Multifunctional protein

A protein and polypeptide chain technology, applied in the field of multifunctional proteins, can solve the problems of limited production, clinical trial failure, research silence and other problems

Inactive Publication Date: 2018-07-06
TIMMUNE BIOTECH INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But research in the field has fallen silent due to failed clinical trials and limited production issues
(Garber K, 2014) In 2009, the bispecific antibody Catumaxomab developed by Trion was approved by the European Union for the treatment of malignant ascites caused by EpCAM-positive tumors, but its clinical application was greatly limited due to the high immunogenicity of murine antibodies (Spasevska I, 2014)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Embodiment 1, the construction of expression multifunctional protein molecular carrier

[0159] 1. Construction of a novel multifunctional protein TiTE-1 targeting CD19-positive tumor cells

[0160] A novel multifunctional protein TiTE-1 targeting CD19-positive tumor cells, which is polymerized from the main peptide chain X1 and the auxiliary peptide chain Y1 ( figure 1 );

[0161] The main peptide chain X1 includes antigen 1 binding domain R1, cytokine or cytokine receptor cytokine binding domain R2 and antigen 2 binding domain R3;

[0162] The auxiliary peptide chain Y1 includes the antigen 3 binding domain R4 and the main peptide chain X linking domain R5.

[0163] The antigen-binding domain (R1) of the main peptide chain (X1) is selected from antiCD19-ScFv (sequence 1), the auxiliary peptide chain connecting domain (R2) is selected from IL15Rαsushi (sequence 2), and the antigen-binding domain (R3) is selected from antiCD3-ScFv (sequence 3 );

[0164] The antigen...

Embodiment 2

[0194] Example 2. Expression and purification of multi-targeted functional multifunctional protein molecules and polypeptides

[0195] 1. Cultivate 293F (invitrogen) at 37°C, 8% CO2, 120rpm to a cell density of 1*10E 6 Cell / ml.

[0196] 2. The vector PCDNA3.1-TiTE-1 constructed in Example 1 was transfected into the cells in the above 1 with PEI, the concentration of the transfected plasmid was 1 mg / L, and the concentration of PEI was 3 mg / L. Culture at 37°C, 8% CO2, 120rpm for 5-6 days.

[0197] 3. Centrifuge the culture product of the above 2 at 4000rpm, collect the culture supernatant, purify the protein with Protein / capto L beads, elute with 500uL pH2.6-3.0 0.1MGly-HCL eluent, and collect the eluate.

[0198] 4. SDS-PAGE detects the collected protein (see image 3 ), it can be seen that the target protein of about 65KD and 30KD is obtained, which is the main peptide chain X and auxiliary peptide chain Y of the multifunctional protein molecule TiTE-1.

[0199] 5. Use the...

Embodiment 3

[0200] Example 3. Multifunctional protein molecules TiTE-1, 15, and 16 mediate CD19 killing by T cells in vitro + Target cell experiment verification

[0201] 1. Set 1*10E 4 Target cell malme-3M-CD19-luc (transfect CD19 antigen (its nucleotide sequence is sequence 32) and Luc gene (its nucleotide sequence is sequence 33) in Malme-3M purchased by ATCC, so that it can express CD19 antigen and Luc gene , the obtained cells (malme-3M-CD19-luc) (50uL) were plated in a 96-well plate, and cultured at 37°C and 5% CO2 for 18-20h.

[0202] 2. After the cells adhere to the wall, absorb and discard the medium, add 50uL of fresh medium and incubate at 37°C, 5% CO2 for 1-3h.

[0203] 3. Stepwise dilute the target protein TiTE-1 obtained in Example 2 to a concentration of 50, 5, 0.5, 0.05, 0.005 ng / uL;

[0204] Experimental group: inject 50uL of 1*10E 5 T cells (normal peripheral blood density gradient centrifugation, get the buffy coat rich in mononuclear cells, 50ng / mLOKT3, 300IU / mLIL2...

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Abstract

The invention discloses a multifunctional peptide chain or protein. The polypeptide chain X provided by the invention includes an antigen 1 binding domain R1, an assistant peptide chain connection domain R2 and an antigen 2 binding domain R3. The assistant peptide chain connection domain R2 is a cytokine binding domain in a cytokine or cytokine receptor. The invention provides a protein, the polypeptide chain X serves as the main peptide chain to compose a heterodimer together with an assistant peptide chain Y. The assistant peptide chain Y consists of an antigen 3 binding domain R4 and a mainpeptide chain X connection domain R5, or the assistant peptide chain Y is the main peptide chain connection domain R5. The multifunctional protein provided by the invention can combine with differenttumor antigens through the antigen binding domains of two tumor related antigens thereof to mediate specific cell killing, and improve the accuracy of targeting. Also a cytokine and cytokine receptorcompound are introduced into the multifunctional protein provided by the invention and can play the role of cytokines.

Description

technical field [0001] The invention relates to the biological field, in particular to a multifunctional protein. Background technique [0002] Bispecific antibody, also known as bifunctional antibody or bivalent antibody, can specifically bind to two different antigens at the same time, has specificity and bifunctionality, and is used in the fields of tumor immunotherapy and autoimmune diseases with broadly application foreground. [0003] Bispecific antibodies do not exist in nature and can only be prepared artificially. Currently, methods for preparing bispecific antibodies mainly include chemical conjugation, hybridoma technology, and recombinant DNA technology. [0004] As early as 30 years ago, Medarex had developed bispecific antibodies, and conducted phase III clinical trials in 2001. But research in this area has been muted due to failed clinical trials and production issues. (Garber K, 2014) In 2009, the bispecific antibody Catumaxomab developed by Trion was ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/0783A61K38/20A61K47/68A61P35/00
CPCC12N5/0636C12N5/0646C07K14/5406C07K14/5443C07K14/7155C07K16/468A61K38/00C07K2319/33A61P35/00C07K16/30C07K16/2803C07K2317/622C07K2317/569C07K16/2854C07K16/2833C07K16/283C07K16/2818C07K16/2827C07K16/2809C07K2319/00C07K2317/73C07K14/70596C07K14/4747C07K2319/02C07K14/70521C07K14/70532C07K2317/31C07K2319/30
Inventor 张凤禹高斌王磊吴亚松魏卿
Owner TIMMUNE BIOTECH INC
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