Chimeric Immunoreceptor Useful in Treating Human Cancers

Abstract

The present invention relates to chimeric transmembrane immunoreceptors, named zetakines, comprised of an extracellular domain comprising a soluble receptor ligand linked to a support region capable of tethering the extracellular domain to a cell surface, a transmembrane region and an intracellular signalling domain. Zetakines, when expressed on the surface of T lymphocytes, direct T cell activity to those specific cells expressing a receptor for which the soluble receptor ligand is specific. Zetakine chimeric immunoreceptors represent a novel extension of antibody-based immunoreceptors for redirecting the antigen specificity of T cells, with application to treatment of a variety of cancers, particularly via the autocrin/paracrine cytokine systems utilized by human maligancy. In a preferred embodiment is a glioma-specific immunoreceptor comprising the extracellular targetting domain of the IL-13R2-specific IL-13 mutant IL-13(E13Y) linked to the Fc region of IgG, the transmembrane domain of human CD4, and the human CD3 zeta chain.

Claims

1. A chimeric immunoreceptor comprising SEQ ID NO:22.

2. A chimeric immunoreceptor encoded by a nucleic acid sequence comprising SEQ ID NO:19 or SEQ ID NO:23.

3. A method for treating human cancer, comprising administering to a human suffering from a glioma that overexpresses IL132 receptor a plurality of T lymphocyte cells expressing an immunoreceptor of claim 1 or claim 2.

4. A vector which comprises a nucleic acid comprising SEQ ID NO: 19 or SEQ ID NO:23.

5. The vector which consists essentially of SEQ ID NO:19.

6. The vector which consists essentially of SEQ ID NO:23.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1: Results of a Western Blot showing that the IL13zetakine Chimeric Immunoreceptor is expressed as an intact glycosylated protein in Jurkat T cells.

[0022] FIGS. 2A through 2B: Results of flow cytometric analysis showing that expressed IL13zetakine chimeric immunoreceptor trafficks to the cell-surface as a type I transmembrane protein.

[0023] FIGS. 3A through 3C: Results of flow cytometric analysis showing the cell surface phenotype of a representative primary human IL13zetakine.sup.+ CTL clone.

[0024] FIGS. 4A through 4F: Results of chromium release assays. FIG. 4A shows that the IL13zetakine.sup.+ CTL clone acquired glioma-specific re-directed cytolytic activity, and FIG. 4B shows the profile of anti-glioma cytolytic activity by primary human IL13zetakine.sup.+ CD8.sup.+ CTL clones was observed in glioma cells generally.

[0025] FIGS. 5A through 5C: Results of in vitro stimulation of cytokine production, showing that IL13zetakine.sup.+ CTL clones are activated for cytokine production by glioma stimulator cells.

[0026] FIGS. 6A through 6C: Results of in vitro stimulation of cytokine production (FIG. 6A, IFN; FIG. 6B, TNF; FIG. 6C, GM-CSF), showing the specific inhibition of IL13zetakine.sup.+ CTL activation for cytokine production by anti-IL13R Mab and rhIL13.

[0027] FIGS. 7A through 7B: Results of growth studies. FIG. 7A shows that IL13zetakine.sup.+ CD8.sup.+ CTL cells proliferate upon co-culture with glioma stimulators, and FIG. 7B shows the inhibition of glioma-stimulated proliferation of IL13zetakine.sup.+ CD8.sup.+ CTL cells by rhIL-13.

[0028] FIGS. 8A through 8C: Flow chart of the construction of IL13zetakine/HyTK-pMG (FIG. 8A, constructoin fo hsp-IL13-IgG4 (SmP)-hinge-Fe-Zeta; FIG. 8B, construction of IL13-Fc; 3pMBPac; FIG. 8C, construction of Il13/HyTK-pMG).

[0029] FIG. 9: Plasmid map of IL13zetakine/HyTK-pMG.

[0030] FIG. 10: Plasmid map of alternative IL13zetakine/HyTK-pMG.

[0031] FIG. 11: Schematic diagram showing structure of IL13 zetakine insert.

[0032] FIGS. 12A through 12I: Nucleic acid sequence of a plasmid DNA vector (upper strand: SEQ ID NO:24; lower strand: SEQ ID NO:25) and the corresponding amino acid sequence of IL13zetakine (SEQ ID NO:17) and HyTK (SEQ ID NO:18).

[0033] FIGS. 13A through 13I: Nucleic acid sequence of an alternate plasmid DNA vector (upper strand: SEQ ID NO:19; lower strand: SEQ ID NO:20) and the corresponding amino acid sequence of IL13zetakine (SEQ ID NO:22) and HyTK (SEQ ID NO:21).

[0034] FIGS. 14A through 14C: Nucleic acid sequence of an alternate plasmid DNA vector (SEQ ID NO:23).

[0035] FIGS. 15A through 15H: Nucleic acid sequence of an alternate plasma DNA vector (upper strand: SEQ ID NO:14; lower strand: SEQ ID NO:16) and the corresponding amino and sequence of IL 13zetakine (SEQ ID NO:17) and HyTK (SEQ ID NO:18).

DETAILED DESCRIPTION

[0036] An ideal cell-surface epitope for tumor targeting with genetically-engineered re-directed T cells would be expressed solely on tumor cells in a homogeneous fashion and on all tumors within a population of patients with the same diagnosis. Modulation and/or shedding of the target molecule from the tumor cell membrane may also impact on the utility of a particular target epitope for re-directed T cell recognition. To date few ideal tumor-specific epitopes have been defined and secondary epitopes have been targeted based on either lack of expression on critical normal tissues or relative over-expression on tumors. In the case of malignant glioma, the intracavitary administration of T cells for the treatment of this cancer permits the expansion of target epitopes to those expressed on tumor cells but not normal CNS with less stringency on expression by other tissues outside the CNS. The concern regarding toxicity from cross-reactivity of tissues outside the CNS is mitigated by a) the sequestration of cells in the CNS based on the intracavitary route of administration and b) the low cell numbers administered in comparison to cell doses typically administered systemically.

[0037] The IL-13R2 receptor stands out as the most ubiquitous and specific cell-surface target for malignant glioma.sup.47. Sensitive autoradiographic and immunohistochemical studies fail to detect IL-13 receptors in the CNS.sup.46; 48. Moreover, mutation of the IL-13 cytokine to selectively bind the glioma-restricted IL-13R2 receptor is a further safeguard against untoward reactivity of IL-13-directed therapeutics against IL-13R1/IL-4+ normal tissues outside the CNS.sup.55; 57. The potential utility of targeting glioma IL-13R2 the design and testing of a novel engineered chimeric immunoreceptor for re-directing the specificity of T cells that consists of an extracellular IL-13 mutant cytokine (E13Y) tethered to the plasma membrane by human IgG4 Fc which, in turn, is fused to CD4TM and the cytoplasmic tail of CD3 zeta. This chimeric immunoreceptor has been given the designation of IL-13 zetakine. The IL-13R2 receptor/IL-13(E13Y) receptor-ligand pair is an excellent guide for understanding and assessing the suitability of receptor-ligand pairs generally for use in zetakines. An ideal zetakine comprises an extracellular soluble receptor ligand having the properties of IL-13(E13Y) (specificity for a unique cancer cell surface receptor, in vivo stability due to it being derived from a naturally-occurring soluble cell signal molecule, low immunogenicity for the same reason). The use of soluble receptor ligands as distinct advantages over the prior art use of antibody fragments (such as the scFvFc immunoreceptors) or cell adhesion molecules, in that soluble receptor ligands are more likely to be stable in the extracellular environment, non-antigenic, and more selective.

[0038] Chimeric immunoreceptors according to the present invention comprise an extracellular domain comprised of a soluble receptor ligand linked to an extracellular support region that tethers the ligand to the cell surface via a transmembrane domain, in turn linked to an intracellular receptor signaling domain. Examples of suitable soluble receptor ligands include autocrine and paracrine growth factors, chemokines, cytokines, hormones, and engineered artificial small molecule ligands that exhibit the required specificity. Natural ligand sequences can also be engineered to increase their specificity for a particular target cell. Selection of a soluble receptor ligand for use in a particular zetakine is governed by the nature of the target cell, and the qualities discussed above with regard to the IL-13(E13Y) molecule, a preferred ligand for use against glioma. Examples of suitable support regions include the constant (Fc) regions of immunoglobins, human CD8, and artificial linkers that serve to move the targeting moiety away from the cell surface for improved access to receptor binding on target cells. A preferred support region is the Fc region of an IgG (such as IgG4). Examples of suitable transmembrane domains include the transmembrane domains of the leukocyte CD markers, preferably that of CD8. Examples of intracellular receptor signaling domains are those of the T cell antigen receptor complex, preferably the zeta chain of CD3 also Fc Rill costimulatory signaling domains, CD28, DAP10, CD2, alone or in a series with CD3zeta.

[0039] In the IL-13 zetakine embodiment, the human IL-13 cDNA having the E13Y amino acid substitution was synthesized by PCR splice overlap extension. A full length IL-13 zetakine construct was assembled by PCR splice overlap extension and consists of the human GM-CSF receptor alpha chain leader peptide, IL-13(E13Y)-Gly-Gly-Gly, human IgG4 Fc, human CD4TM, and human cytoplasmic zeta chain. This cDNA construct was ligated into the multiple cloning site of a modified pMG plasmid under the transcriptional control of the human Elongation Factor-1alpha promoter (Invivogen, San Diego). This expression vector co-expresses the HyTK cDNA encoding the fusion protein HyTK that combines in a single molecule hygromycin phosphotransferase activity for in vitro selection of transfectants and HSV thymidine kinase activity for in vivo ablation of cells with ganciclovir from the CMV immediate/early promoter. Western blot of whole cell Jurkat lysates pre-incubated with tunicamycin, an inhibitor of glycosylation, with an anti-zeta antibody probe demonstrated that the expected intact 56-kDa chimeric receptor protein is expressed. This receptor is heavily glycosylated consistent with post-translational modification of the native IL-13 cytokine.sup.108. Flow cytometric analysis of IL-13 zetakine+ Jurkat cells with anti-human IL-13 and anti-human Fc specific antibodies confirmed the cell-surface expression of the IL-13 zetakine as a type I transmembrane protein.

[0040] Using established human T cell genetic modification methods developed at City of Hope.sup.107, primary human T cell clones expressing the IL-13 zetakine chimeric immunoreceptor have been generated for pre-clinical functional characterization. IL-13 zetakine+ CD8+ CTL clones display robust proliferative activity in ex vivo expansion cultures. Expanded clones display re-directed cytolytic activity in 4-hr chromium release assays against human IL-13R2+ glioblastoma cell lines. The level of cytolytic activity correlates with levels of zetakine expression on T cells and IL-13R2 receptor density on glioma target cells. In addition to killing, IL-13 zetakine+ clones are activated for cytokine secretion (IFN-, TNF-, GM-CSF). Activation was specifically mediated by the interaction of the IL-13 zetakine with the IL-13R2 receptor on glioma cells since CTL clones expressing an irrelevant chimeric immunoreceptor do not respond to glioma cells, and, since activation can be inhibited in a dose-dependent manner by the addition to culture of soluble IL-13 or blocking antibodies against IL-13 on T cell transfectants and IL-13R2 on glioma target cells. Lastly, IL-13 zetakine-expressing CD8+ CTL clones proliferate when stimulated by glioma cells in culture. IL-13 zetakine+ CTL clones having potent anti-glioma effector activity will have significant clinical activity against malignant gliomas with limited collateral damage to normal CNS.

[0041] An immunoreceptor according to the present invention can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. A nucleic acid sequence encoding the several regions of the chimeric receptor can prepared and assembled into a complete coding sequence by standard techniques of molecular cloning (genomic library screening, PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte cell line, and most preferably an autologous T lymphocyte cell line. A third party derived T cell line/clone, a transformed humor or xerogenic immunologic effector cell line, for expression of the immunoreceptor. NK cells, macrophages, neutrophils, LAK cells, LIK cells, and stem cells that differentiate into these cells, can also be used. In a preferred embodiment, lymphocytes are obtained from a patient by leukopharesis, and the autologous T cells are transduced to express the zetakine and administered back to the patient by any clinically acceptable means, to achieve anti-cancer therapy.

[0042] Suitable doses for a therapeutic effect would be between about 10.sup.6 and about 10.sup.9 cells per dose, preferably in a series of dosing cycles. A preferred dosing regimen consists of four one-week dosing cycles of escalating doses, starting at about 10.sup.7 cells on Day 0, increasing incrementally up to a target dose of about 10.sup.8 cells by Day 5. Suitable modes of administration include intravenous, subcutaneous, intracavitary (for example by reservoir-access device), intraperitoneal, and direct injection into a tumor mass.

[0043] The following examples are solely for the purpose of illustrating one embodiment of the invention.

Example 1

Construction of an Immunoreceptor Coding Sequence

[0044] The coding sequence for an immunoreceptor according to the present invention was constructed by de novo synthesis of the IL13(E13Y) coding sequence using the following primers (see FIGS. 8A-8C for a flow chart showing the construction of the immunoreceptor coding sequence and expression vector):

TABLE-US-00001 IL13P1: (SEQIDNO.1) EcoRI TATGAATTCATGGCGCTTTTGTTGACCACGGTCATTGCTCTCACTTGCCT TGGCGGCTTTGCCTCCCCAGGCCCTGTGCCTCCCTCTACAGCCCTCAGGT AC IL13P2: (SEQIDNO.2) GTTGATGCTCCATACCATGCTGCCATTGCAGAGCGGAGCCTTCTGGTTCT GGGTGATGTTGACCAGCTCCTCAATGAGGTACCTGAGGGCTGTAGAGGGA G IL13P3: (SEQIDNO.3) CTCTGGGTCTTCTCGATGGCACTGCAGCCTGACACGTTGATCAGGGATTC CAGGGCTGCACAGTACATGCCAGCTGTCAGGTTGATGCTCCATACCATGC IL13P4: (SEQIDNO.4) CCTCGATTTTGGTGTCTCGGACATGCAAGCTGGAAAACTGCCCAGCTGAG ACCTTGTGCGGGCAGAATCCGCTCAGCATCCTCTGGGTCTTCTCGATGGC IL13P5: (SEQIDNO.5) BamHI TCGGATCCTCAGTTGAACCGTCCCTCGCGAAAAAGTTTCTTTAAATGTAA GAGCAGGTCCTTTACAAACTGGGCCACCTCGATTTTGGTGTCTCGG

[0045] The final sequence (417 bp) was end-digested with EcoRI-BamHI, and ligated into the plasmid pSK (stratagene, LaJolla, Calif.) as ligation 312#3. Ligation 312#3 was mutagenized (stratagene kit, per manufacturer's instructions) to fix a deleted nucleotide using the primers 5: IL13 312#3 mut5-3 (CAACCTGACAGCTGGCATGTACTGTGCAGCCCTGGAATC (SEQ ID NO. 6)) and 3:IL13 312#3 mut3-5 (GATTCCAGGGCTGCACAGTACATGCCAGCTGTCAGGTTG (SEQ ID NO. 7)), and ligation 312#3 as a template, to form ligation 348#1 (IL13zetakine/pSK).

[0046] The coding Human GM-CSFR alpha chain Signal Peptide (hsp) coding sequence was fused to the 5 end of IL13(E13Y) by standard PCR splice overlap extension. The hsp sequence (101 bp) was obtained from the template ligation 301#10 (hsp/pSK) (human GCSF receptor -chain leader sequence from human T cell cDNA), using the primers 5:19hsp5 (ATCTCTAGAGCCGCCACCATGCTTCTCCTGGTGACAAGCCTTC (SEQ ID NO. 8)) (XbaI site highlighted in bold), and 3: hsp-IL13FR (GAGGGAGGCACAGGGCCTGGGATCAGGAGGAATG (SEQ ID NO. 9)). The IL-13 sequence (371 bp) was obtained using the primers 5: hsp-IL13FF (CATTCCTCCTGATCCCAGGCCCTGTGCCTCCCTC (SEQ ID NO. 10)) and 3: IL13-IgG4FR (GGGACCATATTTGGACTCGTTGAACCGTCCCTCGC (SEQ ID NO. 11)), and ligation 312#3 as template. Fusion was achieved using the 101 bp hsp sequence and 371 bp IL13 sequence thus obtained, and the primers 5: 19hsp5 and 3: IL13-IgG4FR, to yeild a 438 bp fusion hsp-IL13 sequence.

[0047] A sequence encoding the IgG4 Fc region IgG4m:zeta was fused to the 3 end of the hsp-IL13 fusion sequence using the same methods. The IgG4m:zeta sequence (1119 bp) was obtained using the primers 5: IL13-IgG4FF (GCGAGGGACGGTTCAACGAGTCCAAATATGGTCCC (SEQ ID NO. 12)) and 3: ZetaN3 (ATGCGGCCGCTCAGCGAGGGGGCAGG (SEQ ID NO. 13)) (NotI site highlighted in bold), using the sequence R9.10 (IgG4mZeta/pSK) as template. The 1119 bp IgG4m:zeta sequence was fused to the hsp-IL13 fusion sequence using the respective sequences as templates, and the primers 5: 19hsp5 and 3: ZetaN3, to yeild a 1522 bp hsp-IL13-IgG4m:zeta fusion sequence. The ends were digested with XbaI-NotI, and ligated into pSK as ligation 351#7, to create the plasmid IL13zetakine/pSK (4464 bp).

Example 2

Construction of Expression Vector

[0048] An expression vector containing the IL13 zetakine coding sequence was created by digesting the IL13zetakine/pSK of Example 1 with XbaI-NotI, and creating blunt ends with Klenow, and ligating the resulting fragment into the plasmid pMGPac (Invirogen) (first prepared by opening with SgrAI, blunting with Klenow, and dephosphorylation with SAP), to yield the plasmid IL13zetakine/pMG. See FIGS. 8A-8C. The hygromycin resistance region of IL13zetakine/pMG was removed by digestion with NotI-NheI, and replaced by the selection/suicide fusion HyTK, obtained from plasmid CE7R/HyTK-pMG (Jensen, City of Hope) by digestion with NotI-NheI, to create the expression vector IL13zetakine/HyTK-pMG (6785 bp). This plasmid comprises the Human Elongation Factor-1 promoter (hEF1p) at bases 6-549, the IL13zetakine coding sequence at bases 692-2185, the Simian Virus 40 Late polyadenylation signal (Late SV40pAN) at bases 2232-2500, a minimal E. coli origin of replication (Ori ColE1) at bases 2501-3247, a synthetic poly A and Pause site (SpAN) at bases 3248-3434, the Immeate-early CMV enhancer/promoter (h CMV-1Aprom) at bases 3455-4077, the Hygromycin resistance-Thymidine kinase coding region fusion (HyTK) at bases 4259-6334, and the bovine growth hormone polyadenylation signal and a transcription pause (BGh pAn) at bases 6335-6633. The plasmid has a PacI linearization site at bases 3235-3242. The hEF1p and IL13zetakine elements derived from IL13zetakine/pMG, and the remaining elements derived from CE7R/HyTk-pMG (and with the exception of the HyTK element, ultimately from the parent plasmid pMGPac). In sum, IL13zetakine/HyTK-pMG is a modified pMG backbone, expressing the IL13zetakine gene from the hEF1 promoter, and the HyTK fusion from the h CMV-1A promoter. A map of the plasmid IL13zetakine/HyTK-pMG appears in FIG. 9. The full nucleic acid sequence of the plasmid is shown in FIGS. 12A-12I. The sequence of an IL13zetakine insert is given as SEQ ID NO:15, below. See also FIG. 11.

TABLE-US-00002 (SEQIDNO:15) atgcttctcctggtgacaagccttctgctctgtgagttaccacacccag cattcctcctgatcccaggccctgtgcctccctctacagccctcaggta cctcattgaggagctggtcaacatcacccagaaccagaaggctccgctc tgcaatggcagcatggtatggagcatcaacctgacagctggcatgtact gtgcagccctggaatccctgatcaacgtgtcaggctgcagtgccatcga gaagacccagaggatgctgagcggattctgcccgcacaaggtctcagct gggcagttttccagcttgcatgtccgagacaccaaaatcgaggtggccc agtttgtaaaggacctgctcttacatttaaagaaactttttcgcgaggg acggttcaacgagtccaaatatggtcccccatgcccaccatgcccagca cctgagttcctggggggaccatcagtcttcctgttccccccaaaaccca aggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggt ggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggat ggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttca acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactg gctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccg tcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagc cacaggtgtacaccctgcccccatcccaggaggagatgaccaagaacca ggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgcc gtggagtgggagagcaatgggcagccggagaacaactacaagaccacgc ctcccgtgctggactccgacggctccttcttcctctacagcaggctaac cgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtg atgcatgaggctctgcacaaccactacacacagaagagcctctccctgt ctctgggtaaaatggccctgattgtgctggggggcgtcgccggcctcct gcttttcattgggctaggcatcttcttcagagtgaagttcagcaggagc gcagacgcccccgcgtaccagcagggccagaaccagctctataacgagc tcaatctaggacgaagagaggagtacgatgttttggacaagagacgtgg ccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaa ggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtg agattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcct ttaccagggtctcagtacagccaccaaggacacctacgacgcccttcac atgcaggccctgccccctcgc.

Example 3

Expression of the Immunoreceptor

[0049] Assessment of the integrity of the expressed construct was first delineated by Wester blot probed with an anti-zeta antibody of whole cell lysates derived from Jurkat T cell stable transfectants.sup.107 cocultured in the presence or absence of tunicamycin, an inhibitor of glycosylation. FIG. 1. Jurkat T cell stable transfectants (Jurkat-IL13-pMG bulk line) were obtained by electroporating Jurkat T cells with the IL13zetakine/HyTK-pMG expression vector, followed by selection and expansion of positive transfectants. 210.sup.6 cells from the Jurkat-IL13-pMG bulk line were plated per well in a 24-well plate with or without 5 g/ml, 10 g/ml, or 20 g/ml Tunicamycin. The plate was incubated at 37 C. for 22 hrs. Cells were harvested from each well, and each sample was washed with PBS and resuspended in 50 l RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 tablet/10 ml Complete Protease Inhibitor Cocktail (Boehringer Mannheim, Indianapolis, Ind.). Samples were incubated on ice for 30 minutes then disrupted by aspiration with syringe with 21 gauge needle then incubated on ice for an additional 30 minutes before being centrifuged at 4 C. for 20 minutes at 14,000 rpm. Samples of centrifuged lysate supernatant were harvested and boiled in an equal volume of sample buffer under reducing conditions, then subjected to SDS-PAGE electrophoresis on a 12% acrylamide gel. Following transfer to nitrocellulose, membrane was allowed to dry O/N at 4 C. Next morning, membrane was blocked in a Blotto solution containing 0.04 gm/ml non-fat dried milk in T-TBS (0.02% Tween 20 in Tris buffered saline pH 8.0) for 1 hour. Membrane was then incubated with primary mouse anti-human CD3 monoclonal antibody (Pharmingen, San Diego, Calif.) at a concentration of 1 g/ml for 2 hours, washed, and then incubated with a 1:3000 dilution (in Blotto solution) of goat anti-mouse IgG alkaline phosphatase conjugated secondary antibody (Bio-Rd ImmunoStar Kit, Hercules, Calif.) for 1 hour. Prior to developing, membrane was washed 4 additional times in T-TBS, and then incubated with 3 ml of phosphatase substrate solution (Biorad ImmunoStar Kit, Hercules, Calif.) for 5 minutes at room temperature. Membrane was then covered with plastic, and exposed to x-ray film. Consistant with the known glycosylation pattern of wild-type human IL-13, the electrophoretic mobility of expressed IL-13(E13Y) zetakine is demonstrative of a heavily glycosylated protein which, when expressed in the presence of tunicamycin, is reduced to an amino acid backbone of approximately 54 kDa.

[0050] The IL-13(E13Y) zetakine traffics to the cell surface as a homodimeric type I transmembrane protein, as evidenced by flow cytometric analysis of transfectants with a phycoerythrin (PE)-conjugated anti human-IL13 monoclonal antibody and a fluorescein isothiocyanate (FITC)-conjugated mouse anti-human Fc (gamma) fragment-specific F(ab)2 antibody. FIGS. 2A-2B. Jurkat IL13zetakine-pMG transfectants were stained with anti-human Fc(FITC) antibody (Jackson ImmunoResearch, West Grove, Pa.), recombinant human IL13R2/human IgG1 chimera (R&D Systems, Minneapolis, Minn.) followed by FITC-conjugated anti human-IgG1 monoclonal antibody (Sigma, St. Louis, Mo.), and an anti-IL13 (PE) antibody (Becton Dickinson, San Jose, Calif.) for analysis of cell surface chimeric receptor expression. Healthy donor primary cells were also stained with FITC-conjugated anti-CD4, anti-CD8, anti-TCR, and isotype control monoclonal antibodies (Becton Dickinson, San Jose, Calif.) to assess cell surface phenotype. For each stain, 10.sup.6 cells were washed and resuspended in 100 l of PBS containing 2% FCS, 0.2 mg/ml NaN.sub.3, and 5 l of stock antibody. Following a 30 minute incubation at 4 C., cells were washed twice and either stained with a secondary antibody, or resuspended in PBS containing 1% paraformaldehyde and analyzed on a FACSCaliber cytometer.

Example 4

Binding of IL13(E13Y) Zetakine to IL13R2 Receptor

[0051] IL-13(E13Y), tethered to the cell membrane by human IgG4 Fc (i.e., IL13(E13Y) zetakine), is capable of binding to its target IL13R2 receptor as assessed by flow cytometric analysis using soluble IL13R2-Fc fusion protein. FIGS. 3A-3C. Cloned human PBMC IL13zetakine-pMG transfectants were obtained by electroporating PBMC with the IL13zetakine/HyTK-pMG expression vector, followed by selection and expansion of positive transfectants.sup.107. IL13zetakine.sup.+ CTL clonal cells were stained with a fluorescein isothiocyanate (FITC)-conjugated mouse anti-human Fc (gamma) fragment-specific F(ab)2 (Jackson ImmunoResearch, West Grove, Pa.), recombinant human IL13R2/human IgG1 chimera (R&D Systems, Minneapolis, Minn.) followed by FITC-conjugated anti human-IgG1 monoclonal antibody (Sigma, St. Louis, Mo.), and a phycoerythrin (PE)-conjugated anti human-IL13 monoclonal antibody (Becton Dickinson, San Jose, Calif.) for analysis of cell surface chimeric receptor expression. Healthy donor primary cells were also stained with FITC-conjugated anti-CD4, anti-CD8, anti-TCR, and isotype control monoclonal antibodies (Becton Dickinson, San Jose, Calif.) to assess cell surface phenotype. For each stain, 10.sup.6 cells were washed and resuspended in 100 l of PBS containing 2% FCS, 0.2 mg/ml NaN.sub.3, and 5 l of antibody. Following a 30 minute incubation at 4 C., cells were washed twice and either stained with a secondary antibody, or resuspended in PBS containing 1% paraformaldehyde and analyzed on a FACSCaliber cytometer.

[0052] Next, the immunobiology of the IL-13(E13Y) zetakine as a surrogate antigen receptor for primary human T cells was evaluated. Primary human T cells were electroporated with the plasmid expression vector. Positive transformants were selected with hygromycin, cloned in limiting dilution, then expanded by recursive stimulation cyles with OKT3, IL-2 and irradiated feeder cells. Clones demonstrating IL 13zetakine expression by Western blot and FACS were then subjected to functional evaluation in 4-hr chromium release assays against a variety of IL-132.sup.+/CD20.sup. glioma cell lines (U251, SN-B19, U138), and the IL-13.sup./CD20.sup.+ B cell lymphocyte line Daudi). These tests showed that IL13zetakine conferred cytolytic activity that was specific for glioma cells (FIG. 4A), and that this specific cytolytic activity is present for glioma cells as a class (FIG. 4B). The cytolytic activity of MJ-IL13-pMG clones was assayed by employing .sup.51Cr-labeled SN-B19, U251, and U138 glioma cell lines (IL132+/CD20) and Daudi (CD20+/IL132) as targets. MJ-IL13 effectors were assayed 8-12 days following stimulation. Effectors were harvested, washed, and resuspeded in assay media: 2.510.sup.5, 1.2510.sup.5, 2.510.sup.4, and 510.sup.3 effectors were cultured in triplicate at 37 C. for 4 hours with 510.sup.3 target cells in 96-well V-bottom microtiter plates. After incubation, 100 l aliquots of cell-free supernatant were harvested and .sup.51Cr in the supernatants was assayed with a -counter. Percent specific cytolysis was calculated as follows:

[00001]$\frac{\left({\mathrm{Experimental}.Math.}^{51}.Math.\mathrm{Cr}.Math..Math.\mathrm{release}\right)-\left({\mathrm{control}.Math.}^{51}.Math.\mathrm{Cr}.Math..Math.\mathrm{release}\right)}{\left({\mathrm{Maximum}.Math.}^{51}.Math.\mathrm{Cr}.Math..Math.\mathrm{release}\right)-\left({\mathrm{control}.Math.}^{51}.Math.\mathrm{Cr}.Math..Math.\mathrm{release}\right)}100$

Control wells contained target cells incubated in the presence of target cells alone. Maximum .sup.51Cr release was determined by measuring the .sup.51Cr released by labeled target cells in the presence of 2% SDS. Bulk lines of stabley transfected human T cells consisting of approximately 40% IL-13(E13Y) zetakine.sup.+ TCR/.sup.+ lymphocytes displayed re-directed cytolysis specific for 13R2.sup.+ glioma targets in 4-hr chromium release assays (>50% specific lysis at E:T ratios of 25:1), with negligable acitivity against IL-13R2.sup. targets (<8% specific lysis at E:T ratios of 25:1). IL-13(E13Y) zetakine+CD8+TCR/.sup.+ CTL clones selected on the basis of high-level binding to anti-IL-13 antibody also display redirected IL13R2-specific glioma cell killing. FIG. 4b.

[0053] IL-13 zetakine-expressing CD8.sup.+ CTL clones are activated and proliferate when stimulated by glioma cells in culture. FIGS. 5A-5C, 6A-6C, 7A-7B. MJ-IL13-pMG Cl. F2 responder cells expressing the IL13 zetakine were evaluated for receptor-mediated triggering of IFN, GM-CSF, and TNF production in vitro. 210.sup.6 responder cells were co-cultured in 24-well tissue culture plates with 210.sup.5 irradiated stimulator cells (Daudi, Fibroblasts, Neuroblastoma 10HTB, and glioblastoma U251) in 2 ml total. Blocking rat anti-human-IL13 monoclonal antibody (Pharmingen, San Diego, Calif.), recombinant human IL13 (R&D Systems, Minneapolis, Minn.), and IL13R2-specific goat IgG (R&D Systems, Minneapolis, Minn.) were added to aliquots of U251 stimulator cells (210.sup.5/ml) at concentrations of 1 ng/ml, 10 ng/ml, 100 ng/ml, and 1 g/ml, 30 minutes prior to the addition of responder cells. Plates were incubated for 72 hours at 37 C., after which time culture supernatants were harvested, aliquoted, and stored at 70 C. ELISA assays for IFN, GM-CSF, and TNF were carried out using the R&D Systems (Minneapolis, Minn.) kit per manufacturer's instructions. Samples were tested in duplicate wells undiluted or diluted at 1:5 or 1:10. The developed ELISA plate was evaluated on a microplate reader and cytokine concentrations determined by extrapolation from a standard curve. Results are reported as picograms/ml, and show strong activation for cytokine production by glioma stimulator cells. FIGS. 5A-5C, FIGS. 6A-6C.

[0054] Lastly, IL-2 independent proliferation of IL13zetakine.sup.+ CD8.sup.+ CTL was observed upon co-cultivation with glioma stimulators (FIG. 7A), but not with IL13 R2 stimulators. Proliferation was inhibited by the addition of rhIL-13 antibody (FIG. 7B), showing that the observed proliferation was dependant on binding of zetakine to the IL-13R2 glioma cell-specific receptor.

Example 5

Preparation of IL-13 Zetakine.SUP.+ T Cells Suitable for Therapeutic Use

[0055] The mononuclear cells are separated from heparinized whole blood by centrifugation over clinical grade Ficoll (Pharmacia, Uppsula, Sweden). PBMC are washed twice in sterile phosphate buffered saline (Irvine Scientific) and suspended in culture media consisting of RPMI 1640 HEPES, 10% heat inactivated FCS, and 4 mM L-glutamine. T cells present in patient PBMC are polyclonally activated by addition to culture of Orthoclone OKT3 (30 ng/ml). Cell cultures are then incubated in vented T75 tissue culture flasks in the study subject's designated incubator. Twenty-four hours after initiation of culture rhIL-2 is added at 25 U/ml.

[0056] Three days after the initiation of culture PBMC are harvested, centrifuged, and resuspended in hypotonic electroporation buffer (Eppendorf) at 2010.sup.6 cells/ml. 25 g of the plasmid IL13zetakine/HyTK-pMG of Example 3, together with 400 l of cell suspension, are added to a sterile 0.2 cm electroporation cuvette. Each cuvette is subjected to a single electrical pulse of 250V/40 s and again incubated for ten minutes at RT. Surviving cells are harvested from cuvettes, pooled, and resuspended in culture media containing 25 U/ml rhIL-2. Flasks are placed in the patient's designated tissue culture incubator. Three days following electroporation hygromycin is added to cells at a final concentration of 0.2 mg/ml. Electroporated PBMC are cultured for a total of 14 days with media and IL-2 supplementation every 48-hours.

[0057] The cloning of hygromycin-resistant CD8+ CTL from electroporated OKT3-activated patient PBMC is initiated on day 14 of culture. Briefly, viable patient PBMC are added to a mixture of 10010.sup.6 cyropreserved irradiated feeder PBMC and 2010.sup.6 irradiated TM-LCL in a volume of 200 ml of culture media containing 30 ng/ml OKT3 and 50 U/ml rhIL-2. This mastermix is plated into ten 96-well cloning plates with each well receiving 0.2 ml. Plates are wrapped in aluminum foil to decrease evaporative loss and placed in the patient's designated tissue culture incubator. On day 19 of culture each well receives hygromycin for a final concentration of 0.2 mg/ml. Wells are inspected for cellular outgrowth by visualization on an inverted microscope at Day 30 and positive wells are marked for restimulation.

[0058] The contents of each cloning well with cell growth are individually transferred to T25 flasks containing 5010.sup.6 irradiated PBMC, 1010.sup.6 irradiated LCL, and 30 ng/mlOKT3 in 25 mls of tissue culture media. On days 1, 3, 5, 7, 9, 11, and 13 after restimulation flasks receive 50 U/ml rhIL-2 and 15mls of fresh media. On day 5 of the stimulation cycle flasks are also supplemented with hygromycin 0.2 mg/ml. Fourteen days after seeding cells are harvested, counted, and restimulated in T75 flasks containing 15010.sup.6 irradiated PBMC, 3010.sup.6 irradiated TM-LCL and 30 ng/ml OKT3 in 50 mls of tissue culture media. Flasks receive additions to culture of rhIL-2 and hygromycin as outlined above.

[0059] CTL selected for expansion for possible use in therapy are analyzed by immunofluorescence on a FACSCalibur housed in CRB-3006 using FITC-conjugated monoclonal antibodies WT/31 (aTCR), Leu 2a (CD8), and OKT4 (CD4) to confirm the requisite phenotype of clones (TCR+, CD4, CD8+, and IL13+). Criteria for selection of clones for clinical use include uniform TCR +, CD4, CD8+ and IL13+ as compared to isotype control FITC/PE-conjugated antibody. A single site of plasmid vector chromosomal integration is confirmed by Southern blot analysis. DNA from genetically modified T cell clones will be screened with a DNA probe specific for the plasmid vector. Probe DNA specific for the HyTK in the plasmid vector is synthesized by random priming with florescein-conjugated dUTP per the manufacture's instructions (Amersham, Arlington Hts, Ill.). T cell genomic DNA is isolated per standard technique. Ten micrograms of genomic DNA from T cell clones is digested overnight at 37 C. then electrophoretically separated on a 0.85% agarose gel. DNA is then transferred to nylon filters (BioRad, Hercules, Calif.) using an alkaline capillary transfer method. Filters are hybridized overnight with probe in 0.5 M Na.sub.2PO.sub.4, pH 7.2, 7% SDS, containing 10 g/ml salmon sperm DNA (Sigma) at 65 C. Filters are then washed four times in 40 mM Na.sub.2PO.sub.4, pH 7.2, 1% SDS at 65 C. and then visualized using a chemiluminescence AP-conjugated anti-florescein antibody (Amersham, Arlington Hts, Ill.). Criteria for clone selection is a single band unique vector band.

[0060] Expression of the IL-13 zetakine is determined by Western blot procedure in which chimeric receptor protein is detected with an anti-zeta antibody. Whole cell lysates of transfected T cell clones are generated by lysis of 210.sup.7 washed cells in 1 ml of RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 tablet/10 ml Complete Protease Inhibitor Cocktail (Boehringer Mannheim). After an eighty minute incubation on ice, aliquots of centrifuged whole cell lysate supernatant are harvested and boiled in an equal volume of loading buffer under reducing conditions then subjected to SDS-PAGE electrophoresis on a precast 12% acrylamide gel (BioRad). Following transfer to nitrocellulose, membranes are blocked in blotto solution containing 0.07 gm/ml non-fat dried milk for 2 hours. Membranes are washed in T-TBS (0.05% Tween 20 in Tris buffered saline pH 8.0) then incubated with primary mouse anti-human CD3 monoclonal antibody 8D3 (Pharmingen, San Diego, Calif.) at a concentration of 1 g/ml for 2 hours. Following an additional four washes in T-TBS, membranes are incubated with a 1:500 dilution of goat anti-mouse IgG alkaline phosphatase-conjugated secondary antibody for 1 hour. Prior to developing, membranes are rinsed in T-TBS then developed with 30 ml of AKP solution (Promega, Madison, Wis.) per the manufacturer's instructions. Criteria for clone selection is the presence of a chimeric zeta band.

[0061] CD8+ cytotoxic T cell clones expressing the IL-13 zetakine chimeric immunoreceptor recognize and lyse human glioblastoma target cells following interaction of the chimeric receptor with the cell surface target epitope in a HLA-unrestricted fashion. The requirements for target IL-13R2 epitope expression and class I MHC independent recognition will be confirmed by assaying each aTCR+, CD8+, CD4, IL-13 zetakine+ CTL clones against IL-13R2+ Daudi cell transfectants and IL-13R2 Daudi cells. T cell effectors are assayed 12-14 days following stimulation with OKT3. Effectors are harvested, washed, and resuspended in assay media; and Daudi cell transfectants expressing IL-13R2. 2.510.sup.5, 1.2510.sup.5, 0.2510.sup.5, and 0.0510.sup.5 effectors are plated in triplicate at 37 C. for 4 hours with 510.sup.3 target cells in V-bottom microtiter plates (Costar, Cambridge, Mass.). After centrifugation and incubation, 100 L aliquots of cell-free supernatant is harvested and counted. Percent specific cytolysis is calculated as:

[00002]$\frac{\left({\mathrm{Experimental}.Math.}^{51}.Math.\mathrm{Cr}.Math..Math.\mathrm{release}\right)-\left({\mathrm{control}.Math.}^{51}.Math.\mathrm{Cr}.Math..Math.\mathrm{release}\right)}{\left({\mathrm{Maximum}.Math.}^{51}.Math.\mathrm{Cr}.Math..Math.\mathrm{release}\right)-\left({\mathrm{control}.Math.}^{51}.Math.\mathrm{Cr}.Math..Math.\mathrm{release}\right)}100$

Control wells contain target cells incubated in assay media. Maximum .sup.51Cr release is determined by measuring the .sup.51Cr content of target cells lysed with 2% SDS. Criteria for clone selection is >25% specific lysis of IL-13R2+ Daudi transfectants at an E:T ratio of 5:1 and a <10% lysis of parental Daudi at the same E:T ratio.

Example 6

Treatment of Human Glioma Using IL-13 Zetakine-Expressing T Cells

[0062] T cell clones genetically modified according to Example 5 to express the IL-13R zetakine chimeric immunoreceptor and HyTK are selected for: [0063] a. TCR/.sup.+, CD4.sup., CD8.sup.+, IL-13.sup.+ cell surface phenotype as determined by flow cytometry. [0064] b. Presence of a single copy of chromosomally integrated plasmid vector DNA as evidenced by Southern blot. [0065] c. Expression of the IL-13 zetakine protein as detected by Western blot. [0066] d. Specific lysis of human IL-13R2.sup.+ targets in 4-hr chromium release assays. [0067] e. Dependence on exogenous IL-2 for in vitro growth. [0068] f. Mycoplasma, fungal, bacterial sterility and endotoxin levels <5 EU/ml. [0069] g. In vitro sensitivity of clones to ganciclovir.

[0070] Peripheral blood mononuclear cells are obtained from the patient by leukapheresis, preferably following recovery from initial resection surgery and at a time at least three weeks from tapering off steroids and/or their most recent systemic chemotherapy. The target leukapheresis mononuclear cell yield is 510.sup.9 and the target number of hygromycin-resistant cytolytic T cell clones is 25 with the expectation that at least five clones will be identified that meet all quality control parameters for ex-vivo expansion. Clones are cryopreserved and patients monitored by serial radiographic and clinical examinations. When recurrence of progression of disease is documented, patients undergo a re-resection and/or placement of a reservoir-access device (Omaya reservoir) for delivering T cells to the tumor resection cavity. Following recovery from surgery and tapering of steroids, if applicable, the patient commences with T cell therapy.

[0071] The patient receives a target of at least four one-week cycles of therapy. During the first cycle, cell dose escalation proceeds from an initial dose on Day 0 of 10.sup.7 cells, followed by 510.sup.7 cells on Day 3 to the target dose of 10.sup.8 cells on Day 5. Cycle 2 commences as early as one week from commencement of cycle 1. Those patients demonstrating tumor regression with residual disease on MRI may have additional courses of therapy beginning no earlier than Week 7 consisting of repetition of Cycles 3 and 4 followed by one week of rest/restaging provided these treatments are well tolerated (max. toxicities <grade 3) until such time that disease progression or a CR is achieved based on radiographic evaluation.

[0072] Cell doses are at least a log less than doses given in studies employing intracavitary LAK cells (individual cell doses of up to 10.sup.9 and cumulative cell numbers as high as 2.7510.sup.10 have been safety administered), ex vivo expanded TILs (up to 10.sup.9 cells/dose reported with minimal toxicity) and allo-reactive lymphocyte (starting cell dose 10.sup.8 with cumulative cell doses up to 51.510.sup.8) delivered to a similar patient population.sup.75-85. The rationale for the lower cell doses as proposed in this protocol is based on the increased in vitro reactivity/anti-tumor potency of IL-13 zetakine+ CTL clones compared to the modest reactivity profile of previously utilized effector cell populations. Low-dose repetitive dosing is favored to avoid potentially dangerous inflammatory responses that might occur with single large cell number instillations. Each infusion will consist of a single T cell clone. The same clone will be administered throughout a patient's treatment course. On the days of T cell administration, expanded clones are aseptically processed by washing twice in 50 cc of PBS then resuspended in pharmaceutical preservative-free normal saline in a volume that results in the cell dose for patient delivery in 2mls. T cells are instilled over 5-10 minutes. A 2 ml PFNS flush will be administered over 5 minutes following T cells. Response to therapy is assessed by brain MRI+/gandolinium, with spectroscopy.

[0073] Expected side-effects of administration of T cells into glioma resection cavities typically consist of self-limited nausea and vomiting, fever, and transient worsening of existing neurological deficits. These toxicities can be attributed to both the local inflammation/edema in the tumor bed mediated by T cells in combination with the action of secreted cytokines. These side-effects typically are transient and less than grade II in severity. Should patients experience more severe toxicities it is expected that decadron alone or in combination with ganciclovir will attenuate the inflammatory process and ablate the infused cells. The inadvertent infusion of a cell product that is contaminated with bacteria or fungus has the potential of mediating serious or life-threatening toxicities. Extensive pre-infusion culturing of the cell product is conducted to identify contaminated tissue culture flasks and minimize this possibility. On the day of re-infusion, gram stains of culture fluids, as well as, endotoxin levels are performed.

[0074] Extensive molecular analysis for expression of IL-13R2 has demonstrated that this molecule is tumor-specific in the context of the CNS.sup.44; 46; 48; 54. Furthermore, the only human tissue with demonstrable IL-13R2 expression appears to be the testis.sup.42. This tumor-testis restrictive pattern of expression is reminiscent of the growing number of tumor antigens (i.e. MAGE, BAGE, GAGE) expressed by a variety of human cancers, most notably melanoma and renal cell carcinoma.sup.109-111. Clinical experience with vaccine and adoptive T cell therapy has demonstrated that this class of antigens can be exploited for systemic tumor immunotherapy without concurrent autoimmune attack of the testis.sup.112-114. Presumably this selectively reflects the effect of an intact blood-testis barrier and an immunologically privileged environment within the testis. Despite the exquisite specificity of the mutant IL-13 targeting moiety, toxicities are theoretically possible if cells egress into the systemic circulation in sufficient numbers and recognize tissues expressing the IL-13R1/IL-4 receptor. In light of this remote risk, as well as the possibility that instilled T cells in some patients may mediate an overly exuberant inflammatory response in the tumor bed, clones are equipped with the HyTK gene which renders T cells susceptible to in vivo ablation with ganciclovir.sup.115-118. Ganciclovir-suicide, in combination with an intra-patient T cell dose escalation strategy, helps minimize the potential risk to research participants.

[0075] Side effects associated with therapy (headache, fever, chills, nausea, etc.) are managed using established treatments appropriate for the condition. The patient receives ganciclovir if any new grade 3 or any grade 4 treatment-related toxicity is observed that, in the opinion of the treating physician, puts that patient at significant medical danger. Parentally administered ganciclovir is dosed at 10 mg/kg/day divided every 12 hours. A 14-day course will be prescribed but may be extended should symptomatic resolution not be achieved in that time interval. Treatment with ganciclovir leads to the ablation of IL-13 zetakine.sup.+ HyTK.sup.+ CD8.sup.+ CTL clones. Patients should be hospitalized for the first 72 hours of ganciclovir therapy for monitoring purposes. If symptoms do not respond to ganciclovir within 48 hours additional immunosuppressive agents including but not limited to corticosteroids and cyclosporin may be added at the discretion of the treating physician. If toxicities are severe, decadron and/or other immunosuppressive drugs along with ganciclovir are used earlier at the discretion of the treating physician.

Example 7

Additional Preferred DNA Vectors

[0076] Additional DNA vectors are shown in FIGS. 13A-13I and 14A-14C. Table I, below contains further information concerning the sequence of FIGS. 13A-13I. See FIG. 10 for a map of this vector.

TABLE-US-00003 TABLE I Plasmid DNA Vector Sequence Contents for SEQ ID NO: 19. Plasmid Location Element Description (bases) hEF1p Human Elongation Factor-1 Promoter 6-549 IL13zetakine IL13 cytokine fused to Fc: 690-2183 Late SV40pAn Simian Virus 40 Late polyadenylation 2230-2498 signal Ori ColE1 A minimal E. coli origin of replication 2499-3245 SpAn A synthetic poly A and Pause site 3246-3432 hCMV-1Aprom Immediate-early CMV enhancer/promoter 3433-4075 HyTK Genetic fusion of the Hygromycin 4244-6319 Resistance and Thymidine Kinase coding regions BGh pAn Bovine growth hormone polyadenylation 6320-6618 signal and a transcriptional pause

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