Methods for Isolating and Proliferating Autologous Cancer AntiGen-Specific CD8+ T Cells

a technology of autologous cancer and antigen-specific cd8, which is applied in the field of isolating and proliferating autologous cancer antigen-specific cd8 + t cells, can solve the problem of long culture period required to produce a sufficient amount of antigen-specific cd8, and achieve the effect of effectively selecting and eliminating cancer cells

Inactive Publication Date: 2015-09-17
NAT CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0016]According to the present invention, it is possible to isolate autologous cancer antigen-specific CD8+ T cells by using the peptide of the CD8 T cell epitope of the autologous cancer antigen present in blood of individual cancer patients instead of the heterologous antigen. Therefore, by using the T cell, which is isolated by the method of the present invention and recognizes the autologous cancer antigen present in an extremely low ratio in a healthy person, it is possible to effectively select and eliminate cancer cells derived from the cancer patient's own cells.

Problems solved by technology

Generally, antigen-specific CD8+ T cells are isolated by using MHC class I / peptide multimer, but the method has a drawback in that due to the high cell death rate caused by cell apoptosis after cell isolation, a long period of culture is required to produce a sufficient amount of antigen-specific CD8+ T cells.

Method used

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  • Methods for Isolating and Proliferating Autologous Cancer AntiGen-Specific CD8+ T Cells
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  • Methods for Isolating and Proliferating Autologous Cancer AntiGen-Specific CD8+ T Cells

Examples

Experimental program
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experimental example

Epitope Screening Process

[0047]CD 8 T cell epitopes of an autologous cancer antigen were selected through algorithm. To evaluate a type of CD8 T cell epitope with which T cells present in blood of a cancer patient react, peripheral blood mononuclear cells (PBMCs) were isolated from blood of the cancer patient, washed, and suspended in CTL medium (RPMI1640 medium+4 mM L-glutamine+12.5 mM HEPES+50 μM 2-mercaptoethanol+3% autoplasma) to become the concentration of 1×106 cell / ml. Then, 1 ml of the suspension was aliquoted in a 14 ml round tube. Peptides for each epitope selected by the analysis with algorithm were added to each tube in the concentration of 1 μg / ml. Thereafter, culture in a CO2 incubator was started. Two days after culture, 1 ml of CTL medium including 50 U / ml IL-2 was added to each tube. On day 7, 9, 11, and 13 of culture, 1 ml of the medium was removed and CTL medium including 50 U / ml IL-2 was added. On day 14 of culture, RPMI1640 medium was added to each tube, and the...

example 1

Selection of Autologous Cancer Antigen and CD8 T Cell Epitope

[0049]Based on journals (Scanlan M J, et al., Immunol Rev. 2002 October 188:22-32; Ramakrishnan S, et al., Cancer research. 1998. 58:622-625; Nakahara Y, et al., Brain Tumor Pathol. 2004. 21(3):113-6) evaluating which type of cancer antigen is suitable for immunotherapy of a cancer depending on the type of cancer, an autologous cancer antigen, which is suitable for immunotherapy of frequently occurring cancer in Korean and hard-to treat cancers (e.g., gastric cancer, lung cancer and pancreatic cancer), is selected. hTERT (GenBank: BAC11010.1), WT1 (GenBank: AAO61088.1), NY-ESO1 (GenBank: CAA05908.1), and MAGE-A3 (NCBI Reference Sequence: NP—005353.1) are typical autologous cancer antigens used in anticancer immunotherapy in various ways, and types of cancers to which those four cancer antigens are applicable are selected and summarized in Table 1 below.

TABLE 1Target antigenPatientsEBVEBNA1, LMP1, LMP2EBV-related TumorsGast...

example 2

Epitope Screening on Clinical Cancer Patient

[0051]To evaluate whether CD8 T cell epitopes of autologous cancer antigens, i.e. hTERT, WT1, NY-ESO1, and MAGE-A3, selected in Example 1 substantially induce proliferation of CD8 T cells present in blood of a clinical cancer patient, epitope screening as depicted in FIG. 2. was performed. hTERT epitope screening was performed on gastric cancer, lung cancer and pancreatic cancer, as a main subject matter. Also, WT1 epitope screening was performed on brain and spinal cancer and lung cancer; NY-ESO1 epitope screening was performed on ovarian cancer and sarcoma; and MAGE-A3 epitope screening was performed on sarcoma and lung cancer as a main subject matter.

[0052]FIG. 3 shows an hTERT epitope screening result using PBMCs obtained from a healthy doner.

[0053]FIG. 4 shows a WT1 epitope screening result using PBMCs obtained from the healthy doner.

[0054]As shown in FIGS. 3 and 4, CD8 T cell epitopes of hTERT and WT1 did not induce T cell response b...

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Abstract

Provided is a method for isolating and proliferating autologous cancer antigen-specific CD8+T cells, and more particularly, a method for selecting an epitope recognized by CD8+ T cells from autologous cancer antigens present in blood of individual cancer patients; and isolating autologous cancer antigen-specific CD8+ T cells by using a peptide of the selected epitope, and a method of massively proliferating CD8+ T cells by using the method. According to the present invention, it is possible to isolate autologous cancer antigen-specific CD8+ T cells by using the peptide of the CD8 T cell epitope of the autologous cancer antigen present in blood of individual cancer patients instead of a heterologous antigen. Therefore, by using T cells recognizing the autologous cancer antigen, it is possible to effectively select and eliminate cancer cells derived from the cancer patient's own cells. Thus, T cells can be applied to treatment and alleviation of cancer diseases without side effects.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This U.S. non-provisional patent application claims priority under 35 U.S.C. §119 of Korean Patent Application No. 10-2014-0029198, filed on Mar. 12, 2014, the entire contents of which are hereby incorporated by reference.STATEMENT REGARDING SEQUENCE LISTING[0002]The Sequence Listing associated with this application is provided in text form in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 510461_SequenceListing_asfiled31215.txt. The text file is 3 KB, was created on Mar. 12, 2015, and is being submitted electronically via EFS-Web, concurrent with the filing of the specification.BACKGROUND OF THE INVENTION[0003]The present invention relates to a method for isolating and proliferating autologous cancer antigen-specific CD8+T cells. More particularly, the present invention relates to a method for selecting an epitope recognized by CD8+ T cell...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/998C12N2501/2302C12N5/0647C12N2501/50C12N2501/52
Inventor KWON, BYOUNG SEKANG, HYUN-GUYKIM, KWANG-HUIKIM, YOUNG-WOOKIM, YOUNG-HOPARK, BYUNG-KIUPARK, SANG-YOONPARK, SANG-JAEEOM, HYEON-SEOKOH, HO-SIKYOO, HEONLEE, DON-GILLEE, SEUNG-HOONLEE, YOUNG-JOOLEE, JIN-SOOCHOI, BEOM-KYU
Owner NAT CANCER CENT
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