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DNA composition against tumor stromal antigen FAP and methods of use thereof

一种组合物、肿瘤的技术，应用在生物化学设备和方法、DNA / RNA疫苗接种、完整细胞/病毒/DNA/RNA成分等方向，能够解决生产成本高等问题，达到表达稳定的效果

Inactive Publication Date: 2012-09-05
THE SCRIPPS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Subunit vaccines have the advantages of being stable, safe, and chemically well-defined; however, they are too expensive to produce

Method used

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  • DNA composition against tumor stromal antigen FAP and methods of use thereof
  • DNA composition against tumor stromal antigen FAP and methods of use thereof
  • DNA composition against tumor stromal antigen FAP and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1. Preparation of DNA composition 1 encoding murine FAP.

[0120] will encode murine FAP (SEQ ID NO: 4, Figure 10 ) cDNA (SEQ ID NO: 3, Figure 9 ; provided by Dr.J.D.Cheng) subcloned into the EcoRI restriction site of pcDNA3.1 / V5-His-TOPO vector (Invitrogen, San Diego, California) to obtain the eukaryotic expression vector pcDNA3.1-FAP (pFAP) (see figure 1 , sub-figure A). Proper expression of the 95 kDa FAP protein from the above vector was demonstrated by Western blotting of lysates from transiently transfected CT26 colon cancer cells, which do not express FAP themselves ( figure 1 , sub-figure B). Transiently transfected D2F2 breast cancer cells were also treated in the same way. For transient transfections, CT26 and D2F2 cells were electroporated as previously described (see Loeffer et al., FASEB, J. 2001, 15:758-767, incorporated herein by reference). FAP protein expression was demonstrated by Western blotting using a polyclonal rabbit anti-mouse FA...

Embodiment 2

[0123] Example 2. Preparation of DNA composition 2 encoding murine FAP, murine IL-2, and murine CCL21.

[0124] By the same general method described in Example 1, cDNA (DNA SEQ ID NO: 5) encoding murine IL-2 from ATCC, accession number 39892, Manassas, Virginia, and from Invitrogen, San Diego, California The cDNA of murine CCL21a (DNA SEQ ID NO: 7) was subcloned into the murine pFAP vector of Example 1. RE88 Salmonella typhimurium was transformed with the generated vector (pFAP / IL-2 / CCL21 ) by the method described in Example 1 to provide a DNA composition 2 solution comprising the following DNA construct: The DNA constructs operably encode murine FAP, murine IL-2, and murine CCL21a, and are incorporated into an attenuated Salmonella typhimurium vector.

Embodiment 3

[0125] Example 3. Evaluation of DNA compositions of the present invention in mouse models of colon cancer and breast cancer.

[0126] Oral inoculation, tumor cell challenge and treatment with doxorubicin. For experiments in the prophylactic setting, the 9 Salmonella typhimurium (S.typhimurium) (AroA - dam - BALB / c mice (n=8) were inoculated 3 times at about 1 week intervals by oral gavage of about 100 μl PBS of Salmonella typhimurium (S. typhimurium) with a vector plasmid encoding murine FAP Transformed Salmonella typhimurium (DNA composition 1 of Example 1), Salmonella typhimurium transformed with vector plasmids encoding murine FAP, IL-2 and CCL21 (DNA composition 2 of Example 2), or Example 1 The above process was carried out by using the method described in Niethammer, et al., Nat. Med., 2002, 8: 1369-1375. About 10 days later, by subcutaneous (s.c.) injection of about 3×10 4 CT26 colon cancer cells or by orthotopic (o.t.) injection in the leftmost second mammary fat p...

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Abstract

A DNA composition effective for eliciting an immune response against tumor cells and tumor metastases comprising a DNA construct that encodes for at least one epitope of fibroblast activation protein (FAP), which is expressible in immune cells, and which is incorporated in a pharmaceutically acceptable carrier. The composition can encode a single epitope of FAP, a polypeptide comprising two or more epitopes of FAP, the entire FAP protein, or any portion thereof that will elicit the desired immune response. In one preferred embodiment, the composition also includes a DNA construct that encodesan immune effector protein, such as a cytokine. The DNA composition of the invention can be used alone or in combination with a chemotherapeutic agent to treat diseases such as tumors and tumor metastases.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Patent Application Serial No. 60 / 815,316, filed June 21, 2006, which is incorporated herein by reference. [0003] government rights [0004] This invention was made with Government support under Grant No. CA83856 awarded by the National Institutes of Health and Grant No. BC031079 awarded by the Department of Defense. The government has certain rights in this invention. field of invention [0005] The present invention relates to deoxyribonucleic acid (DNA) compositions encoding suitable molecules effective to stimulate an immune response against stromal fibroblasts in tumors. In more detail, the present invention relates to DNA compositions encoding a matrix antigen known as fibroblast activation protein (FAP). The present invention also relates to methods of using the DNA composition to inhibit tumor growth and tumor metastasis, and to increase cellular uptake of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68A61K39/00
CPCA61K39/0005A61K2039/53A61K39/0011A61K2039/585A61P35/00A61P35/04A61P37/04A61P43/00
Inventor R·A·雷斯菲尔德M·勒夫勒J·A·克吕格尔A·G·尼萨默
Owner THE SCRIPPS RES INST
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