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Bispecific antibody for anti-CD3 and CA125 antigens

A bispecific antibody and antigen technology, applied in the field of immunology, can solve the problems of low targeting effect, poor tumor penetration, and strengthen the killing effect of tumor cells, and achieve the effect of long half-life in vivo and excellent cytotoxicity

Active Publication Date: 2022-04-12
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Based on the single recognition site in the prior art in the field of tumor treatment or the difficulty of reaching the acting cells with large traditional antibody molecules, the purpose of the present invention is to provide a bispecific nanobody targeting both tumor cells and T cells. Recognize the CA125 antigen of cancer cells, induce the ADCC effect, and at the same time recognize the CD3 antigen of effector T cells, guide T cells to the vicinity of tumor cells, and strengthen the killing effect of effector T cells on tumor cells
And it can overcome the inherent defects of traditional antibodies such as poor penetration into solid tumors and low targeting effect

Method used

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  • Bispecific antibody for anti-CD3 and CA125 antigens
  • Bispecific antibody for anti-CD3 and CA125 antigens
  • Bispecific antibody for anti-CD3 and CA125 antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Preparation of anti-CD3 monovalent Nanobodies.

[0036] 1.1 Immunity of alpacas

[0037] Select a healthy adult alpaca, mix the recombinant human CD3 antigen (manufacturer: abcam, product number: ab229518) and Freund's adjuvant at a ratio of 1:1, and inject 6-7 μg / kg subcutaneously at multiple points on the back Alpacas were immunized four times in total with an interval of 2 weeks. Afterwards, 10 ml of alpaca peripheral blood was collected for the construction of a phage display library.

[0038] 1.2 Isolation of camel-derived lymphocytes

[0039] Lymphocytes were separated from the collected alpaca peripheral blood using the Camel Peripheral Blood Lymphocyte Separation Solution Kit (Tianjin Haoyang Company, Cat. No. LTS1076), each 2.5×10 7 Add 1ml RNA isolation reagent to each living cell, take 1ml for RNA extraction, and store the rest at -80°C.

[0040] 1.3 Total RNA extraction

[0041] Pipette 1ml Tipure Isolation Reagent containing lymphocytes repe...

Embodiment 2

[0068] Example 2. Preparation of Nanobody 7A5.

[0069] 2.1 Amplification of original nanobody strain TG1 and transformation of nanobody recombinant plasmid into Escherichia coli BL21(DE3)

[0070] Inoculate the original strain TG1 Glycerolbacterium containing Nanobody nucleic acid in 5ml of fresh LB-A medium at a ratio of 1:1000, and culture overnight at 37°C and 200rpm. The next day, use the Plasmid mini kit (OMEGA) to extract plasmids according to the instructions. After verification, transform 1 μl of the above plasmid into 100 μl competent cells, mix gently, place on ice for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and cool in an ice bath for 3 minutes. Add 600 μl LB medium to the centrifuge tube, shake and incubate at 37°C for 60 minutes. Take 100 μl of the supernatant, spread it on the LB-A plate with a triangular spreader, and culture it upside down at 37°C overnight.

[0071] 2.2 Induced expression of nanobodies

[0072] The above monoclonal col...

Embodiment 3

[0075] Example 3. Determination of the affinity and activity of Nanobodies with antigens.

[0076] 3.1 Chip antigen coupling

[0077] The antigen was prepared into a 20 μg / ml working solution with different pH sodium acetate buffers (pH 5.5, pH 5.0, pH 4.5, pH 4.0), and a 50 mM NaOH regeneration solution was prepared at the same time, and used in the Biacore T100 protein interaction analysis system instrument The template method is used to analyze the electrostatic binding between the antigen and the surface of the chip (GE company) under different pH conditions. The amount of signal increase reaches 5 times RL as the standard, and the most suitable neutral pH system is selected and adjusted as needed. The antigen concentration was used as the condition during coupling. The chip was coupled according to the template method that comes with the instrument: select the blank coupling mode for channel 1, select the Target coupling mode for channel 2, and set the target to the desi...

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Abstract

The invention discloses an anti-CD3 antigen nano antibody, and a bispecific antibody containing two VHH structural domains. The invention further discloses a preparation method of the anti-CD3 antigen nano antibody. The nano antibody of the anti-CD3 antigen has three unique complementary determining regions (CDR1, CDR2 and CDR3). The bispecific antibody is formed by fusing two different heavy chain sequences, and can be used for respectively recognizing a CD3 antigen and a CA125 antigen on the surface of a T cell. And the bispecific antibody can significantly enhance the activity of antibody-mediated T cells in killing tumor target cells.

Description

technical field [0001] The invention discloses a polypeptide, more specifically an antibody, and belongs to the field of immunology. Background technique [0002] CA125 (Cancer Antigen 125) ovarian cancer-associated antigen was discovered in 1981 and is a related antigen of ovarian epithelial carcinoid. It is secreted by epithelial cells in the embryonic stage, and it is not secreted or rarely secreted under normal circumstances. However, when malignant lesions occur in the ovary, even if there is no clinical manifestation or pathologically difficult to identify, the CA125 value will increase, so it is a better ovarian Cancer diagnosis and screening indicators, and is closely related to the metastasis and prognosis of ovarian cancer. CA125 was initially recognized and confirmed by Bast et al. through the ovarian cell line OVCA433 immunogen, which mediated the response of the mouse monoclonal antibody OC125. CA125 antigen is a glycoprotein with a relative molecular mass of ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C07K16/46A61K39/395A61P35/00C12R1/19
CPCY02A50/30
Inventor 宋海鹏刘原源于建立曹慧古一李飞王准张霞蒋立仲宋亮
Owner 深圳市国创纳米抗体技术有限公司
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