognize and does not bind other unrelated molecules in the sample, for example, a biological sample.

 By the term “therapeutic cell” is meant a cell that is transformed with the chimeric CD4 receptor of the present invention so that it is able to recognize and destroy an HIV-infected cell; preferably, such therapeutic cells are cells of the haemonoietic system.

 By the term “extracellular” is meant at least a part of the molecule present on the cell surface. By the term “intracellular” is meant at least a portion of the molecule present in the cytoplasm of a therapeutic cell. By the terms “transmembrane” is meant at least a portion of the molecule permeating the plasma membrane. The terms "extracellular part", "intracellular part" and "transmembrane part", as used herein, may include flanking amino acid sequences that extend into adjacent compartments.

 By the term “oligomerization” is meant the formation of a complex with other proteins in the form of dimers, trimers, tetramers or higher order oligomers. Such oligomers may be homo-oligomers or hetero-oligomers. The term “oligomerization moiety” means a portion of a molecule that controls the formation of a complex (ie, oligomer).

 By the term “cytolytic” is meant the ability to destroy a cell (eg, an HIV-infected cell or kill an infectious factor (eg, an HIV virus)).

 By the term "immunodeficiency virus" is meant a retrovirus that, in the form of a wild type, is able to infect T cells of a primate host, has morphogenesis and characteristic morphology of the subfamily of lentiviruses. The term includes, without limitation, all HIV and SIV variants, including HIV-1, HIV-2, SIVmac, SIVagm, SIVmnd, SIVsmm, SIVman, SIVmand and SIVcpz.

 By the term "MHC independent" is meant that the cellular cytolytic response does not require the presence of a MHC class II antigen on the surface of the target cell.

 By the term “functional cytolytic signal transducing derivative” is meant a functional derivative (as defined above) that is capable of controlling at least 40%, more preferably 70% or most preferably at least 90%, of the biological activity of a wild-type molecule. As used herein, a “functional cytolytic signal transducing derivative” can act as a direct signal to a therapeutic cell to destroy a receptor-associated agent or cell (for example, in the case of the intracellular portion of a chimeric receptor) or act indirectly by promoting oligomerization with cytolytic signal-transducing proteins (eg , in the case of a transmembrane domain). Such derivatives can be tested for efficacy, for example, using the in vitro assays described above.

By the term “functional derivative that binds the envelope of HIV” is meant a functional derivative (as defined above) that is capable of binding any HIV envelope protein. Functional derivatives can be identified using, for example, the in vitro assays described above.
THERAPEUTIC INTRODUCTION
The transformed cells of the present invention were used to treat viral immunodeficiency. Current methods of administering such transformed cells include adaptive immunotherapy or cell transfer therapy. These methods allow you to return the transformed cells of the immune system into the bloodstream. Rosenberg, Sci. Am. 62 (May 1990); Rosenberg et al., N. Engl. J., Med. 325: 570 (1990).

 The pharmaceutical preparations of the present invention can be administered to any animal that experiences the beneficial effects of the compounds of the present invention. In the first place among these animals is a man, although the present invention is not intended to be limited to them.

Detailed description
The drawings will first be described.
Brief Description of the Drawings
FIG. 1a represents the amino acid sequence of a fusion site between CD4 (residues 1-369) and different receptor chains (SEQ ID NOS: 58-41). An underlined sequence indicates the position of the amino acids encoded within the BamHI site used to create the fusion construct. The beginning of the transmembrane domain is marked by a vertical stripe. The sequence η is identical to the amino terminus sequence s, but differs in carboxy terminus (Jin et al., Proc. Natl. Acad. Sci USA 87: 3319-3323 (1990)). FIG. 1b presents the results of flow cytometric analysis of surface expression of CD4, CD4: s, CD4: γ, and
CD4: η in CVI cells. Cells were infected with a virus expressing CD4 chimeras or CD16 _PI , incubated for 9 hours at 37 ^° C and stained with phycoerythrin-conjugated anti-CD4 Mad Leu3A.

FIG. 2 shows surface expression of CD16 _TM after co-infection with single CD16 _TM (crowded dots), or co-infection of viruses expressing CD4: γ (streaks) or CD4: s (solid line). Rare dots refer to cells infected with CD4: s alone and stained with 3G8 (Fleit et al., Proc. Natl. Acad. Sci USA 79: 3275-3279 (1982)) (anti-CD16 Mad).

FIG. 3 shows the surface expression of CD16 _TM after co-infection with viruses expressing CD16 _TM and then s-chimeras: CD4: s (bold line), CD4: s C11G (solid line), CD4: s (dashed line); CD4: s C11G / C11G (twisted dots); without co-infection (CD16 _TM only, rare spots). Cells were incubated with anti-CD16 Mad 3G8 and Fab'-conjugated goat anti-mouse IgG antibodies with phycoerythrin. The expression level of s-chimeras was almost identical for the different mutants analyzed, and coinfected cells with viruses expressing CD16 _TM and s-chimeras did not significantly alter the surface expression of chimeras.

 FIG. 4a-d shows an increase in free intracellular calcium ions after cross-linking of mutant s-chimeras in a T cell line. Jurkat EE6 cells (Weiss et al., J. Immunol. 133: 125-128 (1984)) were infected with recombinant vaccinia viruses and analyzed by flow cytometry. The presented results relate to the CD4 population passed through the shutter, so only cells expressing the desired chimeric protein are analyzed. The average ratio of violet and blue Inbo-1 fluorescence reflects the intracellular concentration of free calcium in the population as integers and the percentage of responding cells corresponding to the proportion of cells that exceed a predetermined threshold ratio (taken so that 10% of untreated cells are positive). FIG. 4a and 4b show Jurkat cells expressing CD4: S (solid line) or CD16: s (dashed line), which were exposed to anti-CD4 Mag Leu3a (phycoerythrin conjugate), then cross-linked the goat antibody with mouse IgG. The dotted line shows the response of uninfected cells to anti-CD3 MAt OKT3. FIG. 4c and 4d show Jurkat cells expressing CD4: SD15G (solid line); CD4: SC11G / -D15G (streaks); or CD4: SC11G (dots), which were processed and analyzed as in FIG. 4a and 4b.

 FIG. 5a-c show that the CD4: s, CD4: η and CD4: γ receptors allow cytolytic lymphocytes (CTLs) to destroy targets expressing HIV-1 gp120 / 41 molecules. FIG. 5a: black circles are CTLs expressing CD4: s incubated with HeLa cells expressing gp120 / 41; light circles are CTLs expressing CD4: s incubated with uninfected HeLa cells; black squares are uninfected CTLs incubated with HeLa cells expressing gp120 / 41; light squares are uninfected CTLs incubated with uninfected HeLa cells. FIG. 5b: black circles are CTLs expressing CD4: η incubated with HeLa cells expressing gp120 / 41; light circles are CTLs expressing CD4: γ incubated with HeLa cells expressing gp120 / 41; light squares are CTLs expressing CD4; C11G / D15G double mutant s chimera incubated with HeLa cells expressing gp120 / 41. FIG. 5c: Flow cytometric analysis of the CD4 expressed by CTLs used in FIG. 5b. In order to clarify the ratio of the target and the effector, the percentage of cells expressing the CD4 chimera was determined by subtracting the weighted value of the negative (uninfected) population by superimposing histograms; for comparison, uninfected cells were denoted by an arbitrary threshold value in this figure, which gave approximately the same proportion of positive cells among other cell populations, as if by subtraction of histograms.

FIG. 6a-b shows the specificity of CD4 directed cytolysis. FIG. 6a: black circles are CTLs expressing CD4: s incubated with HeLa cells expressing CD16 _PI ; light circles are CTLs expressing CD4 incubated with HeLa cells expressing gp120 / 41; light squares are CTLs expressing _PI CD16, incubated with HeLa cells expressing gp120 / 41. FIG. 6b: black circles are CTLs expressing CD4: s incubated with Raji cells (MHC class II); light circles are uninfected CTL cells incubated with RJ2.2.5 cells (MHC class II Raji mutant); black squares are uninfected CTLs incubated with Raji cells (MHC class II); light squares are CTLs expressing CD4: s incubated with RJ2.2.5 MHC class II cells). The scale of the ordinate is expanded.

 FIG. 7a-b characterizes CD16: s-chimeric receptor. FIG. 7a is a schematic diagram of a CD16: s-fusion protein. The extracellular part, in the form of monomeric CD16 crosslinked with phosphatidylinositol, was attached to the outer part of the transmembrane domain dimer s. The protein sequence (SEQ ID NOSS: 42, 43) at the point of attachment is shown below. FIG. 7b shows the results of a flow cytometric analysis of calcium dynamics after crosslinking of a CD16: s-chimera with either a TCR-positive or a TCR-negative cell line. The average ratio of violet and blue fluorescence (measures the relative ionic concentration of calcium) in antibody-treated cell populations at the zero point of time is shown. Black squares are Jurkat cell responses to anti-CD3 Mab OKT3; black triangles are CD16: s response to anti-CD16 Mab 3G8 crosslinked with the REX33A TCR mutant; light squares is the answer to CD16: s stitched with the Jurkat TCR mutant line JRT3.T3.5; light triangles are the answer to CD16: s-stitching in Jurkat-cells; and dots is a response to non-chimeric CD16 in a REX33A TCR cell line.

FIG. 8a-b shows an analysis of deletions of the cytolytic potential. FIG. 8a shows the endpoint locations of deletions in s. Here, as elsewhere, mutations in s are represented by a conditional residue located in the mutant of the original residue, so that D66 * indicates, for example, the replacement of Asn-66 with a termination codon. FIG. 8b shows the results of a cytolytic analysis of undeleted CD16: s and characteristic s-deletions. Hybridoma cells expressing the surface anti-CD16 antibody were loaded with ⁵¹ Cr and incubated with an increasing number of human cytolytic lymphocytes (CTLs) infected with vaccinia virus recombinants expressing CD16: s chimeras. The percentage of released ⁵¹ Cr is graphically presented as the functional ratio of effector cells (CTLs) to target cells (hybridoma) (e / m). Black circles are cytolysis mediated by cells expressing CD16: s Asp66 * (mfi 940.2); light squares are cytolysis mediated by cells expressing CD16: s Glu60 * (mfi 16.0); light circles are cytolysis mediated by cells expressing CD16: sTyr51 * (mfi 17.4); black triangles are cytolysis mediated by cells expressing CD16: s Phen34 *, (mfi 17.8); and light triangles are cytolysis mediated by cells expressing non-chimeric CD16 (mfi 591). Although CD16: s Asp66 * expression was not observed in this experiment, unlike other fusion proteins, cytolysis using cells expressing CD16: s at equivalent levels in the same experiment yielded almost identical results shown by cells expressing CD16: s Asp66 .

 FIG. 9a-d show that the elimination of potential transmembrane interactions is detected by a short s-segment capable of mediating cytolysis. FIG. 9a is a schematic diagram of monomeric bifid and trifid chimeras. The upper part is a CD16: s-structure truncated at residue 65 and lacking the transmembrane residues Cis and Asp. Below are the CD16: CD5: s- and CD16: CD7: s-constructs and related control constructs. The peptide sequences of the intracellular domains are shown below (SEQ ID NOS: 45-47). FIG. 9b shows the cytolytic activity of the monomeric chimera of deleted mutants. The cytolytic activity of cells expressing CD16: s (black circles; mfi 495) was compared with the activity of cells expressing CD16: 5 Asp66 * (black squares; mfi 527) or with mutants CD16: s Cis11Gly / Asp15Gli / Asp66 * (light squares; mfi 338) and CD16: s Cis11Gly / Asp15Gly / Glu60 * (black triangles; mfi 259). FIG. 9c shows cytolytic activity mediated by tripartite fusion proteins. Black triangles are CD16: s Asp66 *; light squares are CD16: s (48-65); black squares are CD16: 7: s (48-65); light triangles are CD16: 7: s (48-59); light circles are CD16: 5; the black circles are CD16: 7. FIG. 9d shows the mobilization of calcium by the mutant and tripartite chimeras in TCR-negative JRT3. T3.5 mutant Jurkat cell line. Light circles are the response of cells expressing dimeric CD16: s Asp66 *; black squares are the response of cells expressing CD16: sCis11Gly / Asp15Gli / Asp66 *; light squares are the response of cells expressing CD16: sCis11Gly / Acp15Gly / Gle60 *; black triangles are the response of cells expressing CD16: 7: s (48-65); and light triangles are the response of cells expressing CD16: s (48-59).

 FIG. 10a-f show the appearance of individual amino acids in the activity of an 18 amino acid motif transducing a cytolytic signal. FIG. 10a and 10b show cytolytic activity, and FIG. 10c shows the mobilization of the calcium ion mediated by chimeras carrying point mutations near the tyrosine carboxy terminus (Y62). FIG. 10a and 10b represent data collected for cells expressing, respectively, low and high amounts of CD16: s-fusion proteins. Identical designations were used to analyze calcium mobilization and cytolysis and are shown with a separate letter code on the right. Black circles are cells expressing CD16: s (mfi in A, 21; B, 376); black squares are cells expressing CD16: 7: s (48-65) (mfi A, 31; B, 82); light squares are CD16: 7: s (48-65) Glu60Gli (mfi A, 33; B, 92), crosses are CD16: 7: s (48-64) Asp63Asn (mfi A, 30; B, 74) ; black triangles are CD16: 7: s (48-65) Tyr62phen (mfi A, 24; B, 88); light circles are CD16: 7: s (48-65) Glu61Gln (mfi A, 20; D, 62); light triangles are CD16: 7: s (48-65) Tyr62Ser (mfi B, 64). FIG. 10d and 10e show cytolytic activity, and FIG. 10f shows the mobilization of calcium ion by a chimera carrying point mutations near the amino terminus of tyrosine (Y51). Identical designations were used for analysis of calcium mobilization and cytolysis, which are shown on the right. Black circles are cells expressing CD16: s (mfi in D, 21.2; in E, 672); black squares are cells expressing CD16: 7: s (48-65) (mfi D, 31.3; E, 179); black triangles are CD16: 7: s (48-65) Asn48Ser (mfi D, 22.4; E, 209); light squares are CD16: 7: s (48-65) Lei 50 Ser (mfi D, 25.0; E, 142); and the bright triangles are CD16: 7: s (48-65) Tir51phen (mfi D, 32.3; E, 294).

 FIG. 11a-b show the alignment of internal s repeats and compares their ability to support cytolysis. FIG. 11a is a schematic diagram of chimeras formed by dividing the intracellular domain s into three parts and attaching them to the transmembrane domain of the CD16: 7 chimera. The sequences of the intracellular domains are shown below (SEQ ID NOS: 48-50), with separated, framed, residues and linked residues marked with asterisks. FIG. 11b shows the cytolytic efficacy of three s-subdomains. Black circles are cells expressing CD16: s (mfi 476); black squares are CD16: 7: s (33-65) (mfi 68); light squares are CD16: 7: s (71-104) (mfi 114); and the black triangles are CD16: 7: s (104-138) (mfi 104).

 FIG. 12 is a schematic diagram of CD16: FcRyII chimeras.

 FIG. 13a-b show calcium mobilization after crosslinking of chimeras CD4: FcRyII and CD16: FcRyII. FIG. 13a shows the ratio of violet and blue fluorescence emitted by calcium-loaded cells sensitive to the Indo-1 fluorophore, shown as a time function after cross-linking the intracellular portion of the CD16 domain with antibodies, FIG. 13b shows similar analysis results for an increase in the ratio of violet and blue fluorescence of cells bearing CD4: FcRyII chimeras after crosslinking with antibodies.

FIG. 14a-b show the results of cytolysis assays of chimeras CD4: GcRyII and CD16: FcRyII. FIG. 14a shows the percentage of ⁵¹ Cr released from anti-CD16 hybridoma (target) cells when the cells are exposed to an increasing number of cytotoxic T lymphocytes expressing CD16: FcRyII chimeras (effector cells). FIG. 14b shows the results of a similar assay cytotoxically mediated with CD41FcRyII chimeras against target cells expressing HIV envelope glycoproteins.

 FIG. 15a-e show the identification of residues in the A-tail of FcRyII, which is important for cytolysis. FIG. 15a is a schematic diagram of deletion constructs. FIG. 15b and 15c show calcium mobilization and cytolysis using variants of CD16: FcRyII A with a carboxy-terminal deletion. FIG. 15d and 15e show calcium mobilization and cytolysis by tripartite chimeras carrying a gradually decreasing amount of amino terminal in the intracellular A-tail of CD16: FcRyII.

 FIG. 16 (SEQ ID NO: 24) shows the amino acid sequence of the CD3 receptor delta protein; the bounded sequence represents the preferred cytolytic signal of the transducing moiety.

 FIG. 17 (SEQ ID NO: 25) shows the amino acid sequence of the T3 receptor gamma protein; the bounded sequence represents the preferred cytolytic signal of the transducing moiety.

 FIG. 18 (SEQ ID NO: 26) shows the amino acid sequence of the mbi receptor protein, the bounded sequence represents the preferred cytolytic signal of the transducing moiety.

 FIG. 19 (SEQ ID NO: 27) shows the amino acid sequence of a B29 receptor protein; the bounded sequence represents the preferred cytolytic signal of the transducing moiety.

 FIG. 20 shows a schematic diagram of a CD4 chimera. Molecule "A" is CD4 (D1-D4): Ig: CD7; molecule "B" represents CD4 (D1, D2): Ig: CD7; molecule "C" represents CD4 (D1, D2): Ig: CD7: s and molecule "E" represents CD4: s. The intracellular domain of a human CD4 molecule corresponding to amino acids 1-394 of the precursor was linked via a BamHI site to the hinge of the CH1 and CH2 domains of human IgG1 as previously described (Zettlmeissl et al., DNA Cell Biol. 9: 347 (1990) ), with the exception that the cDNA variant of the human Ig sequence was used to allow expression of recombinant vaccinia viruses. A two-domain variant of the CD4 chimera was created by inserting a BamHI adapter at the unique NheI site (corresponding to the 200th amino acid) into the cDNA of the CD4 precursor. Sequences interacting with the membrane consist of 22 residues from the first exon of human membrane-bound IgG1 after residues 146–205 of the CD7 marker. Amino acids 55-163 s-chains serve as a trigger motive for therapeutic constructs (C and D). In tetra-partitioned constructs containing s-chain, intracellular s-chain expression has been documented using a commercial antibody to intracellular domer (Coulter).

FIG. 21 shows cytolysis of a target cell expressing an HIV envelope glycoprotein mediated by a clone of cytotoxic T cells, WH3 expressing, as effector molecules, a variety of CD4 derived chimeras. For cytotoxicity analysis, the CD8 ⁺ , CD4 ^- HLA B44 markers bounded by the WH3 T cell line were maintained in IMD supplemented with 10% human serum as described above. Cells were stimulated with gamma-irradiated (3000 rad) mononuclear cells carrying B44 and phytohemagglutinin (PHA) at a concentration of 1 μg / ml. After one day of stimulation, the PHA was diluted to 0.5 μg / ml by adding fresh medium; after 3 days, the medium was completely changed. Cells grew at least 10 days before use in cytotoxic analysis. Cells were infected with appropriate recombinant vaccinia viruses, as described here for vPE16. The time of infection of the cells in the complete medium was extended by 3-4 hours, after which the cells were collected by centrifugation and resuspended at a density of 1 · 10 ⁷ / ml. 100 μl of these cells was added to each well of a U-shaped microtiter plate, where 100 μl of complete medium was already contained in each well and diluted 2 times with serial operations. Two sample wells did not contain lymphocytes to allow spontaneous release of chromium and to fully measure chromium uptake. Target cells, S3 subline HeLa cells (HeLa-S3, ATCC), were infected, as indicated above, with vPE16 in 10 cm plates. 10 ⁶ infected cells were separated using PBS and 1 mM EDTA, centrifuged and resuspended in 100 5-1 μl ⁵¹ Cr-sodium chromate (1 mCi / ml in PBS) for 1 hour at 37 ^° C. and then washed three times with PBS. 100 μl of labeled target cells were added to each well. The microtiter plate was centrifuged at 750 g for 1 minute and incubated for 4 hours at 37 ^° C. At the end of the incubation period, the cells in each well were resuspended by careful pipetting, the sample was removed to determine the total label included, and the microtiter plate was centrifuged at 750 G for 1 minute. Aliquots (100 μl) of the supernatant were recovered and the label counted on a gamma scintillation counter. The effector: target ratio was refined to determine the percentage of infected cells using flow cytometry.

FIG. 22 shows HIV-1 replication in a transfected cell line. In sublinear human embryonic kidney cell line 293, cell lines stably expressing wild-type CD4 and various recombinant chimeras were detected. An isolate IIIB of the HIV-1 viral strain was obtained with a titer of ≈10 ⁶ infectious particles / ml, measured by the method of dilution to the end point using, as an indicator, the human T-cell line C8166. Infection was carried out at approximately 1 multiple infection (MOI) for 8-12 hours at 37 ^° C. The next day, cells were washed three times with PBS, trypsinized, transferred to new plates and the culture supernatant was selected for p24 titration (day 0). Every 3-4 days, the supernatants of cultured cells were collected and stored until p24 analyzes. Cells were replenished with fresh medium containing hygromycin B at a concentration of 100 μg / ml. Analysis of the culture supernatants was performed using a commercial ELISA-tested p24 HIV-1 antigen ELISA assay kit according to the manufacturer's instructions (Coulter). The results are presented by two independent experiments of approximately the same duration.

 FIG. 23 shows the nucleic acid sequence (SEQ ID NO: 28) and the amino acid sequence (SEQ ID NO: 29) of the D1-D4 domains of the CD4 molecule (CD4 Bam).

 FIG. 24 shows the nucleic acid sequence (SEQ ID NO: 30) and the amino acid sequence (SEQ ID NO: 31) of the D1-D2 CD4 domains (CD4 Nhe).

 FIG. 25 shows the nucleic acid sequence (SEQ ID NO: 32) and the amino acid sequence (SEQ ID NO: 33) of the hinge, the CH2 and CH3 domains of human IgG1 (Igh23 Bam).

 FIG. 26 shows the nucleic acid sequence (SEQ ID NO: 34) and the amino acid sequence (SEQ ID NO: 35) of the CD7 transmembrane domain (TM7 Bam Mlu).

 FIG. 27 shows the nucleic acid sequence (SEQ ID NO: 36) and the amino acid sequence (SEQ ID NO: 37) of the intracellular zeta domain (Zeta Mlu Not).

 FIG. 28 shows the DNA sequence (SEQ ID NO: 51) and the primary amino acid sequence (SEQ ID NO: 52) of the synthetic alpha helix.

EXAMPLE I. Construction of human IgG1: receptor chimeras
The human IgG1 heavy chain sequences were prepared for the pathways of attachment of the Cn3 domain sequences to a cDNA fragment derived from the 3 ′ end of the mRNA of the transmembrane form of the antibody. This 3'-end was obtained using a polymerase chain reaction, using the palatine tonsil cDNA library as a substrate and oligonucleotides having the sequence:
CGC GGG GTG ACC GTG CCC TCC AGC AGC TTG GGC (SEQ ID NO: 7) and
CGC GGG GAT CCG TCG TCC AGA GCC CGT CCA GCT CCC CGT CCT GGG CCT CA (SEQ ID NO: 8),
corresponding to the 5'- and 3'-ends of the corresponding DNA fragments. The 5'-oligonucleotide is complementary to the site in the Sn1 domain of human IgG1, and the 3'-oligonucleotide is complementary to the site of the 5'-sequence encoding the membrane penetrating domain. The PCR product was treated with BStXI and BamHI and ligated between the BStXI and BamHI sites of the semisynthetic IgG1 antibody gene carrying variable and constant regions. After inserting the BstXI into the BamHI fragment, the amplified parts of the construct were replaced with the SmaI site in Cn3 by restriction of the replaced fragment so that only a part of the amplifier between the SmaI site and the 3'-oligonucleotide was obtained in the PCR reaction.

 To create the human IgG1: ζ-chimeric receptor, the heavy chain gene ending at the BamHI site was linked to the BamHI site of the ζ chimera described below so that the antibody sequences form the intracellular portion. Flow cytometry of COS cells transfected with a chimera-encoding plasmid showed high expression of antibody determinants when the expression plasmid encoding the cDNA light chain was transfected, and moderate expression of antibody determinants when the light chain expression plasmid was absent.

 Similar chimeras, including human IgGl fused to the η or λ chain (see below), or any signal transducing portion of the T cell receptor or Fc receptor protein, can in principle be constructed as described above using standard molecular techniques biology.

To create a single transcriptional unit that allows both the heavy and light chains to be expressed from a single promoter, from sequences encoding the heavy and light chains and the 5'-untranslated portion of mRNA encoding a 78 kDa regulatory protein for glucose, also known as grp78, or BiP, created a plasmid encoding a two-cistronic mRNA, grp78 sequences were obtained by PCR from human genomic DNA using primers having the following 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) for the 5 ′ and 3 ′ ends, respectively. Polymerase chain reactions with these oligos were carried out in the presence of 10% dimethyl sulfoxide. The resulting PCR fragment was processed with NotI and HincII and inserted between NotI and HpaI sites located below the sequences encoding human IgG1. The cDNA sequences encoding the kappa light chains of human IgG1 were then inserted below the grp78 leader sequence using the HincII site and another site in the vector. The expression plasmid obtained as a result of these operations consisted of a semisynthetic heavy chain gene following the grp78 leader sequences, kappa light cDNA sequences, and polyadenylation signals obtained from the SV40 DNA fragment. COS cells transfected with an expression plasmid showing a distinct increase in expression of heavy chain determinants were compared with a transfecting plasmid encoding only one heavy chain determinant.

 To create a two-cistron gene comprising a heavy chain / receptor chimera and a light chain located above the heavy chain sequence, any heavy chain / chimera gene described above can be replaced.

EXAMPLE II Construction of chimeric CD4 receptors
Using polymerase chain reaction from a library obtained from the tumor cell line HPB-ALL (Aruffo et al., Proc. Natl. Acad. Sci. USA 84: 8573-8577 (1987b) and from human natural killer cells, ζ cDNA was isolated human (Weissman et al., Proc. N t1. Acad. Sci. USA 85: 9709-9713 (1988b)) and γ-cDNA (Kustler et al., J. Biol. Chem. 265: 6448-6452 (1990) and η cDNA was isolated from a library of murine tinomites (Jin et al., Proc. Natl. Acad. Sci. USA 87: 3319-3323 (1990)), ζ, η, and γ cDNA were attached to the extracellular domain of the created form CD4 having a BamHI site located immediately above the membrane permeating domain (Aruffo et al., Proc, Natl Acad. Sci. USA 84: 8573-8577 (1987b)); Zettlmeissl et al., DNA Cell Biol. 9: 347-353 (1990)), which was attached to the BamHI site naturally present in ζ and η- cDNA with a similar arrangement of several residues above the membrane penetrating domain (SEQ ID NO: 1, 3, 4, and 6.) To form a fusion protein with γ, the BamHI site was inserted into the sequence with the same localization (Fig. 1; SEQ ID NO: 2 and 5). Fusion genes were introduced into a plasmid expressing vaccinia virus carrying the E. coli gpt gene as a selectable marker and inserted into the genome of the vaccinia strain WR as a result of homologous recombination and growth selection in microphenolic acid (Falkner et al., J. Virol. 62: 1849-1854 (1988); Boyle et al., Gene 65: 123-128 (1988)). Flow cytometric analysis showed that vaccinia recombinants control the abundant production of CD4: ζ and CD4: γ fusion proteins on the cell surface, while expression of CD4: η is practically reduced (Fig. 1b). The latter observation is consistent with a recent report that transfection of η cDNA of an expression plasmid into a murine hybridoma cell line yielded virtually disappeared expression than transfection of a comparable ζ expression plasmid (Clayton et al., J. Exp. Med. 172: 1243-1253 (1990)). Immunoprecipitation of cells infected with vaccinia recombinants revealed that fusion proteins are formed from covalent dimers other than the naturally occurring CD4 antigen. The molecular weights of the monomeric CD: ζ and CD: γ fusion proteins and natural CD4 are 63, 55 and 53 kDa, respectively. The large masses of fusion proteins approximately coincide with the greater length of the intracellular part, which exceeds the intracellular part of CD4 by 75 (CD4: ζ-) or by 5 CD4: γ residues.

EXAMPLE III CD4 chimeras can be associated with other receptor chains
Cell surface expression of the macrophage / natural killer cell of human Fc γ RIII (CD16 _TM ) generated in transfectants is facilitated by transfection of mice (Kurosaki et al., Nature 342: 805-807 (1989)) or human (Hibbs et al., Science 246 : 1608-1611 (1989)) γ, as well as human ζ (Lanier et al., Nature 342: 803-805 (1989)).

These messages are confirmed by the expression of chimeras, which also make possible the surface expression of CD16 _TM upon delivery to the target cell either as a result of counter-infection or co-infection with recombinant vaccinia viruses (Fig. 2). In the studied cell line, stimulation of surface expression with ζ was more pronounced than stimulation with γ (Fig. 2), while natural CD4 does not enhance surface expression of CD16 _TM .

EXAMPLE IV Asp ζ-mutants do not co-associate with the Fc receptor
To create chimeras that would not be associated with the existing antigen or Fc receptors, fusion proteins of the ζ mutant were prepared, which lack either an intrammembrane Asp residue or an intrammembrane Cis residue. Flow cytometry showed that the intensity of cell-surface expression by various mutant chimeras was not significantly different from the non-mutant predecessor, and immunoprecipitation experiments showed that the total expression of chimeras was similar. As expected, mutant chimeras lacking a transmembrane cysteine residue do not show the formation of dimers crosslinked by a disulfide bond. Two mutant chimeras lacking Asp were unable to maintain surface expression of CD16 ^TM , while monomeric chimeras lacking a Cis residue but bearing an Asp residue were able to coexpress CD16 _TM , but with lower efficiency than the parent dimer (Fig. 3) .

EXAMPLE V. Mutant receptors retain the ability to initiate a calcium response
To find out if cross-linking of fusion proteins really makes possible the accumulation of free intracellular calcium in a manner similar to the known method for the T cell antigen receptor, human leukemia T cell line cells, Jurkat E6 (ATCC, catalog number TIB 152, American type culture collection, Rockville, MD) was infected with vaccinia recombinants and the cytoplasmic calcium concentration ratio was measured after crosslinking of the extracellular domain with antibodies. Flow cytometric measurements were carried out with cells loaded with calcium sensitive dye Indo-1 (Grynkiewich et al., J. Biol. Chem. 260: 3340-3450 (1985); Rabinovitch et al., J. Immunol, 137: 952-961 ( 1986)).

 FIG. 4a-d show the results of calcium flux experiments for cells infected with CD4: ζ and Asp and Cis mutants ζ. Crosslinking of chimeras reproducibly increases intracellular calcium. Similarly, CD4: η and CD4: γ make possible the accumulation of intracellular calcium in infected cells. Jurkat cells express low levels of CD4 on the cell surface, however, crosslinking of natural CD4 in the presence or absence of CD16: ζ does not change intracellular calcium levels (Fig. 4a-b).

EXAMPLE VI CD4: ζ, η-, and γ-chimeras mediate cytolysis of targets expressing HIV gp120 / 41
To find out whether chimeric receptors could actually trigger cytolytic effector programs, we created a model in the form of a target system: an effector based on recognition of the CD4 envelope complex gp120 / gp41. HeLa cells were infected with recombinant vaccinia viruses expressing gp120 / gp41 (Chakrabarti et al., Nature 320: 535-537 (1986); Earl et al., J. Virol. 64: 2448-2451 (1990)) and labeled ⁵¹ Cr . Labeled cells were incubated with cells of the all-specific CD8 ⁺ , CD4 ^- ) cytotoxic T-lymphocyte line, which was infected with vaccinia recombinants expressing CD4: ζ-, CD: η- or CD4: γ-chimeras, or the double mutant chimera CD4: ζ Cis11Gly : Asp15Gly. FIG. 5a-c show that HeLa cells expressing gp120 / 41 were specifically lysed by cytotoxic T lymphocytes (CTLs) expressing CD4 chimeras. Uninfected HeLa cells were not attacked by CTLs armed with CD4: ζ chimeras, and HeLa cells expressing gp120 / 41 were not recognized by uninfected CTLs. To compare the efficacy of different chimeras, flow cytometry measurements determined the ratios of the effector to the target for the CTL fraction expressing CD chimeras and for the HeLa fraction expressing gp120 / 41. FIG. 5c shows the results of cytometric assays of the CD4 expression of CTLs used in the cytolysis experiments shown in FIG. 5a and 5b. Although the average density of surface CD4: ζ was much higher than the average density of CD4: η, the cytolytic efficiencies of expressing cells were the same or close. For the fraction of targets expressing gp120, the adjusted cytolysis efficiency mediated by CD4: ζ and CD4: η proteins is comparable with the best performance indicators reported for specific T-cell receptor pairs: target: effector (average effector to target ratio at 50% T secretion -cells expressing CD4: ζ was 1.9 ± 10.99, n = 10). The fusion of CD4: γ was less active, as was the fusion of CD4: ζ, due to the absence of transmembrane residues Asp and Cis. However, in both cases, cytolysis was observed (Fig. 5b-c).

To test the possibility that the infection of vaccinia can promote artefactual recognition using CTLs similar experiments cytolysis was performed with target cells infected with recombinants vaccinia expressing the phosphatidylinositol-linked form of CD16 (CD16 _PI) and labeled ⁵¹ Cr, and with CTL infected for control by recombinants expressing either CD16 _PI or CD: ζ. FIG. 6a shows that T cells expressing non-CD4 chimeras do not recognize natural HeLa cells or HeLa cells expressing gp120 / 41, and likewise T cells expressing CD4 chimeras do not recognize HeLa cells expressing other surface proteins encoded cowpox. In addition, cytotoxic T lymphocytes expressing non-chimeric CD4 slightly lysed HeLa cells expressing p120.41 (Fig. 6a).

против клеточной линии Raji B, которая обильно экспрессирует антиген класса II, не наблюдается. Хотя умеренный (≈ 5%) цитолиз наблюдается, мутанты, негативные по классу II клеток Raji, RJ2.2.5 (Accolla, J. Exp. Med. 157:1053-1058 (1983)), показывают близкую восприимчивость с той, какую проявляют Raji-клетки, инкубированные с неинфицированными T-клетками.EXAMPLE VII. Cells carrying MHC class II are not attacked by chimeras
CD4 is believed to interact with a non-polymorphic sequence expressing MHC class II antigen (Gay et al., Nature 328: 626-629 (1987)); Sleckman et al., Nature 328: 351-353 (1987)). Although the specific interaction between CD4 and class II antigen has never been documented on purified proteins, under certain conditions, adhesion between cells expressing CD4 and cells expressing class II molecules has been shown (Doyle et al., Nature 330: 256-259 (1987); Clayton et al., J. Exp. Med. 172: 1243-1253 (1990); Lammare et al., Science 245: 743-746 (1989)). Then it was found out whether it would be possible to detect the destruction of cells carrying class II molecules. FIG. 6b shows that specific cytolysis directed by CD4:

against the Raji B cell line, which abundantly expresses class II antigen, is not observed. Although moderate (≈ 5%) cytolysis is observed, mutants negative for class II cells of Raji, RJ2.2.5 (Accolla, J. Exp. Med. 157: 1053-1058 (1983)) show similar susceptibility to that of Raji cells incubated with uninfected T cells.

EXAMPLE VIII. The sequence required for the induction of cytolysis by zeta-tsupya T-cell antigen / Fc receptor
Although chimeras between CD4 and ζ can arm cytotoxic T lymphocytes (CTLs) to kill HIV gp120 target cells, CD4s have sought an alternative to accurately compare the properties of zeta chimeras introduced into human T cell lines. Such cell lines can express CD4, making it difficult to specifically determine the relationship between the type or level of calcium mobilization and the cytotoxic potential of different chimeras. To circumvent this difficulty, chimeras were created between ζ and CD16, in which the extracellular domain of CD16 was attached to the transmembrane and the intracellular sequence of ζ (Fig. 7a). The fusion gene was introduced into the vaccinia virus expressing a plasmid carrying the E. coli gpt gene as a selectable marker and inserted into the genome of the vaccinia strain WR by homologous recombination and growth selection in microfenolic acid (Falkner and Moss, J. Virol. 62 : 1849 (1988); Boyle and Coupar, Gene 65: 125 (1988)).

 T cell lines were infected with vaccinia recombinants and the ratio of cytoplasmic free calcium was measured after cross-linking of the intracellular domains with antibodies. Spectrophotometric (main part of the population) and flow cytometric (single cell) measurements were performed with cells loaded with Indo-1 dye (Grynkiewicz et al., J. Biol. Chem. 260: 3440 (1985); Rabinovitch et al., J. Immunol. 137: 952 (1986)). FIG. 7b shows analyzes of data obtained on Jurkat cells of a T cell leukemia line infected with vaccinia recombinants expressing a CD16: ζ fusion protein. Cross-linking of chimeras reproducibly increased intracellular calcium, while similar treatment of cells expressing non-chimeric CD16 had little effect on its concentration or was ineffective. When a chimera was expressed in mutant cell lines lacking an antigen receptor, or in REX33A (Breitmeyer, et al., J. lmmunol. 138: 726 (1987); Sancho et al., L. Biol. Chem. 264: 20760 (1989 )), or in Jurkat cells of the JRT3.T3.5 mutant (Weiss et al., J. lmmunol. 135: 123 (1984)), a strong response to cross-linking of CD16 with the antibody was observed. Similar data were obtained for the mutant cell line REX20A (Breitmeyer et al., Supra, 1987; Blumberg et al., J. Biol. Chem. 265: 14036 (1990)) and a negative mutant CD3 / Ti cell mutant was found in this laboratory Jurkat lines. Infection with recombinants expressing CD16: ζ does not restore the anti-CD3 antibody response, showing that the fusion protein does not work while maintaining the intracellular chains of the CD3 complex.

 To assess the ability of chimeras to reorient cell-mediated immunity, cytotoxic T-lymphocytes were infected with vaccinia recombinants expressing CD16 chimeras and used for specific lysis of hybrid cells expressing membrane-bound anti-CD16 antibodies. This assay is a continuation of the hybridoma cytotoxicity assay originally developed to study the effector mechanisms of cells bearing Fc receptors (Graziano and Fanger, J. Immunol. 138: 945, (1987); Graziano and Fanger, J. lmmunol. 139: 35-36 (1987); Shen et al., Mol. Lmmunol. 26: 959 (1989); Fanger et al., Immunol. Today 10:92 (1989)). FIG. 8b shows that the expression of CD16: ζ in cytotoxic T lymphocytes arms CTLs to kill 3G8 hybridoma cells (anti-CD16; Fleit et al., Proc. Natl. Acad. Sci. USA 79: 3275 (1982), whereas CTLs expressing the form of CD16 associated with phosphatidylinositol are inactive, CTLs armed with CD16: ζ also do not kill hybridoma cells expressing an inappropriate antibody.

 To identify the minimum ζ sequences required for cytolysis, a number of deleted mutants were prepared in which deletions were sequentially performed from the carboxyl end of the ζ intracellular domain (SEQ ID NO: 44). The largest intracellular domain of zeta could be cleaved with a small sequence to realize the cytolytic potential; the total length of the CD16: ζ chimera was almost equal in efficiency to the chimera deleted at residue 65, CD16 ζ Asp66 * (Fig. 8b). A significant decrease in cytotoxicity was observed with a deletion at ζ of residue 59 (chimera CD16: ζ Glu60 *), and an additional deletion at residue 50 led to a slight decrease in activity. However, a complete loss of activity was not observed even when the intracellular domain was reduced to three residues of the transmembrane anchor (Fig. 8d).

Since ζ is a dimer bound by a disulfide bond, the only explanation for the conservation of cytolytic activity was that endogenous ζ formed heterodimers with a deleted ζ chimera, thus restoring activity. To test this idea, the residues 11 and 15 of the ζ chain, after Asp and Cis were replaced, respectively, with Gly (Cis11Gly / Asp15Gly) and then immunoprecipitation was carried out. Approximately 2 × 10 ⁶ CVI cells were introduced within one hour into serum free DME medium containing recombinant vaccinia, with a multiplicity of infection (moi) of at least ten. Six to eight hours after infection, cells were separated from the surface of the plates with PBS / 1 mM EDTA and superficially labeled with 0.2 mCi ¹²⁵ I per 2 · 10 ⁶ cells using lactoperoxidase and H ₂ O ₂ according to the method of Clark and Einfield (WBC typing II, pp. 155-167, Springer-Verlag, NY, 1986). Labeled cells were collected by centrifugation and lysed in 1% NP-40, 0.1% SDS, 0.15 M NaCl, 0.05 Tris buffer, pH 8.0, 5 mM MgCl ₂ 5 mM KCl, 0.2 M iodoacetamide and 1 mM PMSF. Nuclei were removed by centrifugation, and CD16 proteins were immunoprecipitated with 3G8 antibody (Flrit et al., Supra, 1982; Medarex) and intramuscular IgG in agarose (Cappel, Durham, NC). Samples were subjected to electrophoresis in 8% polyacrylamide SDS gel under non-reducing conditions or in 10% gel under reducing conditions. These immunoprecipitates confirmed that the chimera CD16: ζ Cis11Gly / Asp15Gli does not associate dimeric structures in disulfide-bonded bonds.

 The cytolytic activity of mutant receptors was also tested. The mutated chimera deleted at residue 65 (CD16: ζ Cis11Gly / Asp15Gli / Asp66,), depending on the analysis conditions, was two to eight times less active in the cytolytic analysis than the compared non-mutated chimera (CD16: ζ Asp Asp66 *), which was usually within a factor of two, or did not differ in activity from CD16: ζ (Fig. 9b). This decrease in the activity of mutant chimeras is comparable to the observed decrease for CD4-chimeras of a similar structure (see above) and is most likely determined by the low efficiency of ζ-monomers compared to dimers. In contrast, Asp Asp cis-mutated chimera deleted at residue 59 did not have cytolytic activity (Fig. 9b), confirming the hypothesis that association with other chains mediated by transmembrane residues of Cis and / or Asp is responsible for a weak persistence of cytolytic activity deletions of more amino-terminal residues than residue 65.

Flow cytometric studies showed that deletion mutants lacking transmembrane residues
Asp and Cis could still contribute to an increase in intracellular calcium free ions as a response to antibody cross-linking in the mutant TCR line of Jurkat cells (Fig. 9d). Similar results were obtained for chimeras expressed in the parent Jurkat line. For the case of CD16: ζ Cis11Gly / Asp15Gly / Glu60 *, these data indicate that the ability to mediate the calcium reaction can be mutationally separated from the ability to maintain cytolysis.

 In order to completely eliminate the possible contribution of ζ transmembrane residues, the transmembrane and first 17 cytoplasmic residues were replaced with sequences encoding the piercing membrane and, accordingly, the first 14 or first 17 cytoplasmic residues of the CD15 or CD7 antigens (Fig. 9a). The obtained tripartite fusion proteins CD16: 5: ζ (48-65) and CD16: 7: ζ (48-65) did not form dimers bound by disulfide bonds, as simpler CD16: ζ chimeras do, since they did not have a cysteine residue in ζ-transmembrane domain. Both tripartite chimeras were able to mobilize calcium in Jurkat and TCR negative cell lines (Fig. 9d) and to fix the cytolytic response in CTLs (Fig. 9c, but data not shown). However, cutting off the ζ part from residue 59 in the CD16: 7: ζ chimera (48-59) invalidates the ability of the trifacted fusion protein to control the calcium response in TCR-positive or negative Jurkat cells or cytolysis in mature CTLs (Figs. 9c and 9d without presentation data).

 To study the contribution of individual residues within the framework of the motif of 18 residues, we obtained a number of mutant variants using site-directed mutagenene and evaluated their ability to mediate receptor driven destruction in low (Fig. 10a and 10d) or high (Fig. 10b and 10e) expression chimeric receptor. FIG. 10a-f show that while a number of relatively conservative substitutions (e.g., substitution of acid residues with their corresponding amides, or tyrosine with phenylalanine) that span residues 59 to 63, led to a moderate compromise of cytolytic efficacy, in general these options retained the ability to mobilize calcium. However, collectively, these residues include an important sub-motif, since their deletion eliminates cytolytic activity. The conversion of Tyr 62 to either Fen or Ser excludes both the cytotoxic and calcium responses. In the amino-terminal portion of 18 residues, the replacement of Tyr 51 with Fen eliminates both calcium mobilization and cytolytic activity, while replacing Lei with Ser at position 50 excludes the calcium response and only partially weakens cytolysis. Without the existence of a connection with individual hypotheses, it is doubtful that the inability of the Lei50Cer mutant to mobilize calcium in a short time by flow-cytometric analysis did not fully reflect its ability to mediate a significant increase in free intracellular calcium ions while lengthening the time of cytolytic analysis. However, for some cytolytic T-cell lines, calcium-insensitive cytolytic activity was reported, and it is possible that a similar phenomenon emphasizing the results presented above was not excluded. Substitution of Acn48 with Ser partially weakens cytotoxicity in some experiments, while in others it has a negligible effect.

 In order to investigate the potential role of the repeated sequence elements, the intracellular ζ domain was divided into three segments, covering residues from 33 to 65, from 71 to 104 and from 104 to 138. Each of these segments was attached to the CD16: Cd7 chimera via the Mlul site, introduced into the distal region anchored into the main membrane sequences of the intracellular domain of CD7 (see below; Fig. 11f). A comparison of the cytolytic efficacy of these three elements showed that they were virtually equipotent (Fig. 11b). The sequence compared shows that the second motif carries eleven residues between tyrosines, and the first and third motifs carry ten residues.

 Although an exact explanation of the T-cell activation process has not been made, it is obvious that the aggregation of the antigenic receptor or receptor chimeras that carry ζ-intracellular sequences triggers the mobilization of calcium, the release of cytokine and granules and the appearance of cell surface activation markers. The active site of the ζ chain in the form of a short linear peptide sequence, apparently very small to have its own enzymatic activity, probably interacts with one or at most several proteins to mediate cell activation. It is also obvious that the mobilization of free calcium alone is insufficient for cellular activation, since the ability to mediate cytolysis can be mutationally separated from the ability to mediate the accumulation of calcium.

 As shown above, the addition of 18 residues, from the intracellular domain of the ζ chain to the transmembrane and the intracellular domain of two unbound proteins, makes it possible to obtain chimeras to redirect cytolytic activity to target cells that bind to the extracellular portion of fusion proteins. Although chimeras bearing a motif of 18 residues are about 8 times less active than full length ζ-based chimeras, a decrease in activity can be attributed to the loss of transmembrane interaction, which usually allows wild-type ζ to form dimers linked by a disulfide bond. That is, ζ-deletion constructs that have the same carboxy-terminus as the motif and do not have Cis and Asp transmembrane residues usually show slightly lower activity than chimeras bearing a motif of 18 residues.

 The cytolytic competent element that we focused on has two tyrosines and no serines or threonines, limiting the possibility of the contribution of phosphorylation to activity. A mutation in any of tyrosines destroys activity, however, although preliminary experiments do not indicate the importance of tyrosine phosphorylation after crosslinking of antigens on the surface of the chimera, the likelihood of such phosphorylation at a low level cannot be ruled out. In addition to the effects noted for the two tyrosine residues, a number of amino acid substitutions at the amino and carboxy-terminus of the motif weakened activity at low receptor density.

 Sequences similar to the ζ activating motif can be found in the cytoplasmic domains of several other transmembrane proteins, including the CD3 δ and γ molecules, the mb1 and B29 proteins associated with the IgM surface, and the Fc ε RI β-γ chains possessing high affinity for IgE (Reth, Nature 338: 383 (1989)). Although the function of these sequences has not been determined, with effective expression, each can autonomously activate T cells, and this activity can be explained by the residual TCR response observed in the zeta-negative mutant cell line (Sussman et al., Cell 52:85 (1988)) .

 By itself, ζ carries three such sequences distributed approximately equally, and an approximate three-section intracellular domain shows that each has the ability to initiate a cytolytic response. The isoform η splicing ζ does not have half the carboxy end of the third motif. Since the removal of half the carboxy-end of the first motive eliminates activity, it seems likely that most of the biological effectiveness can be attributed to the first two motives. Although, with different measurements, η in activity is equal to ζ in stimulating the release of a cytokine mediated by an antigen (Bauer et al., Proc. Natl. Acad. Sci. USA 88: 3842 (1991)), or in the redirection of cytolism (see above), η not phosphorylated in response to receptor stimulation (Bauer et al., see above, 1991). Therefore, phosphorylation requires either the presence of all three motifs, or a third motif, which is a favorable substrate for unidentified tyrosine kinase.

EXAMPLE IX. Transduction of the cytolytic signal by the human Fc receptor
To evaluate the effect of different subtypes of the human Fc receptor, chimeric molecules were created in which the extracellular domains of human CD4, CD5, or CD16 antigens were combined with the transmembrane and subtypes of the intracellular domains of FcRII γ A, A, B1, B2, and C (Ravetch nomenclature and Kinet, Ann. Rev. Immunol. 9: 457 (1991)). So, cDNA sequences corresponding to the transmembrane and cytoplasmic domains, previously described as A, B1 and B2 isoforms of FcRII, were amplified from a preexisting PC23 clone and from a palatine tonsil cDNA library (constructed by standard methods) using the following synthetic oligonucleotide primers:
CCC GGA TCC CAG CAT GGG CAG CTC TT (SEQ ID NO: 18; FcRII A direct);
CGC GGG GCG CCC GCT TTA GTT ATT ACT GTT GAC ATG GTC QTT (SEQ ID NO: 19; FcRII A reverse);
GCG GGG GGA TCC CAC TGT CAA AGC TCC CAG CTC TTC ACC G (SEQ ID NO: 20 FcRII B1 and FcRII B2 reverse);
GCG GGG GCG GCC GCC TAA ATA CGG TTC TGG TC (SEQ ID NO: 21: FcRII B1 and FcRII B2 reverse).

These primers contain cleavage sites, respectively, for the BamHI and NotI enzymes, indented by 6 residues from the 5 ′ end. The NotI site is located immediately behind the antisense stop codon, either CTA or TTA. All primers contain 18 or more residues complementary to the 5 ′ and 3 ′ ends of the corresponding fragments. A cDNA fragment corresponding to the cytoplasmic domain of FcRIIγC, which differs from the IIA isoform in only one amino acid residue (L instead of P at residue 268), was obtained by site-directed mutagenesis using PCR using primers with overlapping sequences:
TCA GAA ACA 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).

 PCR fragments were inserted into the expression vectors of vaccinia virus, which respectively contained the extracellular domains of CD16 or CD4, and then inserted into wild-type vaccinia at the thymidine kinase locus, using selection for co-integration of E. coli gpt to facilitate identification of the necessary recombinants. The identity of all isoforms (shown in Fig. 12) was confirmed by dideoxy sequencing.

The formation of chimeric receptor proteins has also been confirmed by immunoprecipitation studies. Approximately 10 ⁷ JRT3.T3.5 cells were used for infection within one hour of the recombinant vaccinia contained in serum-free IMDM medium with a multiplicity of infection of at least ten. Twelve hours after infection, cells were harvested and superficially labeled with 0.5 mCi ¹²⁵ I per 10 ⁷ cells using the glucose peroxidase / glucose oxidase method (Clark and Einfeld, see above). Labeled cells were collected by centrifugation and lysed with 1% NP-40, 0.1 mM MgCl ₂ , 5 mM KCl, 0.2 M iodoacetamide and 1 mM PMSF. Nuclei were removed by centrifugation, and CD16 fusion proteins were immunoprecipitated with 4G8 antibody and azarose anti-mouse IgG. Samples were subjected to extrophoresis under reducing conditions. All immunoprecipitated chimeric receptor molecules had the expected molecular weights.

 To test the ability of chimeric receptors to mediate an increase in the concentration of free calcium ions in the cytoplasm, recombinant viruses were used to infect the JCRT3.T3.5 TCR mutant Jurkat cells (as described above), and cytoplasmic free calcium was measured in cells (as described above) after crosslinking the receptor of the extracellular domain with a monoclonal antibody 3G8 or Leu-3A (as described above). These experiments found that the intracellular domains FcR γ II A and C are able to mediate an increase in the concentration of free calcium ions in the cytoplasm after crosslinking of the extracellular domains, while the intracellular domains FcR γ II B1 and B2 were inactive under the same conditions (Figs. 13a and 13b) . The CD4, CD5 and CD16 hybrids of FcR γ II A were almost equally involved in enhancing the calcium response (Fig. 13a-b). Other cell lines derived from monocytes and lymphocytes were able to respond to a signal initiated by cross-linking of extracellular domains.

To probe the involvement of different intracellular domains of FcR γ II in cytolysis, human cytotoxic T lymphocytes (CTLs) were infected with vaccinia recombinants expressing CD16 chimeras: FcR γ II A, B1, B2 and C. Then, infected cells were co-cultured with ⁵¹ Cr loaded, hybridoma cells (e.g., 3G8 10-2 cells) that expressed the anti-CD16 antibody on the cell surface. In this assay, cytotoxic T-lymphocytes carrying the CD16 chimera killed the hybridoma target cells (allowing the release of free ⁵¹ Cr), provided that the extracellular domain of the CD16 chimera was attached to the intracellular segment capable of being activated by the lymphocyte effector program; this cytolysis experiment is described in detail below. FIG. 14a shows that CTLs armed with CD16: FcR γ II A and C, but not FcR γ II B1 or B2, have the ability to lyse target cells expressing an anti-CD16 antibody on the cell surface.

 To exclude the possibility that the specificity of cytolysis was determined in some way by interaction with the CD16 half, cytolysis experiments were carried out in which the intracellular FcRII domains were attached to the extracellular domain of CD4. In this case, the target cells were HeLa cells expressing the gp120 / 41 HIV envelope proteins (in particular, HeLa cells were infected with vaccinia vPE16 vector (supplied by the National Institute of Allergy and Infectious Diseases. AIDS Division, Bethesda, MD). As in CD16 -system, target cells expressing the HIV envelope were susceptible to lysis by T cells expressing the CD41: FcR γ II A chimera, but not the FcR γ II B1 or B2 chimera (Fig. 14b).

 The intracellular domains of FcR γ II A do not have a sequence that is sufficiently homologous to any other protein, including members of the common FcR γ / TCR ε family. To determine the sequence of elements responsible for the induction of cytolysis, the resulting deletions on the 5'- and 3'-coding sequences of the intracellular domain (described below and shown in Fig. 15a) were evaluated in the analysis of the efficiency of calcium mobilization and cytolysis (as described above). Experiments in which the amino-terminal part of the intracellular domain was removed, the transmembrane domain of FeR γ II was replaced by the transmembrane domain of the unrelated CD7 antigen in order to exclude the possibility of promoting interaction mediated by the membrane penetrating domain.

 FIG. 15b and 15c show that the removal of 14 carboxy-terminal residues, including tyrosine 298, leads to a complete loss of cytolytic performance and a noticeable decrease in calcium mobilization potential. An additional deletion immediately before tyrosine 282 gave an identical phenotype (Fig. 15b and 15c). Deletion from the N-terminus of the intracellular domain to residue 268 had no noticeable effect on either the calcium profile or the cytolytic potency, while deletion at residue 275 clearly reduced calcium release, but had little effect on cytolysis (Figs. 15d and 15e). An additional deletion at residue 282 gave FcR γ-tails that lost the ability to either mobilize calcium or trigger cytolysis (Figs. 15d and 15e). The "active element", determined using these approximate measurements, is relatively large (36 amino acids) and contains two tyrosines, separated by 16 residues.

EXAMPLE X. Cytolysis due to lymphocytes carrying chimeric CD4 receptors that do not contribute to infection
As discussed above, it is possible to create effector molecules that redirect the cytolytic activity of cytotoxic T-lymphocytes in a manner independent of MHC. For example, a chimera composed of the extracellular domain of CD4 fused by the ζ chain in the WH3 clone of human CTLs kills precisely target cells that display HIV-1 gp120 envelope glycoprotein on their surface. Since the extracellular domain of the CD4 molecule confers susceptibility to HIV infection, armed cytotoxic T lymphocytes can become targets for the virus, leading to a decrease in their potency (Dalgleish et al., Nature 312: 767 (1984); Klatzman et al., Nature 312 : 767 (1984)). To prevent such an outcome, CD4-based chimeric effector molecules were created that are effective specifically for HIV-infected cells killed by the mediated cell, but which do not confer susceptibility to HIV infection.

 The tripartite fusion protein was created by genetically linking the extracellular domain of CD4 (FIG. 23) with the hinge, second and third constant domains of the human IgG1 heavy chain (Zettlmeissl et al., DNA Cell Biol. 9: 347 (1990)) (FIG. 25), which was connected in this case with a portion of the first transmembrane exon of human IgG1 bound to the membrane, located behind the site of the human CD7 antigen consisting of sequences between the base of the Ig-like domain and the stop transfer sequence of the subsequent transmembrane domain (Aruffo and Seed, EMBO J. 6: 3313 (1987)) (Fig. 26). The primary amino acid sequence of the extracellular component of the CD7 segment consists of a proline-rich region resembling a stalk-like structure that the Ig-like domain exposes outside the cell surface (Aruffo and Seed, EMBO J. 6: 3313 (1987)) (Fig. 26). Recombinant vaccinia viruses were prepared to express this and related chimeras; as described above. In particular, recombinant vaccinia viruses were obtained by homologous recombination in CV-1 cells. At least two cycles of plaque visualization were performed with OK14 or Leu3a followed by plaque isolation, which was performed for each strain before obtaining high titer CV-1 cell strains.

 The tripartite chimera (CD4 (D1-D4): Ig: CD7) (Fig. 20, molecule “A”) showed effective cell-surface expression and was tested for its ability to act as an HIV receptor in a vaccinia-based syncytium formation assay (Lifson et al., Nature 323: 725 (1986)); Ashorn et al., J. Virol. 64: 2149 (1990). HeLa cells infected with recombinant vaccinia virus (vPE16) encoding HIV-1 envelope glycoprotein (Earl et al., J. Virol. 64: 2448 (1990)) were co-cultured with HeLa cells infected with CD4 or CD4: ζ or CD4 ( D1-D4): Ig: CD7. HeLa cells (ATCC, Rockville, MD), from 6 cm plates at 50% fusion, were infected in serum-free medium for 1 hour at an approximate multiplicity of 10 (MOI). Cells were further incubated for 5-6 hours in complete medium and then peeled off with phosphate-buffered saline (PBS) containing 1 mM EDTA. Cells expressing the membrane and the CD4 chimera were mixed in a 1: 1 ratio and transferred to 6 cm plates with complete medium. Syncytium was evaluated 6-8 hours after co-cultivation and photographed.

, и химера CD4(D1,D2):Ig:CD7:ζ обладают способностью опосредовать специфическое уничтожение клеток, экспрессирующих ВИЧ-оболочечные белки на своей поверхности. Четырехраздельные химеры были стабильно неспособны опосредовать образование синцития в опыте с коровьей оспой. Мы также продемонстрировали, что единственный вид мотива ζ, показанный на фиг. 11а, является достаточным для придания цитолитической активности химере CD4(D1-D4).Co-cultivation of CD4 and vPE16 resulted in the formation of easily detectable multinucleated giant cells. In addition, a chimera containing the extracellular domain of CD4 fused to the TCR ζ chain (Fig. 27) (CD4 ζ) was able to mediate the formation of syncytium, while cells expressing CD4 (D1-D4): Ig: CD7 did not cell fusion signal. We also tested a construct expressing only the first and second domains of CD4 (Fig. 24), CD4 (D10D4): Ig: CD7 (Fig. 20) molecule “B”), because in another context, the amino termini of the two CD4 domains, as shown, were necessary to create HIV infectivity (Landau et al., Nature 334: 159 (1988)). This molecule was also insensitive to the formation of HIV-induced syncytium. A study of the binding to soluble gp120 labeled with ¹²⁵ I found that both CD4 (D1-D4): Ig: CD7 and CD4 (D1, D2): Ig: CD7 had an exceptional affinity for gp120. Further, we found out whether chimeric molecules that resisted the formation of syncytium would indeed be able to redirect cell destruction if they were endowed with a trigger component, as described above. We fused the intracellular domain ζ with the 3'-end of CD4 (D1-D4): Ig: CD7 and CD4 (D1, D2): Ig: CD7 and prepared the corresponding recombinant vaccinia viruses. These constructs, CD4 (D1-D4): Ig: CD7: and CD4 (D1, D2): Ig: CD7: ζ (FIG. 20, molecules “C” and “D”), expressed in the human WH3 clone of cytotoxic lymphocytes, and tested them for the ability to target and destroy HeLa cells expressing HIV surface envelope glycoprotein (using the methods described above). FIG. 21 shows that the intracellular domain ζ fused to CD4 (D1-D4): Ig: CD7 or to CD4 (D1, D2): Ig: CD7 can confer the ability to kill; constructs lacking a ζ chain were unable to mediate this activity. CD4: ζ, a positive control, mediated a slightly more effective cytotoxicity, and CD4 (D1, D2): Ig: CD7: ζ- a slightly lower effective cytotoxicity than CD4 (D1-D4): Ig: CD7: ζ (Fig. 21) . Nevertheless, it is obvious that the chimera CD4 (D1-D4): Ig: CD7:

and the chimera CD4 (D1, D2): Ig: CD7: ζ have the ability to mediate the specific destruction of cells expressing HIV-enveloped proteins on its surface. Four-partitioned chimeras were stably unable to mediate syncytium production in the experiment with vaccinia. We also demonstrated that the only kind of motive ζ shown in FIG. 11a is sufficient to impart cytolytic activity to the CD4 chimera (D1-D4).

Radioimmunoprecipitation experiments have established that fusion molecules were predominantly, if not always, dimers. In these experiments, protein A agarose granules were used to immunoprecipitate a solubilized extract of metabolically labeled HeLa cells infected with recombinant smallpox vaccine expressing the chimera CD4 (D1-D4): Ig: CD7: ζ and CD4 (D1, D2): Ig: CD7ζ. The immunoprecipitated material was fractionated by polyacrylamide gel electrophoresis under reducing and non-reducing conditions. In particular, approximately 5 · 10 ⁶ HeLa-S3 cells were infected with vEP16, as described above, of the corresponding strain of vaccinia virus. Cells were metabolically labeled with 200 mCi / ml Tran ³⁵ S-tag (ICN Radiochemicals, lrvine, CA) for 6-8 hours in cysteine and methionine-deficient medium, and were separated using PBS containing 1 mM EDTA. Cells were sequentially pelleted and lysed in 150 mM NaCl, 50 mM Tris buffer pH 7.5 5 mM EDTA, 0.5% NP-40, 0.1% SDS, 5 mM EDTA, 1 mM PMSF. After removing the nuclei by centrifugation, one fifth of each cell extract was adsorbed on washed agarose granules conjugated with protein A for 2 hours at 4 ^° C. The granules were washed sequentially with PBS containing 1% NP-40 and eluted into a SDS buffer sample. in the presence or absence of mercaptoethanol. The results of these experiments indicate that most immunoprecipitated chimeras CD4 (D1-D4): Ig: CD7: ζ and CD4 (D1, D2): Ig: CD7: ζ, migrated under non-reducing conditions as dimers with the expected molecular weight.

 In order to directly assess the ability of cells expressing CD4 fusion molecules to provide HIV infection, we conducted long-term studies of infectivity on transfectants expressing CD4 (D1-D4): Ig: CD7 and CD4 (D1, D2): Ig: CD7. Stable transfectants CD4 (D1-D2): Ig: CD7 and CD4 (D1, D2): Ig: CD7 and CD4 were obtained in a subline of 293 cells of an easily transfected cell line isolated from an embryonic human kidney. Chimeric molecules were subcloned into bidirectional vectors in which the hygromycin B gene was inserted under the herpes simplex virus thymidine kinase promoter. 60-70% of the drain cells from a 10 cm dish transfected 10 μg of DNA of this plasmid by calcium phosphate conception. Prior to transfection, plasmids were linearized at the unique SfiI site, and the ends were blunted using T4 DNA polymerase. After 24 hours of transfection, the cells were exfoliated four times and after 48 hours of transfection, they were selected with hygromycin B (Sigma, St. Louis, Mo) at a concentration of 400 μg / ml. Every 3-4 days, the cells were placed in fresh medium containing hygromycin.

 Resistant colonies expanded in volume were selected and their expression was tested by indirect immunofluorescence, after flow cytometry (Coulter, Hialeah, FL) using human anti-Fc IgG conjugated with fluorescein (Organon Teknika, West Chester, PA), or Q4120, antibody interacting with anti-CD (Sigma). Two independent clones were selected from each construct for analysis, comparable in level of CD4 on the cell surface to other cell lines. FIG. 22 shows that upon subsequent exposure to HIV, p24 was detected in cultures of CD4-stable transfectants already 3 days after infection. The presence of polynuclear giant cells and the characteristic sphericity in these cultures were evident already 5 days after infection. After 32 days of cultivation in the parent cell line or in either of two independently obtained isolates transfected with CD4 (D1-D4): Ig: CD7 and CD4 (D1, D2): Ig: CD7 (Fig. 22), no significant p24 levels were observed or explicit formation of multinucleated giant cells.

 At the end of the infectivity studies, the cells were analyzed for expression of CD4 by the cell surface. The surface CD4 epitope density was significantly reduced in infected cultures expressing CD4, constant at low viral modulation, and was not affected in cultures expressing CD4 (D1-D4): Ig: CD7 and CD4 (D1, D2): Ig: CD7. These experiments indicate the possibility of creating chimeric molecules that carry two apical CD4 domains, which, when fused to the ζ chain of the receptor, T cells are capable of targeting and killing HIV-infected cells, but which do not contribute to CD4-mediated HIV infection.

 Additional experiments confirm that there is a physical distance between the extracellular domain of the CD4 molecule and the lipid bilayer, which gives the ability to resist HIV infection. In the first experiment, we constructed a chimeric molecule carrying a deletion of the CD4 stem and transmembrane domain; this deletion removes the proline-rich region of the transmembrane portion of CD7. When this domain was fused to the extracellular domain of CD4, it retained its ability to efficiently anchor the extracellular domain of the CD4 molecule, as determined by measuring the cell surface expression of the CD4 molecule (as described above). However, the potential to resist the formation of syncytium induced by the HIV envelope glycoprotein was lost. Therefore, a deletion of the propyl-rich region of the CD7 molecule, a region suitable for the formation of the α-helical structure, effectively reduced the distance between the extracellular domain of CD4 and the lipid bilayer and impaired the ability of the chimera to resist the formation of syncytium.

 In a second experiment, we demonstrated that the ability to resist HIV-induced syncytium formation can be conferred on the CD4 / CD5 chimera, which was previously considered as a transmembrane anchor for the extracellular domain of CD4, but which was unable to resist HIV-induced syncytium formation. In this experiment, the hinge, CH2 and CH3 domains of the human IgG1 heavy chain were inserted into the CD4 / CD5 molecule; the resulting chimera resisted the formation of syncytium, reaffirming that the distance provided by the immunoglobulin domains is sufficient to confer resistance to HIV-induced formation of syncytium.

 In the third experiment, the CD4 domain lengthened the varying distances from the cell membrane using alpha helices of different lengths. In particular, synthetic oligonucleotides were constructed that presented repeated alpha-helical motifs of lysine and glutamic acid residues flanked by two alanine residues (see FIG. 28 for primary nucleic acid and amino acid sequences). In previous studies, such amino acid sequences were detected with high frequency in alpha helices, confirming that such repeated motifs would borrow the conformation of the alpha helix and that replacing such alpha helices between the transmembrane domain and the extracellular domains of CD4 would eject CD4 from the cell membrane. By varying the length of the alpha-helical segment, the calculation of the predicted distance required for resistance to HIV penetration was carried out on the basis of the known elongation and growth of the alpha-helix. These results are presented in table 1.

 In Table 1, “CD4” corresponds to CD4 (D1-D4), unless otherwise noted; "H", "CH2" and "CH3" correspond to the hinge, regions of the heavy chain CH2 and CH3 of human IgG1, respectively; "CD7tm and stk" correspond to regions of the CD7 transmembrane and stem; "CD7tm (long version)" and "CD7tm (short version)" correspond to the CB7 transmembrane region and the CD7 transmembrane region deleted by a proline-rich domain (as discussed above); "CD5tm" corresponds to the transmembrane region of CD5 and "CD34tm" corresponds to the transmembrane region of CD34. For J-L inputs, the length of the alpha helical region is indicated in angstroms; these values are based on the fact that there are 3.6 residues per turn of the alpha helix, which corresponds to 5.4 angstroms (or 1.5 angstroms per remainder). Accordingly, 16 residues of the alpha helix will eject the extracellular domain of CD4 by almost 24 angstroms. Alpha helices with a length of 48 and 72 angstroms were constructed by concatemerizing the BstY1 fragment into a fragment of a unique BamHI site (see FIG. 28) after selection of clones with the proper orientation.

 Syncytium formation was evaluated in co-cultivation experiments with HeLa cells expressing the HIV envelope glycoprotein from the vaccinia virus vRE-16 construct (see above).

 The expression of thy-1 was measured as follows. Based on the HIV clone hxb.2, a live retrovirus vector was constructed. In this vector, the non-essential nef gene was replaced by a sequence encoding rat thy-1, a molecule efficiently expressed by a cell surface that is anchored into the membrane via a phosphatidinositol bond. The virus obtained from this molecular clone, designated hxb / thy-1, turned out to be infectious, as follows from its cytopathological effects and the formation of p24 in the culture supernatants of infected C8166 cells (human leukemia T-cell line CD4). In addition, upon exposure to hxb / thy-1, HeLa cells transiently transfected with CD4 detected thy-1 expression signals already 18 hours after infection, as if signaling regulated by the nef-like method was expected. Signals encoded by the nef gene typically belong to the class of viral regulatory proteins that are multiple spliced and lack the rev response element. These signals constitutively accumulate in the cytoplasm in the form of products of an early viral gene. It was suggested that thy-1 signals are regulated similarly, that is, they occur early in the life cycle of the virus. In short, by using thy-1 expression as a substitute for viral entry, this system facilitates the analysis of HIV entry. CD4-based different chimeras were transiently transfected into HeLa cells using standard DEAE-dextran methods. Transfected cells were incubated with hxb / thy-1 virus for 48 hours after infection and evaluated by thy-1 expression for 24-48 hours after infection. In these results, shown in table 1, thy-1 expression was measured within 24 hours after infection using a commercial preparation of thy-1 monoclonal antibody (Exact).

 From the data presented in Table 1, we concluded that the extracellular domains of CD4 should optimally protrude from the cell membrane by at least 48 angstroms and preferably at least 72 angstroms in order to resist HIV-1 infection.

 Using a strategy similar to the general strategy described above, it is possible to construct chimeras based on anti-HIV enveloped antibodies that target HIV-infected cells. Examples of such antibodies are described by Gorny et al., Proc. Natl. Acad Sci. USA 86: 1624 (1989) and Marasco et al., J. Clin. Invest. 90: 1467 (1992).

EXAMPLE XI. Using protruding CD4 molecules as a trap for HIV
As shown above, cells carrying CD4 domains protruding from the cell surface resist HIV infection. Accordingly, such CD4-bearing cells are suitable as traps to bind circulating HIV and reduce the titer of viruses in HIV-infected individuals. Preferably, the CD4 domain is present in a cell that naturally passes through the lymph nodes; beneficial host cells include any cell that plays a role in immunocellular cleansing, like any other cell naturally present in the lymph node follicles. Specific examples include, but are not limited to, macrophages, T cells (e.g., helper T cells), B cells, neutrophils, dendritic cells, and follicular dendritic cells.

 Any CD4 domain capable of binding HIV, including the domains D1-D4 and D1, D2 described above, can be used in this method of the present invention. For at least some embodiments (e.g., the variants described above), portions of the chimera of the intracellular and / or transmembrane domain can be selected from any protein or any amino acid sequence.

EXAMPLE XII. Additional triggering proteins of the T-cell receptor and B-cell receptor
In accordance with the present invention, other intracellular and transmembrane signal transducing domains can be obtained from the T-cell-CD-delta and T3-gamma receptor proteins and the B-cell receptor proteins, mb1 and B29. The amino acid sequences of these proteins are shown in FIG. 16 (CD3 delta; SEQ ID N: 24), FIG. 17 (T3-gamma; SEQ ID NO: 25), FIG. 18 (mb1; SEQ ID NO: 26) and FIG. 19 (B29; SEQ ID NO: 27). Sequences of sequences sufficient for transduction of the cytolytic signal (and therefore preferably included in the chimeric receptor of the present invention) are shown in parentheses. Chimeric receptors that include these protein domains are designed and used in the therapeutic methods of the present invention, essentially as described above.

EXAMPLE XIII. The methods of experiments. Smallpox vaccination and radioimmunoprecipitation
About 5 · 10 ⁶ CV1 cells were infected for one hour in serum free DME medium with recombinant vaccinia vaccine with a multiplicity of infection (moi) of at least ten (titer was measured in CV1 cells). After infection, the cells were placed in fresh medium and metabolized for six hours with 200 mCi / ml ³⁵ S-methionine plus cysteine (Tran ³⁵ S-label, ICN; Costo Mesa, CA) in DMEM benzomethionine and cysteine-free medium (Gibco; Grand Island, NY). Labeled cells were separated using PBS containing 1 mM EDTA, collected by centrifugation and lysed in 1% NP-40, 0.1% SDS, 0.15 NaCl, 0.05 Tris buffer pH 8.0, 5 mM EDTA and 1 mM PMSF Nuclei were removed by centrifugation and CD4 proteins were immunoprecipitated with OKT4 antibody and agarose anti-mouse IgG (Cappel, Durham, NC). Samples were subjected to SDS electrophoresis on an 8% polyacrylamide gel under non-reducing (NR) and reducing (R) conditions. Prior to radiography, gels containing ^35s S-labeled samples were impregnated with En ³ Hance (New England Nuclear, Boston, MA). The expression facilitation of the transmembrane form of CD16, CD16 _{TM was} measured by comparing its expression in CV1 cells once infected with CD16 _TM for expression in cells coinfected with viruses encoding CD16 _TM and the ζ or γ chimera. After infection and incubation for six hours or more, cells were peeled from the surface of the plates with PBS, 1 mM EDTA, and expression of CD16 _™ or chimeras was measured by indirect immunofluorescence and flow cytometry.

Calcium Income Analysis
Jurkat cells of subline E6 (Weiss et al., J. Immunol. 135: 125-128 (1984)) were infected with recombinant vaccinia viruses for one hour in serum-free IMDM medium at moi 10 and incubated for three to nine hours in IMDM medium with 10% FBS. Cells were harvested by centrifugation and resuspended at a concentration of 3 × 10 ⁶ cells / ml in complete medium containing 1 mM Indo-1 acetomethoxy ester (Grynkiewicz et al., J. Biol. Chem. 260: 3340-3450 (1985)) (Molecular Samples) , and incubated at 37 ^o C for 45 minutes. Cells loaded with Indo-1 were besieged and resuspended at a concentration of 1 · 10 ⁶ / ml in serum-free IMDM medium at room temperature in the dark. Cells were analyzed for the presence of free calcium ions by simultaneously measuring violet and cyan fluorescence emission using flow cytometry (Rabinovitch et al., J. Immunol. 137: 952-961 (1986)). To initiate calcium outflow, either Leu-3A (anti-CD4) conjugated with phycoerythrin (PE) and then 10 μg / ml of unconjugated anti-mouse IgG are added to the cell suspension at a concentration of zero time point, or to the cell suspension, at a concentration of 1 μg / ml, unconjugated monoclonal antibody 3G8 (anti-CD16) is added, and then 10 μg / ml goat anti-mouse IgG Fab'-conjugated with phycoerythrin PE at zero time point. Histograms of the violet / blue emission ratio were obtained for a population of PE-positive (uninfected) cells, which typically represented 40-80% of all cells. The T-cell antigen receptor response in uninfected cells was triggered using OKT3 antibody, without crosslinking. For experiments with the participation of CD16-chimeric receptors, samples showing a mixture of the basis to reduce intracellular calcium (without antibody) were excluded from the analyzes. The data histogram was sequentially analyzed by converting binary data to ASCII using handwritten (Cooper City, FL) software, after analysis using FORTRAN collector programs. The violet / cyan emission ratio, before adding the second antibody reagents, was used to set the standardized initial ratio to unity and to set the ratio of the resting threshold so that 10% of the resting population would exceed the threshold.

Cytolytic analysis
The human WH3 T cell line, a cytolytic line limited to CD8 ⁺ CD4 ^- HLA B44, was maintained in IMDM, 10% human serum with 100 e / ml IL-2 and periodically stimulated with either non-specific, irradiated (3000 rad) HLA-scattered peripheral blood lymphocytes and 1 μg / ml phytohemagglutinin, or specifically, irradiated mononuclear cells carrying B-44. After one day of nonspecific stimulation, the PHA was diluted to 0.5 μg / ml by adding fresh medium, and after three days the medium was changed. After stimulation, cells were grown for at least 10 days before use in cytotoxicity assays. Cells were infected with recombinant smallpox vaccine with 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 · 10 ⁷ cells / ml. 100 μl of these cells were added to each well of a U-shaped microtiter plate containing 100 μl / well of complete medium. Cells were diluted in two sequential operations. For each sample, two wells did not contain lymphocytes in order to measure spontaneous chromium release and total chromium uptake. Target cells from the subline S3 of HeLa cells were infected in 6.0 or 10.0 cm plates with an approximate multiplicity of infection (moi) of 10 for one hour in serum-free medium, followed by incubation in complete medium for three hours. Then they were peeled from the surface of the plates using PBS containing 1 mM EDTA and counted. An aliquot of 10 ⁶ target cells (HeLa, Raji, or RJ2.2.5 cells for experiments with the CD4 chimeric receptor, and 3G8 10-2 cells; Shen et al., Mol. Immunol. 26: 959 (1989) for experiments with CD16-chimeric receptor) was centrifuged and resuspended in 50 μl of sterile ⁵¹ Cr-sodium chromate (1 mCi / ml, Dupont Wilmington, DE) for one hour at 37 ^° C. with periodic stirring and then washed three times with PBS. 100 μl of labeled cells resuspended in medium at a density of 10 ⁵ cells / ml were added to each well. Raji RJ2.2.5 target cells were labeled in the same manner as HeLa cells. The microtiter plate was centrifuged at 750 · g for 1 minute and incubated for 4 hours at 37 ^° C. At the end of the incubation period, the cells in each well were resuspended by careful pipetting, the sample was removed to count the included label, and the microtiter plate was centrifuged at 750 · g within 1 minute. 100 μl aliquots of supernatants were collected and counted in a gamma scintillation counter. The percentage of destruction was adjusted for the fraction of infected target cells (usually 50-90%) by measuring flow cytometry. For infected effector cells, the effector: target ratio was adjusted according to the percentage of infected cells (usually 20-50% for experiments with the CD4 chimeric receptor and> 70% for experiments with the CD16 chimeric receptor).

Mutagenesis of the ζ sequence in vitro
To create point mutations at amino acid residues 11 or 16 of the ζ sequence, synthetic oligonucleotide primers were prepared, extending from the BamHI site above the ζ transmembrane domain and transforming the Cis to Gly residue (C11G) or the Asp to Gly residue in the natural ζ chain at position 11 at position 15
(D15G) or both (C11G / D15G), and were used in PCR reactions to obtain mutant fragments that were reintroduced into CD4: wild-type ζ constructs.

 To create ζ deletions, the ζ chain cDNA sequences were amplified by PCR using synthetic oligonucleotide primers designed to create a stop codon (UAG) after residues 50, 59 or 65. The primers contained a cleavage site for the Notl enzyme that cuts five or six residues from the 5'-end, usually in the sequence constructed from CGC GGG CGG CCG CTA (SEQ ID NO: 11), in which the last three residues correspond to a stop anticodon. The Notl and stop anticodon sequences are followed by 18 or more residues complementary to the corresponding 3'-end of this fragment. The resulting chimeras were designated, respectively, CD16: ζY51 *, CD16: ζE60 * and CD16: ζD66 *. The BamHI site located above the transmembrane domain and the Notl site were used to generate fragments that were reintroduced into the CD16: wild-type ζ construct. Monomeric ζ-chimeras were created by isolating the ζ-transmembrane and membrane proximal intracellular sequences by splitting the CD4: ζ construct using BamHI and SacI, described above, and inserting the fragment into CD16: ζE60 * and CD16: ζD66, respectively * construct.

Construction of CD16: 7: ζ (48-65) and CD16: 7: ζ (48-59) tripartite chimera
In order to construct CD16: ζ D66 *, the ζ-chain cDNA sequence corresponding to the transmembrane domain was replaced by the corresponding transmembrane and cytoplasmic domain obtained from CD5 and CD7 cDNAs. CD5 and CD7 fragments were obtained by PCR reaction using direct oligonucleotides including The BamHI restriction cleavage site and the corresponding region located immediately above the transmembrane domain, respectively, CD5 and CD7, and then the reverse oligonucleotides, overlapping, respectively, the sequence of CD5 and CD7 and ζ a sequence that contains a SacI restriction cleavage site.

CD5: ζ: CGC GGG GAG CTC GTT ATA GAG CTG GTT CTG GCG CTG CTT CTT CTG (SEQ ID NO: 12)
CD7: ζ: CGC GGG GAG CTC GTT ATA GAG CTG GTT TGC CGC CGA ATT CTT ATC CCG (SEQ ID NO: 13).

 The PCR products of CD5 and CD7 were treated with BamHI and SacI and ligated with CD16: ζ E60 * treated with BamHI and SacI, and the ζ sequence from BamHI to SacI was replaced with a CD7 fragment. To create a CD16: CD5 and CD16: CD7 constructor, CD5 and CD7 fragments were obtained by PCR using an oligonucleotide containing a NotI restriction cleavage site and an encoding stop codon (UAA) after Gln416 and Ala193, respectively, in CD5 and CD7. The PCR fragment of CD5 and CD7 was treated with BamHI and SacI and inserted into the CD16: ζ Asp66 * construct.

In vitro mutagenesis of N-terminal residues within the ζ motif transducing a cytolytic signal
Synthetic oligonucleotide primers extending from the SacI site within the ζ motif and converting the natural residue 48 from Asp to Ser (N48S), residue 50 from Lei to Ser (L50S) and residue 51 from Tyr to Fen (Y51F), were synthesized and used in a CPR reaction to obtain fragments that were reintroduced into a CD16: 7: wild-type ζ (48-65) construct.

In vitro mutagenesis of C-terminal residues within the ζ motif transducing a cytolytic signal
Synthetic oligonucleotide primers extending from the NotI site to the stop codon and converting the natural residue of 60 sec to Gly on Glyn (E60Q), residue 61 to Glu to Gln (E61Q), residue 62 to Tyr to Fen or Ser (Y62F or Y62S ) and residue 63, from Asp to Asn (D63N), were synthesized and used in a PCR to obtain fragments that were subcloned into CD16: wild-type ζ D66 * construct from the BamHI site to the NotI site.

Chimeric constructs CD16: 7: ζ (33-65), CD16: 7: ζ (71-104), CD16: 7: ζ (104-137)
A CD7 transmembrane fragment carrying MluI and NotI sites at the junction between the transmembrane and intracellular domains was obtained by PCR using the oligonucleotide of the following sequence: CGC GGG GCG GCC ACG CGT CCT CGC CAG CAC ACA (SEQ ID NO: 14). The resulting PCR fragment was treated with BamHI and NotI and re-inserted into the CD16: 7: ζ (48-65) construct. ζ fragments encoding residues 33-65, 71-104 and 104-137 were obtained in a PCR reaction using a pair of primers containing MluI sites at the 5'-end of direct primers and stop codons following NotI sites at 5'-end reverse primers. In each case, restriction sites cut six residues from the 5 ′ ends of the primers to ensure restriction enzyme digestion
ζ 33: CGC GGG ACG CGT TTC AGC CGT CCT CGC CAG CAC ACA (SEQ ID NO: 15)
ζ 71: CGC GGG ACG CGT GAC CCT GAG ATG GGG GGA AAG (SEQ ID NO: 16) and
ζ 104: CGC GGG ACG CGT ATT GGG ATG AAA GGC GAG CGC (SEQ ID NO: 17).

Construction of FcR ζ IIA-deletion mutants
The carboxy terminus of FeRIIA deletion mutants was constructed by PCR in the same manner as full length constructs, converting the tyrosine coding sequences at positions 282 and 298 into stop codons (TAA). N-terminal deletions were obtained by PCR amplification of fragments encoding a successively smaller intracellular domain using oligonucleotides that allow the resulting fragments to be inserted between the MluI and NotI restriction sites into a previously constructed expression plasmid encoding the extracellular domain of CD16 fused to the transmembrane later terminated at the MluI site and attached between the transmembrane and intracellular domain.

Other options for implementation
The above examples indicate that the aggregation of ζ, η, or γ chimeras is sufficient to initiate a cytolytic effector cell response in T cells. Knowledge of the expression region of ζ, η or γ, which includes T-lymphocytes, natural killer cells, basophilic granulocytes, macrophages and mast cells, suggests that conserved sequence motifs can interact with a sensitive apparatus common to hematopoietic cells and that it is an important component of protective body forces in the immune system can be mediated by the outcome of receptor aggregation.

 The effectiveness of the cytolytic response and the lack of response to target cells carrying MHC class II receptors indicate that ζ, η and γ based chimeras provide the basis for a genetic effect on AIDS through adaptive immunotherapy. The widespread occurrence of endogenous ζ and η and the evidence with which Fc receptors associated with γ mediate cytotoxicity in various cell types (Fanger et al., Immunol. Today 10: 92-99 (1989)) allow us to consider cell diversity in relation to this goal. For example, neutrophilic granulocytes, which have a very short lifespan (≈4 h) in circulating blood and are very cytolytic, are attractive target cells for the expression of chimeras. Infection with HIV neutrophils probably does not lead to the release of the virus, and the abundance of these cells (prevail over white blood cells) should enhance the body's defenses. Mature T cells, a population currently suitable for retroviral engineering (Rosenberg, Sci Am. 262: 62-69 (1990)), offer another attractive opportunity for body cells. With the addition of recombinant IL-2, the T-cell population during cultivation can relatively easily increase in volume, and with reinfusion, such populations usually have a limited life span (Rosenberg et al., N. Engl. J. Med. 323: 570-578 (1990 )).

 Under appropriate conditions, recognition by HIV cells expressing CD4 chimeras should also be provided with mitogenic stimuli, opening up the possibility that the armed cell population could dynamically respond to the viral burden. Although we focused here on the behavior of fusion proteins in cytolytic T lymphocytes, expression of chimeras in helper lymphocytes could create a cytokine source mobilized by HIV that could neutralize the destruction of the helper cell subgroup in AIDS. A recent description of several options for creating resistance to infection at other stages than virus penetration into the cell (Friedman et al., Nature 335: 452-454 (1988); Green et al., Cell 58: 215-223 (1989); Malim et al., Cell 58: 205-214 (1989); Trono et al., Cell 59: 113-120 (1989); Buonocore et al., Nature 345: 625-628 (1990)), suggests that cells carrying CD4 chimeras could be designed to inhibit viral reproduction as a result of the expression of appropriate agents having an intracellular site of exposure.

 The ability to transmit signals for T-lymphocytes through autonomous chimeras also provides for the ability to regulate in vivo retrovirus-generated lymphocytes. The stimulating effect of crosslinking, mediated, for example, by specific IgM antibodies designed to remove complement binding domains, can allow such lymphocytes to increase quantitatively, while treatment with similar specific IgG antibodies (for example, to recognize the amino acid variation created in the chimeric chain) could deplete the constructed population. In addition, anti-CD4 IgM antibodies do not require additional crosslinking in order to mobilize calcium in Jurkat cells expressing CD4: ζ chimeras. The ability to regulate the cell population without resorting to repeated extracorporeal amplification can significantly expand the scope and effectiveness of the current use proposed for genetically engineered T cells.

 Although the present invention has been described in connection with specific embodiments, it must be borne in mind that further changes may be made, and the present application is intended to introduce changes, designations or alterations to the present invention, including deviations from the present invention that are within the scope of the technology to which the present invention relates and which can be applied to the essential features set forth above, as well as within the framework of the following form for inventions.

Claims (11)

 1. A method of suppressing HIV-infected mammalian cells, wherein said method comprises administering to said mammal an effective amount of therapeutic cells, said therapeutic cells expressing a membrane-bound protein chimeric receptor containing an extracellular region that includes a CD4 fragment, including at least amino acids 1 - 200 CD4 shown in figure 24, which is able to specifically recognize and bind HIV or HIV-infected cells, but which does not mediate ICH-infection.  2. The method according to claim 1, in which the specified fragment of CD4 consists of amino acids 1 to 394 shown in figure 23.  3. The method according to claim 1, wherein said CD4 fragment is separated from the membrane of the indicated therapeutic cell by a distance of at least 48 angstroms or at least 72 angstroms.  4. The method according to claim 1, wherein said receptor comprises a CD7 transmembrane region, a CD5 transmembrane region, or a CD34 transmembrane region shown in Table 1.  5. The method according to claim 1, wherein said CD4 fragment is separated from the membrane of said therapeutic cell by one or more protein alpha helices.  6. The method of claim 1, wherein said therapeutic cells are selected from the group consisting of T cells, B cells, neutrophils, or dendritic cells.  7. Protein recombinant receptor containing an extracellular region that includes a CD4 fragment, including at least amino acids 1 to 200 CD4 shown in figure 24, which is able to specifically recognize and bind HIV or HIV-infected cells, but which does not mediate HIV infection.  8. The receptor according to claim 7, in which the specified fragment of CD4 consists of amino acids 1 to 394 shown in figure 23.  9. The receptor according to claim 7, wherein said CD4 fragment is separated from the host cell membrane by a distance of at least 48 angstroms or at least 72 angstroms.  10. The receptor according to claim 7, wherein said receptor comprises a CD7 transmembrane region, a CD5 transmembrane region, or a CD34 transmembrane region shown in Table 1.  11. The receptor according to claim 7, in which the specified fragment of CD4 is separated from the membrane of the specified therapeutic cell by one or more protein alpha helices.

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