WO1998041613A1

WO1998041613A1 - Targeted cytolysis of cancer cells

Info

Publication number: WO1998041613A1
Application number: PCT/US1998/003797
Authority: WO
Inventors: Gillis R. Otten; Gary B. Greenburg; Denise Casentini-Borocz; Mitchell H. Finer
Original assignee: Otten Gillis R; Greenburg Gary B; Casentini Borocz Denise; Finer Mitchell H
Priority date: 1997-03-14
Filing date: 1998-03-13
Publication date: 1998-09-24
Also published as: AU6537798A

Abstract

Chimeric proteins and DNA encoding chimeric proteins are provided, where the chimeric proteins are characterized by an extracellular domain capable of binding to A33 in a non-MHC restricted manner, a transmembrane domain and a cytoplasmic domain capable of activating a signaling pathway. Binding of A33 to the extracellular domain results in transduction of a signal and activation of a singaling pathway in the cell, whereby the cell may be induced to carry out various functions relating to the signaling pathway. The chimeric DNA may be used to modify lymphocytes as well as hematopoietic stem cells as precursors to a number of important cell types.

Description

TARGETED CYTOLYSIS OF CANCER CELLS

By

Gillis R. Otten, Gary B. Greenburg, Denise Casentini-Borocz and Mitchell H. Finer

INTRODUCTION Technical Field

The field of the invention is the use of chimeric surface membrane proteins for signal transduction. The cells expressing such proteins are configured to recognize and act on cells expressing a tumor antigen, such as that recognized by the A33 monoclonal antibody (mAb) .

Background Regulation of cell activities is frequently achieved by the binding of a ligand to a surface membrane receptor. The formation of the complex with the extracellular portion of the receptor results in a change in conformation with the cytoplasmic portion of the receptor undergoing an alteration which results in a signal being transduced in the cell. In some instances, the change in the cytoplasmic portion results in binding to other proteins, where the other proteins are activated and may carry out various functions. In some situations, the cytoplasmic portion is autophosphorylated or phosphorylated, resulting in a change in activity. Those events frequently are coupled with secondary messengers, such as calcium, cyclic adenosine monophosphate, inositol phosphate, diacylglycerol and the like. The binding of the ligand may result in a particular signal being induced.

There are a number of instances where one might wish to have a signal induced by virtue of employing a particular ligand. For example, one might wish to activate particular T cells, where the T cells then will be effective as cytotoxic agents, or active in secreting effector molecules, such as, interleukins, colony stimulating factors or other cytokines, which results in the stimulation of another cell. The ability of the T cell receptor to recognize antigen is restricted by the nature of Major Histocompatibility Complex (MHC) antigens on the surface of the host cell. Thus, the use of a chimeric T cell receptor in which a non-MHC restricted ligand binding domain is linked directly to the signal transducing domain of the T cell receptor would permit the use of the resulting engineered T cell in any individual, regardless of MHC genetic background. In that manner, one may change the ligand which initiates the desired response, where for some reason, the natural agent may not be as useful.

There is, therefore, interest in finding ways to modulate cellular responses in providing for the use of ligands other than the normal ligand to transduce a desired signal.

Relevant Literature

The T cell antigen receptor (TCR) has a non-covalent association between a heterodimer, the antigen/MHC binding subunit T_χ/ variable component and five invariant chains: zeta (ζ), eta (η) and the three CD3 chains: gamma (γ> , delta (δ) and epsilon (e) (Weiss & Imboden (1987) Adv. Immunol., 41:1-38; Cleavers et al . (1988) Ann. Rev. Immunol., 6:629-662; Frank et al . (1990) Sem. Immunol., 2:89-97). In contrast to the _L alpha/beta heterodimer which is solely responsible for antigen binding, the physically associated CD3-zeta/eta complex does not bind ligand, but is thought to undergo structural alterations as a consequence of ^-antigen interaction which results in activation of intracellular signal transduction mechanisms.

A description of the zeta chain may be found in Ashwell & Klausner (1990) Ann. Rev. Immunol., 8:139-167. The nature of the zeta chain in the TCR complex is described by Baniyash et al . (1988) J. Biol. Chem. , 263:9874-9878 and Orloff et al . (1989) J. Biol. Chem., 264:14812-14817. The heterodimeric zeta and eta protein is described by Jin et al. (1990) Proc. Natl . Acad. Sci . USA, 87:3319-3323. Discussion of the horaodimers and heterodimers may be found in Mercep et al. (1988) Science, 242:571-574; Mercep et al. (1989) Science, 246:1162-1165; and Sussman et al . (1988) Cell, 52:85-95. For studies of the role of the zeta protein, see Weissman et al . (1989) EMBO J., 8-3651-3656; Frank et al . (1990) Science, 249:174-177; and Lanier et al . (1989) Nature, 342:803- 805. For discussion of the gamma subunit of the Fc„RI receptor, expressed on mast cells and basophils, and its homology to the zeta chain, see Bevan and Cunha-Melo (1988) Prog. Allergy, 42:123- 184; Kinet (1989) Cell, 57:351-354; Benhamou et al . , Proc . Natl. Acad. Sci. USA, 87:5327-5330; and Orloff et al . (1990) Nature, 347:189-191.

The zeta(ζ) chain is structurally unrelated to the three CD3 chains and exists primarily as a disulfide-linked homodimer, or as a heterodimer with an alternatively spliced product of the same gene, eta (η) . The zeta chain also is expressed on natural killer cells as part of the FC_yRIII receptor. The gamma chain of the Fc_e receptor is related closely to zeta and is associated with the Fc_eRI receptor of mast cells and basophils and the CD16 receptor expressed by macrophages and natural killer cells. The role in signal transduction played by the cytoplasmic domains of the zeta and eta chains, and the gamma subunit of the Fc_eRI receptor has been described by Irving & Weiss (1991) Cell, 64:891-901; Romeo & Seed (1991) Cell, 64:1037-1046 and etourneur & Klausner (1991) Proc. Natl. Acad. Sci. USA, 88:8905-8909. More recent studies have identified an 18 amino acid motif in the zeta cytoplasmic domain (Reth (1989) Nature, 338:383-384) that, on addition to the cytoplasmic domain of unrelated transmembrane proteins, endows them with the capacity to initiate signal transduction (Romeo et al . (1992) Cell 68:889-897). Those data suggest a T cell activation mechanism in which that region of zeta interacts with one or more intracellular proteins .

The three CD3 chains, gamma (γ> , delta (δ) and epsilon (e) , are polypeptides related structurally and originally were implicated in signal transduction of T cells by studies in which anti-CD3 monoclonal antibodies were shown to mimic the function of antigen in activating T cells (Goldsmith & Weiss (1987) Proc. Natl. Acad. Sci. USA, 84:6879-6883) and from experiments employing somatic cell mutants bearing defects in TCR-mediated signal transduction function (Sussman et al . (1988) Cell 52:85-95). Sequences similar to the active motif found in zeta also are present in the cytoplasmic domains of the CD3 chains gamma and delta. Chimeric receptors in which the cytoplasmic domain of an unrelated receptor has been replaced by that of CD3 epsilon have been shown to be proficient in signal transduction (Letourneur & Klausner (1992) Science, 255:79-82), and a 22 amino acid sequence in the cytoplasmic tail of CD3 epsilon was identified as the sequence responsible. Although the cytoplasmic domains of both zeta and CD3 epsilon have been shown to be sufficient for signal transduction, quantitatively distinct patterns of tyrosine phosphorylation were observed with those two chains, suggesting that they may be involved in similar but distinct biochemical pathways in the cell.

Initiation of activation by the T cell receptor ("TCR") of the phosphatidylinositol-specific phospholipase C is described by Weiss et al. (1984) Proc. Natl. Acad. Sci. USA, 81:416-4173; and Imboden & Stobo (1985) J. Exp. Med., 161:446-456. TCR also activates a tyrosine kinase (Samelson et al . (1986) Cell, 46:1083-1090; Patel et al . (1987) J. Biol. Chem., 262:5831-5838; and Chai et al. (1989) J. Biol. Chem., 264:10836-10842, where the zeta chain is one of the substrates of the kinase pathway (Baniyash et al . (1988) J. Biol. Chem., 263:18225-18230; Samelson et al. (1986) supra) . Fyn, a member of the src family of tyrosine kinases, is reported to coprecipitate with the CD3 complex, making it an excellent candidate for a TCR-activated kinase (Samelson et al. (1990) Proc. Natl. Acad. Sci. USA, 87:4358-4362). In addition, a tyrosine kinase unrelated to fyn has been shown to interact with the cytoplasmic domain of zeta (Chan et al . (1991) Proc. Natl. Acad. Sci. USA, 88:9166-9170).

Letourner & Klausner (1991) Proc. Natl. Acad. Sci. USA 88: 8905-8909 describe activation of T cells using a chimeric receptor consisting of the extracellular domain of the chain of the human interleukin 2 receptor (Tac) and the cytoplasmic domain of either ζ or y . Gross et al . (1989) Proc. Natl. Acad. Sci. USA, 86: 10024-10028 describe activation of T cells using chimeric receptors in which the MHC-restricted antigen-binding domains of the T cell receptor and β chains were replaced by the antigen-binding domain of an antibody. Romeo & Seed (1991) Cell, 64: 1037-1046 describe activation of T cells via chimeric receptors in which the extracellular portion of CD4 is fused to the transmembrane and intracellular portions of y, ζ and η subunits. Letourner & Klausner (1992) describe activation of T cells by a chimeric receptor consisting of the extracellular domain of the IL-2 receptor and the cytoplasmic tail of CD3 epsilon (Science 255:79-82).

Based on the structural similarities between the immunoglobulin (Ig) chains of antibodies and the alpha (α) and beta (β) T cell receptor chains (TJ , chimeric Ig-T_i molecules in which the V domains of the Ig heavy (V_H) and light (V_L) chains are combined with the C regions of α and T β chains have been described (Gross et al . (1989) Proc. Natl. Acad. Sci. USA, 86:1002-10028). The role of the T_x chains is to bind antigen presented in the context of MHC. The _± heterodimer does not possess innate signalling capacity, but transmits the antigen binding event to the CD3/zeta chains present in the TCR complex. Expression of a functional antigen binding domain required co-introduction of both V_H-T_X and \[ -TJ chimeric molecules. The chimeras have been demonstrated to act as functional receptors by the ability to activate T cell effector function in response to crosslinking by the appropriate hapten or anti-idiotypic antibody (Becker et al . (1989) Cell, 58:911 and Gross et al . (1989) Proc. Natl. Acad. Sci. USA, 86:10024). However, like the native T_x chains, the V_H-T_X and V_h-T_L chains do not possess innate signalling capacity, but act via the CD3/zeta complex.

It has been speculated that antigen-specific cytolytic immune cells might have a significant role in the modulation of human diseases in vivo, including cancer. More recently, adoptive T cell immunotherapy for cancer has shown promise in the clinics. Autologous tumor-infiltrating lymphocytes (TIL's) from melanoma patients were expanded ex vivo and reinfused into the patients. Nine of 41 patients showed partial or complete remission (Schwartzentruber et al . (1994) J. Clin. Oncol., 12:1475-83). A statistically significant correlation between greater autologous tumor lysis by the reinfused TIL's and patient responsiveness was demonstrated in the study. In further clinical studies, autologous TIL's were re-infused into patients at doses of up to 3xl0^u cells, twice weekly for 3 weeks, without significant toxicity but with limited efficacy, most likely due to the low number of tumor-specific T cells. The responses with TIL therapies have been limited, however, to a few tumor types.

A significant drawback of all of those T cell adoptive immunotherapies is the prolonged culture time necessary to generate antigen-specific therapeutically relevant numbers of cells. An alternative approach is the genetic modification of patient T cells to express a chimeric receptor conferring the ability of MHC independent lysis of the target cell. HLA-unrestricted chimeric T cell receptors can redirect the antigenic-specificity of T cell populations to recognize antigens of choice. On binding to tumor antigen, the chimeric receptors can initiate T cell activation, resulting in induction of effector functions including cytolysis of the tumor cell. In relation to human colon cancer, an antigen wtucn nas been studied to a great degree is the carcinoembryonic antigen (CEA) . Another antigen of interest is sialylated Tn recognized by, for example, the TAG 72.3 antibody. There are shortcomings associated with the clinical use of these two antigenic systems, including the secretion of these antigens, their expression on many normal cells, and cell-to-cell heterogeneity in expression.

Monoclonal antibody A33 detects a heat-stable, protease-stable, neuraminidase-resistant and periodate-sensitive epitope present on a high molecular weight glycoprotein which does not appear to be related to blood group antigens expressed on colon cancer cells. The antigen detected by the A33 antibody (hereinafter "A33 antigen" or "A33") is restricted to normal colon epithelium in colon cancer and is not detected in a wide range of other normal tissues. Also, the A33 antigen generally is not shed into the circulation. Welt et al . (1990) J. Clin. Oncol., 8:1894-1905.

The A33 antigen has a molecular weight on SDS-PAGE of 43,000 under non-reducing conditions. On the other hand, the protein displayed a molecular weight of approximately 180,000 under native conditions on both size exclusion chomatography and native PAGE. Hence, the molecule may form a homotetramer . Catimel et al . (1996) J. Biol. Chem., 271:25664-25670.

The A33 antigen is a membrane protein which, as indicated hereinabove neither is secreted nor shed at detectable levels into the extracellular tissue spaces or the serum of cancer patients, unlike the mucin-type glycoconjugates of colorectal cancers that are found at high levels in secretions and serum.

The large and small intestinal mucosa are the principal sites of A33 expression. The A33 antigen appears to be specific for tumors of the gastrointestinal tract. For example, A33 was found in 95% of primary and metastatic colorectal cancers with uniform expression through the tumors in most cases. A33 also was found in a certain portion of gastric cancers. Other cancers of the gastrointestinal tract can express A33 but a much lower level and frequency. Other epithelial cancers, sarcomas, neuroectodermal tumors and lymphoid neoplasms generally are A33-negative. Thus, A33 is not a cancer-specific molecule, however, the molecule is limited to specific organs and a large number of gastrointestinal cancers express A33. Garin-Chesa et al. (1996) Int. J. Oncol., 9:465-471.

The A33 antigen has been cloned and the complete amino acid sequence has been deduced. The A33 antigen does not have any overall sequence similarity with known proteins found and cataloged in available databases. The A33 protein has three distinct structural domains, an extracellular region of 213 amino acids, containing two putative immunoglobulin-like domains, a single hydrophobic transmembrane domain and a highly polar intracellular tail containing 4 consecutive cysteine residues. Heath et al . (1997) Proc. Natl. Acad. Sci. USA, 94:469-474.

Antibodies to A33 have been tested as therapeutic reagents for detection and treatment goals. A murine A33 mAb has been tested through phase I and II therapy trials as iodine labeled forms and was found to have anti-tαmor effects without bowel toxicity. Welt et al . (1994) J. Clin. Oncol., 12:1561-1571; Welt et al. (1996) J. Clin. Oncol., 14:1787-1797

King et al . (1995) (Br. J. Cancer, 72:1264-1372) produced a humanized A33 antibody for pre-clinical evaluation. The antibody and fragments thereof were labelled with yttrium. Essentially, the variable regions of A33 responsible for antigen binding were subcloned into the frameworks for heavy and light chains of human antibody. The humanized antibody was secreted by myeloma cells. The antibody localized very efficiently to human colon tumor zenografts in nude mice.

SUMMARY OF THE INVENTION

The triggering of signal transduction leading to cytotoxic function by different cells of the immune system can be initiated by chimeric receptors with antibody-type specificity. The chimeric receptors bypass the requirement for matching at the MHC locus between target cell (i.e. viral-infected, tumor cell etc.) and effector cell (i.e., T cell, granulocyte, mast cell, monocyte, macrophage, natural killer cell etc.). Intracellular signal transduction or cellular activation is achieved by employing chimeric proteins having a cytoplasmic region associated with transduction of a signal and activation of a secondary messenger system, frequently involving a kinase, and a non-MHC restricted extracellular region capable of binding to a specific ligand and transmitting to the cytoplasmic region the formation of a binding complex. Particularly, cytoplasmic sequences of the zeta, eta, delta, gamma and epsilon chains of TCR and the gamma chain of Fc.RI, a tyrosine kinase or other downstream signalling molecule are joined to a molecule other than the natural extracellular region by a transmembrane domain. In such a manner, cells capable of expressing the chimeric protein can be activated by contact with the ligand, as contrasted with the normal mode of activation for the cytoplasmic portion. For example, the extracellular domain can comprise a portion or derivative of an antibody which binds to the antigen recognized by the A33 mAb. The antibody can be polyclonal or monoclonal. A preferred derivative is a single chain antibody which binds to the antigen recognized by the A33 mAb.

DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagrammatic depiction of the structure of single-chain antibodies used in the chimeric receptors of the invention as compared to the structure of native monoclonal antibodies .

Figure 2 depicts the OperVector carrying humanized A33 sequences .

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel DNA sequences, such as DNA sequences as parts of expression cassettes and vectors, as well as presence thereof in cells are provided, where the novel sequences comprise at least three domains which do not naturally exist together: (1) a cytoplasmic domain, which normally trarsduces a signal resulting in activation of a messenger system, (2) a transmembrane domain, which traverses the cell membrane and (3) a non-MHC restricted extracellular receptor domain which serves to bind to a ligand and initiates a signal to the cytoplasmic domain, resulting in activation of the messenger system. A preferred extracellular domain is an antibody or antigen-binding portion thereof, particularly one that binds to the antigen recognized by the A33 mAb.

The cytoplasmic domain may be derived from a protein which is known to activate various messenger systems. The protein from which the cytoplasmic domain is derived need not have ligand binding capability by itself, it being sufficient that such protein may associate with another protein providing such capability. Cytoplasmic regions of interest include the zeta chain of the T cell receptor, the eta chain, which differs from the zeta chain only in its most C-terminal exon as a result of alternative splicing of the zeta m NA, the delta, gamma and epsilon chains of the T cell receptor (CD3 chains) and the gamma subunit of the Fc_eRI receptor, as well as other cytoplasmic regions which are capable of transmitting a signal as a result of interacting with other proteins capable of binding to a ligand, such as, kinases . See U.S. Patent No. 5,359,046, incorporated herein by reference.

A number of cytoplasmic regions or functional fragments or mutants thereof may be employed, generally ranging from about 10 to 500 amino acids, where the entire naturally occurring cytoplasmic region may be employed or only an active portion thereof. Reth (supra) identified motifs within signalling molecules which suggest evolutionary relatedness and duplication events. The cytoplasmic regions of particular interest are those which may be involved with one or more secondary messenger pathways, particular pathways involved with a protein kinase.

Pathways of interest include the phosphatidylinositol-specific phospholipase involved pathway, which is normally involved with hydrolysis of phosphatidylinositol-4, 5-bisphosphate, which results in production of the secondary messengers inositol-1, 4, 5-trisphosphate and diacylglycerol . Another pathway is the calcium-mediated pathway, which may be as a result of direct or indirect activation by the cytoplasmic portion of the chimeric protein. Also, by itself or in combination with another pathway, the kinase pathway may be involved through, for example, phosphorylation of the cytoplasmic portion of the chimeric protein. One or more amino acid side chains, particularly tyrosines, may be phosphorylated. There is some evidence that fyn, a member of the src family of tyrosine kinases, may be involved with the zeta chain.

While usually the entire cytoplasmic region will be employed, in many cases, it will not be necessary to use the entire chain. To the extent that a truncated portion may find use, such truncated portion may be used in place of the intact chain.

Suitable cytoplasmic domains arise also from other molecules that have a signalling role in eliciting a response by the host cell. For example, tyrosine kinases, such as ZAP-70, syk and members of the Janus kinase family, and ancillary molecules that have less than a direct role in signaling, such as CD2 and CD28, or functional portions thereof, can be employed as the cytoplasmic domain of a receptor of interest. In addition, the cytoplasmic portions of growth factor receptors may be used as cytoplasmic domains of chimeric proteins. See PCT publications W096/23814, and W096/23881, incorporated herein by reference.

Generally a desirable response by the host cell is proliferation or expression of differentiated functions. Manifestation of a desirable phenotype, such as cytotoxicity or the expression of cytokines, is obtained and which can be directed to a specific target, such as a cancer cell, by use of a chimeric receptor of interest.

The transmembrane domain may be the domain of the protein contributing the cytoplasmic portion, the domain of the protein contributing the extracellular portion, or a domain associated with a totally different protein. Chimeric receptors of the invention, in which the transmembrane domain is replaced with that of a related receptor, or, replaced with that of an unrelated receptor, may exhibit qualitative and/or quantitative differences in signal transduction function from receptors in which the transmembrane domain is retained. Thus, functional differences in signal transduction may be dependent not only upon the origin of the cytoplasmic domain employed, but also on the derivation of the transmembrane domain. Preferably, a single pass transmembrane will be used. However, it may be convenient to have the transmembrane domain naturally associated with one or the other of the extracellular domain or intracellular domain, in which case the transmembrane domain may not be single pass. Nevertheless, the two domains originating from the same native protein can be advantageous for maximal function, such as conducting a signal.

In some cases it will be desirable to employ the transmembrane domain of the zeta, eta or Fc_sRI gamma chains which contain a cysteine residue capable of disulfide bonding, so that the resulting chimeric protein will be able to form disulfide-linked dimers with itself, or with unmodified versions of the zeta, eta or Fc_eRI gamma chains, or related proteins. In some instances, the transmembrane domain will be selected to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein to minimize interactions with other members of the receptor complex. In other cases it will be desirable to employ the transmembrane domain of zeta, eta Fc_βRI gamma, or CD3-gamma, CD3-delta or CD3-epsilon, to retain physical association with other members of the receptor complex.

The extracellular domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and/or signal transduction. The extracellular domain may be part of a protein which is monomeric, homodimeric, heterodimeric or associated with a larger number of proteins in a non-covalent complex.

Of particular interest are antibodies and antigen-binding portions thereof. In particular, the extracellular domain may consist of an Ig heavy chain which may in turn be covalently associated with Ig light chain by virtue of the presence of CHI and hinge regions or may become associated covalently with other Ig heavy/light chain complexes by virtue of the presence of hinge, CH2 and CH3 domains. In the latter case, the heavy/light chain complex that associates with the chimeric construct may constitute an antibody with a specificity distinct from the antibody specificity of the chimeric construct. Thus, a new antigen finding specificity may occur. Depending on the function of the antibody, the desired structure and the signal transduction, the entire chain may be used or a truncated chain may be used, where all or a part of the CHI, CH2, or CH3 domains may be removed or all or part of the hinge region may be removed. Single-chain antibodies (scAb's) are desirable tor ease of manipulation. As depicted in Figure 1, the most widely known scAb is one where the variable regions of the heavy and light chain are tethered by a molecular linker so that the tripartite molecule folds spontaneously to form the relevant antigen-binding domain. Other forms of single-chain antibodies are contemplated to fall within the scope of the invention so long as antigen binding ability is retained.

scAb's are desirable because a gene thereof can be subcloned in the proper operative relationship with nucleic acids encoding the signal sequence, transmembrane domain and cytoplasmic domains to yield a gene encoding a chimeric molecule of interest.

Antibodies to the antigen recognized by the A33 mAb are preferred, with monoclonal antibodies being more preferred. A scAb directed to the antigen recognized by the A33 mAb is a desired antibody derivative in the practice of the instant invention.

Naturally occurring cell surface receptors or secreted proteins which bind to the antigen recognized by the A33 mAb also may be employed, analogous to the ligands which bind to other cancer-associated antigens. For example human Heregulin (Hrg) , a protein similar in structure to Epidermal Growth Factor (EGF) , has been identified as a ligand for the receptor Her₂ which is expressed on the surface of breast carcinoma cells and ovarian carcinoma calls (Holmes et al . (1992) Science, 256:1205-1210). The murine equivalent is the "Neu" protein (Bargman et al . (1986) Nature, 319:226-230). The extracellular domain of Hrg could be joined to the zeta transmembrane and cytoplasmic domains to form a chimeric construct of the invention to direct T cells to kill breast carcinoma cells.

In addition, multimeric extracellular domains can be used. For example, the extracellular domain may consist of an A33 scAb joined to another cell surface-binding extracellular domain, for example, another scAb which binds another cancer- associated antigen. See, for example, PCT publication W096/24671, incorporated herein by reference.

Where a receptor is a molecular complex of proteins, where only one chain has the major role of binding to the ligand, it usually will be desirable to use solely the extracellular portion of the ligand binding protein. Where the extracellular portion may complex with other extracellular portions of other proteins or form covalent bonding through disulfide linkages, one also may provide for the formation of such dimeric extracellular region. Also, where the entire extracellular region is not required, truncated portions thereof may be employed, where such truncated portion is functional. For example, when the extracellular region of CD4 is employed, one may use only those sequences required for binding of gpl20, the HIV envelope glycoprotein. In the case in which Ig is used as the extracellular region, one may simply use the antigen binding regions of the antibody molecule and dispense with the constant regions of the molecule (for example, the F_c region consisting of the CH2 and CH3 domains) .

In some instances, a few amino acids at the joining region of the natural protein may be deleted, usually not more than 10, more usually not more than 5. Also, one may wish to introduce a small number of amino acids at the borders, usually not more than 10, more usually not more than 5. The deletion or insertion of amino acids usually will be as a result of the needs of the construction, providing for convenient restriction sites, ease of manipulation, improvement in levels of expression or the like. In addition, one may wish to substitute one or more amino acids with a different amino acid for similar reasons, usually not substituting more than about five amino acids in any one domain. The cytoplasmic domain, as already indicated, generally will be from about 10 to 500 amino acids, depending upon the particular domain employed. The transmembrane domain generally will have from about 25 to 50 amino acids, while the extracellular domain generally will have from about 10 to 500 amino acids.

Normally, the signal sequence at the 5' terminus of the open reading frame (ORF) which directs the chimeric protein to the surface membrane will be the signal sequence of the extracellular domain. However, in some instances, one may wish to exchange that sequence for a different signal sequence. However, since the signal sequence will be removed from the protein, being processed while being directed to the surface membrane, the particular signal sequence normally will not be critical to the subject invention. Similarly, associated with the signal sequence will be a naturally occurring cleavage site, which normally also will be the naturally occurring cleavage site associated with the signal sequence or the extracellular domain.

It is within the province of the instant invention to construct receptors carrying plural extracellular and/or cytoplasmic domains . The plural domains may have the same specificity or function, or may have different specificity or function. Thus, a receptor may comprise a domain obtained from CD28, a domain obtained from a jak kinase, a domain obtained from zeta or any combination thereof .

In the embodiments provided herein, various following chimeric constructs containing as the extracellular domain an antibody portion that binds the A33 were produced.

The instant invention is particularly directed to single-chain antibody (scAb) chimeric receptors in which a scAb functions as the extracellular domain of the chimeric receptor although other antibody portions can be found at the extracellular domain of a chimera of interest. In contrast to previously described Ig-T₁ chimeras (Becker et al . , Gross et al . , supra), the scAb chimeric receptors function by bypassing the normal antigen recognition component of the T cell receptor complex and transducing the signal generated on antigen-receptor binding directly via the cytoplasmic domain of the molecule.

A range of scAb chimeric receptors, for example, anti-A33 immunoglobulin-zeta (Ig-ζ) chimeric receptors can be configured.

For example, the IgG heavy chain, or a portion thereof, comprising the VH and any or all of the CH Ig domains is fused to the cytoplasmic domain of the zeta chain via a transmembrane domain. If the VH domain alone is sufficient to confer antigen- specificity (so-called "single-domain antibodies"), homodimer formation of the Ig-ζ chimera is expected to be functionally bivalent with regard to antigen binding sites. Because it is likely that both the VH domain and the VL domain are necessary to generate a fully active antigen binding site, both the IgH-ζ molecule and the full-length IgL chain are introduced into cells to generate an active antigen-binding site. Dimer formation resulting from the intermolecular Fc/hinge disulfide bonds results in the assembly of Ig-ζ receptors with extracellular domains resembling those of IgG antibodies. Derivatives of the Ig-ζ chimeric receptor include those in which only portions of the heavy chain are employed in the fusion. For example, the VH domain (and the CHI domain) of the heavy chain can be retained in the extracellular domain of the Ig-ζ chimera (VH-ζ) . Co-introduction of a similar chimera in which the V and C domains of the corresponding light chain replace those of the Ig heavy chain (VL-ζ) then can reconstitute a functional antigen binding site.

Because association of both the heavy and light V domains generally is required to generate a functional antigen binding site of high affinity, to generate an Ig chimeric receptor with the potential to bind antigen, a total of two molecules typically will need to be introduced into a host cell. Therefore, an alternative and preferred strategy is to introduce a single molecule bearing a functional antigen binding site. That avoids the technical difficulties that may attend the introduction of more than one gene construct into host cells. The "single-chain antibody" (scAb) generally is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule.

Single-chain antibody variable fragments (ScAb) in which the C-terminus of one variable domain (VH or VL) is tethered to the N-terminus of the other VL or VH, respectively, (see Figure 1) via, for example, a 15 to 25 amino acid peptide or linker, have been developed without significantly disrupting antigen binding or specificity of the binding (Bedzyk et al . (1990) J. Biol. Chem., 265:18615; Chaudhary et al . (1990) Proc. Natl. Acad. Sci., 87:9491). The Fv's lack the constant regions (Fc) present in the heavy and light chains of the native antibody. In the methods of the instant invention, the extracellular domain of the single-chain Ig chimeras consists of the Fv fragment which may be fused to all or a portion of the constant domains of the heavy chain and the resulting extracellular domain is joined to the cytoplasmic domain of, for example, zeta, via an appropriate transmembrane domain that will permit expression in the host cell, e.g. , zeta or CD4.

The resulting chimeric molecules differ from the ScAb's in that on binding of A33 Ag the receptors initiate signal transduction via the cytoplasmic domain. In contrast, free antibodies and ScAb's are not cell-associated and generally do not transduce a signal on A33 Ag binding to activate a secondary messenger pathway. The ligand binding domain of the scAb chimeric receptor may be of two types depending on the relative order of the VH and VL domains: VH-l-VL or VL-l-VH (where "1" represents the linker) (See Figure 1) .

The chimeric construct, which encodes the chimeric protein according to the instant invention, will be prepared in conventional ways. Since, for the most part, natural sequences may be employed, the natural genes may be isolated and manipulated, as appropriate, so as to allow for the proper joining of the various domains. Thus, one may prepare the truncated portion of the sequence by employing the polymerase chain reaction (PCR) using appropriate primers which result in deletion of the undesired portions of the gene. Alternatively, one may use primer repair, where the sequence of interest may be cloned in an appropriate host. In either case, primers may be employed which result in termini, which allow for annealing of the sequences to result in the desired open reading frame encoding the chimeric protein. Thus, the sequences may be selected to provide for restriction sites which are blunt-ended or have complementary overlaps. During ligation, it is desirable that hybridization and ligation do not recreate either of the original restriction sites.

If desired, the extracellular domain also may include the transcriptional initiation region which will allow for expression in the target host. Alternatively, one may wish to provide for a different transcriptional initiation region, which may allow for constitutive or inducible expression, depending on the target host the purpose for the introduction of the subject chimeric protein into such host, the level of expression desired, the nature of the target host and the like. Thus, one may provide for expression on differentiation or maturation of the target host, activation of the target host or the like.

A wide variety of promoters has been described in the literature, which are constitutive or inducible, where induction may be associated with a specific cell type or a specific level of maturation. Alternatively, a number of viral promoters are known which also may find use. Promoters of interest include the β-actin promoter, SV40 early and late promoters, immunoglobulin promoter, human cytomegalovirus promoter and the Friend spleen focus-forming virus promoter. The promoters may or may not be associated with enhancers, where the enhancers may be naturally associated with the particular promoter or associated with a different promoter.

The sequence of the open reading frame may be obtained from genomic DNA, cDNA, be synthesized or combinations thereof. Depending on the size of the genomic DNA and the number of introns, one may wish to use cDNA or a combination thereof. In many instances, it is found that introns stabilize the mRNA. Also, one may provide for non-coding regions which stabilize the mRNA.

A termination region will be provided 3 ' to the cytoplasmic domain, where the termination region may be naturally associated with the cytoplasmic domain or may be derived from a different source. For the most part, the termination regions are not critical and a wide variety of termination regions may be employed without adversely affecting expression.

The various manipulations may be carried out in vitro or may be introduced into vectors for cloning in an appropriate host, e.g., E. coli. Thus, after each manipulation, the resulting construct from joining of the DNA sequences may be cloned, the vector isolated and the sequence screened to insure that the sequence encodes the desired chimeric protein. The sequence may be screened by restriction analysis, sequencing or the like. Prior to cloning, the sequence may be amplified using PCR and appropriate primers so as to provide for an ample supply of the desired open reading frame while reducing the amount of contaminating DNA fragments which may have substantial homology to the portions of the entire open reading frame.

The target cell may be transformed with the chimeric construct in any convenient manner. Techniques include calcium phosphate-precipitated DNA transformation, electroporation, protoplast fusion, biolistics, using DNA-coated particles, transfection and infection, where the chimeric construct is introduced into an appropriate virus particularly a non-replicative form of the virus or the like. Target host cells may be transduced in vivo or in vitro.

Once the target host has been transformed, usually, integration will result. However, by appropriate choice of vectors, one may provide for episomal maintenance. A large number of vectors are known which are based on viruses where the copy number of the virus maintained in the cell is low enougn to maintain the viability of the cell. Illustrative vectors include SV40, EBV, adenovirus and BPV.

The constructs may be designed so as to avoid interaction with other surface membrane proteins native to the target host. Thus, for the most part, one will avoid the chimeric protein binding to other proteins present in the surface membrane. To achieve that goal, one may select for a transmembrane domain which is known not to bind to other transmembrane domains, one may modify specific amino acids, e.g. substitute for a cysteine or the like. Alternatively, host cells that do not contain the suspect confounding molecule can be used, for example, a cell that does not express functional T cell receptor.

Once one has established that the transformed host is capable of expressing the chimeric protein as a surface membrane protein in accordance with the desired regulation and at a desired level, one then may determine whether the transmembrane protein is functional in the host to provide for the desired signal induction. Since the effect of signal induction of the particular cytoplasmic domain will be known, one may use established methodology for determining induction to verify the functional capability of the chimeric protein.

For example, TCR binding results in the induction of CD69 expression. Thus, one would expect with a chimeric protein having a zeta cytoplasmic domain, where the host cell is known to express CD69 on activation, one could contact the transformed cell with the cognate ligand and then assay for expression of CD69. An artisan can determine whether ancillary signals are required from other proteins in conjunction with the particular cytoplasmic domain. Thus, the failure to provide transduction of the signal can be attributed solely to the inoperability of the chimeric protein in the particular target host.

A wide variety of target hosts may be employed, normally cells from vertebrates, more particularly, mammals, desirably domestic animals or primates, particularly humans. The subject chimeric constructs may be used for the investigation of particular pathways controlled by signal transduction, for initiating cellular responses employing different ligands, for example, for inducing activation of a particular subset of lymphocytes, where the lymphocytes may be activated by particular surface markers of cells such as neoplastic cells, virally infected cells or other diseased cells, which provide for specific surface membrane proteins which may be distinguished from the surface membrane proteins on normal cells.

The cells may be further modified so that expression cassettes may be introduced, where activation of the transformed cell will result in secretion of a particular product. In such a manner, one may provide for directed delivery of specific agents such as interferons, TNF's, perforins, naturally occurring cytotoxic agents or the like, where the level of secretion can be enhanced greatly over the natural occurring secretion. Furthermore, the cells may be directed specifically to the site using injection, catheters or the like, so as to provide for localization of the response.

The subject invention may find application with cytotoxic lymphocytes (CTL) , natural killer cells (NK) , TIL's or other cells which are capable of killing target cells when activated. Thus, diseased cells, such as cells infected with HIV, HTLV-I, HTLV-II, cytomegalovirus, hepatitis B or C virus, mycobacterium avium, etc., or neoplastic cells, where the diseased cells have a surface marker associated with the diseased state may be made specific targets of the cytotoxic cells.

Alternatively, other effector functions, such as lymphokine release may yield the desired end result.

By providing a receptor extracellular domain, e.g., CD4, which binds to a surface marker of the pathogen or neoplastic condition, e.g., gpl20 of HIV, the cells may serve as therapeutic agents. By modifying the cells further to prevent the expression or translocation of functional class I and/or class II MHC antigens, the cells will be able to avoid recognition by the host immune system as foreign and therefore can be employed therapeutically in any individual regardless of genetic background. Alternatively, one may isolate and transfect host cells with the subject constructs and then return the transfected host cells to the host.

Other applications include transformation of host cells from a given individual with retroviral vector constructs directing the synthesis of the chimeric construct. By transformation of such cells and reintroduction into the patient one may achieve autologous therapeutic applications.

In addition, suitable host cells include hematopoietic stem cells, which develop into effector cells with both myeloid and lymphoid phenotype including granulocytes, mast cells, basophils, macrophages, natural killer (NK) cells and T and B lymphocytes. Introduction of the chimeric constructs of the invention into hematopoietic stem cells thus permits the induction of, for example, cytotoxicity in the various cell types derived from hematopoietic stem cells providing a continued source of cytotoxic effector cells to fight various diseases.

The zeta subunit of the T cell receptor is associated not only with T cells but is present in other cytotoxic cells derived from hematopoietic stem cells. Three subunits, zeta, eta and the gamma chain of the Fc_e receptor, associate to form homodimers as well as heterodimers in different cell types derived from stem cells. The high level of homology between zeta, eta and the gamma chain of the Fc_β receptor, and their association together in different cell types, suggest that a chimeric receptor consisting of an extracellular binding domain coupled to a zeta, eta or gamma homodimer would be able to activate cytotoxicity in various cell types derived from hematopoietic stem cells.

For example, zeta and eta form both homodimers and heterodimers in T cells (Clayton et al . (1991) Proc. Natl. Acad. Sci. USA, 88:5202) and are activated by engagement of the cell receptor complex; zeta and the gamma chain of the Fc_e receptor form homodimers and heterodimers in NK cells and function to activate cytotoxic pathways initiated by engagement of Fc receptors (Lanier et al . (1991) J. Immunol., 146:1571; the gamma chain forms homodimers expressed in monocytes and macrophages (Phillips et al . (1991) Eur. J. Immunol., 21:895), however because zeta will form heterodimers with gamma, it is able to couple to the intracellular machinery in the monocytic lineage; and zeta and the gamma chain are used by IgE receptors (Fc_eRI) in mast cells and basophils (Letourneur et al . (1991) J. Immunol., 147:2652) for signalling cells to initiate cytotoxic function.

Therefore, because stem cells transplanted into a subject via, for example, bone marrow transplantation, exist for a lifetime, a continual source of effector cells is produced by introduction of the chimeric receptors of the invention into hematopoietic stem cells to combat virally infected cells, cells expressing tumor antigens or effectcr cells responsible for autoimmune disorders.

Additionally, introduction of the chimeric receptors into stem cells with subsequent expression by both myeloid and lymphoid cytotoxic cells may have certain advantages in patients with multiple or congenital carcinoma expressing A33 Ag.

The chimeric receptor constructs of the invention can be introduced into hematopoietic stem cells followed by bone marrow transplantation to permit expression of the chimeric receptors in all lineages derived from the hematopoietic system. High titer retroviral producer lines are used to transduce the chimeric receptor constructs, for example α-A33/ζ, into both murine and human T cells and human hematopoietic stem cells through the process of retroviral-mediated gene transfer as described by Lusky et al. in Blood, 80:396 (1992).

For transduction of hematopoietic stem cells, the bone marrow is harvested using standard medical procedures and then processed by enriching for hematopoietic stem cells expressing the CD34 antigen as described by Andrews et al . in J. Exp. Med., 169:1721 (1989). The cells then are incubated with the retroviral supernatants in the presence of hematopoietic growth factors , such as stem cell factor and IL-6.

The bone marrow transplant can be autologous or allogeneic, and depending on the disease to be treated, different types of conditioning regimens are used (see, Surgical Clinics of North America (1986) 66:589).

The recipient of the genetically modified stem cells can be treated with total body irradiation, chemotherapy using cyclophosphamide or both to prevent the rejection of the transplanted bone marrow. In the case of immunocompromised patients, no pretransplant therapy may be required because there is no malignant cell population to eradicate and the patients cannot reject the infused marrow.

In addition to the gene encoding the chimeric receptor, additional genes may be included in the retroviral construct . The included genes can encompass the thymidine kinase gene (Borrelli et al. (1988) Proc. Natl. Acad. Sci. USA, 85:7572) which acts as a suicide gene for the marked cells if the patient is exposed to gancyclovir. Thus, if the percentage of marked cells is too high, gancyclovir may be administered to reduce the percentage of cells expressing the chimeric receptors.

In addition, if the percentage of marked cells needs to be increased, a multi-drug resistance gene can be included (Sorrentino et al . (1992) Science, 257:99) which functions as a preferential survival gene for the marked cells in the patients if the patient is administered a dose of a chemotherapeutic agent, such as taxol . Therefore, the percentage of marked cells in the patients can be titrated to obtain the maximum therapeutic benefit from the expression of the universal receptor molecules by different cytotoxic cells of the immune system of the patient.

The following examples are by way of illustration and not by way of limitation.

EXAMPLE 1

Construction of CD8/ζ chimera

The polymerase chain reaction, PCR (Mullis et al . (1986) "Cold Spring Harbor Symposium on Quantitative Biology", NY, 263-273) was used to amplify the extracellular and transmembrane portion of CD8α (residues 1-187) from pSV7d-CD8α and the cytoplasmic portion of the human ζ chain (residues 31-142 from pGEM3ζ). Some DNA's were obtained from (Littman et al . (1985) Cell, 40:237-246; CD8) and (Weissman et al . (1988) Proc. Natl. Acad. Sci., 85:9709-9713; ζ). Plasmids pSV7d-CD8α and pGEM3zζ were provided by Drs. Dan Littman and Julie Turner (Univ. of CA at SF) and Drs. R.D. Klausner and A.M. Weissman (NIH) , respectively. Primers encoding the 3' sequences of the CD8 fragment and the 5' sequences of the zeta fragment (ζ) were designed to overlap such that annealing of the two products yielded a hybrid template. From that template, the chimera was amplified using external primers containing Xbal and BamHI cloning sites. The CD8/ζ chimera was subcloned into pTfneo (Ohashi et al. (1985) Nature, 316:606-609) and se uenced via the Sanger dideoxynucleotide technique (Sanger et al . (1977) Proc. Natl. Acad. Sci. USA, 74:5463-5467). Antibodies

C305 and Leu4 mAb's recognize the Jurkat T & chain and an extracellular determinant of CD3e, respectively. 0KT8, acquired from the ATCC, recognizes an extracellular epitope of CD8. The anti-ζ rabbit antiserum, #387, raised against a peptide comprising amino acids 132-144 of the murine ζ sequence (Orloff et al . (1989) J. Biol. Chem., 264:14812-14817), was provided by Drs. R.D. Klausner, A.M. Weissman and L.E. Samelson. The anti-phosphotyrosine mAb, 4G10, was a generous gift of Drs. D. Morrison, B. Druker, and T. Roberts. W6/32 recognizes an invariant determinant expressed on human HLA class I antigens. Leu23, reactive with CD69, was obtained from Becton-Dickinson Monoclonal Center (Milpitas, CA) . MOPC 195, an IgG2a, (Litton Bionetics, Kensington, MD) was used as a control mAb in FACS analysis. Ascitic fluids of mAb were used at a final dilution of 1:1000 (a saturating concentration) in all experiments unless otherwise stated.

Cell lines and Transfections

The human leukemic T cell line Jurkat and derivative thereof, J.RT3-T3.5, were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) , glutamine, penicillin and streptomycin (Irvin Scientific) . Chimera-transfected clones were passaged in the above medium with the addition of Geneticin

(GIBCO, Grand Island, NY) at 2 mg/ml. Electroporation of pTfneo-CD8/ζ into Jurkat and J.RT3-T3.5 was performed in a Bio-Rad

Gene Pulser using a voltage of 250 V and a capacitance of 960 μF with 20 μg of plasmid per 10⁷ cells. After transfection, cells were grown for two days in RPMI before plating out in

Geneticin-containing medium. Clones were obtained by limiting dilutions and screened for TCR and CD8/ζ expression by flow cytometry (see below) . The Jurkat CD8 clone, transfected with the wild-type CD8 protein, was provided by Drs. Julia Turner and Dan

Littman.

Flow Cytometry Approximately 1 x 10⁶ cells/condition were stained with saturating concentrations of antibody, then incubated with fluorescein-conjugated goat anti-mouse Ab prior to analysis in a FACScan (Beckton Dickinson) as previously described (Weiss & Stobo, supra) . Cells analyzed for CD69 expression were stained directly with fluorescein-conjugated Leu 23 (anti-CD69 mAb) or MOPC 195 (control mAb) . [Ca^*3]₁ Measurement by Fluorimetry

Calcium sensitive fluorescence was monitored as previously described (Goldsmith & Weiss (1987) Proc. Natl. Acad. Sci. USA, 84:6879-6883). Cells were stimulated with soluble mAb C305 and 0KT8 at saturating concentrations (1:1000 dilution of ascites) . Maximal fluorescence was determined after lysis of the cells with Triton X-100; minimum fluorescence was obtained after chelation of Ca^*2 with EGTA. [?a was determined using the equation [Ca^*2]^ (?_bserved -ξ_ιn )/ d _x -F_observed) , with K_d=250 nM as described (Grynkiewica et al . (1985) J. Biol. Chem., 260:3440-3448) .

Inositol Phosphate Measurement

Cells were loaded wich [³H]myo-inositol (Amersham) at 40 μCi/ml for 3 hr . in phosphate buffered saline, then cultured overnight in RPMI 1640 supplemented with 10% fetal bovine serum. Cells were stimulated for 15 min. with the indicated antibodies at 1:1000 dilution of ascites in the presence of 10 mM LiCl to inhibit dephosphorylation of IP_X. The extraction and quantitation of soluble inositol phosphates were as described (Imboden & Stobo (1985) J. Exp. Med., 161:446-456).

Surface lodinations

Cells were labeled with ^{1 5}I using the lactoperoxidase/glucose oxidase (Sigma) procedure as described (Weiss & Stobo (1984) J. Exp. Med., 160:1284-1299).

Immunoprecipitations

Cells were lysed at 2 x 10⁷ cells/200 ml in 1% NP40 (Nonidet P40) , 150 mM NaCl, and 10 mM Tris pH 7.8 in the presence of protease inhibitors, 1 mM PMSF, aprotinin and leupeptin. Lysis buffer for lysates to be analyzed for phosphotyrosine content was supplemented with phosphatase inhibitors as described (Desai et al. (1990) Nature, 348:66-69). Iodinated lysates were supplemented with 10 mM iodoacetamide to prevent postlysis disulfide bond formation. Digitonin lysis was performed in 1% digitonin, 150 mM NaCl, 10 mM Tris pH 7.8 and 0.12% Triton X-100. After 30 min. at 4°C, lysates were centrifuged for 10 min. at 14,000 rp , then precleared with fixed Staphylococcus aureus (Staph A; Calbiochem-Behring) . Alternatively, lysates of cells stimulated with antibody prior to lysis were precleared with Sepharose beads . The precleared lysates were incubated with protein A Sepharose CL-4B beads which had been prearmed with the immunoprecipitating antibody. Washed immunoprecipitates were resuspended in SDS sample buffer +/- 5% β-mercaptoethanol and boiled prior to electrophoresis on 11% polyacrylamide gels.

Stimulation of cells for assessment of phosphotyrosine content . Cells were stimulated in serum-free medium at

2 x 10⁷ cells/200 μl with antibodies at 1:250 dilution of ascites. After 2 min. at 37°C, the medium was aspirated and the cells lysed in 100 μl of NP40 lysis buffer. Lysates were precleared, then ultracentrifuged and samples resolved by SDS PAGE.

Immunoblots

Gels were equilibrated in transfer buffer (20 mM Tris base, 150 mM glycine and methanol) for 30 min. and transferred to nitrocellulose membranes in a Bio-Rad Western blotting apparatus run at 25 volts overnight. Membranes were blocked in TBST (10 mM Tris HC1 [pH 8], 150 mM NaCl and 0.05% Tween 20) plus 1.5% ovalbumin, then incubated with either mAb 4G10 or rabbit anti-ζ antiserum (#387) . The immunoblots were washed and incubated with a 1:7000 dilution of alkaline phosphatase-conjugated goat anti-mouse or goat anti-rabbit antibody. After 1-2 hours, the blots were washed and developed with nitroblue tetrazolium and 5-bromo-4-chloro-3 -indolyl phosphate substrates as per instructions ( Promega) .

L-2 Bioassay

For stimulation, cells were coated with the indicated antibodies at saturating concentrations (1:1000 dil . of ascites) for 30 min. at 4°C. After removal of unbound antibody, cells were spun onto 24-well tissue culture plates which had been precoated with rabbit anti-mouse Ig (Zymed Labs) and blocked with medium plus 10% FBS. Phorbol myristate acetate, PMA (Sigma), and ionomycin (Calbiochem) were added to final concentrations of 10 mg/ml and 1 mM, respectively. Cell-free supernatants were harvested after 20 hr . of culture and assessed for IL-2 content utilizing the IL-2-dependent CTLL-2.20 cell line in the MTT colorimetric assay as described (Mosmann (1983) J. Immunol. Meth. , 65:55-63).

RESULTS

Characterization of the CD8/ζ Chimera in T Cell Receptor-positive and Receptor-negative Jurkat Cells

The CD8/ζ chimeric construct described previously was transfected via electroporation into both the Jurkat human T cell leukemic line, yielding clone JCD8/ζ2, and a Jurkat-derived mutant, JRT3.T3.5 deficient in full length T_χβ chain transcripts and protein, yielding Jβ-CD8/ζl4. Though JRT3.T3.5 expresses normal levels of T_λα and the CD3 subunits, the deficiency in T β expression results in the absence of TCR expression on the cell surface (Ohashi et al . (1985) Nature, 316:606-609).

Transfection of the chimera into that cell enabled assessment of the ζ signalling phenotype without the complication of the additional TCR chains. Levels of surface expression of the chimera and TCR in stably transfected clones were quantitated by flow cytometry using mAb's which recognize either CD8 (OKT8) or the CD3e subunit of the TCR (Leu 4) . Fluorescence histograms of the clones which both express high levels of CD8/ ζ was observed; that cell was used as a control in all of the experiments.

The three clones express comparable levels of CD8 epitopes and T cell receptors with the exception of Jβ-CD8/zl4, which fails to express surface TCR. Thus the CD8/ζ chimera can be expressed on the cell surface in the absence of the TCR chains. To characterize the structure of the CD8/ζ chimeric protein, cells were surface radioiodinated, lysed in 1% NP40 and subjected to immunoprecipitation with 0KT8 or a normal rabbit antiserum raised against a cytoplasmic peptide sequence of murine ζ.

Under reducing conditions, antibodies against either CD8 or ζ precipitate a single protein of 34-35 kD from the chimera-transfected cell, while 0KT8 precipitates a 29 kD protein representing wild-type CD8 from Jurkat CD8. Although CD8 in its normal environment has an apparent molecular weight of 32-34 kD, (Snow and Terhorst (1983) J. Biol. Chem., 258:14675-14681), preliminary experiments comparing CD8 in Jurkat and a CD8-positive line, HPB.ALL, suggest that the reduction in size of CD8 observed results from a distinct pattern of glycosylation in the Jurkat host.

Under non-reducing conditions, a more complex pattern of proteins is seen in immunoprecipitates of both CD8 and the CD8/ ζ chimera. The complexity is characteristic of CD8 precipitates since homomultimers and heteromultimers have been previously observed (Snow & Terhorst, supra) . The two prominent species immunoprecipitated from JCD8/ζ2 migrating at approximately 70 and 100 kD are likely to represent homodimers and homotrimers of the chimera. As there are no cysteine residues for the formation of disulfide linkages with the ζ portion of the chimera, any disulfide bonds formed in the chimera must occur through CD8.

Therefore, any protein forming a heterodimer with CD8/ζ is likely to form one with the wild-type CD8 and thus should not account for any signalling events specifically attributable to the CD8/ζ chimera.

Non-covalent association of the chimera with endogenous

CD3 gamma <γ) , delta (δ) and epsilon (e) may complicate the interpretation of signals transduced by the chimera. To determine whether removal of the extracellular and transmembrane domains of ζ is sufficient to result in its expression independent of the CD3 chains, cells were surface iodinated and lysed in digitonin, a detergent known to preserve the integrity of the TCR complex.

Immunoprecipitates of the TCR in both Jurkat CD8 and the TCR-expressing chimera-transfectant JCD8/ζ2, show identical patterns characteristic of a CD3 (Leu 4) immunoprecipitate. Though TCR-associated ζ is not well iodinated, as its extracellular domain contains no tyrosine residues for labelling, ζ immunoblots of CD3 immunoprecipitates confirm the presence thereof under such lysis conditions. A small quantity of labelled CD3e is seen in the Leu 4 i munoprecipitate of the TCR deficient cell despite the fact that same mAb failed to stain the cell. The small amount of immunoprecipitated protein seen is likely due to radiolabelling of internal CD3e in a small number of permeabilized or non-viable cells during the labelling procedure.

More importantly, no CD3 chains are detectable in precipitates of the CD8/ζ chimera in either TCR-positive or TCR-negative cells, nor is any chimera apparent in the Leu 4 precipitate of JCD8/ζ 2. Intentional overexposure of the autoradiogram also failed to reveal TCR chains coprecipitating with the chimeras.

To further address the question of co-association of the chimera and TCR chains, the effect of antibody-induced down modulation of the TCR on chimera expression was assessed. Whereas overnight incubation of JCD8/ζ2 with saturating amounts of C305, a mAb against an epitope of the Jurkat T_t chain, resulted in internalization of 94% of the TCR, surface expression of the CD8/ζ chimera was unaffected. By those two independent criteria, no discernible association exists between CD8/ζ and the CD3 y, δ and e chains .

To determine whether a covalent link exists between endogenous ζ and the CD8/ζ chimera, ζ immunoblot analysis was performed comparing ζ and OKT 8 immunoprecipitates in Jurkat CD8 and JCD8/ζ2. The anti-ζ antiserum immunoprecipitates both the chimera and ζ from JCD8/ζ2, but only endogenous ζ from the Jurkat CD8 control. In contrast to the anti-ζ antiserum, 0KT8 immunoprecipitates the chimera but not ζ in JCD8/ζ2, while neither species is detected in Jurkat CD8. Collectively, the results from these experiments and those described above, argue against an interaction between the chimera and endogenous T cell receptor subunits.

Stimulation of CD8/ζ Results in Activation of the Phosphatidylinositol and Tyrosine Kinase Pathways

To determine whether binding of the extracellular domain of CD8/ζ would result in intracellular signalling events, the ability of 0KT8 to elicit an increase in cytoplasmic free calcium ([Ca^J_j) in chimera-transfected cells was examined. A fluorimetry tracing obtained with JCD8/ζ2 on stimulation of the TCR with the anti-T_jβ monoclonal antibody C305 was obtained. With the addition of soluble 0KT8, a substantial increase in calcium ([Ca^*2]^ was seen, suggesting that the cytoplasmic domain of ζ is capable of coupling to signalling machinery which results in the activation of phospholipase C.

The ability of the chimera to transduce a signal in cells lacking surface expression of the TCR chains was examined next. Stimulation of the TCR-negative Jβ-CD8/ζl4 with C305 results in no detectable increase in [Ca^*2]_i(- however, OKT8 still is able to elicit a strong calcium response. The lack of significant increase in [Ca^*^ with 0KT8 stimulation in Jurkat CD8 demonstrates that the ζ portion of the chimera is required for the elicited [Ca^*2^ response.

Since the increase in [Ca^*2], which occurs with TCR stimulation is attributed to increases in inositol phosphates, the ability of CD8/ζ to induce PIP₂ hydrolysis was tested by assessing changes in total soluble inositol phosphates following stimulation with OKT8. Stimulation of CD8/ζ with OKT8 resulted in the generation of inositol phosphates in both chimera-expressing cells. In contrast, no inositol phosphates were noted with stimulation of the wild-type CD8 protein in Jurkat CD8. Stimulation of TCR in Jurkat CD8 and CD8/ζ2 induced increases in inositol phosphates, whereas in the TCR-deficient transfectant, Jβ-CD8/ζl4, no such increase was observed on TCR stimulation. The results are consistent with the calcium fluorimetry data and confirm the ability of the chimera to activate phospholipase C even in the absence of endogenous cell surface TCR chains.

As stimulation of the T cell receptor activates a tyrosine kinase pathway in addition to inositol phospholipid pathway, it was important to determine whether chimera stimulation would result in tyrosine kinase activation. Western blots reveal a small number of tyrosine-phosphorylated proteins existing in all three clones prior to stimulation. On stimulation of Jurkat CD8 and JCD8/ζ2 with C305 (anti-T_jβ) , the tyrosine kinase pathway is activated as demonstrated by the induction of tyrosine phosphorylation of a number of proteins .

As expected, C305 has no effect in the TCR^" transfectant, Jβ-CD8/ζl4. Stimulation of the chimera on both JCD8/ζ2 and Jβ-CD8/ζl4 with OKT8 results in the appearance of a pattern ot tyrosine-phosphorylated bands indistinguishable from that seen with TCR stimulation. In contrast, stimulation through wild-type CD8 in Jurkat does not result in induction of tyrosine phosphoproteins . Thus, the CD8/ζ chimera, in the absence of T_x and CD3 y, δ and e, is capable of activating the tyrosine kinase pathway in a manner analogous to that of an intact TCR.

Since JCD8/2 expresses two discernible forms of ζ at the surface, endogenous ζ and the CD8/ζ chimera, each of which could be stimulated independently, the specificity of receptor-induced ζ phosphorylation was addressed.

Immunoprecipitates of ζ derived from the three clones, either unstimulated or stimulated with C305 or OKT8, were analyzed by western blotting with an anti-phosphotyrosine antibody. A small fraction of the ζ immunoprecipitates were blotted with ζ antiserum to control for differences in protein content between samples. Analysis of the lysate derived from TCR-stimulated Jurkat CD8 cells reveals a typical pattern of ζ phosphorylation with a multiplicity of bands from 16-21 kD most likely representing the varying degree of phosphorylation of the seven cytoplasmic tyrosine residues of ζ.

In that experiment, a small degree of constitutive ζ phosphorylation was detected in Jurkat CD8; however, that was not augmented by stimulation of the wild-type CD8 protein. Whereas phosphorylation of ζ was seen with stimulation of the TCR in JCD8/ζ2 though weaker than that seen in C305-stimulated Jurkat CD8 , no induced phosphorylation of the chimera was apparent. Conversely, stimulation of the CD8/ζ chimeric receptor on both JCD8/ζ2 and Jβ-CD7/ζl4 resulted in a high degree of phosphorylation of the chimera exclusively, seen as an induced broad band from 34-39 kD. The result indicates that the receptor-activated kinase responsible for phosphorylation of ζ recognizes its substrate only in a stimulated receptor complex.

Stimulation of CD8/ζ Results in Late Events of T Cell Activation T cell activation results from the delivery of receptor-mediated signals to the nucleus where they act to induce expression of specific genes. One such gene encodes the activation antigen, CD69, whose surface expression is induced within hours of T cell receptor stimulation and appears to be dependent on activation of protein kinase C (Testi et al . J. Immunol., 142:1854-1860). Although the function of CD69 in T cell activation is not well understood, CD69 provides a marker of distal signal transduction events.

Flow cytometry reveals a very small degree of basal CD69 expression on unstimulated cells. Maximal levels are induced on all cells with phorbol myristate acetate, PMA, an activator of protein kinase. Stimulation of the TCR results in induction of CD69 on Jurkat CD8 and JCD8/ζ2, but not on the TCR-negative clone, Jβ-CD8/ζl4. Moreover, stimulation of cells with OKT8 induced CD69 on both cells expressing the CD8/ζ chimera. Though a minimal degree of CD69 induction was apparent with stimulation of wildtype CD8 protein, the level was no higher than that observed with stimulation of Jurkat CD8 with a class I MHC antibody, w6/32.

Perhaps the most commonly used criterion to assess late activation events is the production of the lymphokine, interleukin-2 (IL-2) (Smith (1986) Science, 240:1169-1176). The IL-2 gene is regulated tightly, requiring the integration of a number of signals for transcription, making IL-2 a valuable distal market for assessing signalling through the CD8/ζ chimera. Stimulation of Jurkat CD8 and JCD8/ζ2 cells with TCR antibodies in the presence of PMA resulted in production of IL-2.

JCD8/ζ2 and Jurkat CD8 cells were stimulated with the indicated mAb or inomycin (1 um) in the presence of PMA (10 ng/ml) . IL-2 secretion was determined by the ability of culture supernatants of stimulated cells to support the growth of the IL-2-dependent CTLL-2.20 cells. Since PMA alone induces no IL-2 production in Jurkat, yet has a small direct effect on the viability of the CTLL 2.20 cells, values obtained with PMA alone were subtracted from each response value, yielding the numbers shown below. Data from two independent experiments are presented.

Table - Induction of IL-2 Production

Treatment IL-2 (Units/ml)

Jurkat CD8 JCD8/ζ 2 Experiment # Experiment #

#1 #2 #1 #2

Unstimulated <0.1 <0.1 <0.1 <0.1

C305 + PMA 13.5 9.1 3.7 2.1

0KT8 + PMA <0.1 <0.1 6.8 7.0 C305+OKT8+PMA

W6/32 + PMA <0.1 <0.1 <0.1 <0.1

Ionomycin+PMA 30.4 4.2 24.2 24.6

Importantly, while treatment with OKT8 on Jurkat CD8 induced no IL-2, similar treatment of JCD8/2 resulted in levels of secreted IL-2 consistently higher than those produced in that cell with TCR stimulation. Jβ-CD8/ζl4 responded more weakly to all experimental stimuli in the assay, but the data were qualitatively similar in that the cell reproducibly secreted IL-2 in response to 0KT8 but not to C305.

The data confirm that in addition to early signal transduction events, later activation events occur on stimulation of the CD8/ζ chimera, thus demonstrating the ability to couple to the relevant signal transduction pathways in a physiologic manner.

EXAMPLE 2

CD4-Zeta Chimeric Receptor In Signal Transduction Construction of CD4-zeta Chimeras

Plasmid pGEM3zeta bears the human zeta cDNA and was provided by Dr. R.D. Klausner and Dr. S.J. Frank (NIH, Bethesda, MD) . The plasmid pBS.L3T4 bears the human CD4 cDNA and was provided by Dr. D. Littman and Dr. N. Landau (University of California at San Francisco) . A BamHi-Apal restriction fragment (approximately 0.64 kb) encompassing the entire human zeta chain coding sequence from residue 7 of the extracellular (EXT) domain was excised from pGEM3zeta and subcloned into the BamHI and Apal restriction sites of the polylinker of pBluescript II SK (+) 9pSK, a phagemid-based cloning vector from Stratagene (San Diego, CA) , generating pSK.zeta. Subsequently, a BamHI restriction fragment encompassing the entire CD4 coding sequence (approximately 1.8 kb) was excised from pBS.L3T4 and subcloned into the BamHI site of pSK.zeta, generating pSK.CD4. zeta. See U.S. Pat. No. 5,359,046.

Single-stranded DNA was prepared from pSK.CD4.zeta (Stratagene pBluescript II protocol) and used as a template for oligonucleotide-mediated directional mutagenesis (Zoller & Smith (1982) Nucleic Acids Res., 10:6487-6500) to generate CD4-zeta chimeras with the desired junctions described below. CD4-zeta fusions 1, 2, and 3 subsequently were sequenced via the Sanger dideoxynucleotide technique (Sanger et al . (1977) Proc. Natl. Acad. Sci., 74:5463-5467), .excised as EcoRI-Apal restriction fragments and cloned into the polylinker of expression vector pIK.1.1 or pIK.l.l.Neo at identical sites.

An EcoRI-BamHI restriction fragment (approximately 1.8 kb) encompassing the entire coding region of CD4 was excised from pSK.CD4.zeta and subcloned between the EcoRI and Bglll sites of the pIK.1.1 or pIK.l.l.Neo polylinker.

The plasmid pUCRNeoG (Hudziak et al . , (1982) Cell, 31:137-146) carries the neomycin gene under the transcriptional control of the Rous sarcoma virus (RSV) 3' LTR. The RSV-neo cassette was excised from PURCNeoG as a Hindi restriction fragment (app. 2.3 kb) , and subcloned between the two Sspl sites of pIK.1.1, generating pIK.l.l.Neo.

pIK.1.1 is a mammalian expression vector constructed by four successive cassette insertions into pMF2, which was created by inserting the synthetic polylinker 5' -Hindlll-Sphl-EcoRI-Aatll-

BglI-XhoI-3' into Kpnl and SacI sites of pSKII (Stratagene), with loss of the Kpnl and SacI sites. First, a BamHI-Xbal fragment containing the SV40 T antigen polyadenylation site (nucleotides 2770-2533 of SV40, Reddy et al . (1978) Science, 200:494-502) and an Nhel-Sall fragment containing the SV40 origin of replication

(nucleotides 5725-5578 of SV40) were inserted by three-part ligation between the Bglll and Xhol sites, with the loss of the

Bglll, BamHI, Xbal, Nhel, Sail and Xhol sites. The BamHI-Xbal and Nhel-Sall fragments were synthesized by PCR with pSV2Neo (Southern

& Berg (1982) J. Mol. Appl . Gen., 1:327-341) as the template using appropriate oligonucleotide primer pairs which incorporated BamHI,

Xbal, Nhel and Sail sites at the respective ends.

Second, an Sphl-EcoRI fragment containing the splice acceptor of the human αl globin gene second exon (nucleotides +143 to +251) was inserted between the SphI and EcoRI sites. The Sphl-EcoRI fragment was synthesized by PCR with pπSVαHP (Treisman et al. (1983) Proc. Natl. Acad. Sci., 80:7428-7432) as the template using appropriate oligonucleotide primer pairs, which incorporated SphI and EcoRI sites at the respective ends. Third, the synthetic polylinker 5 ' -EcoRI-BglII-ApaI-AatII-3 ' was inserted between the EcoRI and the Aatll sites. Fourth, a Hindlll-SacI fragment containing the CMV IE enhancer/prompter (nucleotides -674 to -19, Boshart et al . (1985) Cell, 41:521-530) and a SacI-SphI fragment containing the CMV IE first exon/splice donor

(nucleotides -19 to +170) were inserted by three-part ligation between the Hindlll and SphI sites. The Hindlll-SacI fragment was prepared by PCR with pUCH.CMV (M. Calos, Stanford University, Palo

Alto, CA) as the template using appropriate oligonucleotide primers which incorporated Hindlll and SacI sites at the respective ends. The SacI-SphI fragment was chemically synthesized. RESULTS

Design of CD-i-zβta Chimeras

Three CD4-zeta chimeric receptors (FI, F2 and F3) were constructed from the extracellular (EC) and cytoplasmic (CYT) domains of CD4 and zeta respectively. The transmembrane (TM) domains of the CD4-zeta receptors were derived from zeta (FI, F2) or CD4 (F3) . F2 and F3 possess all four V domains.

FI retains only the VI and V2 of the CD4 EC domain (residues 1-180 of the mature CD4 protein) , the TM domain of zeta (residues 8-30 of the mature zeta chain) and the CYT domain of zeta (residues 31-142 of the mature zeta chain) .

F2 retains the CD4 EC domain comprising all four V regions (residues 1-370 of the mature CD4 protein) , the TM domain of the zeta chain (residues 8-30 of the mature zeta chain) and the CYT domain of zeta (residues 31-142 of the mature zeta chain) .

F3 retains the CD4 EC domain comprising all four V domains (residues 1-371 of the mature CD4 protein) , the TM domain of CD4 (residues 372-395 of the mature CD4 chain) , and the CYT domain of zeta (residues 31-142 of the mature zeta chain) .

Transient Expression of CD4-zeta Receptors

Chimeric receptors FI, F2 and F3, and the native CD4 gene were introduced into an expression vector pIK.1.1 which directs transcription via the CMV promoter/enhancer. To evaluate the structural integrity and cell surface levels of expression of the chimeric receptors, a highly efficient transient expression system was employed. Constructs were introduced by electroporation into the human embryonic kidney cell line, 293 (American Type Culture Collection, ATCC, Rockville, MD) , cells were harvested 24 hours later and subsequently analyzed by FACS employing a FITC-coupled mAb specific for the VI domain of CD4, 0KT4A. Although similarly high levels of surface F2 and F3 were detected by 0KT4A, the level of FI detected by the antibody in the same transient assay was lower .

To address whether FI was present in the membrane and to assess the structure of the chimeric proteins, immunoprecipitation of radiolabelled proteins was carried out. Twenty hours after electroporation of 293 cells with either FI, F2 or F3 , cells were pulse-labelled with ³⁵S-methionine for four hours, lysed in 1% NP40 and subjected to immunoprecipitation by either OKT4A (Ortho Pharmaceuticals, NJ) or a rabbit antiserum raised against a cytoplasmic peptide of murine zeta (obtained from R. Klausner, NIH, MD) . The level of radiolabelled FI relative to either F2 or F3 was significantly higher when anti-zeta antiserum instead of OKT4A was used as the immunoprecipitation agent. The results suggest that the FI receptor may not present the necessary topology for efficient binding of Vl-specific mAb's.

FI and F2 Form Disulfide-Linked Homodimers; F3 is a Monomer

Native zeta exists as a disulfide-linked homodimer or as a heterodimer in which the zeta chain is associated with an alternatively spliced product of the same gene, Eta. FI and F2 both possess the TM domain of zeta and therefore should have the potential to form a homodimer (and possibly a heterodimer with native zeta) via the membrane proximal cysteine residue (position 11 of the mature zeta chain) . In contrast, the transmembrane domain of F3 is derived from CD4 and therefore would be expected to confer the native monomeric state of the native CD4 molecule to the F3 receptor. To determine whether the receptors do form covalent linkages, immunoprecipitates of radiolabelled 293 cells which have been electroporated with each of the constructs under evaluation were analyzed under reducing and non-reducing conditions. Under both reducing and non-reducing conditions, a single protein of approximately 70 kb was immunoprecipitated by OKT4A from 293 cells electroporated with F3. As expected, CD4 also gave rise to a single protein of approximately 60 kd under both reducing and non-reducing conditions.

In contrast, FI and F2 gave rise to proteins of approximately 70 kd and 150 kd, respectively, under non-reducing conditions, approximately double that seen under reducing conditions (approximately 34 kd and 70 kd, respectively) . The results demonstrate that FI and F2 , like native zeta, exist as disulfide-linked homodimers, whereas F3 exists as a monomer, as does native CD4. The data do not rule out the ability of F3 to form a noncovalently associated dimer .

Introduction of CD4-zeta Receptors into a Human T Cell Line

The chimeric receptor genes FI, F2 and F3 , and the native CD4 gene, were introduced into a derivative of pIK.1.1 bearing a selective marker, pIK.l.lNeo. Each construct was introduced stably via electroporation into the human T cell leukemia line, Jurkat, and independent Jurkat clones obtained by limiting dilution and selection of G418. Cell surface expression of the chimeric receptor was assessed by FACS analysis of Jurkat clones employing FITC-coupled OKT4A.

Although native Jurkat cells express a low level of CD4 on the cell surface, transfectants expressing high levels of F2 or F3 were identified readily due to the significantly higher levels of fluorescence observed relative to untransfected cells. Similarly, stable clones expressing high levels of CD4 also were identified. In contrast, none of the clones isolated from cells electroporated with the FI receptor construct revealed levels of 0KT4A-specific fluorescence higher than that seen with native Jurkat cells.

FACS analysis of over 100 Jurkat clones revealed that the F3 receptor has the potential to be expressed stably in Jurkat cells at significantly higher levels (up to 50-fold) than the F2 receptor .

Induction of CD69 Expression On Stimulation of Native and Chimeric Receptors

CD69 (Leu-23) is an early human activation antigen present on T, B and NK lymphocytes. CD69 is detected on the cell surface of T lymphocytes within 2 hours after stimulation of CD3/TCR, reaching a maximal level by 18 to 24 hours. CD69 is therefore, the first detectable cell surface protein induced in response to CD3/TCR-mediated signals and represents a reliable marker of T cell activation. The ability of the CD4-zeta chimeric receptors to specifically mediate CD69 induction in the Jurkat T cell line was investigated. Representative Jurkat clones expressing either F2, F3 or CD4 were selected for functional analysis .

Monoclonal antibodies specific for the Ti α/β or CD3 chains can mimic the effect of antigen and serve as agonists to stimulate signal transduction and T cell activation events. Cells were stimulated with immobilized mAb's specific for (a) the T^ chain Jurkat (C305) , (b) the CD3e chain (OKT3) and (c) the VI domain of CD4 (0KT4A) . W6/32 recognizes an invariant determinant of human HLA class I antigens and was used in some experiments as negative control. CD69 expression was assayed by FACS analysis approximately 18 hours post-stimulation employing FITC-coupled anti-Leu 23 mAb. Unstimulated cells exhibited a very low level of basal CD69 expression but on stimulation with a pharmacological activator of protein kinase C, phorbol myristate acetate (PMA) , maximal expression was induced. Stimulation of native Ti with the C305 mAb or native CD3 with the 0KT3 mAb also resulted in induction to the CD69 marker. However, stimulation by 0KT4A gave rise to a high level of CD69 expression only for those transfectants expressing a chimeric CD4-ζ receptor. Indeed, for a number of transfectants, particularly F3-derived, the level of CD69 induction observed on stimulation was equal to that seen with PMA.

Stimulation of wild-type CD4 with 0KT4A resulted in little or no induction of CD69 when assayed in a number Jurkat CD4- ransfectants . Similarly, treatment of transfectants with the class 1 antibody, w6/32, had no significant effect in the assay. Furthermore, secretion of IL-2 on stimulation with 0KT4A was observed.

The results demonstrate that CD4 chimeric receptors possessing the cytoplasmic tail of zeta function effectively in initiation of T cell activation events. Specifically, chimeric CD4-zeta receptors bearing the CD4 TM domain (F3) mediate T cell activation more efficiently (with respect to CD69 induction) than those bearing the zeta TM domain (F2) , despite the fact that the latter retains the homodimeric form of native zeta.

F3 differs from F2 and native zeta in that it does not exist in the form of a covalent homodimer. The data therefore demonstrate that covalent dimerisation of the chimeric receptor is not essential for initiation of T cell activation as measured by CD69 induction.

EXAMPLE 3

1. Construction of murine A33 chimeric receptor

The amino acid and nucleotide sequences of the A33 chimeric receptor are provided in Table 1. The construction of the DNA fragments encoding this receptor and the retroviral vectors containing these fragments are described below.

Table 1. Amino acid and nucleotide sequences of the A33 chimeric receptor

Table 1A. Amino acid sequence of the A33 chimeric receptor MDMRVPAQLLGLLLLWLPGARCDWMTQSQKFMSTSVGDRVSITCKASQNVRTWAWYQQKPG QSPKTLIYLASNRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWSYPLTFGSGTKL EVKGSTSGSGKPGSGEGSTKGEVKLVESGGGLVKPGGSLKLSCAASGFAFSTYDMSWVRQTPE KRLEWVATISSGGSYTYYLDSVKGRFTISRDSARNTLYLQMSSLRSEDTALYYCAPTTWPFA YWGQGTLVTVSSERKCCVECPPCPAPPVAAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED PEVQFNWYVDGMEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPELQLEESCAEAQDG ELDMALIVLGGVAGLLLFIGLGIFFCVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR (SEQ ID N0:1)

Features :

Length: 643 aa

1-22: signal peptide from Vkappa chain of human Ab 98-6

(Gorny et al . (1989) Proc. Natl. Acad Sci. USA 86:1624-1628) 23-129: A33 VL

130-147: L218 linker

(Whitlow et al . (1994) (Protein Engineering 7: 1017-1026)

148-264: A33 VH

265-490: human IgG2 constant domain (Cgamma2-1 domain deleted); position 282 changed from G to A (denoted "G237A") to decrease affinity for human IgG Fc receptors.

491-507: human IgG2 Ml segment

508:531: human CD4 transmembrane domain

532-643 : intracellular portion of human TCR-associated zeta chain

Table IB. Nucleotide sequence encoding A33 chimeric receptor protein

GCCACCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGT GCCAGATGTGACGTCGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGG GTCAGCATCACCTGCAAGGCCAGTCAGAATGTTCGTACTGTTGTAGCCTGGTATCAACAGAAA CCAGGGCAGTCTCCTAAAACACTGATTTACTTGGCCTCCAACCGGCACACTGGAGTCCCTGAT CGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAATCTGAA GACCTGGCAGATTATTTCTGTCTGCAACATTGGAGTTATCCTCTCACGTTCGGATCCGGGACA AAGTTGGAAGTAAAAGGTTCTACCTCTGGTTCTGGTAAACCCGGGAGTGGTGAAGGTAGCACT AAAGGTGAAGTGAAGCTTGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAA CTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTACCTATGACATGTCTTGGGTTCGCCAGACT CCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACACCTACTATTTA GACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAGTGCCAGGAACACCCTATACCTGCAA ATGAGCAGTCTGAGGTCTGAGGACACGGCCTTGTATTACTGTGCACCGACTACGGTAGTCCCG TTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTGAGCTCAGAGCGCAAATGTTGTGTCGAG TGCCCACCGTGCCCAGCACCACCTGTGGCGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCC AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAC GAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCATGGAGGTGCATAATGCCAAGACA AAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCGTGCAC CAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCC

ATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC CCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAC CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACA CCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC ACACAGAAGAGCCTCTCCCTGTCTCCGGAGCTGCAACTGGAGGAGAGCTGTGCGGAGGCGCAG GACGGGGAGCTGGACATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATT GGGCTAGGCATCTTCTTCTGTGTCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTAC CAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTT TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAG

GAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATG AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACC

AAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO : 2 )

Features : length: 1938 1-6: Kozak consensus (GCCACC)

7-9: initiation codon (ATG) 1936-1938: stop codon (TAA)

1.1 Construction of plasmid pGX9451

Plasmid pGX9451 contains the A33 scAb in the vector described in Figure 3 of Whitlow and Filpula (1991) (Methods: A Companion to Methods in Enzymology 2: 97-105). The A33 scAb was constructed from the VL and VH domains of the A33 hybridoma (U.S. Patent No. 5,160,723) and its overall structure is VL-L218 peptide linker-A33 VH (N-term to C-term) , where L218 is a linker peptide described in Whitlow et al, supra.

1.2 Construction of retroviral vector plasmid pRT43.3pgkA33g2G237A. z

The A33 scAb was modified by adding the signal peptide from the V kappa chain of human mAb 98-6 (Gorny et al . , supra) to the 5' end and by mutating the 3' end to a Sac I site without changing the encoded amino acids .

First, PCR was used to modify the 5' and 3' ends of the A33 scAb by using the A33/218-1 and A33/218-2 primers (Table 2) . Second, the 98-6 Vkappa signal peptide was PCR amplified using the primers F15sig-1 and F15sig-2 (Table 2) from plasmid pRT43.3pgk.F15G237A. This plasmid encodes a chimeric receptor which comprises a 98-6 scAb and a human IgG2 constant domain (Cgamma2-1 domain deleted and position 237 changed from glycine to alanine) in the pRT43.3pgk expression plasmid.

pRT43.3pgk.F15G237A was constructed in the following manner. The CD4-zeta coding region was deleted from pRT43.3pgk. F3 and replaced with the coding sequence encoding the chimeric receptor F15g2G237A (described in Roberts et al . (1994) Blood 84: 2878) which contains the signal peptide and scAB from 98-6, the hinge, CH2 and CH3 domains from human IgG2 , the CD4 transmembrane domain and the intracellular portion of zeta. The CH2 domain residue 237 was then mutated from an glycine to an alanine ("G237A") by site directed oligonucleotide mutagenesis.

The first and second PCR products, described above, were mixed together and a third PCR amplification reaction was performed on these products using primers F15sig-1 and A33/218-2 (Table 2) to give the modified A33 scAB, which then was cut with Ml I and Sac I to produce a 796 bp segment. The Ml I-Sac I fragment was ligated to the 631 bp Sac I-Nsi I segment and the 7449 bp Ml I-Nasi I segment from pRT43.3pgk. F15G237A to produce plasmid pRT43.3pgk.A33g2G237A. z .

Table 2 PCR primers used to modify the A33 scAb A33/218-1: 5 ' -GCC AGA TGT GAC GTC GTG ATG ACC-3' (SEQ ID NO : 3 ) A33/218-2: 5 ' -GCT GAG CTC ACA GTG ACC AGA GTC CCT TGG CC-3 ' (SEQ ID NO: 4)

F15sig-1: 5 ' -CGG AAT TCA CGC GTG CCA CCA TGG ACA TGA-3' (SEQ ID NO: 5) F15sig-2: 5 ' -CAC GAC GTC ACA TCT GGC ACC-3 ' (SEQ ID NO: 6)

1.3 Construction of retrovirus vector plasmid pRT43.2A33g2G237A. z encoding A33 chimeric receptor

The retrovirus vector plasmid pRT43.2 provides for expression under the control of the MMLV LTR. See PCT Publication WO97/07225, incorporated herein by reference. pRT43.2 was cut with Eco RI and Nhe I and the 6624 bp segment was purified. The Eco RI-Nhe I fragment was ligated to the 2249 bp Eco RI-Nhe I segment from pRT43.3pgk.A33g2G237A. z, which contains the complete A33 chimeric receptor to produce the retrovirus vector plasmid pRT43.2A33g2G237A. z . 2. Construction of humanized A33 chimeric receptors

2.1 Amino acid and DNA sequences for humanized A33 scAb

A single chain protein containing humanized A33 VL and VH was designed, Z33 scAb. The amino acid and DNA sequences are shown in Table 3. The sequence contains a signal peptide (aa 1- 22) followed by humanized A33 VL (aa 23-129), a linker peptide (aa 130-147) and humanized A33 VH (aa 148-264) . The humanized A33 VL and A33VH are from King et al . (1995) Br . J. Cancer, 72 (6):1364- 1372.

Table 3 The amino acid and DNA sequence for the Z33 ScAb containing humanized A33VL and humanized A33VH

Table 3A. Amino Acid Sequence MDMRVPAQLL GLLLLWLPGA RCDIQMTQSP SSLSVSVGDR VTITCKASQN VRTWAWYQQ KPGLAPKTLI YLASNRHTGV PSRFSGSGSG TDFTFTISSL QPEDIATYFC QQHWSYPLTF GQGTKVEVKG STSGSGKPGS GEGSTKGEVQ LLESGGGLVQ PGGSLRLSCA ASGFAFSTYD MSWVRQAPGK GLEWVATISS GGSYTYYLDS VKGRFTISRD SSKNTLYLQM NSLQAEDSAI YYCAPTTWP FAYWGQGTLV TVSS (SEQ ID NO : 7) Features:

Length: 264 amino acids

1-22: Signal peptide from mAb 98-6 Vkappa

23-129: identical to 1-107 of huVkappa (King et al . , supra).

Base 108 (R) of the reported humanized A33 Vkappa amino acid sequence was deleted.

130-147: L218 peptide linker

148-264: identical to 1-113 of ha VH (King et al . , supra) NOTE: because of the VH numbering scheme, humanized A33VH is 117 amino acids long .

Table 3B. DNA sequence that encodes the Z33 scAb

CCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGGACGCGTGCC ACCATGGACATGAGGGTGCCCGCCCAGCTGCTCGGGTTGCTGTTGCTCTGGCTCCCCGGAGCC AGGTGCGACATTCAGATGACCCAAAGCCCCTCCAGCCTCAGCGTTAGCGTGGGTGACAGGGTC ACCATCACCTGCAAAGCCTCCCAGAACGTGAGGACAGTCGTGGCCTGGTATCAGCAGAAACCC GGTCTGGCTCCCAAGACCCTCATCTATTTGGCTTCTAATAGACACACAGGGGTTCCTTCTCGC TTTAGCGGATCAGGATCCGGTACTGATTTCACTTTCACAATTAGCAGCCTGCAACCCGAAGAC ATCGCCACATACTTCTGCCAACAGCACTGGTCCTATCCTCTCACTTTCGGACAGGGGACTAAG GTCGAGGTGAAAGGCAGCACATCTGGGTCTGGTAAGCCCGGCTCTGGAGAAGGCAGCACCAAA GGTGAGGTGCAGCTGCTGGAGTCTGGAGGAGGATTGGTCCAACCCGGTGGCAGCTTGCGCTTG TCTTGCGCTGCTAGTGGTTTTGCTTTCAGCACTTATGACATGAGCTGGGTCAGACAGGCCCCC GGCAAGGGCCTTGAATGGGTGGCTACCATCAGCAGCGGCGGCAGCTACACTTACTATCTGGAT AGCGTTAAGGGCAGATTCACCATTTCACGCGACTCCTCCAAAAATACACTGTACCTTCAGATG AACAGCCTTCAGGCAGAAGACAGCGCAATCTACTATTGTGCTCCTACCACAGTTGTGCCCTTC GCCTACTGGGGCCAAGGCACTCTTGTTACCGTGAGCTCATAAGAATTCGCT-3' (SEQ ID NO:8)

Features : Length: 870

55-60: Ml I site (ACGCGT) 61-66: Kozak consensus (GCCACC) 67-69: protein start (ATG) 67-132: Signal peptide

133-453 humanized A33 VL 454-507: L218 linker 508-858: humanized A33 VH 852-857: Sac I site (GAGCTC) 859-861: stop codon (TAA)

862-867: Eco RI site (GAATTC)

The DNA sequence for the humanized A33 was designed so as not to contain codons that are rare in human genes . The DNA sequence was constructed using synthetic DNA oligonucleotides and placed in the OperVector (Operon Technologies) as shown in Figure

2.

2.2 Construction of phosphoglycerate kinase (pgk) promoter- regulated Z33 retrovirus vector plasmid A Z33 chimeric receptor retrovirus vector plasmid, designated pRT43.3pgk.Z33.G237A. z, was produced by replacing the 98-6 scAb in pRT43.3pgk.F15G237A with the Z33 scAb. The OperVector (Fig. 2) was cut with Ml I and Sac I to obtain a 797 bp fragment that contained the Z33 scAb. That fragment was ligated to the 631 bp Sac I-Nasi I segment and the 7449 bp Ml I- Nasi I segment isolated from pRT43.3pgk. F15g237a to produce pRT43.3pgk.Z33.G237A. z, which encodes the Z33 chimeric receptor. The protein sequence of the Z33 chimeric receptor is shown in Table 4A and the corresponding nucleotide sequence is shown in Table 4B.

Table 4 Ami no acid and nucleic acid sequences of the Z33α2G237Az Chi meri c Receptor

Table 4A Amino acid sequence

MDMRVPAQLL GLLLLWLPGA RCDIQMTQSP SSLSVSVGDR VTITCKASQN VRTWAWYQQ

KPGLAPKTLI YLASNRHTGV PSRFSGSGSG TDFTFTISSL QPEDIATYFC QQHWSYPLTF

GQGTKVEVKG STSGSGKPGS GEGSTKGEVQ LLESGGGLVQ PGGSLRLSCA ASGFAFSTYD

MSWVRQAPGK GLEWVATISS GGSYTYYLDS VKGRFTISRD SSKNTLYLQM NSLQAEDSAI

YYCAPTTWP FAYWGQGTLV TVSSERKCCV ECPPCPAPPV AAPSVFLFPP KPKDTLMISR

TPEVTCVWD VSHEDPEVQF NWYVDGMEVH NAKTKPREEQ FNSTFRWSV LTWHQDWLN

GKEYKCKVSN KGLPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS

DIAVEWESNG QPENNYKTTP PMLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH

YTQKSLSLSP ELQLEESCAE AQDGELDMAL IVLGGVAGLL LFIGLGIFFC VRVKFSRSAD

APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP RRKNPQEGLY NELQKDKMAE

AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR (SEQ ID NO : 9)

Features :

Length: 643 amino acids

1-22: signal peptide from kappa light chain of human mAb 98-6

23-129: humanized Z33 Vkappa

130-147 L218 linker peptide 148-264 humanized A33 VH 265-276 human IgG2 hinge 276-490 human IgG2 CH2 (G237A mutation and CH3 ) 491-507 IgG Ml exon 508-531 CD4 transmembrane domain 532-643 intracellular portion of human zeta

Table 4B. DNA sequence ACGCGTGCCACCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCC CAGGTGCCAGATGTGACGTCGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGA CAGGGTCAGCATCACCTGCAAGGCCAGTCAGAATGTTCGTACTGTTGTAGCCTGGTATCAACAG AAACCAGGGCAGTCTCCTAAAACACTGATTTACTTGGCCTCCAACCGGCACACTGGAGTCCCTG ATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAATCTGA AGACCTGGCAGATTATTTCTGTCTGCAACATTGGAGTTATCCTCTCACGTTCGGATCCGGGACA AAGTTGGAAGTAAAAGGTTCTACCTCTGGTTCTGGTAAACCCGGGAGTGGTGAAGGTAGCACTA AAGGTGAAGTGAAGCTTGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACT CTCCTGTGCAGCCTCTGGATTCGCTTTCAGTACCTATGAC

ATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTG GTAGTTACACCTACTATTTAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAGTGCCAG GAACACCCTA TACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCTTGTATTACTGTGCACCGACTACGG TAGTCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTGAGCTCAGAGCGCAAATGTTG TGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGA GCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCATGGAGGTGCATAATGCCAA GACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCGTG CACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCC

CCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC CCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAC CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG

AACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC TCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGAGCTGCAACTGGAGGAGAGC TGTGCGGAGGCGCAGGACGGGGAGCTGGACATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCC TCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTCAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAG GAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA

AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA ( SEQ ID

NO : 10 )

Features : Length: 1944 Ml I (ACGCGT) : 1-6 protein start (ATG) : 13-15 protein stop (TAA) : 1942-1944

2.3. Construction of retroviral vector containing LTR-regulated Z33 chimeric receptor pRT43.3pgk.Z33.G237A.z was cut with Eco RI and Nhe I and a 2249 bp segment was obtained that contains the complete Z33 chimeric receptor coding region. That segment was ligated to the 6624 bp Eco RI - Nhe I segment from the plasmid pRT43.2 to produce the retrovirus vector plasmid pRT43.2. Z33g2G237A. z .

2.4 Construction of retroviral vector containing LTR promoter- regulated Z33 chimeric receptor deleted in the human IgGl CH2 domain

Four segments of DNA were isolated. A 6708 bp Ml I- Apa I segment and a 797 bp Ml I-Sst I segment containing the Z33 scAb were obtained from pRT43.2Z33g2G237A. z . A 743 bp Nsi I-Apa I segment containing the following sequences was also prepared: (1) bp 1-105 encode the 3' portion of the human IgGl CH3 domain (Roberts et al, supra); (2) bp 106-177 encode the CD4 transmembrane domain (amino acids 372-295 of the mature protein (Roberts et al, supra); (3)bp 178-513 encode the intracellular portion of human zeta, amino acids 31-142 (Roberts et al, supra); and (4) bp 517-743 contain the 3' untranslated portion of human

- zeta (Genbank #J04132) . A 304 bp DNA fragment containing the 5' portion of the C gamma 1-3 constant domain and a partial gamma 1 hinge region was synthesized. The sequence of this fragment (cgl3) is shown in Table 5 below. Table 5 Sequence of cα!3

GAGCTCAGACAAAACTCACACATGCCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTAC

ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA

GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC

AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT (SEQ ID NO: 11)

Features : length: 304 bp Sac I site (GAGCTC) : 1-6

Nasi I site (ATGCAT) : 299-304

The cgl3 fragment was digested with Sac I and Nasi I to obtain a 298 bp segment. The four DNA segments were ligated to obtain the plasmid pRT43.2. Z33.dCH2. z . The amino acid sequence and the nucleotide sequence for the Z33dCH2.z chimeric receptor are shown in Table 6.

Table 6 Amino acid and nucleotide sequences for the

Z33dCH2.z chimeric receptor

Table 6A. Amino acid sequence of Z33dCH2.z chimeric receptor

MDMRVPAQLLGLLLLWLPGARCDIQMTQSPSSLSVSVGDRVTITCKASQNVRTWAWYQQKPGL APKTLIYLASNRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQHWSYPLTFGQGTKVEV KGSTSGSGKPGSGEGSTKGEVQLLESGGGLVQPGGSLRLSCAASGFAFSTYDMSWVRQAPGKGL EWVATISSGGSYTYYLDSVKGRFTISRDSSKNTLYLQMNSLQAEDSAIYYCAPTTWPFAYWGQ GTLVTVSSDKTHTCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPELQLE ESCAEAQDGELDMALIVLGGVAGLLLFIGLGIFFCVRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR (SEQ ID NO: 12)

Features :

Length: 532 amino acids

1-22: signal peptide from human mAb 98-6 Vkappa

23-264: Z33 sScAb

265-274 partial hinge domain from human gamma 1 heavy chain

275-379 human gamma 1 CH3 domain

380-396 Ml domain

397-419 human CD4 transmembrane domain

420-532 human zeta intracellular portion

Table 6B. Nucleotide sequence of Z33dCH2.z chimeric receptor ACGCGTGCCACCATGGACATGAGGGTGCCCGCCCAGCTGCTCGGGTTGCTGTTGCTCTGGCTCC CCGGAGCCAGGTGCGACATTCAGATGACCCAAAGCCCCTCCAGCCTCAGCGTTAGCGTGGGTGA CAGGGTCACCATCACCTGCAAAGCCTCCCAGAACGTGAGGACAGTCGTGGCCTGGTATCAGCAG AAACCCGGTCTGGCTCCCAAGACCCTCATCTATTTGGCTTCTAATAGACACACAGGGGTTCCTT CTCGCTTTAGCGGATCAGGATCCGGTACTGATTTCACTTTCACAATTAGCAGCCTGCAACCCGA AGACATCGCCACATACTTCTGCCAACAGCACTGGTCCTATCCTCTCACTTTCGGACAGGGGACT AAGGTCGAGGTGAAAGGCAGCACATCTGGGTCTGGTAAGCCCGGCTCTGGAGAAGGCAGCACCA AAGGTGAGGTGCAGCTGCTGGAGTCTGGAGGAGGATTGGTCCAACCCGGTGGCAGCTTGCGCTT GTCTTGCGCTGCTAGTGGTTTTGCTTTCAGCACTTATGACATGAGCTGGGTCAGACAGGCCCCC GGCAAGGGCCTTGAATGGGTGGCTACCATCAGCAGCGGCGGCAGCTACACTTACTATCTGGATA GCGTTAAGGGCAGATTCACCATTTCACGCGACTCCTCCAAAAATACACTGTACCTTCAGATGAA CAGCCTTCAGGCAGAAGACAGCGCAATCTACTATTGTGCTCCTACCACAGTTGTGCCCTTCGCC TACTGGGGCCAAGGCACTCTTGTTACCGTGAGCTCAGACAAAACTCACACATGCCCACCGTGCC CAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGAG CTGCAACTGGAGGAGAGCTGTGCGGAGGCGCAGGACGGGGAGCTGGACATGGCCCTGATTGTGC TGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTCAGAGTGAA GTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG GGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC CTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGCTAA (SEQ ID NO: 13) Features :

Length: 1611 nucleotides 1-6: Ml I site

7-12: Kozak consensus sequence

13-78: signal peptide from human mAb 98-6 Vkappa

79-804: Z33 sScAb

805-834: partial hinge from human IgGl 835-1149: human IgGl heavy chain CH3 domain

1150-1200: Ml

1201-1272: CD4 transmembrane segment

1273-1608: zeta intracellular portion 3. Transduction of T cells

3.1 Preparation of retrovirus stocks

Replication-defective retrovirus stocks encoding each version of the A33 and Z33 chimeric receptors described above were prepared as described in Finer et al . (1994) Blood 83:43-50).

3.2 Transduction of human T cells

Human peripheral blood mononuclear cells (MNC) were prepared by centrifugation of peripheral blood on Ficoll-Hypaque gradient. MNC were cultured in the presence of 10 ng/ml 0KT3 (anti-CD3, Ortho) to activate T cells. After two days, cells were harvested and recultured in the presence of thawed retrovirus stock plus polybrene and IL-2 for 1-3 days. Cells then were washed and placed back in culture in the presence of 700 IU/ml rIL-2 for ≥ 2 days before being assayed for cell surface expression of the chimeric receptor and for functional responses to the appropriate stimulator cells. At the time of final harvest cultures were >95% CD3+ T cells.

4. Analysis of human T cells transduced with the A33 and Z## chimeric receptors 4.1 Cytolysis of A33+ tumor target cells by human T cells expressing various A33 and Z33 chimeric receptors

4.1.1 Human peripheral blood T cells were transduced with the retrovirus vectors carrying the various chimeric receptor listed in Table 7. The F15 chimeric receptor reacts with the gp41 portion of the HIV-1 env protein and was used as a negative control for specificity. Table 7. Chimeric receptor retrovirus vectors used to transduce human T cells

Chimeric Promoter V domain C domain retrovirus

Receptor vector

Name plasmid

A33 (pgk) human pgk A33 scAb human IgG2 pRT43.3pgk.A

(dCHl, G237A) 33g2G237A.z

A33 (LTR) MMLV LTR A33 scAb human IgG2 PRT43.2.A33g

(dCHl, G237A) 2G237A.Z

Z33 (pgk) human pgk Z33 scAb human IgG2 pRT43.3pgk.Z

(dCHl, G237A) 33g2G237A.z

Z33 (LTR) MMLV LTR Z33 scAb human IgG2 pRT43.2.Z33g

(dCHl, G237A) 2G237A.Z

Z33dCH2 (LTR) MMLV LTR Z33 scAb human IgGl pRT43.2.Z33d

(dCHl, ΔCH2) CH2.Z

F15 (LTR) MMLV LTR 98-6 scAb human IgG2 pRT43.3pgk. F

(dCHl, G237A) 15g2G237A

Transduced T cells were used in CTL assays against Cr-51-labelled SW1222 (A33+) and SNU-1 (A33-) tumor target cells. Table 8 summarizes the results from several experiments. It can be seen that expression of the A33 chimeric receptor or Z33 chimeric receptor confers the ability to lyse A33+ tumor cells on the transduced T cells.

Table 8 Lysis of tumor cells by human T cells transduced with various chimeric receptors

% Specific Lysis³

Target E:T A33 A33 Z33 Z33 Z33 F15 mock

Ratio (pgk) (LTR) (pgk) (LTR) dCH2 control (LTR)

SW1222 40 54,55^c 52,49 38 54,32 32 4 5,7,2

(A33+)

20 43,52 46,31 34 38,32 26 3 3,7,1

10 27,34 34,18 30 24,25 18 1 1,2,8

5 16,21 21,8 20 15,17 10 -1 0,1,1

2.5 9,12 12,2 9 6,11 6 -2 0,0,0

1.25 4, 5 6,1 6 5,6 3 -1

1,0,0

SNU-1 40 4,11 5,2 11 3,17 14 4 4,6,1

(A33-)

20 2,6 2,0 8 2,11 7 1 2,4,3

10 0,2 1,0 4 1,6 3 0 1,3,5

5 -1,0 0,0 2 1,2 1 -1 1,1,7

2.5 -1,0 0,0 1 0,1 0 -1 0,1,6

1.25 -1,-1 0,0 0 0,1 0 -1 0,1,2

^a 4 hr Cr-51 release assays ^b mock = sham transduced with virus- free culture supernatant ^c results from different experiments

4.2 Cytokine production by T cells expressing A33 and Z33 chimeric receptors

Transduced cells were tested for the production of cytokines following stimulation by A33-positive or A33-negative tumor cells. As shown in Table 9a, A33 chimeric receptor- expressing T cells produced interferon gamma and granulocyte- macrophage stimulating factor (GM-CSF) when stimulated by A33- positive tumor cells but not by A33-negative tumor cells. T cells bearing an irrelevant chimeric receptor ("control" in Table 9) did not respond to any of the tumor cells. Similar results were obtained with T cells expressing the Z33 chimeric receptor as shown in Table 9b, demonstrating the specific functionality of the Z33 chimeric receptor expressed in human T cells. Table 9 Cytokine production by A33 chimeric receptor or control chimeric receptor T cells stimulated by A33 positive or A33 negative tumor cells

Table 9a

Interf'eron GM- -CSF gamma (pg/ml) (pg/ml)

T cell Stimulus Donor 1 Donor 2 Donor 1 Donor 2

Chimeric

Receptor

A33 LIM1215 67 124 107 232

SW1222 57 176 56 239

HCT15 3 6 9 12 none 16 25 62 82 control LIM1215 12 5 50 65

SW1222 13 21 43 56

HCT15 12 23 97 69 none 3 21 158 86

CD8-enriched A33 or F15 control T cells co-cultured with or without tumor cell simulators: LIM1215 (A33+) , SW1222 (A33+) HCT15 (A33-) . Culture supernatant was collected the following day and assayed for IFN gamma and GM-CSF by ELISA.

Table 9b Interferon gamma production by T cells expressing the Z33 chimeric receptor

Interferon gamma (pg/ml)

Stimulating cells Z33 T cells Control T cells

SW1222 (A33+) 886 62 SNU-1 (A33-) 51 124 none 91 114

T cells expressing the Z33 or F15 (control) chimeric receptors were stimulated with tumor cells for 24 hours. Supernatant was collected and tested for interferon gamma by ELISA assay.

4.3. Failure to lyse A33+ normal MNC

Freshly isolated lymphocytes from normal volunteer donors were found to stain with the A33 mAb. However, the amount of A33 antigen found on lymphocytes was about 200-fold less than that on tumor cells, as determined by flow cytometry. It was shown by Roberts et al . (Blood (1994) 84:2878) that CD4/zeta chimeric receptor-expressing CTLs were able to recognize and kill target cells that levels of HIV-1 env protein which were undetectable by other methods . When used in a clinical setting, infused A33 chimeric receptor /Z33 chimeric receptor T cells are likely to encounter and contact recipient lymphocytes, which express the A33 antigen at a level that is detectable by flow cytometry. Therefore, it was necessary to determine whether such contact might result in lysis of the recipient lymphocytes, leading to undesirable lymphopenia. Lymphocyte targets were labelled with Cr-51 and added to autologous Z33 effector CTL. As shown in Table 10, the normal lymphocytes from two donors were not killed by either CD8+ or CD4+ Z33+ autologous CTL, indicating that contrary to what might be predicted from Roberts et al, supra, A33 antigen expressing lymphocytes were unable to stimulate the lytic activity of Z33 CTL.

Table 10 Lymphocytes are not killed bv Z33 chimeric receptor- positive CTL

% Lysis of Cr- 51-labelled autologous MNC

CTL Ef fector : MNC Target Ratio Don or CTL 20 : 1 10 : 1 5 : 1 2 . 5 : 1

RM CD8 + 2 2

CD4 + - -2 JC CD8 + 5 6

CD4+ 3 5

The data were corroborated by inhibition assays in which unlabelled MNC were added to a mixture of A33 chimeric receptor CTL and Cr-51 labelled tumor targets. It was expected from Roberts et al, supra, that the unlabelled A33+ MNC would inhibit the lysis of the Cr-labelled A33+ tumor targets. However, as shown in Table 11, MNC failed either to inhibit or to augment the lysis of the tumor targets even when added in 5-fold or 50-fold excess, indicating that in spite of cell surface expression of the A33 antigen, MNC do not stimulate chimeric receptor-dependent cytolysis .

Table 11 Percent Lysis of Cr-51-labβllβd LIM1215 tumor cells by A33 chimeric receptor effector CTL in the presence or absence of unlabelled inhibitor cells

Unlabeled/Labeled Ratio

Effector/ Unlabeled

Target inhibitor 0 0.5: 1 5: 1 50

Ratio

1.25: 1 MNC #1 23 26 17 17

MNC #2 23 22 17 8

LIM1215 21 18 4 -1

SW1222 25 18 5 0

SW892 21 23 19 7

0.5: 1 MNC #1 14 13 14 9

MNC#2 8 14 7 16

LIM1215 13 8 3 -3

SW1222 15 11 4 -1

SW892 14 17 11 2

EXAMPLE 4

Construction of CD4-CD3γ, CD4-CD35, and CD4-CD3e Chimeric Receptors

Cloning of CD3 Chains: Gamma (Y) , Delta (δ) , and Epsilon (e) cDNA sequences encompassing the transmembrane and cytoplasmic domains of the gamma, delta and epsilon chains were isolated by standard PCR techniques from Jurkat cell RNA.

Construction of Chimeric CD4-CD3e, -CD3δ, and -CD3γ Receptors

The PCR products obtained were digested with Nar I and Apa I and the resulting Nar i-Apa i restriction fragments (γ=276 bp, δ=276 bp, e=305 bp) were subcloned into the expression vector pIKl .1CD4 (as described above) between unique Nar I and Apa I sites. Oligonucleotide-mediated deletion mutagenesis was used to generate chimeric receptors with the following compositions: 1. CD4-CD3γ

(i) CD4 extracellular and transmembrane domain (CD4 amino acids 1-395) and CD3γ cytoplasmic domain (CD3γ amino acids 117- 160) ;

(ii) CD4 extracellular domain (CD4 amino acids 1-370) and CD3γ transmembrane and cytoplasmic domains (CD3γ amino acids 83-160) .

2. CD4-CD3δ

(i) CD4 extracellular and transmembrane domain (CD4 amino acids 1-395) and CD3δ cytoplasmic domain (CD3δ amino acids 107- 150);

(ii) CD4 extracellular domain (CD4 amino acids 1-370) and CD3δ transmembrane and cytoplasmic domains (CD3δ amino acids 73-150).

3. CD4-CD3e

(i) CD4 extracellular and transmembrane domain (CD4 amino acids 1-395) and CD3e cytoplasmic domain (CD3e amino acids 132- 185) ;

(ii) CD4 extracellular domain (CD4 amino acids 1-370) and CD3e transmembrane and cytoplasmic domains (CD3e amino acids 98-185) .

EXAMPLE 5

Stem Cell Transduction

By engineering hematopoietic stem cells, a multi-lineage immune response can be mounted against the disease target, such as, cancers expressing A33. After transduction of stem cells followed by bone marrow transplantation, the engineered bone marrow stem cells will produce continually the effector cells abrogating the need for ex vivo cell expansion. Because stem cells are self-renewing, once transplanted, the cells can provide lifetime immunologic surveillance with applications for chronic diseases, such as malignancy.

Effector cells including T cells, neutrophils, natural killer cells, mast cells, basophils and macrophages are derived from hematopoietic stem cells and utilize different molecular mechanisms to recognize the targets. T cells recognize targets by binding of the T cell receptor to a peptide in the context of an MHC molecule on an antigen presenting cel-1. In the previous examples, it was shown that the chimeric receptors of the invention can bypass the MHC-restricted T cell receptor in T cells. Other cytotoxic cells of the immune system recognize targets through Fc receptors. Fc receptors bind to the Fc portion of antibody molecules which coat virally-infected, fungally- infected or parasite-infected cells. In addition, antibodies against tumor antigens induce antibody dependent cellular cytotoxicity (ADCC) against the tumor cell by cytotoxic cells harboring Fc receptors. It was demonstrated that in addition to the capability of chimeric receptors of the invention to by-pass the MHC-restricted T cell receptor, they also are able to bypass the Fc receptor and redirect the cytotoxicity of neutrophils derived from transduced stem cells.

The transduction method used for introducing the chimeric receptors into stem cells was essentially the same as described in Finer et. al., Blood 83:43-50 (1994). On the day prior to the transduction, 293 cells transfected with the thymidine kinase gene were plated at 10⁵ cells/well in a Corning 6-well plate. The cells serve as transient viral producers. On the day of transfection, CD34^* cells were isolated from low density mononuclear human bone marrow cells using a CellPro LC34 affinity column (CellPro, Bothell, WA) . Recovered cells were plated out in Myelocult H5100 media (Stem Cell Technologies Inc., Vancouver, B.C.) containing 100 ng/ml human stem cell factor (huSCF) , 50 ng/ml huIL-3, 10 ng/ml huIL-6 and 10^"δ M hydrocortisone for a period of 48 hours for "pre-stimulation" .

The next day, the 293/TK cells were transfected as described by Finer et . al . , supra. The following day, the CD34^* cells were collected and resuspended in infection media consisting of Iscoves's Modified Dulbecco's Medium (IMDM) , 10% FBS, glutamine, 100 ng/ml huSCF, 50 ng/ml huIL-3, 10 ng/ml huIL-6 and 8 μg/ml polybrene. 3-5 x 10⁵ cells were added in 2 ml total to each well of the transfected 293 cells to initiate the co-culture.

Forty-eight hours later, the CD34^* cells were collected. Briefly, the 2 mis of cell supernatant were removed and additional adherent CD34^* cells were dislodged using an enzyme-free/PBS-based cell dissociation buffer. Cells then were expanded and differentiated in vitro in Myelocut medium with addition of 100 ng/ml huSCF, 50 ng/ml huIL-3 , 10 ng/ml huIL-6 ar.d 10 μM Gancyclovir to inhibit 293 proliferation. The cells will not survive under gancyclovir selection, due to carrying the thymidine kinase gene.

At approximately day 10 after transfection, cells were cultured in 10 ng/ml huSCF and 2 ng/ml huG-CSF. From day 14 onward, the cells were driven toward becoming neutrophils by culture in 10 ng/ml G-CSF alone. Cells were monitored via cytospins and differentials to ascertain the degree of differentiation and maturity of the neutrophils. Between days 16-24, the cells can be used for testing effector function, such as cytotoxicity, and ascertaining the degree of transduction by FACS and PCR analysis.

The differentiated neutrophils express the CD15 antigen, and the neutrophils derived from transduced stem cells also express the human CD4 extracellular domain (derived from CD4- zeta) . In one experiment, approximately 18% of the neutrophils were expressing CD4-zeta and the correction was factored in the calculation of effector : target ratio. The cytotoxicity of the neutrophils was tested according to the following methods .

Cytotoxicity Assay

Ra i target cells, expressing the envelope protein of HIV (gpl60) , were labeled with sodium ⁵¹Cr chromate (Amersham, Arlington Heights, IL) , generally 50 μCi/10⁶ cells for 2 hours. The targets then were washed 3 times to remove loosely bound ⁵¹Cr and resuspended at 10⁵ cells/ml in RPMI 1640, 10% FBS, and glutamine .

Modified CD34-derived neutrophils expressing the CD4-zeta chimeric receptor were plated in triplicate and titrated 1:2 in a final volume of 100 μl. The E:T ratio is dependent on the cell number available but usually was in the range of 100-200:1. A 100 μl portion (10,000 cells) of the target cell solution was added to each well. Plates then were spun for 2 minutes at 500 rpm and allowed to incubate for 5 hours at 37°C and 5% C0₂. ⁵¹Cr released in the supernatant was counted using a Y counter. The percentage of cytotoxicity was calculated as: 100% x EXP-SR/MR-SR, where EXP are the counts released in the presence of effector cells, SR = those spontaneously released and MR = the maximal release achieved when targets are incubated and lysed with a 1% Triton-X solution (Sigma, St. Louis, MO) .

Cytotoxicity against Ra i cells expressing the envelope protein of HIV was observed. Eliciting no response are the same transduced neutrophils against the parental Raji line not expressing HIV envelope and untransduced neutrophils against the envelope expressing Raji cells. The chimeric receptor-bearing neutrophils specifically recognized and killed cells expressing HIV envelope protein. The transduced cells do not recognize the parental Raji cells not expressing HIV envelope and untransduced neutrophils do not kill Raji cells expressing envelope. The data demonstrate the feasibility of redirecting other cytotoxic cell types derived from stem cells beside T cells.

It is evident from the above results that one can provide for activation of various signalling pathways in a host cell by providing for expression of a chimeric protein, which may serve as a surface membrane protein, where the extracellular domain is associated with a ligand of interest, while the cytoplasmic domain, which is not naturally associated with the extracellular domain, can provide for activation of a desired pathway. In that manner, cells can be transformed so as to be used for specific purposes where cells will be activated to a particular pathway by an unnatural ligand. That can be exemplified by using CD4 as the extracellular domain, where binding of an HIV protein can result in activation of a T cell which can stimulate cytotoxic activity to destroy infected cells. Similarly, other cells may be modified, so as to be more effective in disease treatment or to immune effects and the like.

EXAMPLE 6

Human natural killer (NK) cells can be modified genetically to express high levels of CD4ζ using retroviral transduction. In addition, the CD4ζ chimeric receptor is biochemically active as crosslinking of CD4 ζ on NK cells results in tyrosine phosphorylation of CD4ζ and multiple cellular proteins. The CD4ζ chimeric receptor is functionally active, and can direct NK cells to lyse specifically and efficiently either natural killer-resistant tumor cells expressing the relevant ligand, gpl20, or CD4^* T cells infected with HIV.

NK Cells

The human NK3.3 clone has been described previously in Kornbluth et al . (1982) J. Immunol., 129: 2831. Cells were maintained in NK media: RPMI 1640 supplemented with 15% fetal bovine serum, glutamine, penicillin, streptomycin and 15% Lymphocult-T (Biotest, Denville, NJ) . Cell density was maintained at less than 1 x 10^s cells/ml and media were replaced every two days .

Retroviral Transduction of NK cells with CD4 ζ

Retroviral transduction of NK3.3 cells was carried out employing the kat retroviral producer system previously described for transduction of CD8^* T lymphocytes (Roberts et al . (1994) Blood, 84:2878 and Finer et al.(1994) Blood, 83: 43) with the following modifications. 293 cells were plated at 1 x 10⁶ cells per plate in a 6-well plate with 2 ml of media per well (293-1) , and 48 hours later were transfected transiently with 10 ug of retroviral vector encoding CD4ζ, pRTD2.2F3, and 10 ug of packaging plasmid. 24 hrs post transfection, media were replaced with NK media. 4 hrs later, 3 x 10⁶ NK cells were added per transfected 293-1 plate and co-cultivated in the presence of polybrene (2 ug/ml) . After a 24 hour cocultivation period, NK3.3 cells were removed from the 293-1 plate and subjected to a second round of co-cultivation with freshly transfected 293 cells for an additional 24 hrs. Transduced NK3.3 cells then were harvested and allowed to recover for 24 to 48 hrs. in NK media. Stable expression of the CD4 ζ chimeric receptor in transduced NK3.3 was analyzed 15 days post transduction by flow cytometry with FITC- conjugated anti-CD4 mAb's as described below. CD4ζ^* NK cells subsequently were purified by immunoaffinity anti-CD4 mAb-coated flasks (Applied Immune Sciences) .

Antibodies

Anti-FcγRIII mAb 3G8 was from Medarex (West Lebanon, NH) ; anti-CD4 mAb OKT4A was from Ortho Diagnostic Systems (Raritan, NJ) ; sheep affinity-purified F(ab')₂ fragments to mouse IgG; biotin-conjugated F(ab'). fragment goat anti-mouse IgG ware from Cappel (Durham, NC) ; anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology (Lake Placid, NY) ; anti-ζ rabbit anti-serum, #387, raised against a peptide comprising amino acids 132-144 of the human ζ sequence was provided by Dr. L. E. Samelson (NIH) ; and FITC conjugated-antibodies, Gammal, anti-CD16 (-FcyRIII) and anti- CD4 OKT4A mAb's were obtained from Becton-Dickinson (San Jose, CA) . Rabbit anti-human lymphocyte serum was from Accurate Chemical and Scientific Corp. (Westbury, NY) . Anti-gpl20 mAb was from Dupont/NEN Research Products (Wilmington, DE) ; allophycocyanin streptavidin was from Molecular Probes, (Eugene, OR) . MOPC 21 (IgG^ , used as a control i.Ab in three colored FACS analysis, and goat serum were from Sigma (St. Louis, MO) . Anti- human class II (HLA-DP) mAb was from Becton Dickinson (San Jose, CA) . Sheep anti-mouse Ig peroxidase, donkey anti-rabbit Ig peroxidase and the ECL western blotting system were from Amersham (Arlington Heights, IL) .

NK Cell Stimulation and Immunoprecipitation

NK3.3 and CD4ζ^* NK3.3 cells were fasted in RPMI 1640 containing 1 mg/ml BSA for 2-3 hrs. prior to stimulation. Cells then were spun down and resuspended in the same medium at a density of 2xl0⁷ cells/ml. The cell suspensions were incubated with mAb to FcyRIIIA (3G8) or CD4 (OKT4A) for 15 minutes at 4°C, and then washed to remove unbound antibody. Sheep affinity purified F(ab')₂ fragments to mouse IgG then were added at 37°C for 3 minutes to crosslink FcyRIIIA or CD4ζ. For immunoprecipitations, cells were lysed at 2 x 10⁷ cells/200 ml of 1% NP-40, 150 mM NaCl and 10 mM Tris (pH 7.8) in the presence of protease inhibitors (1 mM each of PMSF, aprotinin and leupeptin ) and phosphatase inhibitors (0.4 mM EDTA, NaHC0₃, 10 mM Na₄P₂0₇10H₂O) . After 30 minutes at 4°C, lysates were centrifuged for 10 minutes at 14,000 rpm, and pre-cleared with protein A Sepharose beads . The pre-cleared lysates then were incubated with the immunoprecipitating anti-ζ serum at 4°C for 30 minutes, followed by protein A Sepharose beads at 4°C overnight. Washed immunoprecipitates then were subjected to SDS-PAGE under reducing conditions .

Immunoblot Analysis

Separated proteins were transferred to nitrocellulose membranes. Membranes were subsequently incubated with the primary antibody (anti-phosphotyrosine or anti-ζ antiserum) . Bound antibody was detected with horseradish peroxidase-conjugated sheep antibody to mouse or rabbit IgG , followed by a non-isotopic enhanced chemiluminescence ECL assay (Amersham) .

Flow Cytometry Approximately 1 x 10⁶ cells per condition were washed once with PBS plus 2% FCS, then incubated with saturating concentrations of fluorescein isothiocyanate (FITC) -conjugated 0KT4A for detection of CD4ζ expression, or anti-CD16 for detection of FcyRIIIA expression. FITC-conjugated isotype-matched antibodies served as negative controls. Cells then were analyzed in a FACScan cytometer (Becton Dickinson, CA) . HIV-gpl20 expression was analyzed by staining with mouse anti-gpl20 mAb or isotype negative control, followed by incubation with goat anti- mouse biotin F(ab^')₂, followed by allophycocyanin-streptavidin prior to analysis. Allophycocyanin-stained cells then were analyzed using a Becton Dickinson Facstar Plus.

Cytotoxic assays

Cytotoxicity was determined using a standard 4 hr . chromium-51 (⁵¹Cr) release assay (Matzinger (1991) Immunol. Methods, 145:185) with the following modifications (1991). 1 x 10⁶ target cells (Raji or Raji-gpl20) were incubated with 50 μCi of ⁵¹Cr in 50 μl of media for 2 hrs. at 371. Labeled target cells then were plated into 96-well plates (1 x 10⁴ cells per well) together with unmodified or CD4ζ^* NK3.3 cells at the target: effector ratios indicated and incubated at 37°C for 4 hrs. For control experiments demonstrating CD16-mediated ADCC, effector cells were pre-incubated with a saturating concentration (1/16 dilution) of rabbit anti-human lymphocyte serum for 30 minutes at 4°C prior to addition of target cells. At the end of the 4 hour incubation period, plates were spun at 600 rpm for 2 min. About 100 ul of supernatant were removed from each well and counted in a gamma counter for the assessment of ⁵¹Cr release. Percentage specific lysis was calculated from triplicate samples using the following formula: [ (CPM-SR) / (MR-SR) ] x 100. CPM = cpm released by targets incubated with effector cells, MR = cpm released by targets lysed with 100 μl of 1% Triton X-100 (i.e., maximum release) and SR = cpm released by targets incubated with medium only (i.e. spontaneous ) .

The CEM.NKR human T cell line is described in Byrn et al . (Nature 344:667, 1990). When uninfected or HIV-1 III_B-infected CEM.NKR T cells were employed as target cells, the JAM test was employed for measuring cell lysis (Matzinger, supra) and is based on the amount of [H] thymidine-labeled DNA retained by living cells. In brief, 1 x 10⁶ actively proliferating target cells were labeled with 20 uCi [³H] thymidine overnight. [³H] thymidine-labeled target cells were plated into 96-well plates (1 x 10⁴ cells per well) together with unmodified or CD4 ζ-expressing NK3.3 cells at the effector: target ratios (E:T) ratios indicated. After a 6 hour incubation period, cells were harvested and processed. Percentage specific lysis was calculated from triplicate samples using the following formula: [ (S - E) /S] x 100. E = experimentally retained DNA in the presence of CD8^* effector T cells (in cpm) and S = retained DNA in the absence of CD8^* effector T cells (spontaneous) .

Raji Transfectants Expressing gpl20

Raji is a human B cell lymphoma which expresses high levels of class II MHC. Raji cells expressing low levels of HIV env were generated by co-transfection with the expression vector, pCMVenv, which encodes rev and env (gpl60) from the HXB2 HIV-1 clone and the selection plasmid, pIKl . lneo which confers resistance to G418 (Roberts et al . , supra). G418-resistant clones were isolated and analyzed for expression of the env proteins gpl20 and gpl60 by immunoblotting with an anti-gpl20 mAb. Raji clones positive by immunoblotting then were subjected to FACS analysis to detect surface expression of gpl20.

Efficient Surface Expression of CD4ζ in Retrovirally Transduced NK Cells

The NK cell line 3.3 was originally isolated from human peripheral blood mononuclear cells (PBL). NK3.3 exhibits an NK characteristic cell surface phenotype (CD3^~, CD16^*) , and mediates strong natural killer activity. The CD4ζ chimeric receptor was introduced into NK3.3 cells by retroviral mediated transduction using the kat packaging system (Finer et al . , supra). After transduction, 26% of the transduced NK population expressed CD4 ζ as detected by immunofluorescence of surface CD4. A population in which greater than 85% of the cells expressed high surface levels of chimeric receptor was obtained after immunoaffinity purification of transduced NK cells with anti-CD4 mAb's. Unmodified and CD4ζ-modified NK3.3 cells express comparable levels of FcyRIIIA.

Tyrosine Phosphorylation Induced by CD4ζ Crosβlin ing on NK Cells

Several studies have shown that crosslinking of FcyRIIIA on NK cells induces the tyrosine phosphorylation of the ζ chain (O'Shea et al . (1991) Proc. Natl. Acad. Sci. USA, 88:350; and Vivier et al . (1991) J. Immunol., 146:206) as well as several additional cellular proteins (Liao et al . (1993) J. Immunol., 150:2668; Ting et al . (1992) J. Exp. Med., 176:1751; Azzoni et al. (1992) J. Exp. Med., 176:1745 and Salcedo et al. (1993) J. Exp. Med., 177:1475) . To evaluate the biochemical activity of the transduced chimeric receptor as compared to FcyRIIIA in NK cells, crosslinking of either receptor was achieved by incubating unmodified (NK) or CD4ζ-modified NK3.3 cells (CD4ζ^* NK) with either 0KT4A mAb to CD4 or 3G8 mAb to FcyRIIIA followed by sheep F(ab^')₂ antibodies to mouse IgG.

Both CD4ζ and native ζ were immunoprecipitated from the cell populations by treating cell lysates with anti-ζ serum and the immunoprecipitated supernatants were subsequently analyzed on immunoblots with an anti-phosphotyrosine antibody (4G10) . Tyrosine phosphorylation of CD4ζ, but not native ζ, is induced rapidly by crosslinking of the chimeric ζ receptor on NK cells . That result is consistent with previous studies conducted in T lymphocytes which have shown that crosslinking of chimeric ζ-receptors induces phosphorylation of the chimeric receptor, but not of native ζ present in T cell receptor (TCR) /CD3 complexes. As expected, crosslinking of FcyRIIIA induces rapid tyrosine phosphorylation of native ζ only, in both unmodified and CD4ζ-modified NK3.3 cells .

FcyRIIIA is thought to mediate cellular activation through a tyrosine kinase-dependent pathway, as indicated by the results of previous studies demonstrating rapid tyrosine phosphorylation of cellular proteins upon crosslinking of FcyRIIIA (Laio et al., supra; Ting et al . , supra; Azzoni et al . , supra; and Salcedo et al . , supra). Rapid tyrosine phosphorylation of cellular proteins with molecular masses of approximately 136, 112, 97 and 32 kDa is induced on crosslinking of either FcyRIIIA or CD4ζ receptors on CD4ζ/NK cells. The sizes of the proteins are similar to those previously reported as undergoing phosphorylation on crosslinking of FcyRIIIA (Liao et al . , supra and Ting et al . , supra) .

Similar results were observed for unmodified NK3.3 cells on crosslinking with mAb to FcyRIIIA, but not to CD4. Functional and physical interaction between the ζ subunit and protein kinases such as ZAP-70 and the src-related tyrosine kinase p56^lck is supported by observations in T cells (Karnitz et al . (1992) Mol . Cell Biol., 12:4521; Chan et al . (1992) Cell, 71:649 and Wange et al. (1992) J. Biol. Chem., 267:1685). For NK cells, similar functional associations between p56^lk and FcγRIII have been shown to be mediated through direct interaction with ζ (Azzoni et al . , supra and Salcedo et al . , supra), the subunit also acting as a substrate for p56^lclc in vitro.

The studies described above show that the CD4 ζ chimeric receptor is able to activate the tyrosine kinase signaling pathway in a manner analogous to the FcyRIIIA/ ζ complex in NK cells, presumably due to retention of functional interactions between such protein kinases and the ζ moiety of the chimeric receptor.

CD4ζ + NK Cells Mediate Cytolysis against Natural Killer-resistant Tumor Cells

The ability of CD4ζ to confer NK cells with the ability to kill a NK-resistant tumor cell line expressing low levels of gpl20 was evaluated to assess the anti-tumor potential of NK cells expressing chimeric ζ-receptors. Target cell lines expressing gpl20 were generated from the NK-resistant human Burkitt lymphoma cell line Raji by co-electroporation of pIKneo and pCMVenv. G418-resistant clones were isolated subsequently and analyzed for stable expression of the HIV env proteins gpl20 and gpl60 by western immunoblotting. To detect surface expression of gpl20, it was necessary to employ a highly sensitive allophycocyanin-streptavidin staining procedure with anti-gpl20 mAb.

Unmodified and CD4ζ-modified NK cells were evaluated functionally in a cytotoxicity assay against either normal Raji cells or Raji-gpl20 cells as targets, over a range of effector: target ratios. To compare the efficiency of chimeric receptor-mediated cytolytic activity with that of FcγRIIIA- mediated ADCC, CD4ζ^* NK cells also were tested for the ability to lyse normal Raji cells in the presence of rabb- *^■ anti-human lymphocyte serum.

The results of the studies show that whereas unmodified NK cells exhibit little or no activity toward Raji-gpl20 targets, NK cells expressing CD4ζ exhibit maximal specific lysis as high as 50% over background levels at effector: target ratios of between 25:1 to 50:1. The specific lysis observed is highly sensitive, with values of approximately 20% above background observed at effector: target ratios as low as 0.4:1. Furthermore, the efficiency of CD4ζ-mediated cytolysis appears to be more efficient than FcyRIIIA-mediated ADCC, at all effector to target ratios tested.

It was reported that both CD4ζ and scAbζ chimeric receptors efficiently redirect primary human CD8^* T lymphocytes to target HIV-infected cells (Roberts et al . , supra). It was therefore of interest to compare the cytolytic activity of CD4ζ^* NK cells to that of human PBMC-derived CD8^* T cells expressing CD4ζ (CD4ζ + CD8^* T cells) against the same Raji-gpl20 target cell line. The highly efficient cytolytic activity observed for CD4ζ + NK cells is comparable to that observed for CD4ζ + CD8^* T cells .

CD4ζ + NK Cells Mediate Cytolysis against HIV-infected T Cells

CD4ζ + NK cells can mount an efficient cytolytic response against HIV-infected CD4^* T cells. The NK-resistant CD4^* T cell line CEM.NKR was infected by HIV-1 IIIB as previously described (Byrn et al . (1990) Nature, 344:667). When uninfected (CEM) or HIV-infected CEM-NKR cells (CEM/IIIB) were used as targets in a cytotoxicity assay with unmodified or CD4ζ-modified NK cells as effectors, specific lysis of the virally infected population was observed at effector : target ratios as low as 1.5:1, with maximal lysis as high as 70% above background occurring at effector: target ratios of 50:1.

Since binds to non-polymorphic sites on MHC Class II molecules, one concern with the use of ζ as a chimeric receptor for re-directing NK-mediated cytotoxicity toward HIV-infected cells is the potential for lysis of cells expressing class II. However, despite the fact that Raji cells express high levels of class II MHC, no significant increase in cytolytic activity is observed against Raji cells when NK cells expressing ζ are employed, even at effector: target ratios as high as 50:1. The result is consistent with the notion that the relative affinity of the receptor for MHC class II molecules is inadequate to induce efficient cross-linking of the chimeric receptor, ζ.

Chimeric ζ-receptors in which the ligand binding domain is fused to the cytoplasmic domain of the signal transducing subunit ζ of FcyRIIIA and of TCR, are expressed at high levels on the surface of NK cells on retroviral-mediated transduction. Furthermore, the ζ chimeric receptor can direct NK cells to initiate a highly effective cytolytic response against natural killer-resistant tumor cells expressing low levels of the relevant target ligand gpl20 and against natural killer-resistant T cells infected with HIV. The cytolytic response is highly sensitive and appears comparable to that previously observed for CD4ζ^* and scAbζ^* CD8^* T lymphocytes.

All publications mentioned in the specification are herein incorporated by reference in its entirety to the same extent as if each individual publication was indicated specifically and individually to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims .

Claims

WHAT IS CLAIMED IS:

1. Chimeric DNA encoding a membrane bound protein comprising in reading frame :

DNA encoding a signal sequence; DNA encoding a portion of an antibody which specifically binds A33;

DNA encoding a transmembrane domain; and

DNA encoding a cytoplasmic signal-transducing domain of a protein that activates an intracellular messenger system.

2. DNA according to Claim 1, wherein said cytoplasmic domain is selected from the group consisting of the zeta chain, the eta chain, the CD3 gamma chain, the CD3 delta chain, the CD3 epsilon chain, the gamma chain of a Fc receptor, a tyrosine kinase, the cytoplasmic domain of CD2, the cytoplasmic domain of CD28 and the cytoplasmic domain of a growth factor receptor.

3. DNA according to Claim 2 , wherein the cytoplasmic domain is the gamma chain of the FceRl receptor.

4. DNA according to Claim 1 wherein said extracellular binding domain is the heavy chain of an immunoglobulin or truncated portion thereof.

5. DNA according to Claim 1, wherein said extracellular domain is a single-chain antibody, or portion thereof.

6. DNA according to Claim 2 wherein said cytoplasmic domain is zeta.

7. DNA according to Claim 1, wherein said cytoplasmic domain is CD28, CD2 or truncated portions thereof.

8. An expression cassette comprising a transcriptional initiation region, DNA according to Claim 1 under the transcriptional control of said transcriptional initiation region, and a transcriptional termination region.

9. An expression cassette according to Claim 8, wherein said transcriptional initiation region is functional in a mammalian host.

10. A retroviral RNA or DNA construct comprising an expression cassette according to Claim 9.

11. A cell comprising DNA according to Claim 1.

12. A cell comprising DNA according to Claim 8.

13. A cell according to Claim 11 or 12, wherein said cytoplasmic domain is the zeta chain.

14. A cell according to Claim 11 or 12, wherein said extracellular domain is the heavy chain of an immunoglobulin or truncated portion thereof .

15. A cell according to Claim 11 or 12, wherein said extracellular domain is a single chain antibody or portion thereof.

16. A cell according to Claim 12, wherein said transcriptional initiation region is functional in a mammalian cell and said cell is a mammalian cell.

17. A cell according to Claim 16, wherein said mammalian cell is a human cell.

18. A cell according to claim 17 wherein said cell is a hematopoietic cell.

19. A cell according to claim 17 wherein said cell is a hematopoietic stem cell.

20. A cell according to claim 17 wherein said cell is a T cell.

21. A chimeric protein comprising in the N-terminal to C- terminal direction: a portion of an antibody which binds A33; a transmembrane domain; and a cytoplasmic signal-transducing domain of a protein that activates an intracellular messenger system.

22. A protein according to Claim 21, wherein said cytoplasmic domain is selected from the group consisting of the zeta chain, the eta chain, the CD3 gamma chain, the CD3 delta chain, the CD3 epsilon chain, the gamma chain of a Fc receptor, a tyrosine kinase, the cytoplasmic domain of CD2, the cytoplasmic domain of CD28 and the cytoplasmic domain of a growth factor receptor.

23. A protein according to Claim 22, wherein the cytoplasmic domain is the zeta chain .

24. A protein according to Claim 21 wherein said extracellular binding domain is the heavy chain of an immunoglobulin or truncated portion thereof.

25. A protein according to claim 21 wherein said extracellular binding domain is a single-chain antibody, or portion thereof.

26. A mammalian cell comprising as a surface membrane protein, a protein according to Claim 21.

27. The mammalian cell according to Claim 26, wherein said mammalian cell is a human cell,

27. The mammalian cell of claim 26 wherein said cell is a hematopoietic stem cell.

28. The mammalian cell according to claim 26 wherein said cell is a hematopoietic cell .

29. The mammalian cell according to claim 28 wherein said cell is a T cell.

30. A method of activating cells by means of a secondary messenger pathway, said method comprising: contacting cells comprising as a surface membrane protein, the protein of claim 21, with a cell expressing A33.

31. A method for producing a source of cytotoxic effector cells for killing cells expressing A33 antigen comprising introducing the DNA sequence of claim 1 into cells to form modified cells expressing a protein encoded by said DNA.

32. A method for producing a source of cytotoxic effector cells for killing cells expressing A33 antigen comprising introducing the DNA sequence of claim 1 into cells to form modified cells expressing a protein encoded by said DNA and transplanting said modified cells into a mammal.

33. A method for producing a source of cytokine producer cells comprising introducing the DNA sequence of claim 1 into cells to form modified cells expressing a protein encoded by said DNA.

34. A method for producing a source of cytokine producer cells comprising introducing the DNA sequence of claim 1 into cells to form modified cells expressing a protein encoded by said DNA and transplanting said modified cells into a mammal.

35. The method of claim 31, 32, 33 and 34 wherein said cells are hematopoietic cells.

36. The method of claim 31, 32, 33 or 34, wherein said cells are human cells.

37. The method of claim 35, wherein said hematopoietic cells are stem cells.

38. The method of claim 35 wherein said hematopoietic cells are T cells.

39. The method of claim 31, 32, 33 or 34 and 32 wherein said extracellular domain is a single-chain antibody, and said cytoplasmic domain is zeta.

40. The method of claim 35 wherein said modified hematopoietic stem cells are transplanted by bone marrow transplantation into said mammal .