Tumor suppression using human placental perfusate and human placenta-derived intermediate natural killer cells

A technology of natural killer cells and placental perfusate, applied in blood/immune system cells, antineoplastic drugs, animal cells, etc., can solve problems such as difficult to handle, difficult to maintain tumor targeting and kill tumors

Active Publication Date: 2010-11-03
ANTHROGENESIS LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Despite the favorable properties of NK cells in killing tumor cells and virus-infected cells, handling them and using them for immunotherapy remains difficult, mainly due to the difficulty in maintaining their tumor targeting during culture and expansion. and the ability to kill tumors

Method used

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  • Tumor suppression using human placental perfusate and human placenta-derived intermediate natural killer cells
  • Tumor suppression using human placental perfusate and human placenta-derived intermediate natural killer cells
  • Tumor suppression using human placental perfusate and human placenta-derived intermediate natural killer cells

Examples

Experimental program
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Effect test

Embodiment 2

[0377] 6.2. Example 2: Placental Intermediate Natural Killer Cells from Mixed Placental Perfusate and Cord Blood cell representation

[0378] Donor-matched mononuclear cells (pool) from cord blood and placental perfusate were pooled and washed once with FACS buffer (RPMI-1640 containing 5% FBS) and using the antibodies listed in Table 2, in BDFACSCanto (BD Biosciences) for immunophenotyping. Data were analyzed with FlowJo software (Tree Star).

[0379] Table 2: List of antibodies used in immunophenotyping characterization

[0380] project

[0381] project

[0382] project

[0383] Characterization of the immunophenotype of placental NK cells and peripheral blood (PB) NK cells. Divide NK cells into two main groups: CD56 + CD16 + NK cells and CD56 + CD16 - cell. CD56 + CD16 + NK cells have a large number of cytolytic granules and highly express CD16, and therefore, are capable of eliciting antibody-dependent cell-mediated cytotoxicity (...

Embodiment 3

[0417] 6.3. Example 3: Cytotoxicity of natural killer cells to tumor cells

[0418] This example demonstrates that placental intermediate natural killer cells are cytotoxic to tumor cells. PINK cells from HPP were cytotoxic to acute myeloid leukemia cells as demonstrated in cytotoxicity assays and Luminex assays for cytokine secretion of NK cells.

[0419] In the cytokine secretion assay, NK cells enriched with CD56 microbeads from HPP were mixed with KG-1a acute myeloid leukemia cells at a ratio of 1:1. After 24 hours of culture, supernatants were collected and subjected to Luminex analysis for IFN-γ and GM-CSF secretion. As shown in Figure 2, after co-cultivating CD56-enriched HPP cells with KG-1a cells for 24 hours, increased levels of IFN-γ and GM-CSF were observed.

[0420]Cytotoxicity of PINK cells

[0421] In the cytotoxicity assay using PINK cells, target tumor cells were labeled with carboxyfluorescein succinimidyl ester (CFSE). CFSE is a vital dye that is not t...

Embodiment 4

[0475] 6.4. Example 4: Cytotoxicity of human placental perfusate to tumor cells

[0476] This example demonstrates that human placental perfusate cells are cytotoxic to tumor cells and that all nucleated cells from HPP (TNC-HPP) are more cytotoxic to KG-1a than TNC from matched UCB. All nucleated cells from HPP or umbilical cord blood (UCB) were mixed with KG-1a cells at a ratio of 1:1, 5:1, 10:1, 20:1 or 100:1. After 24 or 48 hours of incubation, cells were harvested and detected for the presence of CFSE by FACS analysis (BD FACSCanto, BD Bioscience). Tumor cells cultured alone were used as controls. Cytotoxicity is defined as: (1-CFSE 样品 / CFSE 对照 )×100%. At a ratio of 100:1, significant cytotoxicity was exhibited. see Figure 5 .

[0477] In a separate experiment, the cytotoxicity of all nucleated cells from HPP was compared to that of all nucleated cells from cord blood. Matched TNC-HPP or UCB were mixed with KG-1a cells at a ratio of 0.78:1, 1.56:1, 3.12:1, 6.25:...

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Abstract

Provided herein are placental perfusate, placental perfusate cells, and placenta-derived intermediate natural killer (PINK) cells, and combinations thereof. Also provided herein are compositions comprising the same, and methods of using placental perfusate, placental perfusate cells, and placenta-derived intermediate natural killer cells, and combinations thereof, to suppress the growth or proliferation of tumor cells, cancer cells, and the like, and to treat individuals having tumor cells. In a preferred embodiment, the composition comprises: solated CD56+, CD16- natural killer cells.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 60 / 995,763, filed September 28, 2008, and U.S. Provisional Patent Application No. 61 / 090,555, filed August 21, 2008, the entire contents of which are incorporated by reference incorporated into this article. 1. Field of invention [0002] The present invention provides tumor cells obtained by combining tumor cells with placental perfusate, cells from placenta, natural killer cells from placenta (eg, from placental perfusate), and / or containing natural killer cells from placenta (eg, from placental perfusate). A method of inhibiting the growth or proliferation of tumor cells by contacting killer cells with a mixture of natural killer cells derived from umbilical cord blood natural killer cells. The invention also provides methods for preparing a unique population of natural killer cells from placenta, eg, from placental perfusate (eg, human placental perfusate). Further, the present invention pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/50C12N5/06A61P35/00A61K35/12C12N5/0783
CPCA61K2035/124C12N5/0646A61K35/50A61P35/00A61K35/17A61K45/06
Inventor 晓奎·张瓦内萨·A·沃斯基纳里安-贝尔斯琳·康尼拉弗·迪利普·帕德里亚
Owner ANTHROGENESIS LLC
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