Characterisation of a humanised bispecific monoclonal antibody for cancer therapy

Bruynck, A.; Seemann, G.; Bosslet, Klaus

doi:10.1038/bjc.1993.84

Cited by 14 publications

(8 citation statements)

References 29 publications

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“…25 The plasmids containing the cDNA of the BW431 /26 V regions were kindly provided by K Bosslet (Behringwerke, Marburg, Germany ). The V L region was amplified from pUC19 -LC431 by polymerase chain reaction (PCR ) using primer BW431-V L -1A (5 0 -CGAGTCTAGA ACCATGGACA TCCAGATGACC-3 0 ) and BW431 -V L -2 (5 0 -CACCAC-TCCC GGGCTTTCCT GAACCGGAAG TGGATCC-TTT GATTTC CACC TTGGTCCCTT G -3 0 ) and the V H region was amplified from pAB -431/ 26-V H -humÁ3 25,26 using primer BW431 -V H -1 (5 0 -AAGCCCGGGA GTG-GTGAAGG TAGCACTAAA GGCCAGGTCC AGCTG-CAGGA GAGC-3 0 ) and BW431-V H -2 ( 5 0 -TTGGATCCGG CCGCACCTGA GGAGACGGTG ACCGT-3 0 ). The V L fragment digested with NcoI and SmaI and the V H fragment digested with SmaI and NotI (restriction enzymes from New England Biolabs, Frankfurt am Main, Germany ) were cloned into the NcoI and NotI sites of the E. coli periplasmatic expression vector pOPE51 33 (kindly provided by S Dübel, Molecular Genetics, University Heidelberg, Germany ) to obtain the scFv scBW431/26 consisting of the N terminal V L region followed by the Linker-218 ( -GSTSGSGKPGSGEGSTKG -) 4 and the V H region (pOPE51 -scBW431 /26).…”

Section: Construction Of the Scfv Fragmentsmentioning

confidence: 99%

“…CEA is expressed in a number of tumors of epithelial origin such as colorectal, gastric and pancreatic carcinomas, lung and endometrial adenocarcinoma, and mucious ovarian carcinoma. 24 The scFv fragment was constructed of the V regions of the CEAspecific humanized mAb BW431 /26, 25,26 which has been successfully used for radioimmunodiagnostics in patients. 27 -29 The human IgG1 Fc domain served as spacer between the scFv fragment and the CD3 signal chain, which can be necessary for an efficient receptor function.…”

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confidence: 99%

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Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo

Schirrmann

Pecher

2002

Cancer Gene Ther

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The gene transfer of tumor -specific chimeric immunoglobulin T -cell receptors ( cIgTCRs ) combining antibody -like specificity with the effector cell function could be an attractive tool in immunotherapy. In this study, we directed the human natural killer ( NK ) cell line YT to tumor cells by gene transfer of a cIgTCR with specificity against the human carcinoembryonic antigen ( CEA ). The cIgTCR was constructed of a CEA -specific humanized single -chain Fv antibody fragment fused to the IgG1 Fc domain and the CD3 chain. YT cells were transfected with the cIgTCR gene by electroporation and cIgTCR -expressing cells were enriched by immunoaffinity purification. cIgTCR -expressing YT cells specifically lysed CEA + colon carcinoma cell lines, which were resistant to the parental YT cell line. The lysis was not inhibited in the presence of soluble CEA. Receptor gene -modified YT cells retained their CEA -specific cytolytic activity after -irradiation in vitro and inhibited the tumor growth in vivo after adoptive transfer into NOD / SCID mice. This gene -modified NK cell line available in unlimited source might be useful in clinical immunotherapy of CEA + cancer.

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Section: Construction Of the Scfv Fragmentsmentioning

confidence: 99%

mentioning

confidence: 99%

Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo

Schirrmann

Pecher

2002

Cancer Gene Ther

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“…A typical clone was selected and further analyzed in detail. FACS analyses revealed that the cell clones specifically react with the antihumBW431/26-scFv idiotypic mAb BW2069/36 13 and with an anti-human IgG1 antibody directed to the CH2/CH3 domain of the receptor. Western blot analysis utilizing the anti-human IgG1 antibody shows under non-reducing conditions (Figure 1, lanes 1-4) a protein band of about 150 kDa whereas under reducing conditions ( Figure 1, lanes 5-8) a band of about 75 kDa was found.…”

mentioning

confidence: 99%

“…MD45 T cell clones transfected with the humBW431/26-CH2/CH3-␥ receptor and untransfected MD45 cells, respectively, were incubated in microtiter plates coated with the anti-BW431/26 idiotypic mAb BW2064/36 13 or, for control reasons, with the anti-idiotypic mAb 9G10 with specificity for the anti-CD30 mAb HRS3. 15 As shown in Figure 2a, incubation of receptor grafted cells with immobilized anti-idiotypic BW2064/36 antibody resulted in increased IL-2 secretion whereas incubation with an immobilized, isotype matched anti-idiotypic control antibody did not.…”

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confidence: 99%

“…The reactivity of humBW431/26 is restricted to malignant and premalignant tissues whereas no reactivity with normal tissues was found apart from a low intrinsic reactivity with the luminal part of the colon mucosa. [10][11][12][13] To obtain a chimeric T cell receptor we fused the DNA coding for the anti-CEA single chain antibody fragment (scFv) in frame to the CH2/CH3 domains of the human IgG1 14 and the transmembrane and intracellular domain of the ␥-signalling chain derived from the Fc⑀RI␥-receptor. 2 The DNA coding for the chimeric receptor is expressed under control of the RSV LTR after transfection into the murine T cell line MD45.…”

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A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA

et al. 1999

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Chimeric T cell receptors with specificity for tumor-associapletely humanized chimeric receptor. After transfection, the ted antigens are successfully used to target T cells to humBW431/26 scFv-CH2CH3-␥ receptor is expressed as tumor cells. The efficacy of this approach, however, is a homodimer on the surface of MD45 T cells. Co-incureduced by soluble antigen that is frequently present in bation with CEA + tumor cells specifically activates grafted high serum concentrations. To overcome this situation, we MD45 T cells indicated by IL-2 secretion and cytolytic constructed an anti-CEA chimeric receptor whose extraactivity against CEA + tumor cells. Notably, the efficacy of cellular moiety is composed of a humanized single chain receptor-mediated activation is not affected by soluble CEA antibody fragment (scFv) derived from the anti-CEA mAb up to 25 g/ml demonstrating the usefulness of this chim-BW431/26 and the CH2/CH3 constant domains of human eric receptor for specific cellular activation by membraneIgG. The intracellular moiety consists of the ␥-signaling bound CEA even in the presence of high concentrations of chain of the human Fc⑀RI receptor constituting a com-CEA, as found in patients during progression of the disease.Keywords: chimeric T cell receptor; scFv; membrane-bound CEAIn a recently developed strategy for the cellular immunotherapy of malignant diseases, the advantages of antibody-based targeting and cell-mediated cytolysis have been combined by grafting effector cells with a chimeric receptor that binds to a tumor-associated antigen. 1-5 The antigen-binding domain of the receptor is composed of a single-chain antibody fragment (scFv) derived from the heavy (VH) and light (VL) chain variable regions of a monoclonal antibody (mAb). The intracellular signalling domain is derived from the cytoplasmic part of a membrane-bound receptor involved in cellular activation. After grafting cytotoxic T cells with the chimeric receptor, an antigen-specific and MHC-unrestricted cellular immune response is directed to cells expressing the particular mAb-defined antigen (for review, Refs 6 and 7).Due to xenogeneic parts of the chimeric receptor, the efficacy in the therapy of malignant diseases, however, will be negatively influenced by the potential of a hostversus-graft reaction. In addition, soluble antigen frequently present in high concentrations in the serum of cancer patients will block the receptor of grafted effector cells thus preventing tumor cell recognition and antigendriven cellular activation upon specific receptor crosslinking. 6,8 This limitation so far restricts the chimeric receptor approach to those patients with very low amounts of soluble antigen unlikely to prevent effector cell-tumor cell interaction. Malignant tumors, however, that express carcinoembryonic antigen (CEA) are frequently accompanied by high serum concentrations of soluble CEA. 9 In this particular situation the application of the chimeric receptor-based strategy requires a receptor whose tumor cell recognition and effector cel...

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An entirely humanized CD3 ζ-chain signaling receptor that directs peripheral blood t cells to specific lysis of carcinoembryonic antigen-positive tumor cells

et al. 2000

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Recombinant T‐cell receptors with antibody‐like specificity for tumor‐associated antigens are successfully used to direct the cytolytic activity of T cells toward tumor cells. Clinical application, however, needs to comply with the low immunogenicity of the recombinant receptor, efficient gene transfer into peripheral blood T cells, and enrichment of receptor‐grafted cells. Here, we address these issues and describe an entirely humanized immune receptor for use in adoptive immunotherapy of colorectal carcinoma. The receptor consists of a single‐chain antibody (scFv) binding domain specific for carcinoembryonic antigen (CEA), the IgG hinge and CH2/CH3 (Fc) joining region, and the transmembrane and intracellular CD3 ζ signaling chain. To express the receptor in peripheral blood T cells, both GALV envelope and MuLV 4070A pseudotyped retrovirus turned out to be equally efficient, with transduction efficiencies of about 5% to 40%, depending on the lymphocyte donor. Furthermore, receptor‐grafted T cells could be 2‐ to 6‐fold enriched by magnetic activated cell sorting, utilizing an antibody directed to the extracellular IgG domain of the receptor. Upon co‐culture with CEA+ tumor cells, receptor‐grafted T cells are specifically and efficiently activated to cytolysis and IFN‐γ secretion, demonstrating their feasibility for the adoptive immunotherapy of CEA+ carcinomas. Int. J. Cancer 88:115–120, 2000. © 2000 Wiley‐Liss, Inc.

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Characterisation of a humanised bispecific monoclonal antibody for cancer therapy

Cited by 14 publications

References 29 publications

Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo

Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo

A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA

An entirely humanized CD3 ζ-chain signaling receptor that directs peripheral blood t cells to specific lysis of carcinoembryonic antigen-positive tumor cells

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