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Plasmid constructs for treating cancer and methods of use

A technology of plasmids and structural domains, applied in chemical instruments and methods, treatment, electrotherapy, etc., can solve the problems of increasing the existence and quantity of tumor-specific T cells

Pending Publication Date: 2021-09-17
安克塞克医疗公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although IL-12 can increase the number of TILs, there is still a need to increase the presence and number of tumor-specific T cells in tumors

Method used

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  • Plasmid constructs for treating cancer and methods of use
  • Plasmid constructs for treating cancer and methods of use
  • Plasmid constructs for treating cancer and methods of use

Examples

Experimental program
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Effect test

example

[0253] CXCL9

example 1

[0254] Example 1. CXCL9 plasmid construction. Mouse CXCL9 (mCXCL9) or human CXCL9 (hCXCL9) nucleic acid sequences were cloned into expression vectors using standard molecular biology techniques. Alternatively, mCXCL9 or hCXCL9 was cloned downstream of mouse (mIL12-2A) or human (hIL12-2A) IL12 p35-P2A-IL12 p40 to generate mIL12~mCXCL9 and hIL12~hCXCL9 ( Figure 1A -B). The IL12 p35-P2A-IL12 p40 construct was prepared essentially as described in WO2017 / 106795 or WO2018 / 229696.

[0255] The resulting plasmid contained IL-12p35, IL-12p40, and CXCL9, all expressed from the same promoter, with an intermediate exon-skipping (P2A) motif, allowing all three proteins to be expressed from a single polycistronic information expression. Using a similar method to prepare mCXCL9~mCherry

example 2

[0256] Example 2. Protein expression. The mIL12-2A, mCXCL9 and mIL12-mCXCL9 expression vectors were transfected into HEK293 cells in vitro. 96 hours after transfection, supernatants were collected and assayed for IL12 and CXCL9 protein expression by ELISA. figure 2 The results shown in , show that despite decreased expression in cells transfected with the mIL12~mCXCL9 expression vector, detectable levels of both IL12 and CXCL9 were produced. Figure 27 High levels of secreted hIL12 and hCXCL9 were shown in cells transfected with hIL-12~hCXCL9 expression vectors.

[0257] Similarly, hIL12-2A, hCXCL9 and hIL12~hCXCL9 expression vectors were transfected into HEK293 cells in vitro. 96 hours after transfection, supernatants were collected and assayed for IL12 and CXCL9 protein expression by ELISA. hIL12 was almost equally expressed by both hIL12-2A (1.59 μg / mL) and hIL12~hCXCL9 (1.37 μg / mL) expression vectors ( Figure 10 A). Compared with cells transfected with hCXCL9 expres...

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Abstract

Described are expression cassettes encoding CD3-half-BiTE, CXCL9 and CTLA-4 scFv. The described expression cassettes can be used to treat cancer in a subject. Methods of delivering the expression cassettes to a tumor by direct intratumoral injection and electroporation are also described.

Description

[0001] Citations to related applications [0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 771,928, filed November 27, 2018, and U.S. Provisional Application Serial No. 62 / 826,439, filed March 29, 2019, each of which One is incorporated herein by reference. [0003] sequence listing [0004] The sequence listing written in file 522631_SeqListing_ST25 is 143 kilobytes in size, created on November 22, 2019, and is incorporated herein by reference. Background technique [0005] Cancer immunoediting is responsible for eliminating tumors and sculpting tumor immunogenic phenotypes that ultimately develop in immunocompetent hosts after tumors escape immune destruction. Immune system-tumor interactions are postulated to occur in three sequential phases: elimination, equilibrium, and escape. Elimination requires destruction of tumor cells by T lymphocytes. In equilibrium, an immune-resistant tumor cell population emerges. During escape, tumors...

Claims

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Application Information

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IPC IPC(8): C12N15/85C07K14/52C07K14/54A61K38/19A61K48/00A61P35/00
CPCA61K48/00A61P35/00C07K14/5434C07K16/2809C07K2319/03C07K2317/622A61K2039/572A61K2039/54A61K2039/505C07K16/2818C07K2317/76A61K39/3955A61K2300/00C12N15/85C07K14/522C07K14/71A61K38/19A61N1/00
Inventor 克里斯托弗·特威蒂阿纳尼达鲁普·穆霍帕德亚戴维·A·坎顿韩妙君埃丽卡·布罗宁
Owner 安克塞克医疗公司
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