CN118598975B - Interleukin-21 mutant and application thereof - Google Patents

Abstract

The present application provides an interleukin-21 mutant and its application, belonging to the field of medical technology, wherein the mutant is a mutation of the 85th and 86th R (arginine) of the amino acid sequence of wild-type IL-21 to G (glycine), the 88th K (lysine) to G (glycine), the 90th R (arginine) to E (glutamic acid), the 5th R (arginine) and the 78th P (proline) are both mutated to C (cysteine), and a disulfide bond is formed between the two mutated C (cysteine). The mutant has a significantly reduced affinity for IL-21 receptors, and increases the expression of fusion heavy chains, improves stability, and reduces aggregation tendency. At the same time, the present application also provides related nucleic acid molecules, immunoconjugates, kits, pharmaceutical compositions, and the use of the interleukin-21 mutant or its immunoconjugate in the preparation of a drug for treating a subject with a solid tumor.

Description

Interleukin-21 mutant and its application

Technical Field

The present invention belongs to the field of medical technology, and in particular, relates to an interleukin-21 mutant and an application thereof.

Background Art

Interleukin-21 (IL-21) is composed of four α-helical structures, belongs to the γc family, and is composed of 134 amino acids. It is a pleiotropic cytokine that regulates the activity of innate and adaptive immune cells. IL-21 is mainly secreted by activated CD4+ T cells, NK cells, TFH cells, and Th17 cells. When IL-21 binds to the IL-21 receptor (IL-21R)/γc receptor complex, it can activate the JAK/STAT signaling pathway and regulate the differentiation, proliferation, activation of various immune cells such as NK, DC, B, T cells, and the production of different cytokines. However, the development of IL-21-based therapies is complex, including two directions of agonism or antagonism, targeting tumors and autoimmune diseases respectively, and including multiple product types such as antibodies, peptides, cell therapy, and oncolytic viruses.

For CD8+ T cells and NK cells, IL-21 can enhance their survival and cell-killing toxicity, and has great potential for synergistic anti-tumor effects. However, since the IL-21R/γc receptor complex is widely expressed on the surface of a variety of lymphocytes and hematopoietic cells, the administration of exogenous recombinant IL-21 is not selective, and IL-2 has a high affinity for IL21R, which will preferentially bind to and activate a large number of non-target cells in the periphery, resulting in a sink effect, resulting in a short half-life and greater therapeutic toxicity.

In addition, the four α-helices of IL-21 are connected by loops of different lengths, among which the CD loop is longer, more flexible, and rich in positively charged basic amino acids, resulting in poor drugability of recombinant IL-21. Therefore, IL-21 needs to be modified and fused to antibody expression to enhance specificity, reduce non-target affinity, and improve drugability.

Interleukin 21 has a high homology with IL-2, IL-4 and IL-15. The fusion of wild-type IL-21 or other modified IL-21 to the C-terminus of the heavy chain of the pembrolizumab antibody will lead to a decrease in the expression of the fused heavy chain, a low expression level of the antibody-fused IL-21, and poor stability. To solve the above problems, the application provides a new interleukin 21 (IL21) mutant fusion protein, which provides a variety of options for clinical application.

Summary of the invention

The present invention provides an interleukin-21 mutant (IL-21) or an immunoconjugate thereof, and use thereof in the preparation of a medicament for treating a subject having a solid tumor.

On the one hand, the present application provides an interleukin-21 (IL-21) mutant, characterized in that, compared with wild-type IL-21, it comprises the following mutations: R (arginine) and K (lysine) in positions 85 to 90 RRQKHR of the amino acid sequence of wild-type IL-21 are mutated to G (glycine) or E (glutamate), R (arginine) at position 5 and P (proline) at position 78 are both mutated to C (cysteine), and a disulfide bond is formed between the two mutated C (cysteine); the amino acid sequence of the wild-type IL-21 is shown in SEQ ID NO.6.

In certain embodiments, the IL-21 mutant is as follows: the R (arginine) at positions 85 and 86 of the amino acid sequence of wild-type IL-21 is mutated to G (glycine), the K (lysine) at position 88 is mutated to G (glycine), the R (arginine) at position 90 is mutated to E (glutamic acid), the R (arginine) at position 5 and the P (proline) at position 78 are both mutated to C (cysteine), and a disulfide bond is formed between the two mutated C (cysteines).

In certain embodiments, the amino acid sequence of the IL-21 mutant is shown in SEQ ID NO:4.

In certain embodiments, the affinity of the IL-21 mutant for the IL-21 receptor (IL-21R) is significantly reduced compared to the affinity of wild-type IL-21 for the IL-21 receptor (IL-21R).

On the other hand, the present application provides an immunoconjugate, which comprises the aforementioned interleukin-21 mutant and an anti-PD-1 antibody.

In certain embodiments, the IL-21 mutant is attached to the Fc of the anti-PD-1 antibody directly or via a linker.

In certain embodiments, the IL-21 mutant is directly attached to the Fc of the anti-PD-1 antibody.

In certain embodiments, the IL-21 mutant is attached to the Fc of the anti-PD-1 antibody by a linker.

In certain embodiments, the IL-21 mutant is attached to the Fc of the anti-PD-1 antibody by a GGGGS (G4S) linker.

In certain embodiments, the IL-21 mutant is linked to the C-terminus of one of the two heavy chains of the anti-PD-1 antibody.

In certain embodiments, the anti-PD-1 antibody may be selected from Pembrolizumab, Nivolumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab.

In certain embodiments, the anti-PD-1 antibody is pembrolizumab.

In certain embodiments, the anti-PD-1 antibody is Pembrolizumab modified by Knob-into-hole technology.

In certain embodiments, the Pembrolizumab modified by Knob-into-Hole technology (technology to prevent heavy chain mispairing) is that the 366th T (threonine) of the amino acid sequence of _CH3 of one heavy chain of Pembrolizumab is mutated to W (tryptophan); the 366th T (threonine) of the amino acid sequence of _CH3 of the other heavy chain is mutated to S (serine), the 368th L (leucine) is mutated to A (alanine), the 407th Y (tyrosine) is mutated to V (valine), and the 349th Y (tyrosine) is mutated to C (cysteine).

In certain embodiments, the immunoconjugate is a fusion protein comprising an IL-21 mutant and an anti-PD-1 antibody.

In certain embodiments, the fusion protein comprises: two light chains, each of which has an amino acid sequence as shown in SEQ ID NO:3; a heavy chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO:1; and a heavy chain fused to an IL-21 mutant, the amino acid sequence of the fused heavy chain is shown in SEQ ID NO:5.

In certain embodiments, the fusion protein increases expression of the fusion heavy chain.

In certain embodiments, the fusion protein has improved stability.

In certain embodiments, the fusion protein has reduced propensity to aggregate.

On the other hand, the present application provides a kit comprising the mutant and/or immunoconjugate.

In certain embodiments, the present application provides a kit comprising the mutant.

In certain embodiments, the present application provides a kit comprising the immunoconjugate.

On the other hand, the present application provides a pharmaceutical composition comprising the mutant and/or immunoconjugate, and optionally a pharmaceutically acceptable carrier or excipient.

In certain embodiments, the present application provides a pharmaceutical composition comprising the mutant, and optionally a pharmaceutically acceptable carrier or excipient.

In certain embodiments, the present application provides a pharmaceutical composition comprising the immunoconjugate, and optionally a pharmaceutically acceptable carrier or excipient.

On the other hand, the present application provides a use of the pharmaceutical composition in the preparation of a medicament for treating a subject with a solid tumor.

In another aspect, the present application provides a method for treating a disease in a subject, the method comprising administering to the subject an effective amount of the immunoconjugate described herein and/or the pharmaceutical composition described herein.

The present application provides interleukin-21 (IL-21) mutants, immunoconjugates of interleukin-21 mutants and anti-PD-1 antibodies, and the use of the interleukin-21 mutants or their immunoconjugates in the preparation of drugs for treating subjects with solid tumors. The affinity of the interleukin-21 mutant for the IL-21 receptor (IL-21R) is significantly reduced compared to the affinity of wild-type IL-21 for the IL-21 receptor (IL-21R). At the same time, the immunoconjugate of the interleukin-21 mutant and the anti-PD-1 antibody increases the expression of the fusion heavy chain, improves stability, and reduces the tendency to aggregate compared to the anti-PD-1-wild-type IL-21 immunoconjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the molecular form of the immunoconjugate.

Figure 2 is a graph showing the results of SDS-PAGE electrophoresis of the immunoconjugate, wherein Figures 2A and 2B are graphs showing the results of SDS-PAGE electrophoresis of samples after protein A affinity purification; Figures 2C and 2D are graphs showing the results of SDS-PAGE electrophoresis of samples after ion exchange purification.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1: Design and construction of immunoconjugates of anti-hPD-1 antibodies and IL-21 mutants

The molecular form of the immunoconjugate of the present invention is shown in FIG1 , and includes two parts: a first monomer and a second monomer.

1. Anti-hPD-antibody-IL-21 mutant immunoconjugate (N022)

1) The first monomer: comprising the light chain and heavy chain of pembrolizumab (prepared by the method described in patent CN104945508B). The CH3 amino acid of the heavy chain of pembrolizumab has been modified as follows: the 366th T (threonine) of the CH3 amino acid sequence of the heavy chain has been mutated to S (serine), the 368th L (leucine) has been mutated to A (alanine), the 407th Y (tyrosine) has been mutated to V (valine), the 349th Y (tyrosine) has been mutated to C (cysteine), and the 447th K (lysine) has been deleted;

2) The second monomer: includes the light chain and heavy chain of pembrolizumab (prepared by the method described in patent CN104945508B), a flexible amino acid linker and an IL-21 mutant. Among them, the amino acid sequence of the CH3 of the heavy chain of pembrolizumab is mutated from T (threonine) at position 366 to W (tryptophan), S (serine) at position 354 to C (cysteine), and K (lysine) at position 447 is deleted. The C-terminus of the pembrolizumab is fused to the IL-21 mutant and connected using the flexible amino acid GGGGSGGGGSGGGGS (3x(4GS)). The IL-21 mutant is as follows: the R (arginine) at positions 85 and 86 of the amino acid sequence of the wild-type IL-21 is mutated to G (glycine), the K (lysine) at position 88 is mutated to G (glycine), the R (arginine) at position 90 is mutated to E (glutamic acid), the R (arginine) at position 5 and the P (proline) at position 78 are both mutated to C (cysteine), and a disulfide bond is formed between the two mutated C (cysteines).

2. Anti-hPD-1 antibody-wild-type IL-21 immunoconjugate (N035)

1) The first monomer: comprising the light chain and heavy chain of pembrolizumab (prepared by the method described in patent CN104945508B). The CH3 amino acid of the heavy chain of pembrolizumab has been modified as follows: the 366th T (threonine) of the CH3 amino acid sequence of the heavy chain has been mutated to S (serine), the 368th L (leucine) has been mutated to A (alanine), the 407th Y (tyrosine) has been mutated to V (valine), the 349th Y (tyrosine) has been mutated to C (cysteine), and the 447th K (lysine) has been deleted;

2) The second monomer: includes the light chain and heavy chain of pembrolizumab (prepared by the method described in patent CN104945508B), a flexible amino acid linker and an IL-21 mutant. Among them, the amino acid sequence of the CH3 of the heavy chain of pembrolizumab is mutated from T (threonine) at position 366 to W (tryptophan), S (serine) at position 354 to C (cysteine), and K (lysine) at position 447 is deleted. The C-terminus of the pembrolizumab is fused to the wild-type IL-21 and connected using the flexible amino acid GGGGSGGGGSGGGGS (3x(4GS)).

The amino acid sequence information of the immunoconjugates of anti-hPD-1 antibody-IL-21 mutant and anti-hPD-1 antibody-wild-type IL-21 used in the examples is shown in Table 1 below.

Table 1: Amino acid sequence information of immunoconjugates

Example 2: Preparation and purity detection of anti-hPD-1 antibody-IL-21 mutant immunoconjugate

The amino acid sequences of N022 and N035 molecules were codon optimized and synthesized, and the target genes were amplified by polymerase chain reaction (PCR). After 1% agarose gel electrophoresis and gel recovery, the heavy chain and light chain genes were constructed into pcDNA3.4 vectors respectively.

The above plasmids were co-transfected into Expi-CHO cells using the PEI transfection method, and the plasmid transfection ratio was 1:1:1. After transfection, the cells were placed at 37 °C, 8% CO ₂ , and 120 rpm for shaking culture for 16-20 hours. After adding 7.5% (v/v) OPM-CHOProFeed (P81279, Aopuma), the shake flask was placed at 32 °C, 120 rpm, and 8% CO ₂ for shaking culture. On the third and fifth days after transfection, 7.5% (v/v) feed was added, and the sugar content was kept above 4 g/L. When the cell viability was less than 60%, the cell supernatant was harvested, and after centrifugation and filtration, the cell supernatant was harvested, and the supernatant molecular expression titer was determined by biochemical analyzer (Credex Bio, Roche).

Protein A affinity purification (Mab SelectSuRe, Cytiva): The harvested cell supernatant was added to a chromatography column treated with an equilibrium solution (20 mM PBS pH=7.4), and the chromatography column was washed with the equilibrium solution to remove nonspecific binding proteins. The chromatography column was then rinsed with 3 column volumes of elution buffer (0.1 M Glycine-Hcl pH=3.0), the eluate was collected, neutralization solution (1 M Tris-Hcl pH=7.4) was added, and the PBS buffer was exchanged using an ultrafiltration concentration tube, and the yield was calculated. The purity of the immunoconjugate was determined using SDS-PAGE (ExpressPlus™ PAGE Gel, GenScript) and capillary electrophoresis (PA800 Plus, SCIEX), respectively. The results are shown in Table 2, Table 3 and Figure 2.

Ion exchange purification (Capto S, Cytiva): Cation exchange chromatography was used to further separate the heterodimer molecules and remove homodimer impurities. The eluate was also exchanged into PBS buffer using an ultrafiltration tube, and the concentration (A280) was determined, and the purity of the immunoconjugate was determined by the same method as above. The results are shown in Table 2 and Figure 2.

Table 2. Immunoconjugate yields and concentrations

As can be seen from Table 2 and Figures 2A and 2B, the yield and concentration of the anti-hPD-1 antibody-wild-type IL-21 immunoconjugate (N035) were high after one-step affinity purification, but the heavy chain band of the second monomer was shallow after SDS-PAGE (reducing) electrophoresis; while the yield of the anti-hPD-1 antibody-IL-21 mutant immunoconjugate (N022) could reach 106.1 mg/L and the concentration was 1.18 mg/mL, and the proportion of the second monomer heavy chain was significantly higher. Combined with non-reducing SDS-PAGE electrophoresis, it was suggested that N035 had fewer correctly paired heterodimer molecules, while N022 had more correctly paired heterodimer molecules.

As can be seen from Figures 2C and 2D, after ion exchange purification, the proportion of the second monomer heavy chain in the anti-hPD-1 antibody-wild-type IL-21 immunoconjugate (N035) increased, and the proportion of heterodimer molecules increased; the second monomer homodimer and small molecular weight miscellaneous bands in the anti-hPD-1 antibody-IL-21 mutant immunoconjugate (N022) were removed.

Table 3 Purity of immunoconjugates purified by protein A affinity (CE-SDS, reduced)

As can be seen from Table 3, compared with the anti-hPD-1 antibody-wild-type IL-21 immunoconjugate (N035), the second monomer heavy chain ratio of the anti-hPD-1 antibody-IL-21 mutant immunoconjugate (N022) was increased by 25.36%.

Example 3: Binding of immunoconjugates to IL-21R

Elisa was used to detect the binding of the immunoconjugate of anti-hPD-1 antibody-IL-21 mutant to IL-21R. IL-21R-his (2 ug/mL) was coated and blocked with 2% BSA. The immunoconjugate was diluted to 100 nM, then diluted 3 times and added to the ELISA plate, 100 uL per well. A positive control (wild-type IL-21 fusion Fc protein (i.e., IL-21-Fc) is a fusion of wild-type IL-21 with the N-terminus of human IgG1 Fc) and a negative control (pembrolizumab) were set up, incubated at 37 °C for 1 h and then washed. Subsequently, the plate was diluted at a ratio of 1:5000, HRP-goat anti-human IgG secondary antibody was added, and the plate was incubated at 37 °C for 1 h and then washed. TMB colorimetric solution was added for 3-5 min, and the color development was stopped by adding the stop solution, and the OD450 was detected by an ELISA reader (Infinite F50, Tecan). The results are shown in Table 4.

Table 4 Antibody binding activity detection

From the above results, it can be seen that the EC ₅₀ of N022 is 12.98, and the EC ₅₀ of IL-21-Fc is 1.70. It can be seen that compared with IL-21-Fc, the activity of N022 binding to IL-21R is significantly reduced. At the same time, the EC ₅₀ of N035 is 5.33, and the EC ₅₀ of N022 is 2.4 times that of N035. It shows that the immunoconjugate of the anti-hPD-1 antibody-IL-21 mutant (N022) of the present application has significantly reduced binding activity to IL-21R than the immunoconjugate of the anti-hPD-1 antibody-wild-type IL-21 (N035).

Example 4: Stability test of hPD-1 antibody-IL-21 mutant immunoconjugate

The thermal stability and aggregation tendency of the immunoconjugate were tested using a multifunctional protein stability analyzer (Uncle, UNCHAINED LABS). After centrifugation, the sample was diluted to 1 mg/mL with PBS (20 mM, pH=7.4) and centrifuged again at room temperature at 12000rpm for 2 min. 10 µL of each diluted sample was added to the Uni tube in duplicate, sealed, and the Tm and Tagg values were tested on the machine. The results are shown in Table 5.

Table 5 Stability results of immunoconjugates

As can be seen from Table 5, the Tm (melting temperature) and Tagg (aggregation temperature) values of the hPD-1 antibody-IL-21 mutant immunoconjugate (N022) are close to those of the monoclonal antibody. The Tm1 (melting temperature 1) of the anti-hPD-1 antibody-wild-type IL-21 immunoconjugate (N035) is similar to that of the monoclonal antibody, but Tm2 (melting temperature 2) is not measured. Tagg266 (aggregation temperature at a wavelength of 266nm for static light scattering) and Tagg473 (aggregation temperature at a wavelength of 473nm for static light scattering) are 4-6 °C lower than those of the monoclonal antibody. This indicates that compared with the anti-hPD-1 antibody-wild-type IL-21 immunoconjugate (N035), the hPD-1 antibody-IL-21 mutant immunoconjugate (N022) has better thermal stability and is not prone to aggregation.

The present invention has been illustrated by various specific embodiments. However, it will be understood by those skilled in the art that the present invention is not limited to the specific embodiments, and that those skilled in the art can make various changes or modifications within the scope of the present invention, and that the various technical features mentioned in various places in this specification can be combined with each other without departing from the spirit and scope of the present invention. Such changes and modifications are all within the scope of the present invention.

Claims (7)

1. An interleukin-21 (IL-21) mutant, characterized in that the mutant is compared to wild-type IL-21: the amino acid sequence of the wild-type IL-21 is shown as SEQ ID NO.6, the amino acid sequence of the mutant is shown as SEQ ID NO. 4, R at positions 85 and 86 of the amino acid sequence of the wild-type IL-21 is mutated to G, K at position 88 is mutated to G, R at position 90 is mutated to E, P at both positions 5 and 78 is mutated to C, and disulfide bonds are formed between the two mutated C.

2. A nucleic acid molecule encoding the interleukin-21 (IL-21) mutant of claim 1.

3. An immunoconjugate characterized in that it is a fusion protein of the interleukin-21 mutant and an anti-PD-1 antibody according to claim 1, wherein,

The interleukin-21 mutant is attached to the Fc of the anti-PD-1 antibody by a GGGGS linker;

the anti-PD-1 antibody is palbociclib monoclonal antibody;

The fusion protein is as follows: two light chains, the amino acid sequences of which are each shown in SEQ ID NO. 3; a heavy chain, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 1; and a heavy chain fused with interleukin-21 mutant, wherein the amino acid sequence of the fused heavy chain is shown as SEQ ID NO. 5.

4. A kit comprising the mutant of claim 1 or the immunoconjugate of claim 3.

5. A pharmaceutical composition comprising the immunoconjugate of claim 3, and optionally a pharmaceutically acceptable carrier or excipient.

6. A pharmaceutical composition comprising the mutant of claim 1, and optionally a pharmaceutically acceptable carrier or excipient.

7. Use of the pharmaceutical composition of claim 5 in the manufacture of a medicament for treating a subject having a solid tumor.