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WO1997016557A1 - TRAITEMENT DE TUMEURS PAR TRANSFERT ADOPTIF DE LYMPHOCYTES T CYTOTOXIQUES SPECIFIQUES DU CD44v - Google Patents

TRAITEMENT DE TUMEURS PAR TRANSFERT ADOPTIF DE LYMPHOCYTES T CYTOTOXIQUES SPECIFIQUES DU CD44v Download PDF

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Publication number
WO1997016557A1
WO1997016557A1 PCT/EP1996/004688 EP9604688W WO9716557A1 WO 1997016557 A1 WO1997016557 A1 WO 1997016557A1 EP 9604688 W EP9604688 W EP 9604688W WO 9716557 A1 WO9716557 A1 WO 9716557A1
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Prior art keywords
fusion protein
nucleic acid
cell
lymphocyte
acid molecule
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PCT/EP1996/004688
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German (de)
English (en)
Inventor
Armin Hekele
Peter Herrlich
Helmut Ponta
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Boehringer Ingelheim International Gmbh
Forschungszentrum Karlsruhe Gmbh
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Priority to AU74946/96A priority Critical patent/AU7494696A/en
Publication of WO1997016557A1 publication Critical patent/WO1997016557A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4223—CD44 not IgG
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/54—Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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.
  • TCR T cell receptor
  • lymphocytes 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).
  • the present invention relates to a fusion protein containing a first
  • the first portion preferably contains a variable domain of an antibody which is specific for variant CD44 (CD44v).
  • 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.
  • scFv single chain antibody molecule
  • the CD44v-specific portion of the fusion protein can also advantageously be traced back to a human or humanized antibody molecule.
  • 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.
  • 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 ⁇ subunit of the T cell receptor or a Part or fragment of this subunit is linked.
  • the part or the fragment of the ⁇ subunit can advantageously contain the cytoplasmic domain and / or the transmembrane domain of the ⁇ subunit.
  • the fusion protein instead of the extracellular domain of the ⁇ subunit, 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.
  • the first and the second part can be linked to one another via a hinge region, e.g. the hinge region of CD8 ⁇ .
  • the present invention also relates to a fusion protein, as described above, for pharmaceutical use.
  • nucleic acid molecule which codes for a fusion protein as described above.
  • a nucleic acid molecule can advantageously be an expression vector which contains the coding information for such a fusion protein.
  • 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.
  • 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.
  • 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.
  • IL-2 interleukin-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.
  • 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.
  • such treatment can be combined with the administration of interleukin-2 (IL-2).
  • IL-2 interleukin-2
  • 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.
  • the first portion of the fusion protein is preferably a humanized antibody molecule or derived from a humanized antibody molecule.
  • 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.
  • this can be the sequence or a partial sequence of the ⁇ 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 ⁇ 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.
  • 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.
  • IO 3 to IO 6 U / kg IL-2 can be injected once or several times parenterally, preferably intravenously or intraperitoneally, or intravenously infused to support the therapy according to the invention.
  • 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 ⁇ chain is sufficient for the destruction event in vitro.
  • New specificities can be generated by exchanging scFv in the retroviral constructs.
  • Fig. 1 Structure and specificity of the scFv (l.lASML): a: ⁇ 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 V ⁇ domain.
  • the CDRs in the deduced amino acid sequence of I. IASML VH and V ⁇ 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): ⁇ : ⁇ expression plasmid pL [scFv (l. LASML): ⁇ : ⁇ ].
  • the plasmid contains the gene for the chimeric scFv (I.LASML): ⁇ : ⁇ 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 CD8 ⁇ chain similar to immunoglobulin (HINGE), and the cDNA for the transmembrane and cytoplasmic domain of the ⁇ chain of the T cell antigen receptor ( ⁇ ) 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.
  • ⁇ + / 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): ⁇ : ⁇ in cytotoxic T lymphocytes.
  • scFv ⁇ 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 ⁇ g / ml aprotinin, 50 ⁇ g / ml leupeptin, 4 ⁇ g / ml pepstatin
  • NP-40 lysis buffer 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM PMSF, 10 mM iodoacetamide, 80 ⁇ g / ml aprotinin, 50 ⁇ g / ml leupeptin, 4 ⁇ g / ml pepstatin
  • 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- ⁇ ) 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): ⁇ : ⁇ in infected cytotoxic T cells.
  • Biotinylated proteins were visualized with horseradish-peroxidase-conjugated streptavidin.
  • C Specific binding of scFv (l. LASML): ⁇ : ⁇ 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.
  • Fig. 3 Targeted of cells by scFv (l ⁇ ASML): a: ⁇ 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).
  • Fig. 4 Adoptive immunotherapy of BSp73AS14 tumor-bearing mice.
  • 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.
  • 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 ⁇ E 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.
  • 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 ⁇ 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: ⁇ .
  • the present invention involves the reprogramming of cytotoxic lymphocytes.
  • 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 V ⁇ 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 V ⁇ 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 Linke ⁇ eptide encoded by 15 amino acids (GGGGS) 3 , separated from each other.
  • Five independent clones of VH and V ⁇ cDNAs were sequenced. All VH and all V ⁇ sequences showed identical antibody domains of the variable regions (37; Fig. IB).
  • the 5'-VH-left-V ⁇ -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 ⁇ chain cDNA (31). It was shown that the ⁇ 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 ⁇ chain.
  • Moritz and co-workers (41, 42) have determined the need for a spacer between the scFv domain and the ⁇ chain in order to ensure the accessibility of the scFv fragment when it is expressed on the T cell surface.
  • the cDNA for the hinge region of the CD8 ⁇ chain (35) was inserted between the scFv domain and the ⁇ chain cDNA.
  • scFv (l. LASML): ⁇ : ⁇ is transcriptionally regulated by the Moloney Murine Leukemia Virus (MoMuLV) -5'-LTR promoter.
  • MoMuLV Moloney Murine Leukemia Virus
  • Fig. IC A schematic representation of the plasmid pL [scFv (l. IASML): ⁇ : ⁇ ] is shown in Fig. IC.
  • 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.
  • VH variable domain of the heavy chain
  • VK variable domain of the light kappa chain
  • 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 CD8 ⁇ hinge region (amino acids 105-163; numbering according to reference 35), followed by the ⁇ chain cDNA (starting with the nucleotides coding for amino acid 28; numbering according to reference 31).
  • the complete construct, designated scFv (IIASML): ⁇ : ⁇ was subcloned into the retroviral vector pLXSN (36), which contains a selectable marker for G418 resistance.
  • Example 2 Expression of scFv (l.lASML): a: ⁇ .
  • 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- ⁇ 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 CD8 ⁇ hinge region.
  • the endogenous ⁇ 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): ⁇ : ⁇ 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): ⁇ : ⁇ ] SN was converted into the corresponding retrovirus by standard calcium phosphate transfection of the helper virus-free ectopic packaging cell line ⁇ E (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 TM (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide or hexadimethrin bromide, Sigma, Deisenhofen, Germany) at a final concentration of 8 ⁇ g / 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.
  • Example 3 Specificity scFv (l.lASML): a: ⁇ .
  • scFv l. LASML
  • ⁇ ⁇ 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.
  • S glutathione
  • GST glutathione transferase
  • 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 ⁇ 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.
  • rat pancreatic carcinoma cells (BSp73AS14) were mixed with CTLs and the cell lysis was determined in a standard LDH release test (Fig.3A).
  • 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.
  • 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 TM, 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.
  • LDH lactate dehydrogenase enzyme
  • Example 5 Efficient inhibition of tumor growth by CTLs expressing chimeric scFv: a: ⁇ 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).
  • Interleukin-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.
  • scFv l. LASML
  • ⁇ ⁇ 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.
  • glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65: 13-24.
  • Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals 0 at least 12 altematively spliced exons. Proc. Natl. Acad Be. USA 89: 12160-12164.
  • B3 (Fv) -PE38KDEL a single-chain immunotoxin that causes complete regression of a human carcinoma in mice. Proc. Natl. Acad. Be. U.S.A. 88: 8616-8620.
  • MOLECULE TYPE Other nucleic acid
  • DESCRIPTION: / desc "PCR Primer”
  • 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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Abstract

L'invention concerne une protéine de fusion contenant une première fraction présentant une affinité spécifique pour une séquence d'aminoacides, codée par un exon variant du gène CD44, et une seconde fraction comprenant la séquence d'aminoacides de la sous-unité du complexe récepteur du lymphocyte T ou d'un récepteur d'immunoglobuline ou d'une partie de cette sous-unité. La première fraction contient de préférence un domaine variable d'un anticorps, spécifique du gène variant CD44(CD44v), notamment du CD44v6. L'invention concerne en outre une molécule d'acide nucléique qui code une protéine de fusion de ce type. La protéine de fusion et la molécule d'acide nucléique selon l'invention s'utilisent dans le traitement de tumeurs par transfert adoptif de lymphocytes T cytotoxiques qui détruisent les cellules tumorales qui expriment, en surface, un ou plusieurs épitopes variants du gène CD44. Une molécule d'acide nucléique de ce type, par exemple, peut être introduite dans des lymphocytes correspondants et les lymphocytes ainsi modifiés peuvent être administrés à un patient.
PCT/EP1996/004688 1995-10-31 1996-10-29 TRAITEMENT DE TUMEURS PAR TRANSFERT ADOPTIF DE LYMPHOCYTES T CYTOTOXIQUES SPECIFIQUES DU CD44v WO1997016557A1 (fr)

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DE19540515.3 1995-10-31
DE19540515A DE19540515C1 (de) 1995-10-31 1995-10-31 Tumortherapie durch adoptiven Transfer CD44v-spezifischer zytotoxischer T-Lymphozyten

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007020405A2 (fr) * 2005-08-12 2007-02-22 Cartela R & D Ab Nouveaux peptides et utilisations associees
WO2014079943A1 (fr) * 2012-11-21 2014-05-30 Karlsruher Institut für Technologie Peptides dérivés de cd44v6 pour le traitement de cancers métastasés
WO2014079931A1 (fr) * 2012-11-21 2014-05-30 Karlsruher Institut für Technologie Peptides dérivés de cd44v6 pour le traitement de cancers du sein
WO2014079940A1 (fr) * 2012-11-21 2014-05-30 Karlsruher Institut für Technologie Peptides dérivés de cd44v6 pour traiter le cancer du pancréas
US10703796B2 (en) 2014-12-05 2020-07-07 Amcure Gmbh CD44v6-derived cyclic peptides for treating cancers and angiogenesis related diseases

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006036445A2 (fr) 2004-09-24 2006-04-06 Trustees Of Dartmouth College Recepteur nk chimerique et traitement anticancereux
US9181527B2 (en) 2009-10-29 2015-11-10 The Trustees Of Dartmouth College T cell receptor-deficient T cell compositions
US9273283B2 (en) 2009-10-29 2016-03-01 The Trustees Of Dartmouth College Method of producing T cell receptor-deficient T cells expressing a chimeric receptor
US9833476B2 (en) 2011-08-31 2017-12-05 The Trustees Of Dartmouth College NKP30 receptor targeted therapeutics
CN110511278B (zh) 2012-05-07 2024-08-09 达特茅斯大学理事会 抗b7-h6抗体、融合蛋白及其使用方法

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WO1992010591A1 (fr) * 1990-12-14 1992-06-25 Cell Genesys, Inc. Chaines chimeriques utilisees comme voies de transduction de signal associe a un recepteur
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WO1992010591A1 (fr) * 1990-12-14 1992-06-25 Cell Genesys, Inc. Chaines chimeriques utilisees comme voies de transduction de signal associe a un recepteur
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WO1994012631A1 (fr) * 1992-11-20 1994-06-09 Isis Innovation Limited Peptide correspondant a l'exon 6 du gene cd44, anticorps specifiques dudit peptide et utilisation de ces anticorps dans le diagnostic des tumeurs
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007020405A2 (fr) * 2005-08-12 2007-02-22 Cartela R & D Ab Nouveaux peptides et utilisations associees
WO2007020405A3 (fr) * 2005-08-12 2007-05-03 Cartela Ab Nouveaux peptides et utilisations associees
WO2014079943A1 (fr) * 2012-11-21 2014-05-30 Karlsruher Institut für Technologie Peptides dérivés de cd44v6 pour le traitement de cancers métastasés
WO2014079931A1 (fr) * 2012-11-21 2014-05-30 Karlsruher Institut für Technologie Peptides dérivés de cd44v6 pour le traitement de cancers du sein
WO2014079940A1 (fr) * 2012-11-21 2014-05-30 Karlsruher Institut für Technologie Peptides dérivés de cd44v6 pour traiter le cancer du pancréas
JP2016501858A (ja) * 2012-11-21 2016-01-21 アムキュア ゲーエムベーハー 転移性癌を処置するためのｃｄ４４ｖ６由来ペプチド
JP2016501857A (ja) * 2012-11-21 2016-01-21 アムキュア ゲーエムベーハー 膵癌を処置するためのｃｄ４４ｖ６由来ペプチド
US9586993B2 (en) 2012-11-21 2017-03-07 Amcure Gmbh CD44v6-derived peptides for treating pancreatic cancer
US9586994B2 (en) 2012-11-21 2017-03-07 Amcure Gmbh CD44V6-derived peptides for treating breast cancer
US9593146B2 (en) 2012-11-21 2017-03-14 Amcure Gmbh CD44v6-derived peptides for treating metastasizing cancer
US10703796B2 (en) 2014-12-05 2020-07-07 Amcure Gmbh CD44v6-derived cyclic peptides for treating cancers and angiogenesis related diseases

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