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Bispecific antibody for anti-CD16 and CEA antigens

A bispecific antibody and nanobody technology, applied in the field of immunology, can solve problems such as enhancing the killing effect of NK cells on tumor cells, and achieve the effects of excellent cytotoxicity, high affinity, and long in vivo half-life

Active Publication Date: 2022-01-11
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 problems that traditional antibody molecules are too large to reach the acting cells, the purpose of the present invention is to provide a bispecific nanobody targeting both tumor cells and NK cells. Recognize the CEA antigen of cancer cells, inhibit the proliferation of tumor cells induced by CEA, and at the same time specifically recognize NK cells, guide NK cells to the vicinity of tumor cells, and strengthen the killing effect of NK cells on tumor cells

Method used

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  • Bispecific antibody for anti-CD16 and CEA antigens
  • Bispecific antibody for anti-CD16 and CEA antigens
  • Bispecific antibody for anti-CD16 and CEA antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Preparation of anti-CD16 monovalent nanobody

[0037] 1.1 Immunity of alpacas

[0038] Select a healthy adult alpaca, mix the recombinant human CD16 antigen (manufacturer: abcam, product number: ab151819) 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.

[0039] 1.2 Isolation of camel-derived lymphocytes

[0040] Lymphocytes were separated from the collected alpaca peripheral blood using the Camel Peripheral Blood Lymphocyte Separation Solution Kit (Tianjin Haoyang Company, Cat. No. LTS1076). 7 Add 1ml RNA isolation reagent to each living cell, take 1ml for RNA extraction, and store the rest at -80°C.

[0041] 1.3 Total RNA extraction

[0042] Pipette 1ml Tipure Isolation Reagent containing lymphocytes repeatedly and le...

Embodiment 2

[0069] Example 2. Preparation of Nanobody 6C5

[0070] 2.1 Amplification of original nanobody strain TG1 and transformation of nanobody recombinant plasmid into Escherichia coli BL21(DE3)

[0071] 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, plasmids were extracted using the Plasmid mini kit (OMEGA) 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.

[0072] 2.2 Induced expression of nanobodies

[0073] The above monoclona...

Embodiment 3

[0076] Example 3. Determination of the affinity and activity of nanobodies and antigens

[0077] 3.1 Chip antigen coupling

[0078] The antigen was formulated 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 the Biacore T100 protein interaction analysis system instrument was used to 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, and the signal increase amount reaches 5 times RL as the standard, select the most suitable neutral pH system and adjust it 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 designed...

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Abstract

The invention discloses a nano antibody for resisting a CD16 antigen, and a bispecific antibody containing two VHH structural domains. The nano antibody of the anti-CD16 antigen has three unique complementary determining regions (CDR1, CDR2 and CDR3). The bispecific antibody is formed by fusing two heavy chains with different sequences and is used for respectively recognizing a CD16 antigen and a carcino-embryonic antigen CEA on the surface of an NK cell. The bispecific antibody can significantly enhance the activity of antibody-mediated NK 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] Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEA is a member of the immunoglobulin superfamily and contains seven domains linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. The seven domains include a single N-terminal Ig variable domain and six domains homologous to Ig constant domains (A1-B1-A2-B2-A3-B3). CEA, originally classified as a protein expressed only in fetal tissues, has now been identified in several normal adult tissues. Overexpression of CEA has been observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers. Therefore, CEA has been identified as a tumor-associated antigen. CEA is readily cleaved from the cell surface and shed from th...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/46C12N15/13C12N15/70C12N15/85C12N1/21C12N5/10A61K39/395A61P35/00C12R1/19
CPCC07K16/283C07K16/3007C12N15/70C12N15/85C12N5/0686A61P35/00C07K2317/31C07K2317/569C07K2317/565C07K2317/56C07K2317/52C07K2317/73C07K2317/92C07K2317/732C07K2317/76A61K2039/507
Inventor 宋海鹏刘原源于建立王准曹慧古一李飞张霞蒋立仲宋亮
Owner 深圳市国创纳米抗体技术有限公司
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