Patent Description:
Interleukin-<NUM> (IL-<NUM>) is a cytokine having a size of about <NUM> to <NUM> kD. The mature peptide of natural human interleukin-<NUM> contains <NUM> amino acids, including <NUM> cysteine residues. Two pairs of intramolecular disulfide bonds respectively formed by the connection of Cys35 and Cys85 and the connection of Cys42 and Cys88 play an important role in maintaining the spatial conformation and biological activity of IL-<NUM>.

Similar to most cytokines, IL-<NUM> plays a role in normal immune responses, such as promoting the development of T cells, B cells and natural killer (NK) cells. IL-<NUM> and IL-<NUM> share the same β chain and γ chain receptors, thus their biological activities are very similar. Due to the different α chain receptors, IL-<NUM> can activate Treg cells and induce Teff and NK cell apoptosis (AICD), thus the clinical application of IL-<NUM> is greatly limited. In contrast, IL-<NUM> of the same family does not have the functions of Treg activation and AICD, thus IL-<NUM> has great therapeutic potential.

The α subunit of the IL-<NUM> receptor has a high affinity for IL-<NUM>. Under physiological conditions, IL-<NUM> mostly forms a complex (IL-<NUM>-Rα) with the α subunit, which enhances the affinity of IL-<NUM> for the β chain subunit and the γ chain subunit of the receptor, and activates T cells and NK cells. Thereby, companies such as ALTOR and Hengrui have utilized the characteristics of the α subunit to formed a complex with IL-<NUM> and α subunit (or its part) through fusion or non-fusion methods, which has shown good biological potency and stability in animal experiments.

The α subunit of IL-<NUM> receptor is expressed on the surface of myeloid cells, including macrophages, antigen-presenting cells, NK cells and T cells. It also plays an important role in anti-tumor in vivo. Therefore, the IL-<NUM>-Rα complex is independent of the α subunit in vivo. Although the stability of IL-<NUM> monomer is improved, it has a subtle functional difference from native IL-<NUM>, which may reveal essential differences in antitumor in vivo.

Human IL-<NUM> variant T95X was described (Geneseq, <NUM> November <NUM>, EBI accession no. GSP:BBN47332, Database accession no. Human IL <NUM>-human alpha-synuclein SP fusion protein sequence is described (Geneseq, <NUM> October <NUM>, EBI accession no. GSP:AZZ02884, and <CIT>). generated an rclL-<NUM> protein consisting of Axn-<NUM>-Ser-<NUM> with a C-terminal His tag and examiner its functions ex vivo in term of the proliferation and antitumor effects on canine noon-B, non-T, large granular natural killer (NK) cells (<NPL>).

IL-<NUM> has higher safety and activity as compared to IL-<NUM>. However, as a protein drug, natural wild-type IL-<NUM> has significant drug development bottlenecks, including low expression levels in prokaryotes and eukaryotes, difficult purification and short half-life, making it difficult to be industrialized. Therefore, it is necessary to study the exogenous expression of IL-<NUM> to obtain high production efficiency. In addition, the N-terminal or C-terminal of IL-<NUM> analog obtained by in vitro renaturation can be coupled to the fatty acid chain of human serum albumin binder, so as to achieve a long-term efficacy, which has certain significance for the treatment of tumors.

In view of the low production efficiency of IL-<NUM> in the prior art, the present disclosure provides an IL-<NUM> analog with high expression level and relatively simple purification process. Further, the present disclosure provides a conjugate of the IL-<NUM> analog, thus as to improve the half-life of IL-<NUM> and the long-term efficacy thereof.

The present inventors have found that the expression of IL-<NUM> in Escherichia Coli could be promoted by adding some amino acids to the C-terminal of IL-<NUM>, and that positively charged amino acids could significantly enhance the expression of IL-<NUM>. The present IL-<NUM> analog is highly expressed in Escherichia Coli, and the expression level is about <NUM> or even more fold higher than that of IL-<NUM> without extra amino acids at the C-terminal. All or most of the present IL-<NUM> analog retains the amino acid sequence of the natural wild-type IL-<NUM>, and there is no significant difference in cell activity in vitro, which lays a foundation for the industrialization of IL-<NUM> protein drugs.

In the present disclosure, a fatty acid chain is linked with the IL-<NUM> analog through in vitro coupling to form a coupling product of IL-<NUM> analog-fatty acid chain. Since the fatty acid chain is a ligand of albumin and can bind to albumin in blood, the IL-<NUM> analog-fatty acid chain coupling product entering the body will form an IL-<NUM> analog-fatty acid chain-albumin complex. On one hand, it can increase the molecular weight to escape from the renal filtration. On the other hand, hydrolysis by intracellular lysosomes can be avoided through the binding of albumin to FcRn which mediates recycling pathway as a protection mechanism, thereby achieving a long-acting mechanism of IL-<NUM> analog-fatty acid chain coupling product. Herein, the conjugate of the present IL-<NUM> analog is not in the form of a drug that is co-expressed with the IL-<NUM> receptor α subunit to form a complex as used by most domestic and foreign companies. Instead, it retains the same cellular biological pattern as native IL-<NUM>, that is, it completely retains its binding to the IL15Rα receptor to participate in signal transduction and to function in vivo.

Hereinafter, the present invention will be further described in conjunction with examples.

In the following examples, the experimental methods without special instructions were usually carried out in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. See, for example, <NPL>). Unless otherwise specified, the reagents used are commercially available or publicly available reagents.

In particular embodiments according to the present disclosure, the positively charged amino acids were leucine (Lys, K), arginine (Arg, R) and histidine (His, H).

As the basis for modification, the amino acid sequence of IL-<NUM> was selected from the group of:.

In particular embodiments according to the present disclosure, the expression of IL-<NUM>, especially in prokaryotic expression systems, could be enhanced by adding one or more amino acids, especially positively charged amino acids, to the C-terminal of IL-<NUM>.

In some particular embodiments, the amino acid sequence of the IL-<NUM> analog could be represented as the following general formula (that is, the amino acid sequence Xa-Yb-Zc was added to the C-terminal of IL-<NUM>):.

In some particular embodiments, the amino acid sequence of the IL-<NUM> analog could be represented as the following general formula (that is, a linker sequence and the amino acid sequence Xa-Yb-Zc were added to the C-terminal of IL-<NUM>):.

In some optional particular embodiments, the linker could be (GGGGS)n, (GS)n or (GAPQ)n, with n being <NUM> to <NUM>.

In some particular embodiments, in the above general formula of the amino acid sequence of IL-<NUM> analogs, Xa comprised LPBTG, wherein B was any amino acid and the linker was (GS)n, with n being <NUM> to <NUM>. In a particular embodiment, the IL-<NUM> analog was obtained by adding a sequence comprising -GS-LPETG to the terminal of the amino acid sequence of IL-<NUM>.

In some particular embodiments, the IL-<NUM> analogs were coupled with fatty acid chains in vitro to improve the long-term efficacy of the IL-<NUM> analogs. The way of in vitro coupling of fatty acid chains could be selected from the group of:.

The wild-type IL-<NUM> nucleotide sequence was synthesized by Sangon Biotech (Shanghai).

The sequence of the C-terminal of IL-<NUM> was altered by introducing a base sequence of different amino acids to be added into the reverse primer. The amino acid sequence, the nucleotide sequence and the primer sequence (used in the construction of IL-<NUM> analogs) of the wild-type IL-<NUM> and constructed IL-<NUM> analogs are shown in Table <NUM>.

The analogs <NUM> to <NUM> were formed by introducing mutations into the wild-type IL-<NUM> to form IL-<NUM> mutants, and then adding the sequence GSLPETGGGSGGSHHHHHH to the C-terminals based on the IL-<NUM> mutants. The analogs <NUM>- to <NUM>- were the mutants of IL-<NUM> formed just after the mutations were introduced, without further adding the sequence GSLPETGGGSGGSHHHHHH to the C-terminals. The nucleotide sequences of the mutated analogs <NUM> to <NUM> and analogs <NUM>- to <NUM>- without adding the sequence GSLPETGGGSGGSHHHHHH to the C-terminals were synthesized by the Genewiz, Inc. , and cloned into the pET41a vectors.

The PCR amplification was classified into three cases as follows.

In the first case, only one reverse primer was required, and a total of <NUM> run of PCR was performed.

In the second case, two reverse primers were required, and a total of <NUM> runs of PCR were performed.

In the third case, three reverse primers were required, and a total of <NUM> runs of PCR were performed.

<NUM>st run of PCR
Sample loading system: <NUM>µL (<NUM> tube)
Primers: forward primer and reverse primer <NUM>
Template: IL-<NUM>
Annealing temperature: <NUM>
Extension time: <NUM>
<NUM>nd run of PCR
Sample loading system: <NUM>µL (<NUM> tube)
Primers: forward primer and reverse primer <NUM>
Template: PCR product of <NUM>st run
Annealing temperature: <NUM>
Extension time: <NUM>
<NUM>rd run of PCR
Sample loading system: <NUM>µL (<NUM> tube)
Primers: forward primer and reverse primer <NUM>
Template: PCR product of <NUM>nd run
Annealing temperature: <NUM>
Extension time: <NUM>
Sample loading system for PCR: <NUM>µL.

Reaction system: <NUM>µL, incubating at <NUM> overnight after mixing.

The molar ratio of vector to fragment was <NUM>:<NUM>. After reacting at <NUM> for <NUM>, it was immediately cooled on ice and transformed into DH5α.

<NUM> of Amp-resistant LB media was added to a <NUM> centrifuge tube and well-grown single colonies were inoculated thereto. A total of <NUM> tubes were inoculated and they were incubated in a shaker under shaking at <NUM>. After incubation for <NUM>, <NUM>µL of the culture was taken as a template for PCR of the bacterial suspension. Reaction system for PCR: <NUM>µL.

After PCR was completed, it was detected by agarose gel electrophoresis. Three positive clones were selected for sequencing (Sangon Biotech).

The positive clones with correct sequencing were inoculated into <NUM> of Amp-resistant LB liquid media with an inoculation volume of <NUM>µL. <NUM>µL of bacterial suspension was added into a <NUM> centrifuge tube. <NUM>µL of <NUM>% glycerol was added for storage. It was labeled with the name, host bacteria and date, capped and stored in a refrigerator at -<NUM>.

The remaining bacterial suspension was collected by centrifugation for plasmid extraction.

The electrophoretogram of wild-type IL-<NUM> was shown in <FIG>, wherein the expression of wild-type IL-<NUM> was virtually undetectable in "T (total)", "S (supernatant)" or "P (pellet)". The electrophoretograms of IL-<NUM> analogs <NUM> to <NUM> were shown in <FIG>, wherein the expression levels of the IL-<NUM> analogs were significantly higher than that of wild-type IL-<NUM>. Further, the expression levels of the IL-<NUM> analogs (analogs <NUM> to <NUM>) with the sequence GSLPETGGGSGGSHHHHHH at the C-terminal were also significantly higher than those of the IL-<NUM> analogs (analogs <NUM>- to <NUM>-) without the sequence GSLPETGGGSGGSHHHHHH at the C-terminal, as shown in <FIG> and <FIG>.

As shown by the HPLC quantitative results in <FIG>, the expression levels of the IL-<NUM> analogs <NUM> to <NUM> were about <NUM>-fold higher than that of the wild-type IL-<NUM>.

The IL-<NUM> analogs <NUM>, <NUM>, <NUM> and <NUM> expressed by the inclusion bodies were dissolved in <NUM> urea solution, and purified by ion exchange and reversed-phase chromatography (for details, see:<NPL>), to obtain relatively pure proteins. The SDS-PAGE electrophoretograms were shown in <FIG>.

The CTLL-<NUM> cell proliferation assay is commonly used to detect the activity of immune cells stimulated by interleukin at the cellular level. Therefore, the biological activity of IL-<NUM> analogs was determined herein by the proliferative effect of the wild-type IL-<NUM> and IL-<NUM> analogs on CTLL-<NUM> cells.

The results were shown in Table <NUM>. There was no significant difference in the cellular activity of the IL-<NUM> analogs and wild-type IL-<NUM>.

The purified IL-<NUM> Analog <NUM> (IL-<NUM>-GS-LPETG-GSGGSHHHHHH) was used in this example. The fatty acid chain (<NUM>) with GGG at the N-terminal was linked to IL-<NUM>-GS-LPETG- GSGGSHHHHHH by a ligation reaction catalyzed by the transpeptidase Sortase A. The reaction was carried out in a Sortase A: IL-<NUM>: fatty acid chain ratio of <NUM>: <NUM>: <NUM>, wherein the reaction buffer was <NUM> Tris-HCl (<NUM> CaCl, <NUM> NaCl, pH <NUM>). After reacting at room temperature for <NUM> hours, purification was carried out. Reversed-phase chromatography C8 (Sepax Technologies, Inc. ) was used for purification to separate the unconjugated IL-<NUM> Analog <NUM> and the unreacted fatty acid chains from the conjugated products. The purity of the final product was determined by UPLC (<FIG>) and LC-MS (<FIG>). The results showed that IL-<NUM> Analog <NUM> had been coupled to a fatty acid chain to form IL-<NUM> Analog <NUM>-<NUM>.

The purified IL-<NUM> Analog <NUM> was coupled to a fatty acid chain with a succinimidyl ester (<NUM>) through the amino group at N-terminal thereof at neutral pH. The reaction was carried out in an IL-<NUM>: fatty acid chain ratio of <NUM>: <NUM>, wherein the reaction buffer was PBS at pH <NUM>. After reacting at room temperature for <NUM> hour, purification was carried out. Reversed-phase chromatography C8 (Sepax Technologies, Inc. ) was used for purification to separate the unconjugated IL-<NUM> and the unreacted fatty acid chains from the conjugated products. The final product was identified by LC-MS. As shown in <FIG>, IL-<NUM> has been coupled to a fatty acid chain to form the IL-<NUM> Analog <NUM>-<NUM>.

The conjugates of IL-<NUM> analogs used in this example were prepared in Example <NUM>.

In this example, biolayer interferomeory (BLI) was used to determine the affinity between the target protein and the receptor. For procedures, see <NPL>. The receptor protein IL15Rα-His used in the experiment was produced by Leto Laboratories Co. The formulation of buffer was: <NUM> HEPES, <NUM> NaCl, <NUM> EDTA, and <NUM>% Tween <NUM>. The receptor IL15Rα-His was pre-immobilized on a HISIK sensor (Pall Fortebio, Catalog# <NUM>-<NUM>), followed by an established process comprising the steps of setting baseline, loading, baseline, association and dissociation. Data acquisition and analysis were carried out using the software Data acquisition <NUM> and Data analysis <NUM> installed with Octet RED96, respectively.

The results for affinity assay of the IL-<NUM> analog and the conjugated products of IL-<NUM> analogs and fatty acid chains to the IL15Rα receptors are shown in Table <NUM>. As compared to the IL-<NUM> analog before conjugation, the affinity of the conjugated products of IL-<NUM> analog and fatty acid chains to the IL15Rα receptor did not change significantly.

Eight C57BL/<NUM> mice were divided into <NUM> groups with <NUM> mice in each group. Blood was collected from two mice at each time point, and blood was collected cyclically. For one group, the mice were injected with IL-<NUM> at <NUM>/kg via tail vein. Blood samples were collected immediately, and then at <NUM>, <NUM>, <NUM> and <NUM> after administration. For the other group, the mice were injected with long-acting IL-<NUM> at <NUM>/kg via tail vein. Blood samples were collected immediately, and then at <NUM>, <NUM> and <NUM> after administration. At each time point, <NUM> to <NUM>µL of blood was collected from eye orbit. Then serum was collected for ELISA assay of IL-<NUM>.

The calculation formula of half-life is: t<NUM>/<NUM> = <NUM>/k, k = (Inc<NUM>-Inc)/t. Upon calculation, the half-life of IL-<NUM> was about <NUM>, and the half-life of long-acting IL-<NUM> could reach <NUM>.

Fifteen C57BL/<NUM> mice aged <NUM> to <NUM> weeks were randomly divided into <NUM> groups, namely the reagent control group (PBS), the IL-<NUM> group and the long-acting IL-<NUM> group, with <NUM> mice in each group. On Day <NUM>, the mice were subcutaneously inoculated with B16-F10 cells on the back of the neck, with <NUM>×<NUM><NUM>/<NUM>µL/mouse. On Days <NUM> to <NUM>, the PBS group and the IL-<NUM> group were administered intravenously (i. ) for <NUM> consecutive days, respectively, at a dose of <NUM>µg/<NUM>µL/mouse (IL-<NUM> group) or <NUM>µL/mouse (PBS group). The IL-<NUM> analog conjugate group was administered twice in the same way on Day <NUM> and Day <NUM>, with the same dosage of <NUM>µg/<NUM>µL/mouse each time. Tumor sizes were observed from Day <NUM> and continued for <NUM> days.

Claim 1:
An IL-<NUM> analog, wherein the amino acid sequence of the IL-<NUM> analog comprises the amino acid sequence of IL-<NUM>, and one or more amino acids added to the C-terminal of the amino acid sequence of IL-<NUM>,
wherein the one or more amino acids added to the C-terminal of the amino acid sequence of IL-<NUM> are selected from the group of:
GSGSGS-HHHHHH, GS-HHHHHH, PLASTKKR, LPKSAKKK, KKKKKKK, GAPQGAPQ-LVESAHHH, GS-LVSSAHHK, GS-LIEHHRRK, GS-IVEHRKKK, GS-VPKTGRRR, GS-LVASGKK, GS-HRKSGHHH, GS-LPKTGRHK, KKKTGRRH, LPRSGRHK, LVETHHHH, VRPETHHH, KKK, RHHHH, KRETHHHHH, GS-LPETG-GSGGSHHHHHH HLETGKKK, HVESGRRR, RRHTGKKK, HVKTGHHH, HVKSGRHH, HVKSSHRH, GSGSGSGSGS-LVKSGHHH, RPKSGHHK, KKC, LHKAGKHH, K, and KK, and wherein the amino acid sequence of IL-<NUM> is shown in SEQ ID NO. <NUM>.