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Polypeptide, preparation method of polypeptide, bispecific antibody and application of bispecific antibody

A multi-specific antibody and antibody technology, applied in the field of medicine, can solve the problems of complicated reaction mechanism and purification steps, limited clinical application of multi-specific antibodies, large randomness, etc., and achieve the effect of simple and efficient preparation and high clinical application prospects

Active Publication Date: 2022-02-25
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006]The existing multispecific antibody construction methods have many inherent defects, high cost, and slow progress in clinical transformation
The preparation of multispecific antibodies by chemical methods requires chemical bonding of antibody molecules and nanoparticles in organic solvents. The reaction mechanism and purification steps are complicated. The clinical application of multispecific antibodies is limited; and the genetic background of double hybridomas is derived from two hybridoma cells of the parent, which must produce two kinds of heavy chains and two kinds of light chain molecules, and the random combination of these light and heavy chains is at least Ten types. In theory, only the combination of light and heavy chain homologous pairing and heavy chain and heavy chain heterologous pairing can produce the desired bispecific antibody. Therefore, this preparation method is random and inefficient. Product production Problems such as purification and product stability are also difficult to solve; the method of gene recombination also has complicated reaction mechanism and purification process, high industrial production cost, many defects, high cost, and slow progress in clinical transformation

Method used

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  • Polypeptide, preparation method of polypeptide, bispecific antibody and application of bispecific antibody
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  • Polypeptide, preparation method of polypeptide, bispecific antibody and application of bispecific antibody

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preparation example Construction

[0061] The present invention develops a new method for preparing multispecific antibodies, including the antibody combination provided by the above technical solution and other applicable monoclonal antibody drugs.

[0062] The present invention also provides a method for preparing an antibody drug for inhibiting tumor growth, the object is a mammal or a tumor cell of the mammal, and the mammal is preferably a rodent, an artiodactyla, an odd ungulate Hoofed animals, lagomorphs, primates, etc. The primate is preferably a monkey, ape or Homo sapiens. The subject may be a patient suffering from a tumor, or an ex vivo tumor cell of a patient suffering from a tumor. Based on this, the research of the present invention finds that the bispecific antibody drug has a more obvious tumor treatment effect.

[0063]According to the present invention, after changing the type and combination of antibodies, the tumors include nasal and sinus malignant tumors, nasopharyngeal cancer, oral can...

Embodiment 1

[0067] Example 1 Preparation of poly(L-glutamic acid) homopolymer (PGLU)

[0068] Dissolve γ-benzyl-L-glutamate-N-internal carboxylic anhydride monomer (BLG-NCA) in anhydrous N,N-dimethylformamide (DMF), stir to dissolve and add 1.0 mL of n-hexane Amine (n-HA), sealed, stirred and reacted for 72h at a temperature of 25°C. Then add acetic anhydride to react for 6 hours. After the reaction, the obtained reaction solution was settled into 2.0 L of ether, followed by filtration and washing with ether, and vacuum-dried at room temperature for 24 hours to obtain the intermediate product poly(γ-benzyl-L -glutamate) (PBLG).

[0069] 10.0 g of the above-prepared poly(γ-benzyl-L-glutamate) was dissolved in 100 mL of dichloroacetic acid, and under stirring conditions, 30 mL of hydrogen bromide / glacial acetic acid solution with a mass content of 33% was added, The reaction was stirred for 1 h at a temperature of 30 °C. Afterwards, the obtained reaction solution was settled into 1.0L of...

Embodiment 2

[0071] Example 2: Preparation of multivalent Fc-binding polypeptide PGLU-Fc-III-4C

[0072] Prepare the polypeptide of formula (II) structure, wherein, R 1 for -(CH 2 ) 5 CH 3 , x=0.875, y=120, R 2 for H ions, R 4 is acetyl, L 1 with L 2 for -CH 2 CH 2 -.

[0073] Dissolve poly(L-glutamic acid) homopolymer (PGLU) and N,N'-disuccinimidyl carbonate (N,N'-Disuccinimidyl carbonate) in DMF under anhydrous conditions, add triethylamine After reacting for 48 hours, Fc-III-4C and triethylamine were added, and after reacting for 2 hours at 30°C, the reactant was first dialyzed with DMF for 12 hours, and then dialyzed in water between pH 6.8-7.4 (adjusted with 1M NaOH) for 72 hours. After the product was lyophilized, the obtained PGLU-Fc-III-4C was analyzed by nuclear magnetic resonance with deuterated water as a solvent, and the results are shown in figure 2 , the results showed that Fc-III-4C was successfully grafted to the side chain of PGLU, and the synthesis process cou...

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Abstract

The invention relates to the technical field of biology, in particular to a polypeptide, a preparation method of the polypeptide, a bispecific antibody and application of the bispecific antibody. The polypeptide is an Fc binding polypeptide, and can be universally, simply and conveniently connected with a monoclonal antibody to prepare a bispecific / multispecific antibody. The polyvalent Fc binding polypeptide PGLU-Fc-III-4C or PASP-Fc-III-4C is prepared by bonding side carboxyl groups of polyglutamic acid (PGLU) or polyaspartic acid (PASP) with Fc-III-4C polypeptide which has general high affinity to an immune globulin (antibody) constant region (Fc), and the polyvalent Fc binding polypeptide can react with various monoclonal antibodies in different proportions in an aqueous solution to obtain a bispecific / multispecific antibody. The preparation method of the bispecific / multispecific antibody has the following advantages: the bispecific / multispecific antibody is prepared by direct incubation in a water environment, the reaction condition is mild, and the activity of the monoclonal antibody can be retained to the greatest extent; and the method is simple, efficient, controllable in monoclonal antibody proportion and high in universality, and has an industrialization prospect.

Description

technical field [0001] The present invention relates to the technical field of medicine, in particular to a polypeptide, a preparation method thereof, a bispecific antibody and applications thereof. Background technique [0002] CAR-T (Chimeric antigen receptor T cell, chimeric antigen receptor T cell) cell therapy is a very promising cancer treatment method. It uses gene modification technology to activate T cells with specific antigen recognition domains and The genetic material of the signal is transferred to the T cell, so that the T cell is directly activated by binding to the specific antigen on the surface of the tumor cell, and directly kills the tumor cell by releasing perforin, granzyme B, etc., so as to achieve the purpose of treating the tumor. At present, 6 CAR-T drugs have been approved by the US Food and Drug Administration (FDA), and are widely used in the treatment of blood diseases and myeloma. However, CAR-T therapy is not suitable for patients who need i...

Claims

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Application Information

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IPC IPC(8): C08G69/48C08G69/10C07K16/46A61K39/44A61K47/64A61P35/00A61P35/02
CPCC08G69/48C08G69/10C07K16/44C07K16/2818C07K16/2878A61K39/44A61K47/645A61P35/00A61P35/02C07K2317/31A61K2039/505
Inventor 汤朝晖薛福鑫沈娜黄月陈学思
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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