Method and composition for altering a t cell mediated pathology

A composition and cell technology, applied in the field of immunology and immunotherapy, can solve the problems of difficult production of TCR molecules and low production level

Inactive Publication Date: 2004-01-14
FAVRILLE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as mentioned above, the production of these TCR molecules is difficult and the production levels are low

Method used

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  • Method and composition for altering a t cell mediated pathology
  • Method and composition for altering a t cell mediated pathology
  • Method and composition for altering a t cell mediated pathology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Long:

[0154] Tumor samples obtained from peripheral lymph nodes were biopsied under sterile conditions according to clinically prescribed methods, and the samples were used to produce patient idiotype-specific recombinant V α and V β - Ig chimeric protein. Store the remaining lymph node biopsy material in a tissue cell bank in liquid nitrogen for future use.

[0155] Cell isolation: Single cell suspensions of patient lymph node biopsy samples were obtained by forcing patient biopsy lymphoma tissue through a disposable 0.38 mm steel mesh and simultaneously immersed in sterile PBS. Dispersed cells were washed twice with PBS, then resuspended and counted. A 10% ratio of the cells was processed for total RNA extraction and the remaining cells were stored in liquid nitrogen after resuspension in RPMI 1640 tissue culture medium containing 30% fetal bovine serum and 10% DMSO. All clinical sample processing was performed in a biological safety cabinet.

[0156] Total...

Embodiment 2

[0158] Cloning and sequencing of PCR products: According to the manufacturer's recommendations, from multiple reactions, determined to contain V α and V β The PCR product of the tumor-specific sequence of the chain was cloned directly into the plasmid pCR2.1-TOPO, and then introduced into Top10 competent E. coli (E. coli) cells (Invitrogeh). Twenty-four miniprep DNA plasmids were prepared from carbenicillin-resistant colonies using the QIAPrep Spin Miniprep Kit (Qiagen) and quantified by spectrophotometry. 200 ng of each plasmid were sequenced using the Cy5 / Cy5.5 Dye Primer Cycle Sequencing Kit (Visible Genetics). After the sequencing reaction was completed, the samples were electrophoresed on the OpenGene DNA Automated Sequencing System (Automated DNASequencing System), and the data were processed with the GeneObjects software package (Visible Genetics). Additional analysis, including sequence alignment, was performed using SEQUENCHER Version 4.1.2 DNA analysis software (...

Embodiment 5

[0180] Production of TCR / Ig Chimeric Proteins in High-5 Insect Cells: High-5 insect cells (BTI-TN-5B1-4) secrete higher levels (2-20×) of recombinant immunoglobulins than Sf-9 cells and are selected for TCR / Ig chimeric protein production cell line. High-5 cells (1.0-2.0×10 6 cells / ml) at 5×10 5 Cells / ml were seeded in ESF921 medium (Expression Systems LLP) in 1 liter disposable culture flasks with vented closures. The culture bottle was shaken at 140-150 rpm at 28°C, and the volume of the culture medium in the culture bottle was adjusted within a specified time so that it did not exceed 500 ml. When the cell density reached 1.5-2.5 cells / ml in 500ml culture medium, the cells in the culture flask were infected with a high-titer recombinant baculovirus stock solution at a multiplicity of infection (MOI) of about 0.5:1 (pfn:cells). The flasks were then shaken at 140-150 rpm at 28°C. Cell viability was checked daily and cultures were harvested when viability did not drop to ...

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Abstract

The present invention provides a method for altering a T cell mediated pathology in a patient. This method comprises administering a composition comprising at least one and/or two chimeric proteins. Each chimeric protein comprises at least a portion of either the Valpha or Vbeta region of a TCR from particular T cells from a patient having a T cell mediated pathology, and an immunoglobulin constant region. The genes encoding Valpha and/or Vbeta regions and the genes encoding immunoglobulin constant regions are isolated and inserted into an expression vector. The chimeric proteins are produced by introducing the expression vectors into insect cell lines. The chimeric proteins are purified using antibody affinity columns, and then chemically conjugated to an immunogenic carrier, keyhole-limpet hemocyanin (KLH). Since the conjugates comprises chimeric proteins made specifically from particular T cells from a patient having T cell mediated pathology, when it is administered to such a patient, with or without a cytokine, such as granulocyte-macrophage-CSF, or a chemokine, it can induce immune responses to alter such a T cell mediated pathology.

Description

[0001] related application [0002] This application claims U.S. Provisional Application No. 60 / 224,723, entitled "Method for producing an Idiotypic Vaccine", entitled "Expression Vectors for Production of Recombinant Immumoglobulin Vector)" and U.S. Provisional Application No. 60 / 266,133 entitled "Method and Composition for Altering a T Cell Mediated Pathology" number priority. field of invention [0003] The present invention relates generally to the fields of immunology and immunotherapy. More specifically, the present invention relates to methods and compositions for altering T cell mediated pathologies such as T cell malignancies and / or autoimmune diseases. Background of the invention [0004] The present invention relates generally to the fields of immunology and immunotherapy. More specifically, the present invention relates to methods and compositions for altering T cell mediated pathologies such as T ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/385A61K39/395A61P35/00A61P37/00C12N15/866
Inventor D·P·戈德R·J·肖佩斯
Owner FAVRILLE
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