CN114409790B

CN114409790B - Method for improving antibody drug efficacy and antibody drug

Info

Publication number: CN114409790B
Application number: CN202210329044.3A
Authority: CN
Inventors: 邱晓彦; 何峙峤; 黄歆梅; 姜文华; 张生华
Original assignee: Beijing Aisaiji Biomedical Technology Co ltd; Peking University
Current assignee: Guangzhou Aisaiji Biomedical Technology Co ltd; Peking University
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-09-13
Anticipated expiration: 2042-03-31
Also published as: CN114409790A

Abstract

The invention discloses a method for improving the efficacy of an antibody medicament and the antibody medicament, wherein the antibody modification method comprises the following steps: (1) when the antibody is used for treating cancer or infectious diseases, the sialylation modification at the CH1 domain Asn162 site of the antibody is removed; or (2) when the antibody is used for treating chronic inflammatory diseases, a sialylation modification is added to the CH1 domain Asn162 site of the antibody. The modification is a research result that the sialylation modification based on a CH1 structural domain can inhibit T cells from playing a role, and by removing the inhibition, the T cells can help antibody drugs kill tumors or resist infection, or inhibit the activity of the T cells to reduce the attack of an immune system on the T cells and further help the antibody drugs to treat chronic inflammatory diseases such as autoimmune diseases, and the like, so that better treatment effect compared with the original antibody drugs can be obtained, the modification method is simple, the effect is obvious, and the clinical application prospect is wide.

Description

Method for improving antibody drug efficacy and antibody drug

Technical Field

The invention relates to the technical field of immunotherapy, in particular to a method for improving the efficacy of an antibody medicament and the antibody medicament.

Background

Antibodies for the treatment of diseases, also known as antibody drugs. The antibody drug provides a brand new way for treating various diseases such as cancer, autoimmune diseases, virus infection and the like by virtue of high specificity, effectiveness and safety of the combination of the antibody drug and a target antigen, is developed rapidly, is a drug with the highest composite growth rate in the current biological drugs, and becomes a hotspot of global drug research and development.

Currently, more than 50 monoclonal antibody drugs (hereinafter abbreviated as mabs) are approved by FDA and european union pharmaceutical product administration (EMA) for marketing, and more than forty mabs are in phase iii clinical studies, and about 80 mabs are expected to be marketed by 2023 at the rate of approving 4 mabs per year.

The main previous research efforts in improving the effector functions of antibodies have been directed to humanizing antibodies, developing miniaturized antibodies, and combining them with certain chemotherapeutic agents to show synergistic effects. For example, clinical application results show that the combination of antibody drugs such as Rituxan, Herceptin, Avastin, Erbitux and the like and chemotherapeutic drugs has higher curative effect than the combination of the drugs used alone (screening Nevesu, antibody targeted drugs for treating tumors, 13 th No. 22: 1861-1682 at the end of 11 months in the journal of China's tumor prevention and treatment 2006). With the intensive research on antibody drugs, it is found that the effector function of a naked antibody depends on the binding of the antibody and the corresponding antigen and the Fc structure of the antibody, so that the improvement of the effector function of the antibody can also be realized by modifying the Fc of the antibody to increase the ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (Complement-dependent cytotoxicity) effects, binding to immunoconjugates, constructing bispecific antibodies, and binding to T cells of patients to construct CAR-T cells and the like (Shen, history review and prospect of tumor antibody treatment, book No. 1: 1-5 of 1 month No. 30 of 2016 of China pharmacology and toxicology).

In addition, glycosylation modification analysis of monoclonal antibody drugs has been developed at various levels, including qualitative and quantitative characterization at the intact protein level, protein fragments, glycopeptides, and sugar chain level. At present, it is clear that glycosylation modification of a monoclonal antibody drug has different influences on various aspects such as stability, effectiveness, safety and the like, however, since glycosylation modification itself is complex and glycosylation modification function is unclear, current research is basically limited at a laboratory level.

Disclosure of Invention

The invention aims to provide a novel method for improving the drug efficacy of an antibody, which is suitable for the antibody for treating cancer, infection resistance or chronic inflammatory diseases such as autoimmune diseases and the like based on the research result of glycosylation modification of an Fab region of an IgG heavy chain of the antibody.

The invention provides a method for improving the drug efficacy of an antibody, wherein the antibody comprises a CH1 structural domain, and the antibody is modified in the following modes (1) or (2):

(1) when the antibody is used for treating cancer or infectious diseases, the sialylation modification at the CH1 domain Asn162 site of the antibody is removed;

(2) when the antibody is used for treating chronic inflammatory diseases, sialylation modification is added to the CH1 domain Asn162 site of the antibody.

Alternatively or preferably, in the above method, the method for removing sialylation modification described in mode (1) is:

digesting the antibody with neuraminidase; or

When the antibody is a genetic engineering antibody, mutating an N glycosylation site gene on Asn162 in the CH1 structural domain of the antibody to remove a sialylation modification function; or

When the antibody is a genetic engineering antibody, the sialyltransferase gene in the engineering cell is knocked out in the process of preparing the genetic engineering antibody.

Alternatively or preferably, in the above method, when the antibody in the mode (2) is a genetically engineered antibody, the method for adding a sialylation modification comprises: exogenously transferring N glycosyl transferase gene into the engineering cell and/or exogenously transferring sialic acid transferase gene into the engineering cell.

In the above method, the engineered cell is preferably a CHO cell.

In the above method, the chronic inflammatory disease in the mode (2) may be an autoimmune disease.

In the modification (1) of the above method, the antibody may preferably be Herceptin (Herceptin), sitoxib (situximab) or rituximab (Rituxan). These cancer treating antibodies all have sialic acid modification at position Asn162 of the CH1 domain.

In the modification (2) of the above method, the antibody may preferably be an anti-inflammatory therapeutic antibody, which is deficient in sialic acid modification at Asn162 in the CH1 domain.

The invention also provides a novel antibody drug obtained by the modification of any one of the methods.

In the present invention, the antibody is any antibody comprising a CH1 domain, and may be, for example, one or more of a full-length antibody, a domain antibody, a Fab, and a F (ab') 2. The antibodies may be of human or non-human origin. The full length antibody may be an IgG.

Among them, Fab is composed of VL (light chain variable region), VH (heavy chain variable region), CL (light chain constant region), and CH1 (heavy chain constant region domain 1).

The gene engineering antibody is an antibody molecule expressed by using recombinant DNA and protein engineering technology to process, modify and reassemble the gene of the coded antibody according to different requirements and transfecting proper engineering cells. The engineered cell is a recipient cell.

The infectious diseases refer to diseases caused by infection of microorganisms such as viruses, chlamydia, mycoplasma, rickettsia, bacteria, fungi, spirochetes, protozoa, worms and the like.

The chronic inflammatory disease refers to the excessive activation of the immune system by various endogenous and exogenous factors, wherein the chronic inflammatory disease mainly causes cell damage caused by lymphocytes and macrophages, and simultaneously causes a complex reaction accompanied with vascular reaction, fluid retention and leukocyte retention in extravascular tissues, and a fibrotic reaction accompanied with tissue necrosis or one of tissue repair links. The chronic inflammatory diseases comprise autoimmune diseases and immune inflammatory diseases, wherein the autoimmune diseases are diseases which cause self-tissue damage by the immune reaction of an organism to autoantigens, such as rheumatoid arthritis, systemic lupus erythematosus, autoimmune hepatitis, Sjogren syndrome and the like; immunoinflammatory diseases are diseases in which the body has damaged its own tissues due to excessive immune response to exogenous stimuli, such as inflammatory bowel disease, chronic nephritis, asthma, chronic gastrointestinal tract, psoriasis, atopic dermatitis, and the like.

The neuraminidase, Sialidase (SA), is capable of cleaving the glycosidic bond at the end of the glycoconjugate to a sialic acid residue to hydrolyze the sialylglycoconjugate, and can remove sialic acid modifications from the antibody. Sialidases include exosialidase, endo-sialidase and transsialidase. Exosialases can hydrolytically remove sialic acid from the ends of glycoconjugates; the sialic acid endonuclease mainly cuts off glycosidic bonds on macromolecular sugar chains and generates fragments which can be explained by exonuclease; transfer sialidases can transfer sialic acid from the end of the glycoconjugate to a new glycoconjugate.

The N-glycosyltransferase (NGT), is capable of adding a sugar from an activated donor, e.g., UDP-Glc/UDP-Gal/GDP-Man, etc., to an Asn (X is any amino acid other than proline) of a peptide sequence having Asn-X-Ser/Thr. After the engineering cell expresses N glycosyltransferase by exogenously transferring the N glycosyltransferase gene into the engineering cell, sialylation modification of Asn162 site of CH1 structural domain of the antibody can be carried out in the cell.

The sialyltransferase is a type II transmembrane glycoprotein containing disulfide bonds, and is capable of transferring sialic acid from an activated glycosyl donor to a terminal galactose or acetylgalactosamine of an acceptor sugar chain, thereby synthesizing a sialylated sugar chain. After the engineering cell expresses sialyltransferase by exogenously transferring the sialyltransferase gene into the engineering cell, sialylation modification of Asn162 site of CH1 structural domain of the antibody can be carried out in the cell.

The CHO cell is Chinese Hamster Ovary (CHO), the expression system is gold standard mammal engineering cell of the antibody, and the glycosylation mechanism is similar to human IgG glycosylation mechanism.

The method for improving the drug efficacy of the antibody has the following beneficial effects:

through research on sialylation modification of the CH1 domain of an antibody drug, we found that sialylation at the Asn162 site can increase the inhibitory effect of the antibody on T cells, and in tumor treatment, although therapeutic antibodies can directly kill tumors by binding with target molecules on tumor cells, if sialylation modification of the sugar chain at the Asn162 site can inhibit T cell activation and proliferation by binding with sialic acid receptors (Siglec) on the surface of T cells, tumor immune escape is promoted. The method removes sialylation modification from the Asn162 site of the CH1 structural domain of the antibody drug for treating tumor, and relieves the inhibition effect of the antibody drug on T cells, thereby enhancing the killing effect of the T cells on the tumor and further achieving the purpose of enhancing the drug effect.

In chronic inflammatory diseases, the body generates abnormal immune response to cause self-tissue damage, and the sialylation modification is added to the Asn162 site of the CH1 structural domain of the antibody drug to further inhibit the action of autoreactive T cells, so that the attack of the T cells on the body can be reduced, and the therapeutic effect of the antibody drug can be indirectly enhanced.

By simply adding or removing the sialylation modification of Asn162 site of CH1 structural domain, the therapeutic effect of the antibody drug can be effectively improved, the operation is simple, the effect is obvious, and the antibody drug has wide clinical application prospect.

Drawings

FIG. 1 is the result of secondary mass spectrometry glycopeptide analysis of SIA-IgG in example 1, showing Asn162 site sialylation modification of oncogenic IgG.

FIG. 2 is a comparison of the amino acid sequences of the heavy chain polypeptide of the cancer-derived wild-type IgG and the mutant heavy chain and the changes in the size and weight of the tumors in vivo after the cancer-bearing mice of example 1 are treated, wherein WT represents a group of mice injected with an expression Vector containing the heavy chain of the wild-type IgG, CH1-mu represents a group of mice injected with an expression Vector containing the mutation at the CH1 domain 162 site, and Vector represents a group of mice injected with an empty Vector group containing no target gene.

FIG. 3 shows the effect of the recombinant cancer-derived antibody rCIgG in example 1 on the growth of tumors in tumor-bearing mice after removal of sialylation modification at Asn162 site of CH1 domain, wherein a is the result of detection of sialic acid modification of rCIgG by RP215, B is the change in size of tumors in each group of mice, c is a photograph of tumors taken out from each group of mice after the experiment was completed, d is the size distribution of tumors in each group of mice, and e is the content of T cells and B cells in peripheral blood of each group of mice.

FIG. 4 is an electrophoretogram of the commercial antibody drug of Table 1 and IVIG detected by Western blot in example 2 for RP215, wherein the bands are shown with the antibody sialylated at Asn162 site recognized by RP 215.

FIG. 5 is a graph showing the changes in the size of the tumor growth with time in different groups of tumor-bearing nude mice of example 2 injected with Pertuzumab (Control-Pertuzumab) or Herceptin (Herceptin), respectively, on the ordinate of the volume (cm) ³ ) The abscissa is the number of days.

Fig. 6 is a photograph of tumors taken out 16 days after injection of different groups of tumor-bearing nude mice with Pertuzumab (Pertuzumab) and Herceptin mab (Herceptin) in example 2.

FIG. 7 is a line graph of the change in tumor size of groups of mice measured in vivo on different days in example 3.

FIG. 8 is a photograph comparison of tumors taken out from each group of mice in example 3.

FIG. 9 is the size distribution of tumors taken from each group of mice in example 3.

FIG. 10 shows the distribution of tumor size and tumor weight after killing in each group of mice in example 3.

FIG. 11 shows the comparison of the sizes of tumors taken from each group of mice after killing in example 3.

FIG. 12 is a graph showing the effect of site-directed mutagenesis of the humanized antibody 162 of example 4 on tumor growth inhibition in tumor-bearing mice, compared to unmutated and control antibodies, showing the change in tumor volume over time on the left and the size of the tumor removed on the right.

FIG. 13 shows the changes in body weight and sole thickness of mice in the control and experimental groups in example 5.

FIG. 14 shows the change of ankle thickness and ankle width of control and experimental mice in example 5.

FIG. 15 shows the ankle volume changes and the clinicopathological score statistics of the control and experimental mice in example 5.

FIG. 16 is a graph showing the degree of swelling of joints in the same sites in two groups of mice in example 5.

Detailed Description

The technical solutions of the present invention are explained and illustrated in detail below with reference to the accompanying drawings and preferred embodiments so that those skilled in the art can better understand and implement the technical solutions.

The instrumental reagents used in the examples were all conventional in the art unless otherwise specified.

Example 1 Effect of modification of sialic acid at Asn162 site of CH1 Domain of tumor SIA-IgG on tumor growth

A first part:

RP215 is a monoclonal antibody produced by one of the hybridoma cells obtained by immunizing mice with a protein extracted from an ovarian cancer cell line and is capable of specifically recognizing IgG molecules.

Please refer to the patent: CN201510776518.9, CN201810330585.1, SIA-IgG is a class of IgG with unique sialic acid modification, mainly expressed on tumor stem cells. Tumor stem cells can be modified by unique sialylation of SIA-IgG to promote the maintenance of dryness of the tumor stem cells and the immune escape of the tumor, respectively.

The inventor previously over-expresses the wild type SIA-IgG and the recombinant SIA-IgG with the mutation of the 162 site of the CH1 domain (S160A and N162C) and the recombinant SIA-IgG with the mutation of the 297 site of the CH2 domain (N297Q) in 293 cells respectively, and then detects the recognition condition of the RP215 to the wild type IgG and the mutant type IgG by using a Western blot method, and the result shows that the RP215 can only recognize the mutation of the 297 site of the wild type and the CH2 domain and can not recognize the mutation of the 162 site of the CH1 domain 162, and verifies that the recognition site of the RP215 is Asn 162. Through secondary mass spectrometry glycopeptide analysis, an N-sugar chain exists at the CH1 structural domain 162 site of the SIA-IgG, and the tail end of the SIA-IgG is modified by sialic acid and can be recognized by RP215, which is shown in figure 1. Through previous studies, we determined that sialylation at the CH1 domain 162 site of IgG expressed by tumor stem cells has a strong correlation with tumors, but the mechanism thereof is not clear.

In this example, different modified lung cancer cell lines were inoculated subcutaneously into three groups (control group, wild type group, mutant group, 6 mice per group) of nude mice by subcutaneous injection of cell suspension, and the nude mice were normally bred.

The different groups of lung cancer cell line modification methods are as follows:

control group (Vector): the empty vector, which is a eukaryotic expression vector-PCDB, was transfected into lung cancer cell lines, which did not include any gene of interest.

Wild type group (WT): transfection of a plasmid containing the heavy chain gene of wild-type IgG into lung cancer cell lines allowed overexpression in lung cancer cell lines, the expression product of which was recognized by RP215, with sialylation modification at CH1 domain 162 as confirmed. The wild-type IgG heavy chain gene is an IgG gene sequence expressed by tumor cells.

Mutant group (CH 1-mu): a plasmid containing a mutant heavy chain gene mutated at CH1 domain 162 site, which is an Asn162 mutant of wild-type IgG heavy chain overexpressed in lung cancer cell lines, mutated at sites S160A, N162C, was transfected into lung cancer cell lines. Referring to FIG. 2, the amino acid sequences of the CH1 domain of Wild Type (WT) and mutant (CH 1 mu) are shown above. The mutant heavy chain gene Asn162 has no sialic acid modification.

All groups of mice are raised under the same condition, the sizes of the tumors in the mice are detected in vivo in the raising process, and the tumors in the mice are taken out for measurement after the experiment is finished.

The size and size of the tumors in the mice of the different groups are shown in the lower part of fig. 2, and the results show that the size and weight of the tumors in the mice of the wild type group are significantly larger than those in the other two groups, and no significant difference exists between the other two groups (see statistics of data on tumor volume and weight on the right side of fig. 2). The results show that the mutant IgG heavy chain has a reduced effect on promoting tumor growth compared to the wild-type group with sialylation modification at Asn162 of CH1 domain, comparable to the control group. Therefore, after the mutation of the Asn162 site of the CH1 structural domain of the tumor-derived IgG heavy chain cannot be modified by sialylation, the tumor growth promoting function of the IgG heavy chain can be obviously weakened.

A second part:

next, we performed the sialic acid modification without changing the amino acid sequence of the wild-type CH1 domain, but only removing the N-sugar chain terminal at Asn162 site, to observe its effect on the change in tumor growth.

In the experiment, genes of a heavy chain and a light chain of SIA-IgG are inserted into an expression vector to obtain a recombinant vector, and the recombinant vector is transferred into an engineering CHO cell to be cultured and secreted to obtain a recombinant cancer-derived antibody rCIgG. Western blot detection of rCIgG using RP215 revealed that rCIgG was recognized by RP215, as shown in a in FIG. 3. Indicating that the site Asn162 of the CH1 structural domain has sialic acid modification.

The rCIgG was digested with Neuraminidase (Neuraminidase) to obtain rCIgG with sialic acid modifications removed, labeled rCIgG + N, which was verified with RP215 and not recognized by RP 215.

The lung cancer cell line is inoculated to the left side and the right side of a nude mouse subcutaneously by a mode of subcutaneous injection of cell suspension, and the lung cancer cell line is cultured until the tumor diameter is increased to 2 cm, so that the lung cancer model tumor-forming mouse is constructed.

The tumorigenic mice are divided into three groups, each group comprises 5 mice, a control group is injected with PBS, a wild group is injected with rCIgG, an experimental group is injected with rCIgG + N, the injection modes and the injection doses of the three groups of mice are the same, the injection doses of the three groups of mice are tail vein injections, the injection doses are 5mg/kg, the injection doses are twice a week, the injection doses are 2 weeks, and the feeding conditions are the same. Measuring the sizes of the tumors of each group of mice in vivo during the experiment, monitoring the growth conditions of the tumors, and extracting after the experiment is finishedTaking peripheral blood of mouse, detecting CD4 ⁺ T cell, CD8 ⁺ Percentage of T cells and B cells, mice were sacrificed and tumors were removed from the mice and measured for size and weight.

The experimental results are shown in b-e in FIG. 3, b shows the change of the size of the tumor in the mice of different groups with time during the experimental process, c is the comparison of the size of the tumor taken out of the mice of different groups after the experiment is finished, d is the distribution of the volume and weight of the tumor in the mice of different groups, and e is the CD4 in the mice of different groups ⁺ T cell, CD8 ⁺ Percentage of T cells and B cells.

As can be seen from the figure, rCIgG can promote the tumor growth in vivo more significantly compared with the PBS control group, the tumor promotion effect in the tumor tissue is reduced after sialic acid is removed (rCIgG + N) (b-d), the infiltration of T cells in the tumor tissue is increased correspondingly, and the detection result shows that CD4 is increased ⁺ Increased T cells, CD8 ⁺ T cells increased and B cells increased, substantially similar to the PBS group (e). The removal of the sialylation modification at the Asn162 site of the CH1 domain was shown to reduce the tumorigenic effect of the recombinant oncogenic antibody rCIgG.

Example 2 commercial antibody CH1 Domain Asn162 site modified without sialylation for better efficacy

This example performed CH1 domain sialylation modification analysis of 10 commercial antibodies in table 1 using two mabs, RP215 and recombinant antibody BD11, as tools. BD11 is a humanized chimeric antibody composed of CDRs (complementarity determining regions) of RP215 and human FRs (framework regions) that specifically recognizes sialylation modification at Asn162 in CH1 domain of IgG heavy chain. See figure 4 and table 1 for analytical results.

Since the sialylation modification occurs at position 162 of the IgG heavy chain CH1, the IgG heavy chain and light chain of the antibody drug can be separated by reduction electrophoresis, so that whether the sialylation modification occurs in the IgG heavy chain (55 kD) can be seen when the detection is carried out by using RP215 or BD 11. FIG. 4 shows that the reductive electrophoresis results show that Ustekinumab, Avastin, Rituximab, Pertuzumab, Omalizumab and Nivolumab all obtain IgG heavy chain with 55kD after reduction.

After 10 commercial antibody drugs are detected by using RP215 and BD11, Western blot results in FIG. 4 show that Omalizumab, Belimumab, Rituximab and Herceptin all have sialylation modification at Asn162 site of CH1 domain; ustekinumab, Avastin, Siltuximab, Pertuzumab, Obimutuzumab, Nivolumab were either free of sialylation modification or very weakly sialylated. FIG. 4 shows the top and bottom Western blot results, showing the RP215 binding result and BD11 binding result, respectively, for mutual confirmation.

Table 1: results of sialylation detection of domain Asn162 of 10 commercial antibodies CH1

The breast cancer cell MDA-MB-231 is inoculated into fat pads of the second pair of mammary glands on the right side of two groups (6 mice in each group) of nude mice in a mode of subcutaneous injection of cell suspension, and the breast cancer cell MDA-MB-231 is cultured until the tumor diameter is increased to 2 cm, so that the breast cancer model is constructed.

Antibody drugs were injected around the tumor at a dose of 5mg/kg, respectively, with Herceptin (Herceptin) injected in one group and Pertuzumab (Pertuzumab) injected in the other group, once every three days.

FIG. 5 shows the change of tumor size in nude mice 0-16 days after the injection of two antibody drugs, respectively (averaged per group), and the experimental results show that the growth of tumor size in nude mice injected with Pertuzumab (labeled as Control-Pertuzumab in the figure) is smaller than that in nude mice injected with herceptin over time.

Fig. 6 shows that the tumor removed from the sacrificed mice 16 days after the injection of the antibody drug shows that the in vivo injection of Pertuzumab (Pertuzumab) has stronger inhibitory effect on the growth of breast cancer than Herceptin (Herceptin), and even 2 mice have the tumor disappeared in vivo.

It can be seen that in commercial antibodies, also for the treatment of breast cancer, antibodies with no sialylation modification at domain Asn162 of antibody CH1, such as pertuzumab, are more potent than antibodies with sialic acid modification at domain Asn162 of CH1, such as herceptin.

Example 3 removal of sialylation modification at Asn162 site of CH1 Domain in commercial antibodies can improve drug efficacy

The herceptin was incubated with neuraminidase at 37 ℃ for an appropriate time to remove the sialylation modification at site Asn162 of domain CH1 of herceptin to obtain a sialic acid modified herceptin.

Next, a comparative experiment of injection treatment was performed on the nude mice with tumor formation.

Nude tumorigenic mice, 8 per group, were injected with Herceptin (Herceptin), asialo-modified Herceptin (Herceptin + neuroaminidase), potuzumab (pertuzumab), and PBS buffer, respectively. The experimental period is 22 days, the injection dosage is 50mg/kg, and the injection is performed every three days. The sizes of tumors in the mice of each group are measured in vivo on different days in the experimental process, the mice are sacrificed after the experiment is finished, the tumors in the mice are taken out, and the sizes and the weights are measured.

Fig. 7 is a linear graph of the change in tumor size (averaged per group) for each group of mice on different days, fig. 8 is a photograph comparison of tumors taken from each group of mice, fig. 9 is the distribution of tumor sizes taken from each group of mice, fig. 10 is the distribution of tumor sizes and tumor weights after killing for each group of mice, and fig. 11 is a pair-by-pair comparison of tumor sizes taken from each group of mice after killing.

The experimental results show that after the sialylation modification of the H1 domain of the herceptin is removed, the effect of the herceptin is obviously better than that before the removal, and the effect of the herceptin is not obviously different from that of the Peretol bead, which indicates that the removal of the sialylation modification can improve the tumor treatment efficacy of the herceptin.

Example 4 Effect of absence or modification of sialylation of recombinant antibodies on antitumor Effect

In this example, the humanized antibody SIG-001 described in patent CN 202210009222.4 was used as an experimental tool to examine the effect of sialylation modification at Asn162 site of CH1 domain of SIG-001 on the antitumor effect in a lung squamous carcinoma animal model.

Tumor model: lung squamous carcinoma PDX model, tumor-bearing mice: NOD-SCID mice. The total amount of 30 drugs is divided into three groups, each group comprises 10 drugs, the amount of the drug injection in each group is the same, 5mg/kg, and the injection is injected 3 times per day for 25 days.

Negative control group: mIgG is injected;

normal antibody group: injection of SIG-001 with sialylation modification at the Asn162 site of the CH1 domain of the antibody;

mutant antibody group: injection of SIG-001 ^mu The antibody CH1 domain Asn162 site is mutated and cannot be modified by sialylation.

During the experiment, tumor volumes were measured in vivo and recorded. After the experiment was completed, the mice were sacrificed and the tumors were removed.

The experimental results are shown in FIG. 12, the left side shows the change of tumor volume in each group of mice, which is the result of averaging after measurement of each group of mice, and the right side shows the comparison of tumor size of each group of mice. Compared with a negative control group, the normal antibody group and the mutant antibody group have certain inhibition effects on tumor growth, the inhibition effect of the mutant group is more obvious, and tumors in 3 mice disappear. Thus, the humanized antibody SIG-001 which cannot be sialic acid modified by mutation at Asn162 site in CH1 domain ^mu The anti-tumor effect of the original antibody SIG-001 is obviously enhanced.

Example 5 antibody drug addition of sialylation modification significantly inhibited adjuvant-induced progression of rheumatoid arthritis disease in mice

Rheumatoid Arthritis (RA) is a systemic autoimmune disease mainly characterized by joint disease, and is mainly manifested by chronic, progressive, and aggressive inflammation of joint sites. In this example, the therapeutic effects of the antibody drug IVIG with and without sialic acid modification were examined using an adjuvant-induced (AIA) rheumatoid arthritis model mouse as a study. The AIA mice were divided into two groups, experimental and control, 10 mice each.

IVIG is human venous blood IgG and has been used clinically. About 1-5% of the commercial IVIG products carry Asn162 site sialic acid modification, and bulk IVIG purchased in this example was enriched by RP215 affinity chromatography to obtain CH1 domain Asn162 site sialylated modified IVIG for injection into experimental mice. IVIG without sialic acid modification at the Asn162 site of the CH1 domain was injected into control mice. The two groups of the injection are injected at a dose of 5mg/kg for 3 times a day for 25 days.

In the experimental process, the weight, sole thickness, ankle width and ankle volume of the mouse are measured in vivo, and each group records the measured data and carries out clinical pathological scoring. After the experiment, mice were sacrificed and their lymph nodes and joints were observed.

Results of the experiments see FIGS. 13-16, in which Non SIA-IgG represents IVIG without sialic acid modification at Asn162 site of CH1 domain (control group) and SIA-IgG represents IVIG with sialylation modification at Asn162 site of CH1 domain (experimental group). Data measured simultaneously within each group were averaged.

FIG. 13 shows the changes in the body weight and sole thickness of the control and experimental mice during the course of the experiment, showing that the body weight of both the experimental and control mice increased with the treatment time, and the sole thickness of the experimental mice increased to a lesser extent than the control, indicating that SIA-IgG significantly reduced the swelling of the sole of the AIA mice.

FIGS. 14 and 15 show the ankle thickness, width and volume changes of the control and experimental mice during the course of the experiment, showing that the ankle thickness and width of both the experimental and control mice decreased after the increase with time, but overall the SIA-IgG increased to a lesser extent, indicating that SIA-IgG was able to significantly reduce the ankle swelling of the AIA mice. Figure 15 statistics of clinical pathology scores also show that IVIG with sialylation modification, i.e. SIA-IgG, was more effective in treatment.

FIG. 16 is a display of the joints in the same area of two groups of mice, and it can be seen that the control group treated with Non SIA-IgG had a larger joint volume and more significant swelling, while the experimental group treated with SIA-IgG had a smaller joint volume and less swelling.

It can be seen that, in the same antibody drug, the treatment effect on rheumatoid arthritis is better when the CH1 domain Asn162 site carries sialic acid modification.

The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are all included in the scope of the present invention.

Claims

1. A method for preparing an antibody drug for improving the efficacy of the antibody drug, wherein the antibody comprises a CH1 domain, and the antibody is modified in the following (1) or (2) manner:

(1) when the antibody is used for treating cancer, the sialylation modification at the Asn162 site of the CH1 domain of the antibody is removed;

(2) when the antibody is used for treating chronic inflammatory diseases, a sialylation modification is added to the CH1 domain Asn162 site of the antibody.

2. The method according to claim 1, wherein the method for removing sialylation modification in mode (1) is:

digesting the antibody with neuraminidase; or

Mutating the N glycosylation site gene on Asn162 in the CH1 structural domain of the antibody to remove the sialylation modification function; or

3. The method according to claim 1, wherein in the mode (2), when the antibody is a genetically engineered antibody, the sialylation modification is added by: exogenously transferring N glycosyl transferase gene into the engineering cell and/or exogenously transferring sialic acid transferase gene into the engineering cell.

4. The method according to claim 1, wherein the chronic inflammatory disease in the mode (2) is an autoimmune disease.

5. The method of claim 1, wherein in the alteration of (1), the antibody is herceptin, stoxib, or rituximab.