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Anti-EpCAM antibodies

A technology of antibody and antigen, applied in the field of binding protein

Inactive Publication Date: 2012-07-04
奥菲技术科学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, the therapeutic potential of anti-EpCAM antibodies is clear and the challenge is to develop anti-EpCAM antibodies that exhibit alternative or improved properties compared to antibodies known in the art, especially the now apparent leader, MT201

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0415] Example 1: Novel Antibody

[0416] Considering the need for further tumor-specific antibodies that can be used in cancer therapy, a human antibody was identified that specifically recognizes colon cancer cell lines such as HT29. This antibody can specifically bind EpCAM. A single-chain form of this antibody was cloned into the pHOG21 plasmid containing c-myc and a 6×His-tagged epitope ( Figure 8 ). TG1 bacteria were transformed and scFv was induced by IPTG to express. Binding of purified scFv was confirmed by flow cytometry using an Easycyte flow cytometer.

[0417] sequence

[0418] The nucleotide sequences of the heavy and light chains of the cloned antibodies were sequenced. This antibody was named 3-17I(scFv). The nucleotide and amino acid sequences of the light and heavy chains of 3-17I (scFv) are in figure 1 and shown in Table 1. The CDR regions of the 3-17I light and heavy chains are shown in Table 1.

[0419] This antibody was also produced in its ...

example 2

[0432] Example 2: Binding of 3-17I IgG to KatoIII cells

[0433] Flow cytometry was used to analyze the binding of 3-17I IgG to EpCAM positive cells. Anti-EpCAM chimeras (murine variable regions / human constant regions) and fully human antibodies, MOC31 and MT201, respectively, were used as positive controls.

[0434] The amino acid sequences of the complete heavy and light chains of the MT201 IgG and MOC31 IgG antibodies used are shown below. Constant regions are in bold italics.

[0435] MT201 IgG heavy chain (amino acid sequence)

[0436] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNK

[0437] YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDMGWGSGWRPYYYYGMD

[0438] VWGQGTTVTVSS

[0439]

[0440] MT201 IgG light chain (amino acid sequence)

[0441] ELQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWYQQKPGQPPKLLIYWASTRESGVPDR

[0442] FSGSGSGTDFTLTISSLQPEDSATYYCQQSYDIPYTFGQGTKLEIK

[0443]

[0444] These sequences were deduced from the amino acid sequence of MT201, w...

example 3

[0455] Example 3: Binding properties of 3-17I IgG

[0456] To determine the ability of 3-17I to bind EpCAM, Western blot analysis, ELISA and BIAcore assays were used.

[0457] Western blot analysis

[0458]To analyze the binding specificity and cross-reactivity of antibodies 3-17I, MT201 and MOC31 to human and cynomolgus EpCAM, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS -PAGE) and Western blot analysis. All antibodies were in the IgG format and were produced by transiently transfected HEK-293 cells. Antibodies were then purified in Protein A Sepharose (GE Healthcare Uppsala, Sweden) followed by fractionation on two coupled molecular sieve columns, Superdex 200 and Superdex 75. The resulting IgG product exhibited a monomeric purity of >95%. Recombinant human and cynomolgus EpCAM antigens were also produced in HEK-293 cells and purified in one step on protein A sepharose columns. The purity of the tested antigen preparation is higher than 90%. Both Ep...

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Abstract

Disclosed are antibodies that bind to Epithelial Cell Adhesion Molecule (Ep CAM) and display certain advantages over known antibodies which bind to Ep CAM, for example, the antibodies of the invention show good affinity, good cross-reactivity profiles and excellent ADCC and CDCC activity. Antibodies comprising specific heavy and light chain CDRs are disclosed. The invention thus relates to these antibodies and all uses thereof, in particular in the treatment of cancer. The present invention thus provides new antibody-based compositions, methods and combined protocols for treating cancer. Advantageous immunoconjugate compositions and methods using the new anti-Ep CAM antibodies are also provided.

Description

technical field [0001] The present invention relates to binding proteins to epithelial cell adhesion molecule (EpCAM) and all uses thereof. In particular, the invention relates to antibodies or antibody fragments that bind to EpCAM and methods of use thereof, including cancer therapy. Background technique [0002] In 2000, approximately 22 million people worldwide suffered from cancer and 6.2 million died from the disease. There will be 10 million more new patients per year, and this number will increase by 50% in the next 15 years (WHO, World Cancer Report. Bernard W. Stewart and Paul Kleihues, eds. IARC Press, Lyon, 2003). Deaths caused by cancer account for 13% of all human deaths. According to the American Cancer Society, in 2007, 7.6 million people worldwide died of cancer. [0003] The most common current cancer treatment options are generally limited to invasive surgery, radiation therapy, and chemotherapy, which can lead to potentially serious side effects, nonspe...

Claims

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Application Information

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IPC IPC(8): C07K16/30
CPCC07K2317/622C07K2317/565C07K2317/92C07K2317/21C07K2317/734C07K2317/732A61K2039/505C07K2317/56C07K16/30A61P35/00
Inventor 拉维尼娅·黛安娜·奇科尔塔什·贡纳松迪德里克·派于斯燕妮·玛加丽塔·卡尔松雷姆科·阿尔贝特·格里普谢尔盖·米哈伊洛维奇·基普里亚诺夫
Owner 奥菲技术科学研究院
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