WO2015110002A1 - Human interleukin ii mutant and application thereof - Google Patents

Abstract

The invention provides a human interleukin II mutant and use thereof in the preparation of medicament. The mutant comprises any one or more mutations at site of 37, 38, 41-45, 61, 62, 65, 68, 72, 80, 81, 85, 86 and 92 in the amino acid sequence of human interleukin II, and preferably comprises SEQ ID NO.3.

Description

Human interleukin II mutant and its application Technical field

The invention relates to a human interleukin II mutant, in particular to a human interleukin II mutant with low toxicity, high efficiency and small side effect and its use in preparing biomedicine.

Background technique

Interleukin-2 (IL-2, interleukin II) is a lymphokine secreted by T lymphocytes (human interleukin II is a protein polypeptide chain consisting of 133 amino acids with a molecular weight of 15KD, and its amino acid sequence is as in the present invention. The sequence listings shown in SEQ ID No. 1 and SEQ ID No. 2) are one of the most important molecules for maintaining normal function in the immune system. Interleukin II can promote T cell maturation, maintain T cell activity, and promote the activity of cytotoxic T lymphocytes, and transform it into tumor infiltrating lymphocytes (TIL), promote the activity of natural killer (NK) cells, and induce killing. The growth of tumor lymphokine cells and killer cells (CTL). Interleukin II enhances human immune function, activates natural killer cells, and amplifies T lymphocytes. It can induce and activate lymphokine-activated cell killing (LAK) and tumor-infiltrating lymphocytes, all of which kill cells infected with foreign microorganisms. And the role of cancerous cells. The effect of interleukin II is described in detail in WO99/60128A1.

Because interleukin II has significant immune enhancement, it has good antiviral, anticancer effects and broad clinical application potential. By promoting the maturation and proliferation of T cells and NK cells, interleukin II has the effect of inducing anti-tumor immunity. It has been found that interleukin II has obvious effects on renal cell carcinoma, melanoma, non-Hodgkin's lymphoma and colorectal cancer. It has different degrees of curative effect on liver cancer, soft-neck cancer, head and neck squamous cell carcinoma, bladder cancer, lung cancer, etc., and has been approved by the FDA for clinical treatment of melanoma and renal cell carcinoma. Interleukin II has also been widely used in clinical cancer treatment and viral infection in China.

There have been a large number of reports on IL-II pharmaceutical preparations such as the aqueous preparations or lyophilized powders disclosed in US Pat. No. 5,358,708, US Pat. No. 4,604,377, US Pat.

However, at the same time, interleukin II has many toxic side effects in the clinic. At low doses, interleukin II promotes lymphocyte killing of tumors with minimal function, but it significantly promotes regulatory T cell function, increasing The number of peripheral blood regulatory T cells (T suppressor cells) (CD4+CD25+ cells) (JEM 201:723-735) further inhibits the function and proliferation of infiltrating lymphocytes and inhibits anti-tumor immune responses (JI 26:85- 93); At high doses, interleukin II can cause pain, high fever, nausea, vomiting, pulmonary edema, vascular leakage, and even death (JCO 21:3127-3132). Due to its dual function of killing tumor cells and promoting inhibition of cells, the clinical application of interleukin II is currently limited due to its serious toxicity. It is estimated that only about 20% of patients with related diseases can be treated with interleukin II.

Summary of the invention

In order to solve the obvious defects of human interleukin II side effects, the present invention provides a human interleukin II mutant with low toxicity and high efficacy and side effects, while maintaining the function of activating natural killer cells and tumor infiltrating lymphocytes, thereby expanding its clinical application. .

A first aspect of the present invention provides a human interleukin II mutant comprising a sequence which is a human interleukin II amino acid sequence, and an alpha and/or beta receptor in human interleukin II A sequence in which the binding point is mutated; wherein the mutation at the binding site to the α receptor reduces the affinity of the human interleukin II to the α receptor; the mutation at the binding site to the β receptor enhances the affinity for the β receptor.

In a preferred embodiment of the invention, the binding site is selected from the group consisting of 37, 38, 41-45, 61, 62, 65, 68, 72, 80, 81, 85, 86 of the amino acid sequence of human interleukin II. Any one or more of 92 points.

In a preferred embodiment of the invention, the mutation has a binding point of at least 3, more preferably at least 5, more preferably at least 6.

In a preferred embodiment of the invention, the mutated binding site comprises at least the 65, 80, 81, 85, 86, 92 amino acid sequence of the human interleukin II amino acid sequence.

In a preferred embodiment of the invention, the mutation comprises a deletion or substitution of an amino acid, and preferably the corresponding amino acid in the human interleukin II sequence is replaced by a different amino acid.

In a preferred embodiment of the invention, the mutation comprises at least: amino acid Pro at position 65 of the amino acid sequence of human interleukin II is replaced by amino acid Arg.

In a preferred embodiment of the invention, the mutation comprises at least: the amino acid Leu at position 80 of the amino acid sequence of human interleukin II is replaced by the amino acid Phe.

In a preferred embodiment of the invention, the mutation comprises at least: human interleukin II amino acid The amino acid Arg at position 81 of the sequence is replaced by the amino acid Asp.

In a preferred embodiment of the invention, the mutation comprises at least: the amino acid Leu at position 85 of the amino acid sequence of human interleukin II is replaced by the amino acid Val.

In a preferred embodiment of the invention, the mutation comprises at least: the amino acid Ile at position 86 of the human interleukin II amino acid sequence is replaced by the amino acid Val.

In a preferred embodiment of the invention, the mutation comprises at least: the amino acid Ile at position 92 of the amino acid sequence of human interleukin II is replaced by the amino acid Phe.

In a preferred embodiment of the invention, the human interleukin II mutant comprises the amino acid sequence of SEQ ID No. 3 of the Sequence Listing.

In a preferred embodiment of the invention, the human interleukin II mutant is the amino acid sequence of SEQ ID No. 3 of the Sequence Listing or the fusion protein comprising the amino acid sequence of SEQ ID No. 3.

A second aspect of the invention provides a method of synthesizing the human interleukin II mutant, comprising:

Gene synthesizing the amino acid sequence of the human interleukin II mutant and recombining the gene into an expression vector;

The host is transfected with an expression vector for production of a human interleukin II mutant.

In a preferred embodiment of the invention, the host may be any eukaryotic cell capable of receiving the expression vector.

The eukaryotic cell may be any one or several of animal cells, plant cells, and human cells, and is preferably any one or more of animal cells and human cells, such as muscle cells, liver cells, red blood cells, Ovarian cells, etc.

A third aspect of the invention provides a medicament comprising at least the human interleukin II mutant.

In a preferred embodiment of the invention, the medicament is preferably in the form of an injection, such as a solution, an emulsion, a suspension, a powder (more preferably a lyophilized powder) or the like.

In a preferred embodiment of the invention, the drug may also be included in the drug.

In a preferred embodiment of the present invention, the medicament may further comprise an adjuvant such as a pH adjuster, an antifreeze, a surfactant (such as a solubilizer, a suspending agent, etc.), a cosolvent, an antioxidant, and the like. Bacterial agents, osmotic pressure regulators (such as sodium chloride, glucose), fillers.

In a preferred embodiment of the invention, the medicament may further comprise a second active ingredient, which may be any one or more of a therapeutically active ingredient, a stimulating active ingredient.

The active ingredient for reducing irritation is preferably any one or more of an analgesic agent, a desensitizing agent, and the like.

The medicament according to the present invention may be any one or more of drugs for treating viruses, bacilli and parasitic infections, drugs for treating cancer and its complications, immunomodulators, vaccines, and drugs for treating autoimmune diseases. Kind.

Wherein, the virus, bacillus and parasitic bacteria may be hepatitis B virus, influenza virus, tuberculosis virus, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes virus, rabies virus, HIV, Candida albicans and the like.

The cancer may be kidney cancer, melanoma, breast cancer, bladder cancer, liver cancer, lymphoma, rectal cancer, lung cancer, and the like.

The autoimmune disease may be hyperthyroidism, diabetes, muscle weakness, cirrhosis, rheumatoid arthritis, systemic vasculitis, pemphigus, dermatomyositis, autoimmune hemolytic anemia, ulcers, and the like.

It is to be understood that the various aspects of the invention described above and various preferred embodiments thereof can be arbitrarily combined by those skilled in the art without limitation, and such combinations are also included in the scope of the present invention.

The human interleukin II mutant of the present invention mutates at a site binding to the α receptor compared to the protoplast, and reduces the affinity of the α receptor (CD4CD25) and interleukin II, thereby further affecting the promotion of regulatory T cells. The ability to attenuate alpha receptor-binding interleukin II gene mutations will have a reduced function of promoting T cell proliferation inhibition. At the same time, the mutants of the present invention reduce the binding of alpha receptors to lung epithelial cells, thereby reducing and eliminating the toxic side effects of high doses causing pulmonary edema.

Beta receptor binding point mutations are mainly expressed on the surface of natural killer (NK) cells, lymphokine cells and killer cells. By mutations at the 80, 81, 85, 86 and 92 amino acid positions, structurally altered interleukin II enhances binding to beta receptor binding, thereby increasing and promoting the ability of immune cells to kill tumor cells.

The experimental results show that the present invention reduces the function of inducing regulatory T cells by site-directed mutagenesis of the human interleukin II amino acid sequence, reduces or eliminates its toxic and side effects, and retains the property of interleukin II on natural killer cell immune activity. Therefore, the mutant of interleukin II can stimulate the immune system, enhance the function of inhibiting tumor growth, and increase its effect of inhibiting the growth of solid tumors in vivo, thereby improving its clinical efficacy and application range.

The invention can be used as a medicine or vaccine alone for the treatment and prevention of diseases, or with other treatment methods Combinations, such as use as an immunomodulator after surgery.

DRAWINGS

Figure 1 is a comparison of the immunoreactive effects of the human interleukin II mutant (mutat IL-2) and human interleukin II (native IL-2); wherein, Figure 1A shows NK cell activity, and Figure 1B shows CD8 cell activity. 1C is the activity of CD4 and CD25 cells, and FIG. 1D is the comparison of the number of CD25 cells;

Figure 2 is a comparison of the effects of injection of the human interleukin II mutant (mutat IL-2) and human interleukin II progenitor (native IL-2) on pulmonary edema in mice;

Figure 3 is a comparison of the antitumor effects of the human interleukin II mutant (mutat IL-2) and human interleukin II progenitor (native IL-2).

detailed description

In one embodiment of the present invention, the SEQ ID No. 2 (human interleukin II protagonist) and SEQ ID No. 3 (inventive human interleukin II mutant) of the sequence listing of the present invention are compared with the protoplast The mutation positions and alternative amino acids are as follows:

Table 1. Mutation sites and substitution amino acids of the interleukin II mutant of the present inventors of the present invention

Site	Protozoa amino acid	Mutant amino acid
			65	Pro	Arg
80	Leu	Phe
			81	Arg	Asp
85	Leu	Val
			86	Ile	Val
92	Ile	Phe

First, a gene sequence was designed based on the human interleukin II protoplast amino acid sequence (NM_00586), and then the gene sequence was mutated. The mutated gene having the N-terminal polyhistidine tag was synthesized by the overlap extension method and further cloned into the multifunctional expression vector. The vector was transfected into Chinese hamster ovary cells, and the cells were cultured in a 37 ° C, 5% CO 2 incubator.

After 72 hours, the supernatant was taken and the supernatant contained the human interleukin II mutant. Fixed The recombinant protein was purified by chemical ion chromatography, and the purified protein was confirmed by electrophoresis.

Mutant interleukin II promotes inhibition of (CD4CD25+) T cells, NK cells and CD8+ T cell activity Determination

The mice were injected with interleukin II progeny or interleukin II mutant at a daily dose of 20 μg for 3 days. Blood was collected on day 4, and peripheral blood cells were stained with CD25, CD8, NK1.1 and CD4 antibody staining. Finally, flow cytometry and data analysis were performed.

As shown in Fig. 1, as shown in Figs. 1A and 1B, the mutant of the present invention (mutat IL-2) did not significantly decrease the effect on promoting NK and CD8 cells compared to native IL-2. The number of NK and CD8 cells was significantly increased compared to the blank control, which was 2-3 times the number of blank control groups, indicating that the mutant of the present invention retains the function of interleukin II progenitor on NK and CD8 cells.

Meanwhile, as shown in FIG. 1C and FIG. 1D, in the case of injecting the mutant of the present invention, there was no significant difference between the CD25 content and the blank control group, and the amount of the native CD25 was 3-4 times that of the mutant and the blank control group. It is shown that the mutant of the present invention eliminates the binding to CD25, thus greatly reducing the promoting effect on regulatory T cells.

Effect of Mutant and Protoplast Interleukin II Protein on Pulmonary Edema in Animals

The mice were injected with interleukin II protoplasts or interleukin II mutants at a daily dose of 80 micrograms for a total of 3 days. On the 7th day, the lungs were collected and weighed.

As shown in Fig. 2, compared with the blank control group, the mutant of the present invention did not increase the lung weight under high-dose conditions, indicating that pulmonary edema was not caused, and the lung weight was compared with the injection of high-dose protozoa. The mutant of the present invention and the blank control group have significant differences. This specification of the mutant of the present invention eliminates the toxic side effects of high dose of protozoa causing pulmonary edema.

Solid tumor immunotherapy of mutant and protoplast interleukin II protein

Renal cancer cells were inoculated subcutaneously in normal 6-week-old female mice.

From day 5, tumor-bearing mice were given daily high-dose (50 μg/mouse) interleukin II progeny or interleukin II mutant injection for a total of 7 days, and the tumor size was measured daily using an electric caliper.

As shown in Figure 3, compared with interleukin II progeny, the effect of interleukin II mutant on tumor immunotherapy was significantly improved (P < 0.001), and the tumor weight was only 1/10 or even lower than that of the original body; The difference in tumor weight was significantly lower in the living body compared with the blank control group.

The specific embodiments of the present invention have been described in detail above, but only by way of example, the present invention It is not limited to the specific embodiments described above. Any equivalent modifications and substitutions to the invention are also within the scope of the invention. Accordingly, equivalents and modifications may be made without departing from the spirit and scope of the invention.

Claims (10)

1. A human interleukin II mutant characterized by comprising a sequence which is a mutation in the amino acid sequence of human interleukin II at a point of binding to the alpha and/or beta receptor of human interleukin II The sequence; wherein the mutation at the binding site to the alpha receptor reduces the affinity of the human interleukin II to the alpha receptor; the mutation at the binding site to the beta receptor enhances the affinity for the beta receptor.
2. The human interleukin II mutant according to claim 1, wherein the binding site is selected from the group consisting of 37, 38, 41-45, 61, 62, 65, 68, 72 of the amino acid sequence of human interleukin II. Any one or more of the 80, 81, 85, 86, 92 sites.
3. The human interleukin II mutant according to claim 1, wherein the mutation has at least three binding points.
4. The human interleukin II mutant according to claim 3, wherein the mutation has a binding point of at least 6.
5. The human interleukin II mutant according to any one of claims 1 to 4, wherein the mutation-binding site comprises at least 65, 80, 81, 85 of the amino acid sequence of human interleukin II. 86, 92 points.
6. The human interleukin II mutant according to claim 5, wherein the mutation comprises at least one or more of the following:

   The amino acid Pro at position 65 of the human interleukin II amino acid sequence is replaced by the amino acid Arg;

   The amino acid Leu at position 80 of the human interleukin II amino acid sequence is replaced by the amino acid Phe;

   The amino acid Arg at position 81 of the human interleukin II amino acid sequence is replaced by the amino acid Asp;

   The amino acid Leu at position 85 of the human interleukin II amino acid sequence is replaced by the amino acid Val;

   The amino acid Ile at position 86 of the human interleukin II amino acid sequence is replaced by the amino acid Val;

   The amino acid Ile at position 92 of the human interleukin II amino acid sequence is replaced by the amino acid Phe.
7. The human interleukin II mutant according to claim 1, wherein the human interleukin II mutant comprises the amino acid sequence of SEQ ID No. 3 of the Sequence Listing.
8. A method of synthesizing the human interleukin II mutant of claim 1, comprising:

   Gene synthesizing the amino acid sequence of the human interleukin II mutant and recombining the gene into an expression vector;

   The host is transfected with an expression vector for production of a human interleukin II mutant.
9. A medicament, characterized in that the medicament comprises at least the human interleukin II mutant of claim 1.
10. The medicament according to claim 9, wherein the medicament further comprises a component selected from the group consisting of a carrier protein, an adjuvant, and a second active ingredient.

Images