WO2022078185A1 - Interleukin-2 mutant - Google Patents

Abstract

Disclosed in the present invention is an IL-2 mutant. On the basis of human IL-2, the amino acid sites associated with binding to IL2Rα are subjected to one or more mutations of substitution, deletion and addition to obtain an IL-2 mutant with a reduced binding capacity to IL2Rα, wherein the amino acid sites associated with binding to IL2Rα are positions 30-75 of wild-type IL-2. The interaction between the IL-2 mutant of the present invention and the IL2Rα is reduced, and the expression quantity is increased.

Description

An interleukin-2 mutant technical field

The invention belongs to the field of molecular biology, and particularly relates to an interleukin-2 mutant.

Background technique

Interleukin-2 (IL-2), discovered in 1976 and then known as T cell growth factor (TCGF), is a globular glycoprotein that plays an important role in maintaining the normal function of T lymphocytes and NK cells . Natural IL-2 is a polypeptide consisting of 133 amino acid residues, with a molecular weight of about 15kD, and three cysteine residues located at positions 58, 105 and 125, respectively. Post-translational modifications include Thr glycosylation at position 3, and cysteine residues at positions 58 and 105 form disulfide bonds, which are mainly composed of four α-helices and some linking sequences (loops) that are essential for their function. ) composed of higher-order structures (Bazan et al., Science 257, 410-413 (1992)).

IL-2 is mainly produced by activated T cells, it can promote the proliferation and differentiation of T cells, maintain T cell activity; stimulate the generation, proliferation and activation of natural killer (NK) cells, and induce cytotoxic T lymphocytes (CTL) and induces and activates lymphokine-activated killer (LAK) and tumor-infiltrating lymphocytes; promotes the expression of cytokines and cytolytic molecules by T cells, and promotes the proliferation of B cells (Waldmann et al., Nat Rev Immunol 6, 595-601 (2009) )); these cells have or indirectly have the effect of killing foreign microbial infected cells and cancerous cells, so IL-2 has good antiviral, anticancer effects and wide clinical application potential.

IL-2 mediates its effects by binding to the IL-2 receptor (IL2R), which consists of three subunits, α (CD25), β (CD122), and γ (CD132) receptor subunits. α receptors are mainly expressed on the surface of T suppressor cells (Treg) and some endothelial cells (endothelial cells), while β and γ receptor subunits are highly expressed on effector T cells (Teff) and NK cells. The affinity of IL-2 for the complex form of different receptor subunits is different. IL-2 has the highest affinity for the complex composed of α, β and γ receptor subunits. The affinity of the complex composed of IL-2 is moderate (about 100-fold lower), and IL-2 can transmit signals after binding to the combination of both forms of receptor subunits (Minami et al., Annu Rev Immunol 11, 245-268 ( 1993)). However, clinically, under the condition of low dose of IL-2, it will preferentially bind to high-affinity receptors on the surface of Treg cells, which will produce immunosuppression and fail to achieve the therapeutic effect. High doses of IL-2 can neutralize the immunosuppression caused by Treg activation by activating a large number of effector T cells, and at the same time, there will be more toxic side effects, as well as activation induced cell apoptosis.

Based on the antitumor effect of IL-2, high-dose IL-2 (aldesleukin) was approved by the FDA in 1992 for the clinical treatment of melanoma and renal cell carcinoma. However, patients receiving high doses of IL-2 often experience severe side effects, including cardiovascular, pulmonary edema, hepatic, gastrointestinal, neurological, and hematological events, most of which can be compounded by vascular (or capillary) leakage It is also an indicator for evaluating the side effects of IL-2 treatment in clinical and animal experiments. VLS is caused by the expression of high-affinity receptors (α, β and γ subunits) of IL-2 on endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)), so attenuation or elimination of Alpha receptor binding will reduce the function of IL-2 to promote T-suppressive cell proliferation activity, and also reduce the binding to endothelial cell alpha receptors, thereby reducing or eliminating the toxic and side effects caused by IL-2 therapy.

The binding sites of IL-2 and α receptor subunits are mainly at amino acid positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68 and 72 (Rickert.M et al. (2005). )Science 308:1477-1480), Merck and Roche or other scientific institutions made some mutations around these IL-2 surface amino acids that bind to α receptor subunits, such as Merck's mutants (R38W, F42K, WO2008003473A2) , reducing the interaction with the alpha receptor subunit to achieve effector T cell activation to enhance efficacy; while Roche's IL-2 mutants (F42A, Y45A and L72G, US 2016/0208017A1), which do not interact with alpha receptors Binding and, but can normally bind to β and γ receptor subunits, and can exert effects, is currently in clinical practice.

In the published patent application CN111018961A, two methods are used to eliminate the binding to IL2Rα receptors. One is to introduce additional disulfide bonds inside IL-2, which can not only make IL-2 more structurally stable, but also It can also form a barrier to destroy the binding plane with the α receptor; the other is to use a blocking module to form a disulfide bond between the α receptor binding surface of IL-2 to form a new molecular blocking module/IL- 2 heterodimer (heterodimer). This complex cannot bind to endogenous alpha receptors in the body, but can bind to beta and gamma receptor subunits.

Therefore, reducing or eliminating the interaction of IL-2 with the alpha receptor subunit may be an important aspect of treatment efficacy and reducing the side effects of treatment in cancer patients.

SUMMARY OF THE INVENTION

In view of the fact that IL-2 drugs in the prior art will first bind to high-affinity receptors, resulting in immunosuppression, the therapeutic effect cannot be achieved, and the use of high-dose IL-2 will cause more toxic side effects and cellular In the case of apoptosis, the technical problem to be solved by the present invention is to provide an IL-2 mutant with reduced binding to high-affinity receptors.

A first aspect of the present invention provides an IL-2 mutant. On the basis of IL-2, one or more mutations of substitution, deletion and addition of amino acid sites related to IL2Rα binding are performed to obtain An IL-2 mutant with reduced binding ability to IL2Rα; wherein, the amino acid sites related to IL2Rα binding are positions 30-75 of wild-type IL-2. Optionally, the amino acid site related to the binding of IL2Rα is positions 35-75 of wild-type IL-2; alternatively, the amino acid site related to the binding of IL2Rα is positions 37-75 of wild-type IL-2.

Further, the substitution and addition of amino acid residues that select mutations that can make the IL-2 mutant structure tend to be stable and/or less energetic.

Further, one or more mutations of substitutions, deletions and additions are made to one or more amino acids in positions 35-45.

Further, amino acids at positions 35-37 and 41-43 were substituted, and amino acids at positions 38-40 were deleted.

Further, the amino acid at position 45 was also substituted.

Optionally, amino acid position 35 is replaced by K35G or K35M; amino acid position 36 is replaced by L36G or L36H; amino acid position 37 is replaced by T37G; amino acid position 41 is replaced by T41G or T41L; amino acid position 42 is replaced by T41G or T41L The substitution of amino acid is F42G or G42D; the substitution of amino acid 43 is K43G; the substitution of amino acid 45 is Y45G.

In another specific embodiment, one or more of substitutions, deletions and additions are made to one or two amino acids in positions 31-32.

Further, amino acids at positions 31-32 were substituted.

Further, the 31st amino acid is replaced by Y31G; the 32nd amino acid is replaced by K32G.

In another embodiment, cysteine at position 125 is also mutated to an amino acid with a smaller side chain.

Further, amino acids with smaller side chains include alanine and glycine.

Further, the amino acid sequence of the wild-type IL-2 is shown in SEQ ID NO.1.

Alternatively, the amino acid sequence of the IL-2 mutant is shown in SEQ ID NO.3-7.

A second aspect of the present invention provides an isolated polynucleotide encoding an IL-2 mutant as described above.

A third aspect of the present invention provides an expression vector comprising an isolated polynucleotide as described above.

A fourth aspect of the present invention provides a host cell comprising an isolated polynucleotide as described above.

A fifth aspect of the present invention provides a composition comprising the IL-2 mutant as described above and a pharmaceutically acceptable carrier.

The sixth aspect of the present invention provides the use of the IL-2 mutant as described above in the manufacture of a medicament or a preparation for treating a disease.

A seventh aspect of the present invention provides the use of an IL-2 mutant as described above in the manufacture of a composition for stimulating the immune system of an individual.

An eighth aspect of the present invention provides a method of producing an IL-2 mutant, the method comprising culturing a host cell as described above under conditions suitable for expressing the IL-2 mutant.

In the IL-2 mutant of the present invention, one or more mutations of substitution, deletion and addition of amino acid sites related to IL2Rα binding in IL-2 are performed, thereby reducing the binding with IL2Rα, and can also increase IL-2Rα binding. 2 The expression levels of mutants. The IL-2 mutant in the present invention is a new direction for reducing VLS or reducing or eliminating the toxic and side effects caused by IL-2 treatment.

Description of drawings

Figure 1 is a schematic diagram of the three-dimensional structure of IL-2 and IL2Rα (PDB ID 2b5i).

Fig. 2 is the sequence comparison diagram (part) of IL-2 mutant and IL-2wt C125A in the embodiment of the present invention.

Fig. 3 is an SDS-PAGE electropherogram of IL-2 mutants in the examples of the present invention. Among them, "reduction" means adding the reducing agent 2-mercaptoethanol to the loading buffer; "non-reducing" means not adding the reducing agent 2-mercaptoethanol to the loading buffer.

FIG. 4 is a CTLL2 cell proliferation experiment of IL-2 mutants in the examples of the present invention.

Detailed ways

The present invention will be further described below with reference to the examples, and it should be understood that these examples are only for the purpose of illustration and are not intended to limit the protection scope of the present invention.

The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as molecular cloning such as Sambrook: conditions described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions. recommended conditions. The reagents used, unless otherwise specified, are commercially available or publicly available reagents.

Herein, the relative amino acid positions of IL-2 mutants and wild-type IL-2 are calculated based on the amino acid sequence of wild-type IL-2 (such as SEQ ID NO. 1).

Wild-type IL-2 (IL-2wt, SEQ ID NO. 1):

The nucleotide sequence of wild-type IL-2 is shown in SEQ ID NO.2.

Herein, "IL2Rα" refers to interleukin-2 receptor α, also known as "α receptor subunit"; "IL2Rβ" refers to interleukin-2 receptor β, also known as "β receptor subunit"; " IL2Rγ" refers to interleukin-2 receptor γ, also known as "γ receptor subunit"; "IL2Rβγ" refers to the complex formed by interleukin-2 receptor β and receptor γ, also known as "β and γ receptor". body subunit complex".

As used herein, "mutation" includes substitutions, deletions and additions to amino acids.

In a specific embodiment of the present invention, in order to reduce or eliminate the binding of IL-2 to α receptors, the amino acid sites in IL-2 that are related to the binding of IL2Rα, that is, positions 30-75, are mutated. The base sequence for mutation can be IL-2 wild type, or it can be 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, Sequences of 95%, 96%, 97%, 98%, 99% and more than 99% homology.

In a specific embodiment, one or more of substitutions, deletions and additions are made to one or more of amino acids at positions 35-45 to reduce binding of the IL-2 mutant to IL2Rα.

In another specific embodiment, one or more of substitutions, deletions and additions are made to one or more of amino acids 31-32 and 35-45 to reduce the interaction of the IL-2 mutant with IL2Rα. combine.

Specifically, through the three-dimensional structure of the binding of IL-2 and IL2Rα, it was found that IL-2 is located at the 37th, 38th, 41st, 42nd, 43rd, 44th, 45th, 61st, 62th, 65th, 68th and 72nd amino acid positions and IL2Rα. Therefore, through bioinformatics and protein engineering design, mutations, deletions, and additions were performed at the 30th to 75th amino acids to change the binding of IL-2 to IL2Rα.

In some embodiments, the amino acid sequences of the obtained IL-2 mutants are shown in Table 1:

Table 1 Amino acid sequence of IL-2 mutants obtained by design

The nucleotide sequences encoding the amino acid sequences of the IL-2 mutants designed and obtained in Table 1 are shown in Table 2:

Table 2 Nucleotide sequences of IL-2 mutants obtained by design

Expression hosts can be E. coli or mammalian cells.

Example 1 Preparation of IL-2 and mutants

In this example, IL-2wt(C125A) and the mutant were expressed respectively, and purified and prepared relying on the HPC4 tag carried at the C-terminal of the molecule.

1.1 Expression plasmid construction

Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to synthesize the gene with IL-2wt (C125A, SEQ ID NO.1) and mutant, and cloned into the pTT5 universal vector. DH10B was transformed, sequenced and maintained to obtain the desired IL-2 wild-type and mutant plasmids.

1.2 Plasmid extraction and HEK293 cell preparation

1.2.1 Plasmid extraction

According to the operation methods mentioned in "Qiagen Mini-prep Kit" and "Qiagen Endofree Maxi-prep Kit", IL-2wt (C125A) and IL-2 mutant were prepared.

1.2.2 HEK293 cell preparation

Freshly passaged HEK293 cells (National Research Council, Canada) at a density of 1-1.2* ¹⁰⁶ /ml were used for transient expression.

1.3 Transient expression of HEK293

1.3.1 Reagent preparation

A) G418 solution: Weigh 250mg Geneticin ^TM , add 4.5ml ultrapure water to dissolve, dilute to 5ml ultrapure water, filter with 0.22um membrane, store at -20℃

B) PEI solution: Weigh 50mg PEI, add 45ml ultrapure water to dissolve, adjust pH to 7.0 with 1M NaOH, dilute to 50ml ultrapure water, filter with 0.22um membrane, and store at -20°C

C) Medium: Add 10ml Pluronicd ^™ F-68 and 500ul G418 to 1L FreeStyle ^™ 293Expression Medium

D) Plasmids were prepared in advance in 2 ml endotoxin-depleted centrifuge tubes.

E) Prepare a cell suspension freshly passaged to 1-1.2×10 ⁶ cells/ml according to the volume required for transfection

1.3.2 Preparation of transfection reagent-plasmid complex

Solution A: Plasmid 1μg/mL + Opti-MEM ^TM 33.3ul/mL

Liquid B: PEI 2μg/mL+Opti-MEM ^TM 33.3ul/mL

Pour solution B into solution A, mix and incubate for 10 min, then add 1 mL of cell suspension, which can be scaled up in equal proportions.

1.3.3 Expression culture and harvest

After culturing for 5 days at 115 rpm, 36.8°C, 5% CO ₂ , the cell supernatant was collected by centrifugation at 8500 rpm for 15 min.

1.4 Purification preparation

All IL-2wt(C125A) and IL-2 mutants are C-terminally tagged with HPC4 and can therefore be affinity purified using HPC4 antibody-conjugated media followed by gel filtration chromatography (superdex200) for further purification The protein with higher purity was obtained. After purification, it was estimated that the expression level of IL-2wt C125A was ~9.9 mg/L, and the expression levels of 384 and 386 were 18.6 mg/L and 19.6 mg/L, respectively. SDS-PAGE analysis was performed according to the method described in "Molecular Cloning", and the results are shown in Figure 3.

Example 2 Determination of the affinity of hIL2 mutants to IL2Rβ and IL2Rα by biolayer interferomeory (BLI), respectively

1. Experimental materials

The proteins used in the experiments were all produced by our company, and IL2Rα-his, IL2Rβ-Fc/Fc and IL2 mutants were obtained by transient expression and affinity purification of HEK293. Buffer formulation (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.1% BSA and 0.05% Tween 20); ProA sensor (Pall Fortebio, Catalog #18-5010); HISIK sensor (Pall Fortebio, Catalog #18) -5120); BLI equipment is Octet RED96 produced by Pall Fortebio Company; Data acquisition and analysis work were carried out with Data acquisition 11.0 and Data analysis 11.0 software respectively.

2. Experimental method

2.1 IL2Rβ-Fc/Fc preparation

IL2Rβ-Fc/Fc was diluted with buffer to a concentration of 10ug/ml, added to the second column of the 96-well assay plate, and the control program was set to Loading, 600s.

2.2 IL2Rα-his preparation

Dilute IL2Rα-his with buffer to a concentration of 10ug/ml, add it to the third column of the 96-well assay plate, and set the control program to Loading, 600s.

2.3 Sample Preparation

The IL2 mutant was diluted to 100nM with buffer, then 1:1 serially diluted down 6 gradients (7 gradients in total) to a concentration of 1.625nM and a concentration of 0, added to the 96-well assay plate 5-9 columns, respectively, control program Medium is set to Association, 200s. Buffer was added to columns 1, 4, 10, and 11 of the 96-well assay plate, and glycine with pH 1.7 was added to column 12. The sample volumes of the above samples and solutions were both 200ul.

2.4 Detection of affinity with IL2Rβ-Fc/Fc

Take 8 ProA sensors and place them in A-H of the first column of the sensor bracket, and set the detection conditions in the Data acquisition 11.0 software as follows: 1 Pre-wet: Baseline, 60s, position: 1 column. 2 cycle detection: Column 2: loading, 600s; Column 4: Baseline1, 60s; Sample Columns 5-9: Association, 200s, Column 10: Dissociation, 600s; Column 11: Neutralization; Column 12: Regeneration .

2.5 Detection of binding to IL2Rα

Take 8 HISIK sensors and place them in A-H in the second column of the sensor bracket, and set the detection conditions in the Data acquisition 11.0 software as follows: 1 Pre-wet: Baseline, 60s, position: 1 column. 2 cycle detection: Column 3: loading, 600s; Column 4: Baseline1, 60s; Sample Columns 5-9: Association, 200s, Column 10: Dissociation, 600s; Column 11: Neutralization; Column 12: Regeneration .

2.6 Data Analysis

Data were analyzed using Data analysis 11.0 software. The background was subtracted with 0 concentration as the control, and the KD value was calculated by Fitting curve.

3. Results

The results are shown in Table 3. None of the designed IL-2 mutants bind to the IL2Rα receptor, but all maintain the binding to IL2Rβ.

Table 3 Affinity of IL-2 mutants to IL2Rα and IL2Rβ receptors

protein name	Affinity to IL2Rα (M)	Affinity to IL2Rβ (M)
IL-2wt C125A	8.21E-09	2.23E-07
IL-2 mutant 383	N.B	2.40E-07
IL-2 mutant 384	N.B	1.50E-07
IL-2 mutant 385	N.B	7.26E-07
IL-2 mutant 386	N.B	1.90E-07

N.B:Non binding, not binding

Example 3 Proliferation experiment of T cells

The proliferation assay of CTLL-2 (T cell) is a commonly used assay to measure the activity of interleukin-stimulated immune cells at the cellular level. Therefore, the biological activity of IL-2 mutants was examined here by proliferation experiments of CTLL-2 cells.

1. Preparation of CTLL-2 cells: Resuspend the cells in culture medium containing FBS and Rat-T-Stim.

2. Loading: Inoculate cells in a 96-well culture plate, 0.1 ml per well. At the same time, the protein of the IL-2 mutant 386 of the sample to be tested (ie, the protein prepared in Example 1) was diluted by multiples, and 0.1 ml was added to each well, and each dilution concentration was set to 3 replicate wells. A culture medium control well (100ul cells+100ul culture medium) was also set up. Incubate for 72 hours at 37 degrees, 5% _CO2 .

3. MTS addition: add 20 μl to each well

AQueous One Solution Reagent, 37 degrees, 5% CO ₂ incubate for 2 to 4 hours.

4. Determination: Measure the absorbance value (A) at a wavelength of 490 nm with an enzyme label analyzer and calculate the EC50 value.

5. Results: As shown in Figure 4, all IL-2 mutants had CTLL2 cell proliferation activity, indicating that the mutation did not seriously affect the signaling function of the β and γ receptor subunit complexes. The proliferative activity of the mutant on CTLL2 cells is lower than that of the positive control, because the IL-2 mutant 386 does not bind to the IL2Rα receptor subunit, while the positive control can enhance the binding to IL2Rβ after binding the IL2Rα receptor. more active.

Claims (17)

1. An IL-2 mutant, characterized in that, on the basis of human IL-2, one or more mutations in substitution, deletion and addition of amino acid sites related to IL2Rα binding are performed to obtain the ability to bind to IL2Rα Reduced IL-2 mutant; wherein, the amino acid sites related to IL2Rα binding are positions 30-75 of wild-type IL-2.
2. The IL-2 mutant according to claim 1, characterized in that amino acid residues that can make the structure of the IL-2 mutant tend to be stable and/or less energetic after the selected mutation are replaced and added.
3. The IL-2 mutant of claim 1, wherein one or more of substitutions, deletions and additions are made to one or more amino acids in positions 35-45.
4. The IL-2 mutant according to claim 3, wherein amino acids at positions 35-37 and 41-43 are substituted, and amino acids at positions 38-40 are deleted.
5. The IL-2 mutant according to claim 4, wherein the amino acid at position 45 is further substituted.
6. The IL-2 mutant of claim 5, wherein the amino acid at position 35 is replaced by K35G or K35M; the amino acid at position 36 is replaced by L36G or L36H; the amino acid at position 37 is replaced by T37G; The substitution of amino acid at position 42 is T41G or T41L; the substitution of amino acid at position 42 is F42G or G42D; the substitution of amino acid at position 43 is K43G; the substitution of amino acid at position 45 is Y45G.
7. The IL-2 mutant of claim 1, wherein the cysteine at position 125 is further mutated to an amino acid with a smaller side chain.
8. The IL-2 mutant of claim 7, wherein the amino acids with smaller side chains include alanine and glycine.
9. The IL-2 mutant of claim 1, wherein the amino acid sequence of the wild-type IL-2 is shown in SEQ ID NO.1.
10. The IL-2 mutant according to claim 1, wherein the amino acid sequence of the IL-2 mutant is shown in SEQ ID NO.3-7.
11. An isolated polynucleotide, characterized in that it encodes the IL-2 mutant according to any one of claims 1-10.
12. An expression vector, characterized in that it comprises an isolated polynucleotide as claimed in claim 11 .
13. A host cell, comprising the isolated polynucleotide of claim 11.
14. A composition, characterized by comprising the IL-2 mutant according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.
15. Use of the IL-2 mutant according to any one of claims 1 to 10 in the preparation of a medicament or a preparation for treating a disease.
16. Use of an IL-2 mutant according to any one of claims 1 to 10 in the manufacture of a composition for stimulating the immune system of an individual.
17. A method of producing an IL-2 mutant, comprising culturing the host cell of claim 13 under conditions suitable for expressing the IL-2 mutant.

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