CN111840544A

CN111840544A - Application of defucose anti-HER 2 antibody in cancer patient carrying Fc gamma R2A131R genotype

Info

Publication number: CN111840544A
Application number: CN201910356158.5A
Authority: CN
Inventors: 侯理理; 张亮; 谢玖清; 胡盼
Original assignee: Shanghai Bangyao Biological Technology Co ltd
Current assignee: Shanghai Bangyao Biological Technology Co ltd
Priority date: 2019-04-29
Filing date: 2019-04-29
Publication date: 2020-10-30

Abstract

The application discloses an application of a defucose anti-HER 2 antibody in a cancer patient carrying Fc gamma R2A131R genotype. The defucose anti-HER 2 antibody is produced by a cell that knocks down the FUT8 gene; the amino acid sequence of the light chain of the defucose anti-HER 2 antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain of the defucose anti-HER 2 antibody is shown as SEQ ID NO. 3. The defucose anti-HER 2 antibody has better ADCC activity than that of a defucose anti-HER 2 antibody no matter whether the Fc gamma R2A genotype is H/H type, or H/R type, or R/R type, and particularly has obvious advantages of ADCC of the defucose anti-HER 2 antibody when the Fc gamma R2A genotype is R/R type or H/R type.

Description

Application of defucose anti-HER 2 antibody in cancer patient carrying Fc gamma R2A131R genotype

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a defucose anti-HER 2 antibody in a cancer patient carrying Fc gamma R2A131R/R or 131H/R allelic genes.

Background

The world health organization reports show that global cancer cases will present a rapidly growing situation, from 1400 million people in 2012, increasing year by year to 1900 million people in 2025, and up to 2400 million people by 2035. The developing countries in africa, asia and central and south america have the most severe forms of cancer. In 2012, there are 1400 new cancer cases worldwide and 820 deaths.

Cancer morbidity and mortality in china are rising all the time, have become the main cause of death since 2010, and have become a major public health problem in china. According to the Chinese cancer statistical report in 2015, 4,292,000 new cancer cases and 2,814,000 cancer deaths are predicted in 2015, and the incidence and mortality of lung cancer are the highest. The morbidity and mortality of gastric, esophageal and liver cancers is also high.

HER2 is a member of the epidermal growth factor receptor family. The human HER2 gene maps to chromosome 17 (17q21-22) and expresses a single-chain transmembrane glycoprotein with a molecular weight of 185,000 daltons. The structure of HER2 is highly homologous to the Epidermal Growth Factor Receptor (EGFR), which is a member of the EGFR family. This family also includes HER1(ErbBl), HER3(ErbB3) and HER4(ErbB4), which act in dimeric or oligomeric forms. HER2 receptor dimerization can induce tyrosine kinase activation, so that the C-terminal tyrosine residue of the receptor is autophosphorylated, an intracellular signal cascade reaction is excited, the activation of downstream signal molecules is promoted, and various biological effects such as cell growth, differentiation, transfer, adhesion and the like are caused; when HER2 is overexpressed, it can cause uncontrolled cell growth and proliferation, malignant transformation, and tumor infiltration and metastasis. HER2 is overexpressed in a variety of tumor cells, including breast, ovarian, prostate, lung, stomach, esophageal, colon, and the like. And most studies suggest that HER2 expression is correlated with tumor stage.

Herceptin, the main component of which is Trastuzumab (Trastuzumab), is a targeted drug against HER2 developed by Genentech, Inc. in the United states. It is a human recombinant monoclonal antibody (MAb) directed against the Ectodomain (EDC) of HER2/neu protein, in which the murine complementarity determining region (i.e., clone 4D5) is inserted into the human IgGl framework. Herceptin was approved in 1998 in the united states for HER 2-overexpressed breast cancers, including metastatic breast cancer and metastatic gastric cancer. Since the market, the single-drug objective response rate of herceptin was 15-26%.

One important mechanism by which herceptin acts is by utilizing the constant domains (Fc) of its antibodies to trigger antibody-dependent cellular (mediated) cytotoxicity (ADCC) effects and Fc-mediated immunopotentiation by binding to leukocyte receptors (fcyr). Thus, the binding of an antibody to a receptor directly affects the potency of the antibody. Two factors influence the ability of Fc to bind Fc γ R: the Fc gamma R has certain difference due to the polymorphism of the Fc gamma R; secondly, the sugar structure mode of the Fc region of the antibody shows that the antibodies with different glycosylation characteristics can show different ADCC effects, so that the optimized glycosylation of the Fc segment of the antibody can achieve the effect of improving the ADCC effect of the antibody.

ADCC results in the specific lysis of antigen-positive tumor cells. Compared with the effect of second-line afatinib + vinorelbine and herceptin + vinorelbine after the auxiliary or first-line high fucose anti-HER 2 antibody of a HER2 positive breast cancer patient, namely the herceptin develops, the effect of the afatinib group is obviously inferior to that of the herceptin group. Small molecule Tyrosine Kinase Inhibitors (TKIs), such as afatinib, are highly potent and irreversible inhibitors against HER proteins with full kinase activity (EGFR, HER2, HER4), while afatinib blocks all heterohomodimers of the HER family, herceptin still shows strong advantages over such potent TKIs, this clinical study strongly supports the in vivo role of herceptin, i.e. the high fucose anti-HER 2 antibody, probably more dependent on its ADCC effect.

Fc γ R plays an important role in immune regulation, mediating ADCC, endocytosis, phagocytosis, and release of inflammatory factors and antigen presentation. Fc γ R is classified into 3 types: fc γ R1(CD64), Fc γ R2(CD32), and Fc γ R (CD 16). Specific genes, each located in the same region of the long arm of the chromosome, encode several different genotypes, including Fc γ R1A, Fc γ R1B, Fc γ R1C, Fc γ R2A, Fc γ R2B1, Fc γ R2B2, Fc γ R2B3, Fc γ R2C, Fc γ R3A, and Fc γ R3B. Single Nucleotide Polymorphism (SNP), i.e., a polymorphism in a nucleic acid sequence due to a change in a single nucleotide base, is generally considered to be distinguished from point mutations in that SNPs occur at a frequency of more than 1%. SNPs in the coding region of a gene often result in amino acid changes that in turn affect the function of the protein. The histidine to arginine mutation at Fc γ R2A 131 site (H131R) affected the antibody and its binding.

Disclosure of Invention

The removal of fucose from the glycosylation site of the Fc region of the high fucose anti-HER 2 antibody increases the ADCC activity due to the increased affinity of the Fc γ R2A molecule on the cell membrane of the antibody and some effector cells such as NK cells, macrophages, etc. The invention finds that the affinity of the defucose anti-HER 2 antibody for Fc gamma R2A131R genotype is particularly obviously enhanced. In order to improve the tumor treatment effect and/or expand the tumor application range, the invention aims to provide the application of the defucose anti-HER 2 antibody in a cancer patient carrying Fc gamma R2A131 genotype.

The defucose anti-HER 2 antibody is produced by a cell that knocks down the FUT8 gene; the amino acid sequence of the light chain of the defucose anti-HER 2 antibody is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain of the defucose anti-HER 2 antibody is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4.

Further, a CH2 domain is included; the amount of fucose in the CH2 domain is zero.

Further, a sugar-based structure is included, the sugar-based structure including a first sugar-based structure consisting of 3 six-carbon sugars and 4N-acetylhexosamine; the proportion of the first glycosyl structure in the glycosyl structure is 60% -95%; the six-carbon sugar comprises one or more of galactose, mannose and glucose; the N-acetylhexosamine includes N-acetylgalactosamine and/or N-acetylglucosamine.

Further, the six carbon sugar is mannose; the N-acetylhexosamine is N-acetylglucosamine.

Further, the glycosyl structure further comprises a second glycosyl structure, a third glycosyl structure, a fourth glycosyl structure, a fifth glycosyl structure and/or a sixth glycosyl structure; the glycosyl composition of the second glycosyl structure, the third glycosyl structure, the fourth glycosyl structure, the fifth glycosyl structure and the sixth glycosyl structure and the proportion of the glycosyl structures are shown in the following table:

glycosyl structure	Glycosyl composition	Ratio of
			Second glycosyl structure	4 six carbon sugars and 4N-acetylhexosamine	2％-15％
A third glycosyl structure	3 six carbon sugars and 3N-acetylhexosamine	0-10％
			Fourth glycosyl structure	5 six carbon sugars and 2N-acetylhexosamine	0-10％
Fifth glycosyl structure	5 six carbon sugars and 4N-acetylhexosamine	0-10％
			Sixth glycosyl structure	3 six carbon sugars and 5N-acetylhexosamine	0-10％

Further, sugar chains corresponding to the first, second, third, fourth, fifth and sixth sugar structures, respectively, are shown in the following table:

wherein circles in the sugar chain represent the six-carbon sugar; black squares represent the N-acetylglucosamine; black hexagons represent galactose; black circles indicate mannose.

Further, the cell is a CHO cell, and the knocking-out is realized by knocking out a gene responsible for encoding fucose, namely, a FUT8 gene in the CHO cell by using CRISPR cas9 gene editing technology.

Further, the patient is HER2 positive; the cancer comprises one or more of breast cancer, esophageal cancer, gastric cancer, lung cancer, bronchial cancer, pancreatic cancer, colon cancer, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, and head and neck cancer.

Further, the medicament is for HER2 positive metastatic cancer.

Further, the medicament is used for the metastasis of HER2 positive malignant tumor, and the metastasis of HER2 positive malignant tumor comprises one or more of skin metastasis, visceral metastasis and lymphatic metastasis.

The invention has the following beneficial effects:

1. the affinity of the defucose anti-HER 2 antibody and the extracellular region ECD of HER2 was comparable to the affinity of the high fucose anti-HER 2 antibody and the extracellular region ECD of HER 2.

2. The affinity of the defucose anti-HER 2 antibody and the Fc gamma R2A131H genotype protein is 10 times that of the high fucose anti-HER 2 antibody, and the affinity of the defucose anti-HER 2 antibody and the Fc gamma R2A131R genotype protein is 16 times that of the high fucose anti-HER 2 antibody, and the increase of the affinity is the basis for the exertion of higher ADCC effect in vivo.

3. The genotype of the defucose anti-HER 2 antibody in a HER2 positive patient Fc gamma R2A is Fc gamma R2A131R/R or Fc gamma R2A131H/R, which shows stronger tumor killing effect than the high fucose anti-HER 2 antibody.

The defucose anti-HER 2 antibody is a specific glycosylation optimized anti-HER 2 antibody, the ADCC anti-tumor effect of the herceptin antibody approved for treating HER2 over-expressed breast cancer and gastric cancer is enhanced by 10-50 times, so that more excellent anti-tumor effect can be achieved, better effectiveness and lower drug resistance are generated on HER2 positive tumors, and the genotype of HER2 positive cancer patient Fc gamma R2A is Fc gamma R2A131R/R or Fc gamma R2A 131H/R.

Detailed Description

The protection of the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

The defucose anti-HER 2 antibody of the present invention has the same amino acid sequence as herceptin, but is expressed in CHO cells in which the FUT8 gene is knocked out, and thus is afucose trastuzumab, and is represented by "FUC-trastuzumab".

The control high fucose anti-HER 2 antibody used in the present invention is trastuzumab, the main component of herceptin, and is denoted by "FUC + trastuzumab".

CHO-S cells in the present invention were obtained from Invitrogen, and all other cells were purchased from ATCC (American type culture Collection). The relevant reagents are commercially available.

The defucose anti-HER 2 antibody and the high fucose anti-HER 2 antibody of the present invention are identical to those of the prior patent 2016111168043.

Example 1 comparison of the affinity of the defucose anti-HER 2 and high fucose anti-HER 2 antibodies to the receptors human Fc γ R2A131R/R genotype receptor protein and Fc γ R2A131R/R genotype receptor protein

The experiment was performed on a Biacore T200 mobile phase PBS buffer (8.0 g NaCl, 0.2g KCl, KH)₂PO₄0.24g，Na₂HPO₄×12H₂O3.628 g in 800ml of distilled water, pH 7.4 adjusted with hydrochloric acid, and volume of distilled water to 1000ml), all the samples were dissolved in the above PBS buffer.

The defucose anti-HER 2 antibody and the high fucose anti-HER 2 antibody were immobilized by amino coupling on a CM5 sensor chip at a flow rate of 10ul/min, and the surface of the CM5 chip was activated with an equal volume of mixed solution of EDC at a concentration of 0.1mol/L and NHS at a concentration of 0.1 mol/L.

Fc γ R2a131R or Fc γ R2a131H employ multi-cycle kinetics. Respectively preparing Fc gamma R2A131R or Fc gamma R2A131H with different concentrations, setting the flow rate of an experiment to be 30ul/min, the sample injection time to be 120s, adopting a 5M NaCl regeneration solution, and the dissociation time to be 30 s. The results of the experiment are shown in table 1.

TABLE 1 affinity of defucose anti-HER 2 and high fucose anti-HER 2 antibodies to different genotypes Fc gamma R2A131H/R, respectively

From the experimental results, we can see that: the affinity of the defucose anti-HER 2 antibody and the Fc γ R2a131R genotype protein was 16 times that of the high fucose anti-HER 2 antibody, and the affinity of the defucose anti-HER 2 antibody and the Fc γ R2a131H genotype protein was 10 times that of the high fucose anti-HER 2 antibody. This increase in affinity is the basis for the development of a higher ADCC effect in vivo.

Example 2 comparison of ADCC Activity of different genotype donors on SK-BR-3 cells mediated by a defucose anti-HER 2 antibody and a high fucose anti-HER 2 antibody

The PBMC source respectively selects three donors of Fc gamma R2A131(H/H), Fc gamma R2A131(H/R) and Fc gamma R2A131 (R/R).

The technical method comprises the following steps:

HER2 positivity differentiated in RPMI 1640 medium containing 2% Fetal Bovine Serum (FBS)The tumor cell of (2) to (10)⁴The method comprises the steps of inoculating a well to a 96-well plate, adding defucose anti-HER 2 antibody and high fucose anti-HER 2 antibody with different concentrations, placing the well in an incubator at 37 ℃ for half an hour, adding freshly separated human Peripheral Blood Mononuclear Cells (PBMC) according to the ratio of 20:1, namely adding 20 ten thousand PBMC into each well, placing the well in the incubator at 37 ℃ for 4 hours, detecting the activity of lactate dehydrogenase (lactate dehydrogenase) by using a Roche cytotoxicity detection kit, and setting three multiple wells for all detection. Finally, the absorbance at OD490 was measured by reading on a microplate reader. The maximum release amount was determined by adding triton x100, the spontaneous release amount was determined by adding only target cells (cell lines shown in table 2) and effector cells (PBMCs) without adding antibodies, and the minimum release amount was determined by adding only target cells, and the specific cytotoxicity was calculated as: cell lysis (%) (experimental group release-spontaneous release)/(maximum release-minimum release) × 100%. The results of the comparison are shown in Table 2.

TABLE 2 comparison of ADCC Activity of defucose anti-HER 2 and high fucose anti-HER 2 antibodies in SK-BR-3 tumor cells (PBMC of different genotype origin)

For donors with Fc γ R2A genotype 131(H/H) with ADCC effect on SK-BR-3 cells, the EC50 of the high fucose anti-HER 2 antibody was 3.3 times that of the defucose anti-HER 2 antibody; for donors with Fc γ R2A genotype 131(H/R) with ADCC effect on SK-BR-3 cells, the EC50 of the high fucose anti-HER 2 antibody was 13.3 times that of the defucose anti-HER 2 antibody; for donors with Fc γ R2A genotype 131(R/R) with ADCC effect on SK-BR-3 cells, the EC50 of the high fucose anti-HER 2 antibody was 32 times that of the defucose anti-HER 2 antibody.

According to example 2, in the tumor highly expressing HER2 molecule (IHC3+), since the activity of the defucose anti-HER 2 antibody is much greater than that of the high fucose anti-HER 2 antibody, it can also be applied to some tumor patients highly expressing HER2 molecule (IHC3+), but the Fc γ R2A genotype of these patients is H/R or R/R type, resulting in the tumor which has lower affinity of the high fucose anti-HER 2 antibody and NK effector cells and may not respond to the high fucose anti-HER 2 antibody.

In summary, the defucose anti-HER 2 antibody has better ADCC activity than the defucose anti-HER 2 antibody for no matter whether the Fc gamma R2A genotype is H/H type, or H/R type, or R/R type, and particularly has obvious advantages of ADCC of the defucose anti-HER 2 antibody for the FCR2A genotype is R/R type, or H/R type.

Sequence listing

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<120> use of defucose anti-HER 2 antibody in patients with cancer bearing Fc gamma R2A131R genotype

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Claims

1. Use of a defucose anti-HER 2 antibody as an active ingredient in the manufacture of a medicament for treating a patient with a cancer having an Fc γ R2A131R genotype, said defucose anti-HER 2 antibody being produced by a cell in which an FUT8 gene is knocked out; the amino acid sequence of the light chain of the defucose anti-HER 2 antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain of the defucose anti-HER 2 antibody is shown as SEQ ID NO. 3.

2. The use of claim 1, wherein: the amino acid sequence of the light chain of the defucose anti-HER 2 antibody is encoded by the nucleotide sequence shown in SEQ ID NO. 2; the amino acid sequence of the heavy chain of the defucose anti-HER 2 antibody is encoded by the nucleotide sequence shown in SEQ ID NO. 4.

3. Use according to claim 1 or 2, characterized in that: the Fc gamma R2A131R genotype is Fc gamma R2A131R/R genotype or Fc gamma R2A 131R/H genotype.

4. Use according to any one of claims 1 to 3, wherein: the defucose anti-HER 2 antibody comprises a CH2 domain; the amount of fucose in the CH2 domain is zero.

5. The use according to any one of claims 1 to 4, wherein: the defucose anti-HER 2 antibody comprises a glycosyl structure comprising a first glycosyl structure consisting of 3 six carbon sugars and 4N-acetylhexosamine; the proportion of the first glycosyl structure in the glycosyl structure is 60% -95%; the six-carbon sugar comprises one or more of galactose, mannose and glucose; the N-acetylhexosamine includes N-acetylgalactosamine and/or N-acetylglucosamine.

6. The use of claim 5, wherein: the six carbon sugar is mannose; the N-acetylhexosamine is N-acetylglucosamine.

7. The use of claim 5, wherein: the glycosyl structure further comprises a second glycosyl structure, a third glycosyl structure, a fourth glycosyl structure, a fifth glycosyl structure and/or a sixth glycosyl structure; the glycosyl composition of the second glycosyl structure, the third glycosyl structure, the fourth glycosyl structure, the fifth glycosyl structure and the sixth glycosyl structure and the proportion of the glycosyl structures are shown in the following table:

8. the use of claim 7, wherein: sugar chains corresponding to the first, second, third, fourth, fifth and sixth sugar structures are shown in the following table:

9. Use according to any one of claims 1 to 8, wherein: the cell is a CHO cell, and the knockout is to knock out a gene responsible for encoding fucose, namely FUT8 gene, in the CHO cell by using CRISPR cas9 gene editing technology.

10. Use according to any one of claims 1 to 9, wherein: the patient is HER2 positive; the cancer comprises one or more of breast cancer, esophageal cancer, gastric cancer, lung cancer, bronchial cancer, pancreatic cancer, colon cancer, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, and head and neck cancer.