CN110151987B - Application of symmetrical IgG and composition thereof in preparation of medicines for treating and preventing tumors - Google Patents

Abstract

The invention discloses application of symmetrical IgG and a composition thereof in preparing medicines for treating and preventing tumors, wherein the symmetrical IgG can be widely used for preventing and treating various malignant tumors, has relatively low medicine cost, wide application, no obvious side effect and wide clinical application prospect.

Description

Application of symmetrical IgG and composition thereof in preparation of medicines for treating and preventing tumors

Technical Field

The invention belongs to the technical field of tumor immunotherapy medicaments, and in particular relates to application of symmetrical IgG and a composition thereof in preparation of medicaments for treating and preventing tumors.

Background

The incidence rate of tumor in China, especially malignant tumor, is increased increasingly, the death rate is high and is not low, the new tumor is nearly 400 ten thousand per year, more than 200 ten thousand of people dying from the tumor are dead, and a new method for effectively preventing and treating the tumor is urgently needed. Tumor occurrence and development are caused by two factors, namely, the uncontrolled growth and division of tumor cells on one hand and the failure of the immune function of the organism on the other hand, the tumor cells cannot be normally identified and inhibited from growing.

Current methods of treatment for cancer mainly include surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. Radiotherapy and chemotherapy have certain treatment effects on cancers, but have serious side effects, and the overall five-year survival rate of patients is not obviously improved. Targeted therapies are therapies that have recently emerged against molecular targets of tumor cells that have particular therapeutic effects on certain tumors that are expressed by the target molecule, but have a small proportion of patients with this particular target, and that rebound or have other side effects much after tumor inhibition. Immunotherapy is the most recently developed therapy, which has obvious inhibition effect on various cancers, but the current immunotherapy is mostly cellular immunity, i.e. the purpose of killing tumor is achieved by reactivating antitumor lymphocytes. There are few current methods of treating tumors using humoral immunity. In the method of humoral immunity treatment of tumor, some tumor specific antibodies can carry antitumor drugs to achieve a certain effect by injecting specific antitumor antibodies to kill tumor cells. However, most immunotherapeutic drugs are currently in clinical trials or preclinical studies, and there are no very many mature drugs on the market.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides the application of the symmetrical IgG and the composition thereof in preparing medicaments for treating and preventing tumors, can widely prevent and treat various malignant tumors, has relatively low medicament cost, wide application, no obvious side effect and wide clinical application prospect.

One of the technical schemes adopted for solving the technical problems is as follows:

the invention provides application of symmetrical IgG in preparing medicines for treating and/or preventing tumors.

Where asymmetric IgG refers to an IgG having one glycosylation (including various glycosyl components) in both Fab ends of the IgG, the glycosylation also includes an Fc end, thereby making the two side structure of the IgG asymmetric, and thus referred to as asymmetric immunoglobulin (asymmetric IgG, asy IgG, glycosylated IgG, non-precipitative IgG), the asymmetry appears as a double band when running Western gelatin electrophoresis, and the double band becomes a single band after digestion with glycosylase. In contrast, the heavy and light chains on both sides of an IgG that are not glycosylated are identical at both Fab ends and are symmetrical, and are therefore referred to as symmetrical immunoglobulins (symmetrical IgG, syigg, non-glycosylated IgG, deglycosylated IgG, precipitant IgG).

The invention also provides application of the composition comprising the symmetrical IgG in preparing medicines for treating and/or preventing tumors, wherein the composition can contain other medicines or substances with medicinal activity besides the symmetrical IgG, such as tumor preventing medicines, tumor treating medicines, antibacterial medicines, antiviral medicines, immunopotentiators and the like.

In one embodiment: the tumor comprises solid tumor and blood tumor.

In one embodiment: the tumor includes primary tumor and metastasis.

In one embodiment: the tumor is at least one of melanoma, breast cancer, colon cancer, rectal cancer, skin cancer, lung cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, liver cancer and prostate cancer.

In one embodiment: the prevention includes preventing tumorigenesis, preventing tumor recurrence, preventing tumor invasion, preventing tumor metastasis, i.e. preventing tumorigenesis, tumor recurrence, tumor invasion, tumor metastasis before occurrence of tumor genesis, tumor recurrence, tumor invasion, tumor metastasis; the treatment includes inhibiting tumor growth, inhibiting tumor invasion, inhibiting tumor metastasis, i.e., inhibiting, reducing or even completely eliminating the extent of tumor growth, tumor invasion, tumor metastasis after tumor growth, tumor invasion, tumor metastasis have occurred.

In one embodiment: the medicine also comprises auxiliary materials. The auxiliary materials are pharmaceutically acceptable diluents, solvents, excipients, absorbents, wetting agents, adhesives, disintegrants, lubricants, solubilizers, emulsifiers, suspending agents, surfactants, film forming agents, propellants, antioxidants, flavoring agents, fragrances, bactericides, preservatives and the like; the auxiliary materials also comprise pharmaceutically acceptable drug carriers, namely, the auxiliary materials can carry the drugs, can change the mode of the drugs entering a human body and the distribution in the human body, control the release speed of the drugs to achieve controlled release or slow release, and carry the drugs to target organs and other systems in a targeted manner, and comprise liposome, microsphere, microcapsule, solid dispersion, micelle, microemulsion, gel, slow release type carrier, controlled release type carrier, targeted type carrier, nanoparticle material and the like.

The symmetrical IgG and the composition comprising the symmetrical IgG can be directly prepared into a preparation by a pharmaceutically acceptable method, or the preparation can be prepared into the preparation by a pharmaceutically acceptable method after adding the pharmaceutically acceptable auxiliary materials; the formulation may be in solid form or liquid form, such as tablets, capsules, pills, granules, injections, and the like.

In one embodiment: the administration route of the medicine comprises intravenous administration, subcutaneous administration, local administration of tumor, oral administration and intracavity administration, and the cavity of the intracavity administration comprises peritoneal cavity, pleural cavity and cavity in central nervous system. Accordingly, the symmetrical IgG, compositions comprising symmetrical IgG, can be prepared as formulations suitable for the above routes of administration.

In one embodiment: the dosage of the medicine is 0.01-0.5 g per kilogram of body weight according to symmetrical IgG. The medicine is taken once in three weeks, and four times calculate a treatment course. At least two courses of treatment are performed. The dosage, frequency and duration of administration may be determined by the therapeutic effect.

In one embodiment: the medicament is administered in combination with at least one additional therapeutic modality, which may be administered prior to, during, or after administration. Such additional modes of treatment include surgical treatment, chemotherapy, radiation therapy, targeted therapy, immunotherapy, and the like.

For individuals who are likely to develop tumors, the same method can be used for long-term administration to achieve the effect of preventing tumor generation.

For the postoperative tumor patients, the same method is used for administration, so that the effect of preventing tumor recurrence and metastasis can be achieved.

The second technical scheme adopted by the invention for solving the technical problems is as follows:

a method of preventing and/or treating a tumor by removing asymmetric IgG from a patient or reducing the amount of asymmetric IgG in a patient. Specifically:

a method for preventing and/or treating tumor comprises establishing extracorporeal blood circulation, continuously removing asymmetric IgG from blood of patient, or reducing asymmetric IgG content in blood. For example, patient blood is introduced into a device by extracorporeal blood circulation, where asymmetric IgG in the blood is removed, or the content of asymmetric IgG in the blood is reduced, and then the blood is released back into the patient.

A method for preventing and/or treating tumor comprises removing asymmetric IgG from blood or reducing the content of asymmetric IgG in blood after extracting blood from patient, and introducing blood back into patient.

In one embodiment: methods for removing or reducing the level of asymmetric IgG in blood include separating IgG from other plasma proteins using Protein G, then treating IgG by adsorption to canavalin a (ConA), adsorption to antibodies, and the like to separate symmetric IgG from asymmetric IgG, thereby removing asymmetric IgG, and then reinfusion of symmetric IgG into blood. In the example of ConA adsorption, the Fab end of asymmetric IgG is glycosylated, while ConA sepharose couples concanavalin (ConA) to cross-linked activated sepharose, conA binds to various types of saccharides, glycoproteins, and glycolipids with mannitol and glucose residues, so that ConA sepharose separates the glycosylated asymmetric IgG from the non-glycosylated symmetric IgG at the Fab end. Or specific antibody adsorption is adopted, and anti-asymmetric IgG antibody is adopted to separate the asymmetric IgG. The auxiliary materials used in the adsorption method can be repeatedly used after adsorbed asymmetric IgG is washed away. ConA mainly adsorbs IgG rich in high mannose and glucose, and similarly, other lectins can adsorb sialylated glycosyl groups and the like.

In one embodiment: the method is used in combination with at least one additional therapeutic modality, which may be used prior to, during, or after the use of the method. Such additional modes of treatment include surgical treatment, chemotherapy, radiation therapy, targeted therapy, immunotherapy, and the like.

The reagents, instruments, etc. according to the present invention are commercially available unless otherwise specified. The methods according to the present invention are conventional in the art unless otherwise specified.

Compared with the background technology, the technical proposal has the following advantages:

in earlier studies, we found that both symmetrical and asymmetrical IgG were present in normal human blood. Whereas in the blood of tumor patients, asymmetric IgG is significantly elevated. We have shown that such glycosylated asymmetric immunoglobulins can bind to other immunoglobulins, which do not react with the corresponding antigen through the antigen recognition region of the Fab end variable region, but possibly through the glycosyl group carried thereby, to other IgG which compete with specific antibodies against tumor antigens for receptors on leukocytes, but do not elicit subsequent immune response and thus do not produce killing of the targeted antigen cells, so that such immunoglobulins act to block killing of tumor cells by humoral and cellular immunity. We have further found that this reaction is non-specific and may not recognize specific antigens through the variable region of IgG, but rather through the reaction of the glycosyl groups on its Fab fragments with the Fc-terminus of other immunoglobulins, whereby asymmetric IgG blocks the immune response chain and classical ADCC, ADCP or CDC reactions are inhibited, thus protecting the antigen. Thus, an asymmetric IgG is also referred to as a "blocking antibody". The mechanism of the immunosuppressive effect of asymmetric IgG on tumors is shown in figure 1.

Our research shows that the tumor microenvironment can produce more asymmetric IgG, on one hand, the asymmetric IgG can react with the anti-tumor IgG molecules produced by the organism, and the killing effect of the organism on the tumor is blocked. Meanwhile, the asymmetric IgG can also be combined with Fc receptor of immune benefit cells to competitively inhibit tumor specific antibody from combining with the Fc receptor, and further block ADCC and ADCP reactions, so that tumor cells can evade immune cells from attacking tumors. On the other hand, asymmetric IgG may react with mannose receptor and macrophages and induce the transition of M1 type macrophages to M2 type macrophages, thereby promoting immune escape of the tumor, rendering tumor cell growth uninhibited by the immune system. We find that the injection of Fab segment glycosylated asymmetric IgG into human body can not inhibit tumor cell, but can block the anti-tumor immune reaction of human body, and can promote tumor generation, growth and metastasis, so that the promotion of asymmetric IgG to tumor is proved.

However, we found that increasing symmetrical IgG in a tumor part or whole body of a tumor patient, or decreasing asymmetrical IgG in a tumor part or whole body of a tumor patient, can reverse the action of asymmetrical IgG, and achieve the purposes of preventing, inhibiting and treating the occurrence, growth and metastasis of tumors.

In order to achieve the above purpose, a specific scheme provided by the invention is to prepare symmetrical IgG into medicine, and increase the symmetrical IgG in tumor local or tumor patient after administration. Our study demonstrates that symmetrical IgG administration can significantly inhibit tumor development, progression and metastasis. The same results were obtained in animal models we used including melanoma, colorectal cancer, breast cancer and skin cancer in mice, including tumors injected from tail vein, subcutaneous tumors, tumors induced with chemical agents, and tumors spontaneously generated in mice by genetic engineering. In addition, two cases of advanced tumor patients are tested, so that the expected cancer patients (one lung cancer and one esophagus cancer) living only for three months originally live for more than one year and the disease is stable. Symmetrical IgG has therapeutic effects if applied at the early stage of tumorigenesis, and can be used without growing tumor to prevent tumorigenesis.

In addition, the invention also provides a specific scheme for establishing the extracorporeal blood circulation, and continuously removing or reducing the content of the asymmetric IgG from the blood of a patient; or after the blood of the patient is extracted, the asymmetric IgG in the blood is removed or the content of the asymmetric IgG is reduced, and then the blood is input back into the patient, so that the effect of removing the asymmetric IgG or reducing the content of the asymmetric IgG can be achieved in the whole body of the patient and the microenvironment of the tumor, and the effects of preventing, inhibiting and treating the occurrence, growth and metastasis of the tumor can be achieved. Since this therapy only removes the asymmetrical IgG in the patient's blood, which has an inhibitory effect on the immune system, without adding any other drugs, there is no side effect on the patient.

While not wishing to be bound by any particular theory, we propose that symmetrical IgG may function by one or more of the following mechanisms: the symmetrical IgG can reduce the content and proportion of the asymmetrical IgG in the tumor microenvironment, change the balance of two kinds of IgG in the tumor part, reduce the blocking effect of the asymmetrical IgG on the immune system, break the immune escape mechanism of the tumor, make the immune system of the human body reactivate the immune response of the body to the tumor, inhibit the growth of the tumor and strengthen the killing effect on tumor cells; or the symmetrical IgG may neutralize the asymmetrical IgG, thereby reversing the immune system inhibition by the asymmetrical IgG. Symmetrical IgG may also function by stimulating the production of cytokines including IL4, IL6, IL10, tnfα, etc. by leukocytes including lymphocytes, dendritic cells, macrophages, monocytes and other various leukocytes.

In conclusion, the invention provides evidence that the symmetrical IgG can inhibit the growth, development and metastasis of tumor cells by stimulating the function of the human immune system, and the symmetrical IgG has obvious inhibition effect on the growth and metastasis of tumors in a large number of animal experiments; experiments performed in several volunteer tumor patients also received good results. The medicine has low cost, wide application and no obvious side effect, so the symmetrical IgG is expected to become a low-cost and high-efficiency medicine for preventing and treating tumor, and has wide clinical application prospect.

Drawings

The invention is further described below with reference to the drawings and examples.

FIG. 1 is used to illustrate the mechanism by which elevated asymmetric IgG inhibits tumor immunity in a tumor microenvironment.

FIG. 2A is a graph showing the molecular weight difference between symmetrical IgG and asymmetrical IgG.

Fig. 2B is used to demonstrate that symmetrical IgG can inhibit tumor growth in a breast cancer subcutaneous tumor model.

Fig. 2C is used to demonstrate that symmetrical IgG is able to suppress tumor weight in a subcutaneous tumor model of breast cancer.

Fig. 2D is used to demonstrate that symmetrical IgG is able to inhibit tumor metastasis in a colon cancer lung metastasis model.

Fig. 2E is used to demonstrate that symmetrical IgG is able to inhibit the weight of tumor metastases in a colon cancer lung metastasis model.

Fig. 2F is used to demonstrate that symmetrical IgG is able to inhibit tumor metastasis area ratio in a colon cancer lung metastasis model.

Fig. 3A is used to demonstrate that symmetrical IgG almost completely prevented metastasis of tumors in a colon cancer lung metastasis model with a small number of cell inoculations.

Fig. 3B is used to demonstrate that symmetrical IgG is able to inhibit tumor metastasis area ratio in a colon cancer lung metastasis model with a small number of cell inoculations.

Detailed Description

The following is a detailed description of the present invention by way of examples:

experimental example 1: anti-tumor effect of symmetrical IgG

Human IgG was separated into asymmetric IgG (Asy IgG) and symmetric IgG (Sy IgG) by ConA adsorption and purified by rProtein G as follows:

human IgG flowed by gravity through the ConA column, conA bound to mannose glycosyl groups, adsorbing glycosylated asymmetric IgG (Asy IgG), whereas non-Fab glycosylated symmetric IgG (Sy IgG) flowed out of the column, collecting concentrated non-glycosylated symmetric IgG (Sy IgG). Washing the column with balanced salt solution to remove residual Sy IgG, and competitively binding ConA with alpha-methyl mannose solution to separate glycosylation-modified asymmetric IgG (Asy IgG) from ConA, eluting the column, and collecting and concentrating to obtain Asy IgG. To prevent ConA residues in the isolated Asy and Sy IgG, we passed the Asy and Sy IgG through the rProtein G column, respectively, to which both Asy and Sy IgG bound, and from which residual ConA flowed. After washing the column, asy IgG and Sy IgG were eluted from the rProtein G column using an acidic eluent, and concentrated to obtain purified Asy IgG and Sy IgG, respectively, for subsequent experiments.

Silver staining results showed a clear molecular weight difference between Asy IgG and Sy IgG (fig. 2A).

In the breast cancer subcutaneous tumor model (fig. 2B, 2C) and the colon cancer lung metastasis model (fig. 2D, 2E, 2F), mice were divided into three groups, and the same volumes of PBS (control), sy IgG (50 mg/kg), asy IgG (50 mg/kg) were injected into the tail vein, once every five days, four times in total. As a result, it was found that symmetrical IgG showed a significant antitumor effect compared to the control group, whereas asymmetrical IgG did not show an antitumor effect, and there was a very significant statistical difference between the two.

In the colon cancer lung metastasis model, the inoculation number of tumor cells is 1-5 multiplied by 10 ⁵ In the case of (2), symmetrical IgG has an anti-tumor effect. Intravenous injection of 1X 10 into tail of mouse ⁵ The following day of tumor cell inoculation, 1mg (Sy IgG/Asy IgG) was injected per tail vein (mice each average body weight 20g, equivalent to 50 mg/kg) once every five days for a total of four injections. The control group was injected with the same volume of PBS. In comparison to the control group, symmetrical IgG injection was found to almost completely prevent tumor metastasis, whereas asymmetrical IgG had almost no anti-tumor metastasis effect (fig. 3).

Experimental example 2: clinical trial

One patient with advanced lung squamous cell carcinoma, 63 years old male, the illness state after operation and radiotherapy and chemotherapy is still not controlled, doctor judges that the expected life time of the patient is 3 months, the patient voluntarily uses the symmetrical IgG immunotherapy, intravenous infusion is carried out for 18 months, the illness state is not worsened, the patient still survives, and no side effect is found.

Another example of patients with advanced esophageal cancer (squamous cell carcinoma), 56 years old men, had lung metastases, and after radiotherapy and chemotherapy the cancer could not be controlled, doctors expected that the patient's survival time was less than 3 months, and under the strong requirements of patients, the above immunotherapy was used, and by intravenous drip infusion, the patients had survived for 15 months until now, the tumors did not develop further, and no side effects were found.

The foregoing description is only illustrative of the preferred embodiments of the present invention, and therefore should not be taken as limiting the scope of the invention, for all changes and modifications that come within the meaning and range of equivalency of the claims and specification are therefore intended to be embraced therein.

Claims (6)

1. Use of symmetrical IgG in the manufacture of a medicament for the treatment of a tumor, which is breast cancer, colon cancer, lung squamous carcinoma or esophageal carcinoma; the symmetrical IgG is IgG with both Fab ends unglycosylated.

2. Use of a composition comprising symmetrical IgG for the manufacture of a medicament for the treatment of a tumor, which is breast, colon, lung squamous carcinoma or esophageal carcinoma; the symmetrical IgG is IgG with both Fab ends unglycosylated.

3. Use according to claim 1 or 2, characterized in that: the treatment includes inhibiting tumor growth, inhibiting tumor invasion, and inhibiting tumor metastasis.

4. Use according to claim 1 or 2, characterized in that: the administration route of the medicine comprises intravenous administration, subcutaneous administration, local administration of tumor, oral administration and intracavity administration.

5. Use according to claim 1 or 2, characterized in that: the dosage of the medicine is 0.01-0.5. 0.5g per kilogram of body weight according to the symmetrical IgG.

6. Use according to claim 1 or 2, characterized in that: the medicament is used in combination with at least one additional therapeutic modality.