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Cells expressing th1 characteristics and cytolytic properties

A cell activity and cell technology, applied to cell culture active agents, animal cells, vertebrate cells, etc., can solve problems such as CD4+ cell processing obstacles, problems with recognizing epitopes and TCRs, and complex genetic diversity

Inactive Publication Date: 2013-04-24
IMMUNOVATIVE THERAPIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are significant hurdles in harnessing naturally occurring tumor-specific CD4+ cells for processing
The genetic diversity of HLA class II associated with CD4+ helper cells in any given patient population is much more complex than that of HLA class I found in CD8+ cytolytic (killer) T-cells, making recognition of epitopes and TCRs more complex. something wrong
Furthermore, CD4+ T-cells expand poorly in vitro compared to CD8+ cells, and culture conditions significantly affect their properties
Finally, there is a lack of realistic animal models based on tumor-specific CD4+ cells
These factors have limited the use of CD4+ T cells for translational research and have limited their clinical application

Method used

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  • Cells expressing th1 characteristics and cytolytic properties
  • Cells expressing th1 characteristics and cytolytic properties
  • Cells expressing th1 characteristics and cytolytic properties

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0059] Preparation of activated cells in culture (CAC)—CD4+ T-cells were cultured for 9 days in the presence of CD3 / CD28 ClinEx Vivo Dynal beads obtained from Invitrogen. (Location). Cells were grown in Life Cell Culture Flasks (Baxter) at 37°C and 5% CO 2 were grown and restimulated with beads on days 3 and 6 of culture. After 9 days in culture, the beads can be removed and these cells are termed CACs.

[0060] Preparation of cells in formulation buffer (CFB) - CACs were placed into cRPMI medium for washing. The time is recorded to indicate the start of the formulation protocol. Cells in cRPMI were centrifuged, supernatant removed, and cells resuspended in cRPMI buffer. Cell viability was determined by using the trypan blue assay. The percentage of viable cells was determined using the total cell number and concentration of viable cells. If the sample has greater than 80% cell viability, the steps continue for reactivation and formulation of the cells.

[0061] CAC cel...

example 1

[0068] Example 1 - Purification of CD4+ cells and cell phenotype. Normal donor peripheral blood was obtained and CD4+ cells from the buffy coat were purified using magnetic beads and columns obtained from Mitenyi Biotec (Auburn, CA). Figure 1 is a flow cytometry plot of side scatter (measured size and density of cells) versus CD4. Figure 1 illustrates that CD4+ cells constitute approximately 46% of the total population (N=22). After positive selection for CD4+ cells, the result was 98.69% pure CD4+ cells (N=22). as available from Figure 1B As can be seen, the CD4+ population after passage contains lymphocytes and monocytes.

[0069] Because each batch is produced from a different donor, antigenic markers are tested at the beginning and end of production. Test CD4+ cells ( Figure 2A ) to activated cells in culture (CAC) ( Figure 2B ) cell phenotype. The results illustrate a very consistent change from a naive CD4+ phenotype (CD45RA+) to a memory phenotype (CD45RO+)....

example 2

[0077] Example 2 - Direct killing by activated Th1 / killer cells. To test whether CFB could have a direct effect on tumor cells, the cells were tested for their ability to kill the ARH77 cell line. This cell line is an established myeloma cell line. ARH77 cells were stained in CFSE to distinguish ARH77 cells from CFB. CFB was mixed with stained ARH77 cells for 18 hours with different effector (CFB) and target (ARH77). After this time, 7AAD was added and cells were acquired using a FC500MPL flow cytometer. The experiment was repeated on different batches of CAC as well as on fresh and thawed CFB. ARH77 alone, ARH77 with DynaBeads, CD4+ cells or ARH77 with CAC had no significant death (less than 10%) data, not shown. Figure 5 The results shown demonstrate that CFB, but not CAC, CD4+ cells or beads, has a direct killing effect on the ARH77 cell line.

[0078] The nature of the direct killing effect was examined to see if the effect was due to the perforin-granzyme pathway....

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Abstract

A novel cell type has been generated that has both Thl characteristics and cytolytic activity. These Thl / killer cells are CD4+ cells purified from peripheral blood and manipulated to have Thl characteristics such as production of IFN-gamma combined with cytolytic activity similar to cytotoxic T-cells (CTL). The CTL activity is targeted toward diseased cells, not normal cells. The cytolytic activity of the Thl / killer cells is mediated by Granzyme B-Perforin mechanism and results in apoptotic death of diseased cells. Methods of producing and using these Thl / killer cells include isolating CD4+ cells from peripheral blood, activating the CD4+ T- cells to form Thl / killer cells and administering these Thl / killer cells with the cytolytic activity to a patient wherein theThl / killer cells are allogeneic to the patient.

Description

Background technique [0001] Therapeutic cancer vaccinations are a type of immunotherapy. Immunotherapy is a new treatment modality emerging to combine chemotherapy, radiation therapy and surgery as a class of drugs for the treatment of cancer. Immunotherapy approaches attempt to harness the power of the immune system to treat disease. Therapeutic vaccination is a type of immunotherapy that is a potentially curative treatment against existing neoplastic, viral and bacterial pathogens. Therapeutic vaccines generally consist of an antigen source derived from the disease of interest and an adjuvant designed to enhance the desired immune response. In some therapeutic vaccine regimens, live immune cells (derived from the host (autologous), or derived from a donor (allogeneic)) are a component of the therapeutic vaccine. Live autologous and allogeneic immune cells (eg, dendritic cells, NK cells, and T-cells) can also be used in immunotherapy regimens for the treatment of cancer. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/12A61K35/17A61K39/00
CPCA61K35/17A61K2039/57C12N2501/515C12N5/0636C12N5/0638A61K2039/5158C12N2501/51A61P31/14A61P31/18A61P31/20A61P35/00A61P35/02A61P37/04A61K35/12
Inventor 迈克尔·哈-诺伊
Owner IMMUNOVATIVE THERAPIES
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