WO1997016557A1 - TREATMENT OF TUMOURS BY ADOPTIVE TRANSFER OF CD44v-SPECIFIC CYTOTOXIC T-LYMPHOCYTES - Google Patents

Abstract

The invention concerns a fusion protein containing a first portion having a specific affinity for an amino acid sequence which is coded by a variant exon of the CD44 gene, and a second portion which contains the amino acid sequence of a subunit of the T-cell receptor complex or an immunoglobulin receptor or part of this subunit. Preferably, the first portion contains a variable domain of an antibody which is specific for variant CD44 (CD44v), in particular CD44v6. The invention further concerns a nucleic acid molecule which codes for such a fusion protein. The substances according to the invention are suitable for treating tumours by the adoptive transfer of cytotoxic T-lymphocytes which destroy tumour cells expressing one or a plurality of variant epitopes of the CD44 gene on their cell surface. For example, a nucleic acid molecule according to the invention can be introduced into corresponding lymphocytes and the lymphocytes thus modified can be administered to a patient.

Description

Tumor therapy through adoptive transfer of CD44v-specific cytotoxic T lymphocytes

The invention relates to the therapy of tumors by adoptive transfer of cytotoxic T-lymphocytes which destroy tumor cells which express one or more variant epitopes of the CD44 gene on their cell surface.

Combating multiple (metastatic) solid tumors is currently a critical problem in cancer therapy. Many laboratories and clinics are attempting to revive the immune defense against the tumors. Such attempts are based on the assumption that reactivity to tumor antigens exists but has become ineffective. Expansion and reinjection of tumor infiltrating lymphocytes, administration of missing components such as cytokines or costimulatory surface molecules, or improvement in tumor antigen presentation are experimental approaches which are believed to lead to tumor destruction (1-6).

In an alternative approach to mobilizing anti-tumor immune responses, lymphocytes are made to destroy tumor cells regardless of their TCR specificity (TCR = T cell receptor). As one way to accomplish this, bispecific antibodies have been generated that have affinity for both lymphocyte leader molecules and tumor antigen and are believed to bring lymphocytes into contact with tumor cells (7,8). However, because antibodies can only penetrate solid tumors poorly (9), it would be desirable as a further possibility to transfer the tumor specificity to lymphocytes, the cells that naturally penetrate into tissue and can carry out antigen-specific cytolysis. Mergers of structures that determine specificity, e.g. an antibody-minimigen, with components of the TCR complex have therefore been proposed (10). The central role of the ζ subunit of the TCR complex for signal transmission has recently been recognized, using chimeric receptors consisting of the cytoplasmic domain of the ζ chain and the extracellular and transmembrane domain of other proteins (21- 25).

Before you can reprogram a tumor patient's lymphocytes, a host of questions need to be answered. For example, which epitope should lymphocytes be targeted at? Another question is, which antibody affinity (of the miniantibody in the TCR fusion) is needed or is sufficient to cause the destruction of tumor cells? Because resting lymphocytes, in addition to the If the desired affinity for a tumor antigen requires sufficient signal transmission to the nucleus to achieve expansion and, more importantly, its differentiation into CTLs, it must also be clarified whether the genetically produced fusion proteins can signal at all or can provide signals of sufficient strength to sufficiently activate primary 5 peripheral lymphocytes.

The tumor antigen CD44 represents a family of surface molecules with extreme variability, which is generated by alternative splicing (11-15). Metastatic tumors from animals and humans often express certain CD44 isoforms. In particular, epitopes encoded by exon v6 appear to be correlated with tumor progression and poor prognosis (11, 16-19). In the rat, the 1.1ASML antibody, which is specific for a v6-encoded epitope, interferes with the metastatic spread of pancreatic cancer (20).

is The task, the solution of which is the subject of the present invention was

Providing improved methods for adoptive immunotherapy of tumors and means for such methods.

The present invention relates to a fusion protein containing a first

20 portions that have a specific affinity for an amino acid sequence encoded by a variant exon of the CD44 gene, and a second portion that contains the amino acid sequence of a subunit of the T cell receptor complex or an immunoglobulin receptor or a part of this subunit. The first portion preferably contains a variable domain of an antibody which is specific for variant CD44 (CD44v). Such

The domain can be provided in the form of a single chain antibody molecule (scFv) in which the variable region of the light chain (VL) and the variable region of the heavy chain (VH) of the antibody are linked to one another via a flexible linking region. The CD44v-specific portion of the fusion protein can also advantageously be traced back to a human or humanized antibody molecule. In particular,

Preferred forms are those in which the antibody portion of the fusion protein has a specific affinity for an amino acid sequence encoded by the variable exon v5 and / or v6. A specific affinity for an epitope in the amino acid sequence KWFENEWQGKNPPT (rat) or QWFGNRWHEGYRQT (human) is very particularly preferred. An advantageous way of carrying out the invention is to construct a

35 fusion protein, the antibody portion of which contains an amino acid sequence as shown in Figure IB. Also particularly preferred is a fusion protein, the first portion of which is derived from the antibody VFF-18, which is secreted by a hybridoma cell line which was sold on June 7, 1994 under the file number DSM ACC2174 to the DSM-Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany. This antibody is specific for an epitope within the amino acid sequence QWFGNRWHEGYRQT.

A preferred embodiment of the present invention is a fusion protein, in which the first portion, which has a specific affinity for an amino acid sequence encoded by a variant exon of the CD44 gene, with the ζ subunit of the T cell receptor or a Part or fragment of this subunit is linked. The part or the fragment of the ζ subunit can advantageously contain the cytoplasmic domain and / or the transmembrane domain of the ζ subunit. In such an embodiment, instead of the extracellular domain of the ζ subunit, the fusion protein can then have the first portion which has a specific affinity for an amino acid sequence which is encoded by one or more variable exon (s) of the CD44 gene , contain. In a further embodiment, the first and the second part can be linked to one another via a hinge region, e.g. the hinge region of CD8α. The present invention also relates to a fusion protein, as described above, for pharmaceutical use.

Another object of the present invention is a nucleic acid molecule which codes for a fusion protein as described above. Such a nucleic acid molecule can advantageously be an expression vector which contains the coding information for such a fusion protein. Typically, the sequence coding for the fusion protein is arranged in such an expression vector in such a way that it is functionally related to one or more regulatory sequences, so that with the aid of this expression vector the fusion protein can be found in a suitable host cell, e.g. a eukaryotic culture cell, or else, particularly preferably, can be expressed in a lymphocyte.

Accordingly, the invention further relates to a host cell which contains a nucleic acid molecule or an expression vector as described in the preceding paragraph.

Another object of the invention is a method for producing a fusion protein as described above, which is characterized in that a host cell into which a corresponding vector has been introduced is cultivated and the fusion protein formed is isolated. Another object of the present invention is a T lymphocyte which expresses a fusion protein as described above. Preferably, the fusion protein is inserted into the cell membrane of this T lymphocyte and the portion with the specific CD44v affinity is exposed to the outside. Another object of the invention is such a T lymphocyte for pharmaceutical use. The use for the treatment of cancer or for the treatment and / or prophylaxis of metastatic diseases is particularly preferred. Such use is particularly advantageous when the disease is squamous cell, breast, colon, stomach or pancreatic carcinoma. In particular, such treatment can be combined with the administration of interleukin-2 (IL-2).

The invention further relates to a method for producing such a T lymphocyte, which is characterized in that a nucleic acid molecule or an expression vector is introduced into a T lymphocyte as described.

Accordingly, the use of such a nucleic acid molecule or expression vector for producing such a modified T lymphocyte is also the subject of the invention.

Another object of the invention is a nucleic acid molecule or expression vector, as described above, for pharmaceutical use. The use for the treatment of cancer or for the treatment and / or prophylaxis of metastatic diseases is preferred. Such a use is particularly advantageous if the disease is a squamous cell, breast, colon, stomach, cervical or pancreatic carcinoma or a malignant lymphoma. In particular, such treatment can be combined with the administration of interleukin-2 (IL-2).

The present invention can be carried out as described in the examples or, with knowledge of the technical teaching of this description, using methods known per se. The production of antibody molecules which are specific for variant epitopes of CD44 is described in the prior art. In addition to the literature points already mentioned, reference is also made here to WO 91/17248, WO 95/00851, WO 95/04547 and PCT / EP9502126, to which reference is hereby made in full. In particular, WO 95/00851 describes the production of v5- and v6-specific antibodies and of antibodies which are specific for a transition epitope which is coded jointly by exons v7 and v8. Methods for cloning the variable regions of such antibodies and for producing derived antibody molecules, for example chimeric, humanized or single-chain antibody molecules, are also known in the prior art. Here In addition to the references already mentioned, EP 0239400, EP 0519596, EP 0368684, EP 0438310, WO 92/07075 and WO 92/22653 may be mentioned. For the application of the teaching according to the invention in the therapy and prophylaxis of human cancer diseases, the first portion of the fusion protein is preferably a humanized antibody molecule or derived from a humanized antibody molecule.

When the teaching according to the invention is used in the therapy and prophylaxis of human diseases, the second portion of the fusion protein preferably contains an amino acid sequence of a subunit of the human T cell receptor complex or of a human immunoglobulin receptor or a part of this subunit. In particular, this can be the sequence or a partial sequence of the ζ subunit of the human T cell receptor (51).

Fusion proteins with v5-specific affinity in the sense of the present invention or nucleic acids which code therefor are advantageous because exon v5 is expressed in a number of cancers at a very early stage. V6-specific fusion proteins or nucleic acids which code therefor can be used, for example, for the treatment of squamous cell, colon, stomach, pancreatic or breast carcinomas. Treatment of squamous cell carcinoma of the head and neck area with cytotoxic T lymphocytes which express a v6-specific fusion protein on their cell surface is very particularly preferred. Fusion proteins with specificity for the transitional epitope v7 / v8 or nucleic acids that code for them are particularly advantageous for the treatment of cervical carcinomas.

The present invention can be carried out, for example, by constructing an expression vector which allows the expression of a fusion protein in human T-lymphocytes, the first portion of this fusion protein being a single-chain antibody construct which is derived from the CD44v6-specific antibody VFF-18 is derived, and the second part of the fusion protein contains the cytoplasmic domain and the transmembrane domain of the ζ subunit of the human TCR, the two parts being linked via a hinge region. Such an expression vector can then be introduced into lymphocytes which have been obtained from the blood or tumor tissue of a tumor patient, and the lymphocytes which have been modified in this way are injected or infused back into the patient. Methods for obtaining and expanding such lymphocytes are known in the prior art (52, 53, 2, 48). Therapeutic doses of the lymphocytes reprogrammed according to the invention can be in the range from IO ⁶ to IO ¹² lymphocytes per injection or infusion. Intravenous administration is preferred. Therapeutic applications can be done once or repeatedly, for example daily application on several Consecutive days. The therapy according to the invention can advantageously be connected to a surgical intervention, for example the removal of a solid tumor, in order to combat tumor cells remaining in the body (minimal residual disease). In this case, the lymphocytes can be obtained from the tumor tissue removed. Particularly advantageously, IO ³ to IO ⁶ U / kg IL-2, preferably IO ⁴ to IO ⁵ U / kg, can be injected once or several times parenterally, preferably intravenously or intraperitoneally, or intravenously infused to support the therapy according to the invention.

The retroviral transfer of the scFv: α: ζ fusion construct described in the examples led to the surface expression of the protein. The fusion protein showed the correct antigen specificity on the surface of the T cells. The reprogrammed CTL line shows a changed target cell specificity. While maintaining cytotoxic activity against the P815 mastocytoma against which it was generated, it now also destroys tumor cells expressing v6 epitope in vitro. The affinity and possibly the signal transmission via the ζ chain is sufficient for the destruction event in vitro. This is in agreement with comparable experiments in which other antigens and either the ζ chain or the homologous γ chain of the high-affinity FcεRI were used as fusion partners for the scFv fragments (10, 41, 42, 47-49). In the mouse, the reprogrammed CTLs lead to a significant delay in the tumor growth of xenotransplants.

Knowing the examples below, it is possible to generate antigen-selective CTLs with other specificities, especially for CD44 epitopes that are formed by human cervical carcinomas or by breast or colorectal carcinomas (16-18,

25 44, 50). New specificities can be generated by exchanging scFv in the retroviral constructs.

30

Description of the pictures

Fig. 1: Structure and specificity of the scFv (l.lASML): a: ζ construct. (A) Amino- 35 acid sequences containing the epitope of the monoclonal antibody (mAb) I.IASML. The epitope is contained in amino acids 318-331 of the CD44v4-v7 isoform of the rat (clone pMeta-1; sequence data see ref. 11). The approximate extent of the epitope was determined by competition analysis with synthetic peptides. (B) Nucleotide and deduced amino acid sequence of the scFv (l.lASML) gene. The sequence shows the cDNA fragment (bp 1-357) encoding the VH domain, the synthetic linker (bp 358-402) and the cDNA fragment (bp 403-735) encoding the Vκ domain. The CDRs in the deduced amino acid sequence of I. IASML VH and Vκ domains and the synthetic linker peptide are underlined. Sequence positions bp 736-746 are contributed by the plasmid pWW152. (C) Schematic representation of the scFv (l. LASML): α: ζ expression plasmid pL [scFv (l. LASML): α: ζ]. The plasmid contains the gene for the chimeric scFv (I.LASML): α: ζ surface receptor, which is inserted into the retroviral vector pLXSN. The leader sequence of the heavy immunoglobulin chain (SP), the PCR-amplified cDNA fragment VH of mAb I. IASML, a sequence which encodes the 15 amino acid linker (LINKER), the PCR-amplified cDNA fragment VK from I.IASML, a sequence which codes for the hinge region of the CD8α chain similar to immunoglobulin (HINGE), and the cDNA for the transmembrane and cytoplasmic domain of the ζ chain of the T cell antigen receptor (ζ) are indicated as boxes . The expression of the fusion gene is transcriptionally regulated by the Moloney Murine Leukemia Virus 5'-LTR promoter. The neo ^r gene that mediates G418 resistance is encoded by the same plasmid. The arrows indicate the transcription start points. ψ + / gag refers to sequences of the Moloney Murine Sarcoma virus and MoMuLV genome that are required for packaging.

Fig. 2: Expression of scFv (l.lASML): ά: ζ in cytotoxic T lymphocytes. (A)

Expression of scFv (l.lASML): α: ζ in cytotoxic T lymphocytes. Cell lysate (NP-40 lysis buffer: 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM PMSF, 10 mM iodoacetamide, 80 μg / ml aprotinin, 50 μg / ml leupeptin, 4 μg / ml pepstatin) were prepared from parent cI96 or CAYZ.007. Protein aliquots (cI96: lane 1; CAYZ.007: lane 2) were separated in a 10% SDS-PAGE under reducing conditions and subjected to a standard immunoblot analysis. An incubation with mAb H146-968 (anti-ζ) was followed by an incubation with horseradish-peroxidase-conjugated second reagent (P260, DAKO, Hamburg, Germany), which reacted with HI 46-968. (B) Cell surface localization of scFv (l. LASML): α: ζ in infected cytotoxic T cells. 2 x IO ⁷ viable CTLs (CAYZ.007: lanes 1 and 2; cI96: lane 3) were labeled with sulfo-NHS-biotin (Pierce, Rockford, IL, USA), lysed in 1 ml NP-40 lysis buffer and subjected to immunoprecipitation with the anti-ζ mAb H146-968 (lanes 1 and 3) or a control mAb (lane 2). Immune complexes were precipitated by treatment with protein G agarose, separated in a 10% SDS-PAGE under reducing conditions and transferred to a PVDF (polyvinylidene difluoride) membrane (Millipore Bedford, MA, USA). Biotinylated proteins were visualized with horseradish-peroxidase-conjugated streptavidin. (C) Specific binding of scFv (l. LASML): α: ζ to rat CD44 splice variants that carry v6 epitope. 5 x IO ⁷ CAYZ.007 cells were lysed in NP-40 lysis buffer. Nuclear-free supernatants were pre-clarified with glutathione agarose and then incubated with GST # CD44v4-v7 (lane 2). GST # CD44v4-v7 is a bacterially expressed fusion protein consisting of the S. japonicum glutathione S-transferase and sequences specified by the rat CD44 variant exons v4-v7. Immune complexes were precipitated by treatment with glutathione agarose and separated under reducing conditions in a 10% SDS-PAGE. In a standard immunoblot analysis, the ζ proteins were detected by reaction with anti-ζ mAb Hl 46-968 (see Fig. 2A). Incubations with GST (instead of GST # CD44v4-v7; lane 3) or with glutathione agarose alone (lane 1) served as controls. Positions and molecular weights of standard proteins are indicated. Arrows indicate the endogenous ζ chain or the ζ chimera.

von Zellen durch scFv(lΛASML):a:ζ exprimie- rende CTLs. (A) CAYZ.007 und Eltern-cI96-Zellen wurden auf ihre zytolytische Aktivität gegenüber verschiedenen Zielzellen in einem 6-stündigen LDH-Freisetzungstest untersucht. BSp73 AS 14 ist eine transfizierte Rattenpankreaskarzinomzellinie, die die CD44v4-v7-Iso- form der Ratte in großer Menge exprimiert; NIH3T3#CD44v4-v7 sind infizierte murine Fi- broblasten, die Ratten-CD44v4-v7 exprimieren. Die spezifische LDH-Freisetzung (in Pro¬ zenten) ist gegen das E/T- Verhältnis (Effektor/Zielzell-Verhältnis) aufgetragen. (B) Kom¬ petitive Suppression der BSp73AS14-Lyse durch mAk I. IASML. BSp73AS14-Zellen wur¬ den mit CAYZ.007-CTLs bei einem konstanten Effektor/Zielzellen- Verhältnis von 4 : 1 in der Gegenwart von steigenden Mengen des mAk I. IASML inkubiert. Ein Zytotoxizitäts- test, der in der Gegenwart eines isotypgleichen Antiköφers irrelevanter Spezifitat (anti- Gallium-Chelat; siehe Ref. 12) durchgeführt wurde, diente als Kontrolle. Die prozentspezi¬ fische LDH-Freisetzung ist gegen die Konzentration des Kompetitors aufgetragen.Fig. 3: Targeted

of cells by scFv (lΛASML): a: ζ expressing CTLs. (A) CAYZ.007 and parent cI96 cells were examined for their cytolytic activity against different target cells in a 6-hour LDH release test. BSp73 AS 14 is a transfected rat pancreatic carcinoma cell line which expresses the CD44v4-v7 isoform of the rat in large quantities; NIH3T3 # CD44v4-v7 are infected murine fibroblasts that express rat CD44v4-v7. The specific LDH release (in percent) is plotted against the E / T ratio (effector / target cell ratio). (B) Competitive suppression of BSp73AS14 lysis by mAb I. IASML. BSp73AS14 cells were incubated with CAYZ.007-CTLs at a constant effector / target cell ratio of 4: 1 in the presence of increasing amounts of mAb I. IASML. A cytotoxicity test, which was carried out in the presence of an isotype-like antibody of irrelevant specificity (anti-gallium chelate; see ref. 12), served as a control. The percent specific LDH release is plotted against the concentration of the competitor.

Fig. 4: Adoptive immunotherapy of BSp73AS14 tumor-bearing mice.

Athletic BALB / c nude mice who carried palpable sc BSp73AS14 xenografts (pancreatic carcinoma of the rat) with a size of 20-50 mm ³ received daily intravenous injections of either 3 x IO ⁷ cI96 (gray columns; medium tumor volume lumens at the start of the treatment: 30 mm ³ , n = 7), 3 x IO ⁷ CAYZ.007 (black columns; average tumor volume at the start of the treatment: 34 mm ³ , n = 10), or PBS (PBS = phosphate buffered saline = isotonic saline buffer) (white columns; mean tumor volume at the start of the treatment: 28 mm ³ , n = 7). The tumor size was added to the determined times. The average tumor volume is plotted against time (in days). Treatment was stopped after 7 days of injection. Values are mean values ± SD. The differences in tumor volume that were observed were statistically significant (Student's t test, p <0.025).

Fig. 5: Enhanced anti-tumor activity of CAYZ.007 in the presence of IL-2.

Athletic BALB / c nude mice carrying palpable sc BSp73AS14 xenografts (pancreatic carcinoma of the rat) with a size of 20-50 mm ³ were either given daily iv injections of PBS (white columns; mean tumor volume at the beginning of the Treatment: 32 mm ³ , n = 9), daily ip injections of IO ⁴ U human rEL-2 (gray columns; mean tumor volume at the start of treatment: 34 mm ³ , n = 5) or daily iv injections of 3 x IO ⁷ CAYZ.007 in combination with ip injections of IO ⁴ U human rIL-2 (black columns; mean tumor volume at the start of treatment: 29 mm ³ , n = 6). The tumor size was determined at the times indicated. The average tumor volume is plotted against time (in days). Values are mean values ± SD. Treatment was stopped after 7 days of injection.

Examples

Cell lines and animals

The BSp73 variant ASpSVMetal-14 (BSp73AS14) and the hybridoma cell line (11) producing the mAb I. IASML (gamma 1 / kappa) were cultivated in RPMI 1640 medium (Life Technologies, Gaithersburg, MD, USA), which was fetal Calf serum was supplemented (Life Technologies, Gaithersburg, MD, USA). Clone 96 (cI96) is a C57BL / 6 (H-2 ^b ) -derived permanent cytotoxic T cell line with H-2 K ^d -restricted specificity for P815 (H-2 ^d ) mastocytoma cells (26, 27). CI96 and its infectants (e.g. CAYZ.007) were found in DMEM (Life Technologies, Gaithersburg, MD, USA) containing 10% fetal calf serum, 2 mM L-glutamine, 100 mM Hepes, 50 mM 2-ME and 100 U / ml of mouse rIL-2 was cultivated. X63Ag8-653 transfectants that secreted IL-2 (28), the retroviral packaging cell lines ΩE and PA317 (29, 30), their transfectants and infectants and the murine fibroblast cell line NIH3T3 and their infectants were in DMEM with 10% fetal calf serum L-glutamine, 100 mM Hepes and 50 mM 2-ME cultured. BALB / c nu / nu (H-2 ^D ) mice were from Charles River, Sulzfeld, Germany, related, kept in filter-top cages, and used for experiments at the age of 8-10 weeks.

Antibodies and interleukin 2

Hl 46-968 is a hamster IgG which is directed against the C-terminal peptide (amino acids 151-164; numbering according to reference 31) of the mouse ζ chain (32). Human rIL-2 (1.8 x 10 ⁷ U / mg) was obtained from Chiron GmbH, Ratingen, Germany.

Example 1: Construction of the expression plasmid scFv (l.lASML): a: ζ.

The present invention, with which metastatic tumors can be destroyed, involves the reprogramming of cytotoxic lymphocytes. In order to convey tumor cell specificity to a CTL line, a single-chain mini antibody was fused to the TCR-Kette chain and a corresponding construct was expressed in the CTL line. The fusion protein retained the antigen-binding specificity of the antibody. The system which has been used here as an example contains the monoclonal antibody I.IASML, which recognizes an epitope which is encoded by the variant exon v6 of the rat CD44 gene (the sequence which contains the epitope is) shown in Fig. IA), and a highly metastatic rat pancreatic adenocarcinoma that expresses this epitope (11). cDNA fragments which encode the VH and Vκ domains of I. IASML were isolated by reverse transcription of hybrid mRNA, followed by cDNA amplification using the polymerase chain reaction (33). The amplified cDNA sequences were inserted into the plasmid pWW152 (34), the I.IASML VH and Vκ cDNAs being arranged in such a way that they are in an open reading frame, but by a sequence which is suitable for an artificial one Linkeφeptide encoded by 15 amino acids (GGGGS) ₃ , separated from each other. Five independent clones of VH and Vκ cDNAs were sequenced. All VH and all Vκ sequences showed identical antibody domains of the variable regions (37; Fig. IB). The 5'-VH-left-Vκ-3 'design used here to assemble the antigen-determining structure of a single-chain peptide has previously been used successfully in bacterial expression (34, 38-40) . It was shown that a bacterially expressed scFv (1.1 ASML) is indeed able to recognize the v6 epitope. The 3 'end of the scFv (l. LASML) gene encoding the minimal antigen recognition site was fused to a truncated mouse ζ chain cDNA (31). It was shown that the ζ chain protein of the T cell antigen-receptor complex could transmit the signal, when antigen binding was mediated by the scFv fragment. Coding sequences located at the outer 5 'end of the fusion gene specify a signal peptide of the heavy immunoglobulin chains, which ensures efficient transport of the recombinant receptor to the cell surface. The fusion protein is integrated into the plasma membrane through the transmembrane region of the ζ chain. Moritz and co-workers (41, 42) have determined the need for a spacer between the scFv domain and the ζ chain in order to ensure the accessibility of the scFv fragment when it is expressed on the T cell surface. Correspondingly, the cDNA for the hinge region of the CD8α chain (35) was inserted between the scFv domain and the ζ chain cDNA. The complete chimeric gene was inserted into the retroviral expression plasmid pLXSN (36). The expression of scFv (l. LASML): α: ζ is transcriptionally regulated by the Moloney Murine Leukemia Virus (MoMuLV) -5'-LTR promoter. A schematic representation of the plasmid pL [scFv (l. IASML): α: ζ] is shown in Fig. IC.

The detailed procedure was as follows. Total RNA was derived from l. lASML hybrid cells produced. Then the first strand cDNA synthesis of the variable domain of the heavy chain (VH) and the variable domain of the light kappa chain (VK) was carried out, the oligonucleotides 5'-AGATCCAGGGGCCAGTGGATAGA-3 '(CHFOR), specifically for the Ig-gamma-1 constant region of the mouse, and 5'-GGATACAGTTGCG-GCCGCATCAGC-3 '(CKFOR), specifically for the kappa-constant region of the mouse. CDNAs of the variable domain were amplified by PCR, the primers 5'-ATTATAAGCTTCAGGT G / CA / CAA / G CTGCAG G / C AGTC A / T GG-3 '(VHBACK) and 5 ^, -TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3 ^, (VHl - FOR; 33) for VH and 5'-GACATTCAGCTGACCCAG T / A CT C / GC / AA / C / T-3 ¹ (VK BACK) and 5'-GTTAGATCTCCA G / AC / T TT G / T GT G / CCG / C-3 '(VKFOR) were used for VK. The primers contain appropriate restriction sites at their ends to allow cloning of the PCR products into the modified pBluescript KS ⁺ vector pWWl52 (Stratagene Cloning Systems, La Jolla, CA, USA; modifications described in reference 34), which allows a 15th - Amino acid linker encoded (GGGGS) ₃ . The cDNA encoding the CD44v6-specific scFv (1.1. ASML) was placed 3 'in a sequence coding for a leader peptide of a heavy chain of an immunoglobulin and then ligated to the cDNA which corresponds to the CD8α hinge region (amino acids 105-163; numbering according to reference 35), followed by the ζ chain cDNA (starting with the nucleotides coding for amino acid 28; numbering according to reference 31). The complete construct, designated scFv (IIASML): α: ζ, was subcloned into the retroviral vector pLXSN (36), which contains a selectable marker for G418 resistance. Example 2: Expression of scFv (l.lASML): a: ζ.

In order to produce retroviral particles from the retroviral expression vector pL [scFv (l .1 ASML): α: ζ], the plasmid was introduced into the amphotropic packaging line PA317 (30). Infected clones were selected in the presence of G418 and the expression of the chimeric Kette chain was identified by Western blot analysis. The retroviral supernatant of a clone that produced a high titer was used to infect the murine cytotoxic T lymphocyte line cI96 (26, 27). Stable infectants (designated CAYZ.001 to 103) were selected in the presence of G418. Immunoblot analyzes of lysates prepared from representative clones using the anti-ζ mAb Hl 46-968 showed bands with apparent molecular weights of 50-70 kD (example shown in FIG. 2A, lane 2), which in lysates, that were produced from the parent cell line cI96 cannot be detected (lane 1). The size of the 50 kD band corresponds to the unmodified chimeric surface receptor. The identity of the slower migrating gangs has not been investigated. They could represent N-glycosylated products (41). An N-glycosylation consensus motif (NST) is in the CD8α hinge region. The endogenous ζ chain is detected as a band of 16 kD apparent molecular weight in lysates from both infected and parent CTLs (Fig. 2A, lanes 1 and 2). Comparable results were obtained with various infected cell lines. Clone CAYZ.007 was used for further studies. The correct membrane insertion of scFv (l. LASML): α: ζ in the clone CAYZ.007 CTLs was confirmed by surface biotinylation followed by immunoprecipitation (Fig. 2B). Bands of 50 kD and 70 kD apparent molecular weight are only observed after biotinylation of infected cells (lane 1), but not of parent cells (lane 3).

The detailed procedure was as follows. The vector construct pL [scFv (l. L-ASML): α: ζ] SN was converted into the corresponding retrovirus by standard calcium phosphate transfection of the helper virus-free ectopic packaging cell line ΩE (29). Transfectants were selected for stable integration of proviral DNA in the presence of the neomycin analog G418 sulfate (G418, Geniticin, Life Technologies, Gaithersburg, MD, USA) at a concentration of 1 mg / ml. Retroviral supernatants from pools of stably transfected producers were supplemented with Polybren ™ (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide or hexadimethrin bromide, Sigma, Deisenhofen, Germany) at a final concentration of 8 μg / ml and used, to infect the helper virus free packaging cell line PA317 (30). Infected clones were selected in medium containing G418 (1 mg / ml). Retroviral titers of cell culture supernatants from these packaging cell lines (in the order of 10 ⁵ CFU / ml) were determined by infection of NIH3T3 cells and counting of the G418-resistant clones. Supernatants of this Producer lines were used to infect the murine CTL cell line cI96 (26, 27) in the presence of polybrene (8 μg / ml). Infected CTL clones (CAYZ. #) Were selected by growth in culture medium containing 1 mg / ml G418.

Example 3: Specificity scFv (l.lASML): a: ζ.

The affinity of scFv (l. LASML): α: ζ for the rat CD44 epitope encoded by exon v6 was determined by incubating cell lysates with bacterially expressed glutathione (S) transferase (GST) fusion protein, which v6 epitope contains (GST # CD44-v4-v7), followed by precipitation of the immune complexes with glutathione-agarose. GST # -CD44v4-v7 is produced by inserting exon v4-v7 sequences from rat CD44 (nucleotide positions 753-1246; numbering according to reference 11) into the singular Smal site of the bacterial expression vector pGEX2T (Pharmacia Biotech, Uppsala, Sweden) . Western blot analysis of GST # CD44v4-v7 precipitates under reducing conditions using the ζ chain specific mAb Hl 46-968 showed bands of 50-70 kD apparent molecular weight (Fig. 2C, lane 2), which were in size correspond to the chimeric receptor and its post-translational modification product. These bands do not occur in immunoprecipitations carried out with GST (lane 1) or glutathione agarose alone (lane 3). The affinity for bacterially expressed v6 epitope could also be demonstrated with an ELISA.

Example 4: Lytic activity of infected CTLs.

In order to investigate the specificity of the infected CTLs, to recognize and destroy tumor cells which express the v6 epitope on their surface, rat pancreatic carcinoma cells (BSp73AS14) were mixed with CTLs and the cell lysis was determined in a standard LDH release test (Fig.3A). CTLs expressing the chimeric scFv: α: ζ protein efficiently lysed BSp73AS14 target cells. With an appropriate E / T ratio, up to 100% of the BSP73AS14 cells were eliminated. Parent cI96 CTLs showed no cytolytic activity against BSP73AS14 cells. This specificity of lysis by retrovirally infected CTLs was also observed with NTH3T3 fibroblasts transfected with rat CD44v4-v7 by retroviral gene transfer. NTH3T3 # CD44v4-v7 cells express the v6 epitope on their cell surface and are effectively destroyed by CAYZ.007 infectants (Fig. 3A). No cell lysis was detected when the parent NIH3T3 fibroblasts were used as target cells for CAYZ.007 CTLs, or when NTH3T3 # CD44v4-v7 cells were incubated with the parent cI96 CTLs. In order to further confirm the specificity of the target cell lysis by the infected CTLs, cytotoxicity tests were carried out in the presence of varying amounts of specific competitors. At a constant E / T ratio of 4 to 1, the specific LDH release of BSP73AS14 cells increased with the addition of increasing amounts of mAb I. IASML (Fig. 3B) or bacterially expressed soluble antigen (GST # CD44v4-v7). The addition of either a control antibody of the same isotype, but irrelevant specificity (mAb 3-9; see ref. 12), or bacterially expressed GST alone does not influence the LDH release.

The cytotoxicity test was carried out in detail as follows. The cytotoxicity of CTLs against various target cells was measured using a non-radioactive cytotoxicity test (CytoTox96 ™, Promega, Madison, WI, USA) in accordance with the manufacturer's instructions. The test is based on the colorimetric quantification of stable cytosolic lactate dehydrogenase enzyme (LDH), which is released into the culture medium after T cell lysis. Different amounts of effector lymphocytes were added to IO ⁴ target cells in 0.1 ml phenol red-free medium in 96-well microtiter plates with a U-bottom and incubated for 6 hours in a water-saturated atmosphere at 5% CO2. The LDH content was determined in 50 μl aliquots of cell-free culture supernatant (EXP). Spontaneous LDH release of target cells (TSR) and maximum LDH release of target cells (TMR) after lysis with 0.8% Triton X-100 (100% LDH release) were determined. The spontaneous LDH release of each effector CTL concentration used in the test was also measured (ECSR). The absorption values correspond to triple determinations and were corrected for the LDH activity which is contributed by the serum in the culture medium. The corrected values were used to calculate the specific lysis using the following equation:% specific lysis = (EXP-ECSR-TSR) x (TMR-TSR) ^"1. TSR was 20% of TMR or less.

Example 5: Efficient inhibition of tumor growth by CTLs expressing chimeric scFv: a: ζ protein in vivo.

The reprogrammed CTLs not only destroy target tumor cells in vitro, but also act in vivo. Interference with tumor growth in vivo was examined by subcutaneously injecting rat BSp73AS14 tumor cells into athymic nude BALB / c mice and growing the tumors to a size of 20-50 mm ³ were. Then either PBS, parenteral cI96 CTLs or genetically modified CTLs (over a period of 7 days with 3 x IO ⁷ cells per animal and day) were injected intravenously daily. In the control animals that received PBS injections, the tumor volume doubled every 2.5 days (Fig. 4). Iv injections from parental cI96 CTLs did not affect tumor growth (Fig. 4). Daily IV injection of CTLs expressing the scFv: α: ζ fusion protein significantly delayed tumor growth (Fig. 4). The experiment was stopped on day 13. The average size of the tumors on day 13 in CAYZ.007-treated animals was only 50% of those in all control groups. None of the animals developed lymph nodes or lung metastases at this early point in the observation. All treatments were well tolerated by the animals and no weight loss or other signs of systemic toxicity were observed. The differences in tumor size on all days were statistically significant (Student's T test, p <0.025).

Interleukin-2 (IL-2) is the most important growth factor for cytotoxic T-lymphocytes. CI96 and its infectants survive and proliferate in cell culture only in the presence of IL-2. We therefore investigated whether systemic administration of IL-2 would increase the anti-tumor effect of CAYZ.007. Nude mice carrying 20-50 mm ³ BSP73AS14 tumors received daily iv injections of 3 x IO ⁷ CAYZ.007 in combination with ip injections of 10 ⁴ U human rIL-2. (CAYZ.007 poliferate in cell culture in the presence of human rIL-2). Control animals received either iv injections of PBS or only ip injections of IO ⁴ U human rlL-2. The animals were treated again for 7 days and tumor growth was monitored by caliper measurements. The data shown in Fig. 5 clearly show the increased suppression of tumor growth by scFv (l. LASML): α: ζ expressing CTLs in the presence of IL-2. While the average tumor volume in the two control groups increased more than 25-fold in one week, the tumors in the animals treated with CAYZ.007 hardly increased twice in the same time. Systemic administration of BL-2 itself has no growth suppressive effect on BSP73AS14 tumors.

The procedure for the animal experiments was as follows. The growth of tumors in athymic BALB / c-nu / nu mice was induced by sc inoculation of tumor cells (5 x 10 ⁵ cells) in the middle dorsal region. Animals which carried tumors in the size of 20-50 mm ³ were subjected to various treatments, as described in the figure legends 4 and 5. The tumor load was determined daily using calipers. The tumor volume was calculated using the following equation: Tumor volume = 4/3 x π xr. To avoid unnecessary suffering avoided, the animals were killed when the tumors had reached 1.5 cm ^{3 in} size. All animals were examined for metastases in lymph nodes and lungs.

Statistical significance of the differences in tumor size between CAYZ.007-treated animals and control groups was measured, using the Student's t test after logarithmic transformation of the raw data.

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(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Boehringer Ingelheim International GmbH

(B) ROAD: PO Box 200

(C) LOCATION: Ingelheim am Rhein

(E) COUNTRY: Germany

(F) POSTAL NUMBER: 55216

(G) TELEPHONE: + 49- (0) -6132-77-2770 (H) TELEFAX: + 49- (0) -6132-77-4377

(A) NAME: Forschungszentrum Karlsruhe GmbH

(B) ROAD: x

(C) LOCATION: Karlsruhe

(E) COUNTRY: Germany

(F) POSTAL NUMBER: 76021

(ii) DESCRIPTION OF THE INVENTION: Tumor therapy by adoptive transfer of CD44v-specific cytotoxic T lymphocytes

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GAG GTA CAA CTG CAG GAG TCA GGA CCT GGC CTC GTG AAA CCT TCT CAG 48 Glu Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

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GCA AGT CAC GGC TAC GGT AGT AGC GGG TTT GTT TAC TGG GGC CAA GGG 336 Ala Ser His Gly Tyr Gly Ser Ser Gly Phe Val Tyr Trp Gly Gin Gly 100 105 110

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130 135 140

CTG CCT GTC AGT CTT GGA GAT CAA GCC TCC ATC TCT TGC AGA TCT AGT 480 Leu Pro Val Ser Leu Gly Asp Gin Ala Ser Ile Ser Cys Arg Ser Ser 145 150 155 160

CAG AGC CTT GTA CAC ATT AAT GGA AAC ACC TAT TTA CAT TGG TAC CTG 528 Gin Ser Leu Val His Ile Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu

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CAG AAG CCA GGC CAG TCT CCG AAG CTC CTG ATC TAC AAA GTT TCC AAC 576 Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn

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CGA TTT TCT GGG GTC CCA GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA 624 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205

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Lys Asn Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gin Phe Phe 65 70 75 80

Leu Asn Leu Asn Ser Val Thr Thr Glu Asp Thr Ser Thr Tyr Tyr Cys

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Ala Ser His Gly Tyr Gly Ser Ser Gly Phe Val Tyr Trp Gly Gin Gly 100 105 110

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gin Leu Thr Gin Ser Ala Leu Ser 130 135 140

Leu Pro Val Ser Leu Gly Asp Gin Ala Ser Ile Ser Cys Arg Ser Ser 145 150 155 160

Gin Ser Leu Val His Ile Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu

165 170 175

Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Met Tyr Lys Val Ser Asn 180 185 190

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205

Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val 210 215 220

His Phe Cys Ser Gin Ser Thr His Asp Pro Pro Thr Phe Gly Gly Gly 225 230 235 240

Thr Lys Leu Glu Ile Lys Ala Leu

245

Claims

claims 1. Fusion protein containing a first portion which has a specific affinity for an amino acid sequence which is encoded by a variant exon of the CD44 gene, and a second portion which is the amino acid sequence of a subunit of the T cell receptor complex or one Contains immunoglobulin receptor or a part of this subunit. 2. Fusion protein according to claim 1, characterized in that the first portion contains at least one variable domain of an antibody which is speci¬ fish for variant CD44. 3. Fusion protein according to claim 2, characterized in that the variable region of the light chain (VL) and the variable region of the heavy chain (VH) of the antibody in this fusion protein are linked to one another via a flexible linking region. 4. Fusion protein according to claim 2 or 3, characterized in that the Antikör¬ is human or humanized. 5. Fusion protein according to one of claims 1 to 4, characterized in that it has a specific affinity for an amino acid sequence which is encoded by the variable exon v5 and / or v6. 6. Fusion protein according to one of claims 1 to 5, characterized in that it has a specific affinity for an epitope in the amino acid sequence KWFENEWQGKNPPT or QWFGNRWHEGYRQT. 7. Fusion protein according to one of claims 1 to 6, characterized in that it contains an amino acid sequence according to Figure IB. 8. Fusion protein according to one of claims 1 to 7, characterized in that the subunit of the T cell receptor is the ζ subunit. 9. Fusion protein according to one of claims 1 to 8, characterized in that the part of the ζ subunit contains the cytoplasmic domain and / or the transmembrane membrane. 10. Fusion protein according to one of claims 1 to 9, characterized in that the first and the second portion are linked to one another via a hinge region. 1 1. Fusion protein according to claim 10, characterized in that the hinge region is the hinge region of CD8α. 12. nucleic acid molecule which codes for a fusion protein according to any one of claims 1 to 11. 13. Expression vector containing a nucleic acid molecule according to claim 12. 14. Host cell containing a nucleic acid molecule according to claim 12 or an expression vector according to claim 13. 15. A method for producing a fusion protein according to any one of claims 1 to 11, characterized in that a host cell according to claim 14 is cultivated and the fusion protein formed is isolated. 16. T lymphocyte which expresses a fusion protein according to any one of claims 1 to 11. 17. A method for producing a T lymphocyte according to claim 16, characterized in that a nucleic acid molecule according to claim 12 or an expression vector according to claim 13 is introduced into a T lymphocyte. 18. Use of a nucleic acid molecule according to claim 12 or an expression vector according to claim 13 for the production of a T-lymphocyte according to claim 16. 19. Nucleic acid molecule according to claim 12 or expression vector according to claim 13 for pharmaceutical use. 20. Use of a nucleic acid molecule according to claim 12 or expression vector according to claim 13 for the treatment of cancer. 21. Use of a nucleic acid molecule according to claim 12 or an expression vector according to claim 13 for the treatment and / or prophylaxis of metastatic diseases. 22. T lymphocyte according to Anspmch 16 for pharmaceutical use. 23. Use of a T lymphocyte according to Anspmch 16 for the treatment of 5 cancer. 24. Use of a T lymphocyte according to Claim 16 for the treatment of meta-static diseases. 25. Use according to one of claims 20, 21, 23 or 24, characterized gekenn¬ characterized in that the disease is a squamous cell, breast, colon, gastric or pancreatic carcinoma. 26. Fusion protein according to one of claims 1 to 11 for pharmaceutical use. 27. Use of interleukin-2 (IL-2) for the production of a pharmaceutical composition for medical treatment in combination with a fusion protein according to Anspmch 26, a lymphocyte according to Anspmch 22 or a nucleic acid or 20 an expression vector according to Claim 19. 28. Use according to one of claims 20, 21, or 23 to 25, characterized gekenn¬ characterized in that IL-2 is additionally administered.