ABSTRACT
The cancer-testis antigen NY-ESO-1 is a potential target antigen for immune therapy expressed in a subset of patients with multiple myeloma. We generated chimeric antigen receptors (CARs) recognizing the immunodominant NY-ESO-1 peptide 157-165 in the context of HLA-A*02:01 to re-direct autologous CD8(+) T cells towards NY-ESO-1(+) myeloma cells. These re-directed T cells specifically lysed NY-ESO-1(157-165)/HLA-A*02:01-positive cells and secreted IFNγ. A total of 40% of CCR7(-) re-directed T cells had an effector memory phenotype and 5% a central memory phenotype. Based on CCR7 cell sorting, effector and memory CAR-positive T cells were separated and CCR7(+) memory cells demonstrated after antigen-specific re-stimulation downregulation of CCR7 as sign of differentiation towards effector cells accompanied by an increased secretion of memory signature cytokines such as IL-2. To evaluate NY-ESO-1 as potential target antigen, we screened 78 bone marrow biopsies of multiple myeloma patients where NY-ESO-1 protein was found to be expressed by immunohistochemistry in 9.7% of samples. Adoptively transferred NY-ESO-1-specific re-directed T cells protected mice against challenge with endogenously NY-ESO-1-positive myeloma cells in a xenograft model. In conclusion, re-directed effector- and central memory T cells specifically recognized NY-ESO-1(157-165)/ HLA-A*02:01-positive cells resulting in antigen-specific functionality in vitro and in vivo.
Subject(s)
Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Immunotherapy , Multiple Myeloma/therapy , Neoplasm Proteins/immunology , Peptide Fragments/immunology , Receptors, Antigen, T-Cell/immunology , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/transplantation , Cell Line, Tumor , Genetic Therapy , Humans , Immunologic Memory , Interferon-gamma/genetics , Interferon-gamma/metabolism , Interleukin-2/genetics , Interleukin-2/metabolism , Mice , Multiple Myeloma/immunology , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Peptide Fragments/genetics , Peptide Fragments/metabolism , Receptors, Antigen, T-Cell/genetics , Receptors, CCR7/genetics , Receptors, CCR7/metabolism , Transduction, GeneticABSTRACT
The EB1/RP1 family is a new protein family that is characterized by the ability of its members to serve as interacting partners for the adenomatous polyposis coli (APC) tumour suppressor protein and tubulin. Data obtained with highly conserved yeast homologues suggest that the EB1/RP1 protein family promotes cytoplasmic microtubule dynamics and contributes to the sensor mechanism controlling the cytokinesis checkpoint during mitosis. However, the precise function of this protein family in mammalian cells has not been elucidated so far and remains unclear. Here, we report on the genomic localization of the RP1 gene and the characterization of the corresponding promoter. The RP1 gene was found to be encoded on chromosome 18q21, a locus which is altered or deleted in up to 50% of all patients with colorectal cancer. Promoter analysis revealed that the RP1 gene is under the control of a strong promoter that was 10 times more active in mammalian cells when compared to SV40 promoter. Members of the cyclic AMP response element binding protein family (CREB1 and CREB2) could be identified as transcription factors binding specifically within the RP1 promoter sequence.
Subject(s)
Chromosomes, Human, Pair 18/genetics , DNA-Binding Proteins/genetics , Eye Proteins , Genes, APC , Promoter Regions, Genetic/genetics , Trans-Activators/genetics , Transcription, Genetic , Base Sequence , Cell Line , Chromosome Mapping , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Humans , Microtubule-Associated Proteins , Molecular Sequence Data , RNA, Messenger/metabolism , T-Lymphocytes , Tumor Cells, CulturedABSTRACT
Mutations in the adenomatous polyposis coli (APC) gene are linked to the dysplastic transformation of colorectal polyps and represent an early step in the development of colorectal tumors. Ninety-four percent of all mutations result in the expression of a truncated APC protein lacking the C-terminal region. The C-terminal region of the APC protein may have a tumor suppressor function as its absence appears to be linked to the development of dysplastic lesions. Recently, we discovered and characterized a protein called RP1 which binds specifically to the C-terminal region of the APC protein. We show now that RP1 and the other known members of the EB/RP family (EB1 and RP3) also bind directly to tubulin, both in vitro and in vivo. Immunohistochemical analyses reveal a distinct staining pattern during interphase as well as an association of RP1/EB1 with mitotic microtubule structures. The previously described puncta of the APC protein at the leading edge of membrane protrusions contact microtubule fibers that contain RP1 or EB1.
Subject(s)
Carrier Proteins/genetics , DNA-Binding Proteins/genetics , Eye Proteins , Microtubule-Associated Proteins/genetics , Multigene Family , Proteins/genetics , Tubulin/metabolism , Adenomatous Polyposis Coli/genetics , Amino Acid Sequence , Genetic Code , Humans , Immunohistochemistry , Mitosis/physiology , Molecular Sequence Data , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism , Tumor Cells, CulturedABSTRACT
Synopsis The phase inversion emulsification is a convenient method of preparing fine-disperse and long-term stable oil-in-water emulsions, which are stabilized with nonionic emulsifiers. On the basis of EACN-values (equivalent alkane carbon numbers) the calculation of phase inversion in concentrates (CAPICO) is possible, which yields emulsifier and oil mixing ratios corresponding to a given phase inversion temperature (PIT). The CAPICO-method is illustrated for the example of a cosmetic oil-in-water lotion containing an oil mixture, glyceryl monostearate and a fatty alcohol ethoxylate. Of special interest is the influence of silicone oils on the PIT. At a constant emulsifier oil ratio the complete phase behaviour of this emulsion system is represented in a temperature/water content graph. Optimum emulsification results are obtained if during PIT emulsification a microemulsion or a lamellar phase is passed. The emulsions were characterized by particle sizing, and emulsion stability against sedimentation was evaluated by ultrasonic velocity changes. A fine-disperse and long-term stable oil-in-water emulsion was prepared by a time and energy-saving two-step hot-cold process.
