CN114230654B - Interleukin-2 capable of covalently crosslinking CD25 and application thereof in autoimmune disease treatment - Google Patents
Interleukin-2 capable of covalently crosslinking CD25 and application thereof in autoimmune disease treatment Download PDFInfo
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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Abstract
The present invention relates to sites capable of covalent cross-linking of human interleukin-2 and CD25, human interleukin-2 mutants with non-natural amino acids inserted via site-directed mutagenesis and long acting human interleukin-2 mutants modified with these mutants. Interleukin-2 and its mutant, which produce covalent cross-linking activity with CD25, can activate regulatory T cells in a biased and lasting manner, and can play a long-acting immunosuppressive role with less or no other effector cells. The invention further relates to the use of such site-directed mutagenesis or modification of interleukin-2, such as the use as a stable, long-acting immunosuppressant for the treatment of various autoimmune diseases. The IL-2 mutant obtained by the invention can generate irreversible covalent crosslinking after being combined with CD25, and greatly enhances the recycling efficiency of the internalized IL-2 along with the CD25, thereby increasing the existence time and the action efficiency of the IL-2 on Treg cells.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and in particular relates to interleukin-2 inserted with unnatural amino acid at fixed points, which can generate covalent crosslinking with CD25 receptor after contacting with the receptor, thereby having the function of specifically activating and regulating T cells. The invention further relates to the application of the site-directed modification interleukin-2 in treating autoimmune diseases, such as safe and effective systemic lupus erythematosus, rheumatoid arthritis, graft versus host rejection disease and the like.
Background
Interleukin-2 (Interleukin-2) is a pleiotropic cytokine, and has the activity of activating regulatory T cells (Treg) and effector cells, and is one of the important cytokines for immune regulation. IL-2 plays a biological role by binding to IL-2 receptors (IL-2R) on cell membranes, IL-2R consists of alpha, beta, gamma three molecular subunits, wherein the alpha chain (CD 25) has no signaling function and can only form high affinity binding with IL-2 as a three molecular complex together with beta and gamma chains (Kd=10pM); while the β chain (CD 122) and the γ chain (CD 132) are both in the type I cytokine receptor superfamily, they can bind with affinity to IL-2 in the absence of α chain (kd=1 nM, and thus transmit downstream signals (r.spolski et al, 2018). The three receptor subunits are expressed in different cell subsets, CD25 is continuously expressed on the surface of regulatory T cells (Treg) for a long period of time, and natural killer cells (NK) and cd8+ T killer cells are normally expressed with CD25 under normal conditions, so that the sensitivity of Treg cells to IL-2 is greater than NK without stimulation by foreign antigens, and cd8+ T equivalent cells (Boyman et al, 2006). The prior studies indicate that low dose IL-2 activates Treg cells by a bias, inhibits autoimmunity, and thus plays a role in treating autoimmune diseases.
Previous studies have shown that IL-2, upon binding to the IL-2Rαβγthree receptor complex, undergoes rapid endocytosis and isolation in the primary endosome. Wherein, IL-2Rβγ enters into secondary endosome and lysosome, and is degraded; IL-2Rα (CD 25) is circulated to the surface of cell membrane in the primary endosome, and plays a biological role again. The intracellular transport mode of IL-2 depends on its affinity for the tri-receptor subunit, a portion of which can circulate with CD25 to the cell surface to continue its biological role, while a large portion degrades with the entry of IL-2Rβγ into the lysosome. This mechanism brings important research direction for improving the effect of IL-2. For example, a series of high affinity CD25 mutants have been developed to increase the time of presence of IL-2 on the surface of cell membranes (Rao et al, protein engineering (12), 1081-1087, 2003). Our earlier studies have also found that site-specific modification of polyethylene glycol at different sites on IL-2 can affect the recycling pathway of the modified product to varying degrees, thereby significantly affecting the duration of the IL-2 cell membrane. Nevertheless, because IL-2 binding to CD25 remains a non-covalent reversible bond and mutants still suffer from short half-life, purely mutational enhancement of IL-2 affinity to CD25 is not an effective approach to obtaining IL-2 agonists biased to activate tregs.
If the irreversible covalent binding between the IL-2 and the CD25 can be generated, the recycling rate of the IL-2 along with the CD25 is expected to be greatly improved, so that the sustainable effect of the IL-2 on the CD25 high-expression cells, such as Treg cells, is improved, the bias and the effectiveness of the IL-2 activated Treg cells are enhanced, and the method can be used for treating various autoimmune diseases.
Disclosure of Invention
To solve the above problems, the present invention provides IL-2 mutants that can be covalently crosslinked with CD25, and their use in the treatment of autoimmune diseases.
First, the present invention provides a site that allows covalent binding of human interleukin-2 to CD25, the selected modification site being selected from the group consisting of: the sequence shown in SEQ ID NO: r38 of 1, L72, or a combination thereof. The invention selects to insert unnatural amino acid fluorine-L-tyrosine (FSY) with near reactivity at a specific site of IL-2, which keeps inert in a natural state, and can generate covalent bonding reaction with the imidazole ring side chain on histidine when the imidazole ring side chain is separated from the histidine sufficiently close to the imidazole ring side chain, so as to generate stable and irreversible covalent crosslinking. The selected site is sufficiently close to the H120 position of CD25 from the spatial structure of IL-2 binding to CD25 to promote covalent cross-linking reactions.
The invention also provides site-directed mutagenesis of human interleukin-2, which is an amino acid or amino groups at said specific siteThe acid is mutated to an unnatural amino acid, which isThe unnatural amino acid FSY shown, the specific site is selected from the group consisting of: the sequence shown in SEQ ID NO: r38 of 1, L72, or a combination of both sites.
A site-directed mutated human interleukin-2 which hybridizes to the sequence set forth in SEQ ID NO:1 is distinguished by the sequence: in SEQ ID NO:1 is mutated to FSY at the N-th amino acid of the sequence shown in SEQ ID NO:1 is represented by the following formula (II):
the direction from R1 to R2 is the direction from N end to C end of the amino acid sequence, wherein the amino acid at the N position is selected from one or two of the amino acids at the R38 position and the L72 position,
r1 is SEQ ID NO:1 to the amino acid residues 1 to N-1 of the sequence shown in 1,
r2 is SEQ ID NO:1 from the n+1 position to the C-terminal amino acid residue of the sequence shown in figure 1,
the invention also provides the human interleukin-2 modified by PEG and mutated at the specific site, wherein the molecular weight of the modifier introduced at the modification position is in the range of 5kDa,10kDa,20kDa or 40kDa.
Specifically, the human interleukin-2 mutated at a specific site modified by PEG is a mutant R38-FSY with the mutation of R38 site into FSY; mutant L72-FSY in which L72 is mutated to FSY; mutants of double mutation of R38 and L72R 38/L72-FSY;5kDa-PEG modified R38-FSY,5K-PEG-R38FSY;10kDa-PEG modified R38-FSY,10K-PEG-R38FSY;20kDa-PEG modified R38-FSY,20K-PEG-R38FSY;40kDa-PEG modified R38-FSY,40K-PEG-R38FSY;5kDa-PEG modified L72-FSY,5K-PEG-L72FSY;10kDa-PEG modified L72-FSY,10K-PEG-L72FSY;20kDa-PEG modified L72-FSY,20K-PEG-L72FSY;40kDa-PEG modified L72-FSY,40K-PEG-L72FSY.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of said human interleukin-2 or said modified interleukin-2, and a pharmaceutically acceptable carrier.
The invention also provides the use of said interleukin-2 or said modified interleukin-2 in the manufacture of a medicament for immunomodulation and a variety of autoimmune diseases.
The invention also provides the use of interleukin-2 or said modified interleukin-2 in the manufacture of a medicament for graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus, autoimmune disease diabetes, dermatomyositis, scleroderma, multiple sclerosis, myasthenia gravis, demyelinating disease, primary adrenocortical atrophy, chronic thyroiditis, chronic non-specific ulcerative colitis, chronic active hepatitis, cachexia and atrophic gastritis, autoimmune glomerulonephritis, pulmonary renal hemorrhagic syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, autoimmune alopecia.
Compared with the existing research drugs, the IL-2 derivatives obtained based on the invention have the technical effects and advantages that are mainly represented by one or more of the following:
the IL-2 derivative obtained by the invention has stronger therapeutic effectiveness and safety: the IL-2 mutant obtained by the invention can generate irreversible covalent crosslinking after being combined with CD25, and greatly enhances the recycling efficiency of the internalized IL-2 along with the CD25, thereby increasing the existence time and the action efficiency of the IL-2 on Treg cells. Whereas effector cells, such as cd8+ T cells, traditional cd4+ T cells, lack CD25 expression thereon and are therefore not responsive to the effects of such IL-2 mutants. This biasing greatly enhances the bias and activity of such IL-2 derivatives on tregs, thereby enhancing their effectiveness and safety in the treatment of autoimmune diseases.
The IL-2 derivative obtained by the invention has long-acting property and durability: based on covalent crosslinking activity, the IL-2 derivative can generate stable covalent crosslinking with CD25 on the cell surface, so that the in vivo time of the derivative is prolonged, and the effect of the derivative is long-acting and durable.
Drawings
FIG. 1 shows the design of the FSY amino acid insertion site and verification of crosslinking efficiency. The corresponding position of the selection site is mutated into TAG stop codon, a mutant expression vector is prepared, the mutant expression vector and auxiliary plasmid pEVOL-FSY are co-transferred into escherichia coli OrigamiB (DE 3), unnatural amino acid is added into a culture medium, and IL-2 with the unnatural amino acid inserted into the site is obtained for modification through induction expression. Crystal Structure of AIL-2 and CD25 Complex (PDB 2 ERJ). The selected site is a sufficiently close spatial distance from the H120 site of CD 25. B in vitro level of crosslinking activity verification. And (3) incubating the mutants with different sites mutated into FSY with the same amount of CD25 protein in vitro, adding a loading buffer solution with a reducing agent for heating, and carrying out coomassie brilliant blue staining and Western blot detection on the denatured samples. The results show that, at the in vitro level, mutants with mutation at the R38 site to FSY (R38-FSY) and mutants with mutation at the L72 site to FSY (L72-FSY) can produce CD25 crosslinking activity, while F42 site does not produce crosslinking activity. And C, mass spectrum verification. Mass spectrometry of the crosslinked bands described in B confirmed that covalent crosslinking of the FSY amino acids to the H120 site in CD25 occurred. Wherein X is indicated as FSY amino acid. D cell level crosslinking efficiency validation. L72-FSY was incubated with YT cells expressing CD25, and Western blot detection was performed on the denatured cell lysates. The crosslinking activity of L72-FSY is dose and time dependent.
FIG. 2 shows in vitro activity verification of L72-FSY. (left) L72-FSY vs wild-type IL-2 (WT-IL-2) CD25 binding activity. Shown as a bio-film interference-binding line graph. pSTAT5 assay of L72-FSY versus wild-type IL-2 (WT-IL-2). The affinity constant and half-effective concentration of (right) in vitro activity. The results indicate that mutation of the L72 site on IL-2 to FSY amino acid slightly reduces the affinity with CD25 and has no significant effect on cell activity.
Figure 3 shows that IL-2 with crosslinking activity significantly prolonged its presence on Treg cells. A L72-FSY or WT-IL-2 was incubated with isolated pure Treg cells or CD8+ T cells for 1.5 hours, and the cells were washed three times with a biased acid buffer and incubation continued. The cell surface was directly incubated with anti-IL-2 antibody, and the IL-2 residue on the surfaces of Treg cells and CD8+ T cells was detected. B the CD25-YT cells were incubated with WT-IL-2 or L72-FSY labeled with AF555 fluorescent molecules for fluorescence confocal detection. It was observed that after incubation, L72-FSY had partially cycled to the cell surface, at higher levels than WT-IL-2, and had developed confocal with CD 25. C with A treated Treg cells and CD8+ T cells, L72-FSY was observed to give a more durable and higher level of pSTAT5 activation signal than WT-IL-2 after cell washing.
Figure 4 shows that L72-FSY has a durable and potent Treg-biased activation effect in vitro. L72-FSY or WT-IL-2 was incubated overnight with isolated pure Treg cells or CD8+ T cells, pickled, and cultured for an additional 3 days in the absence of cytokines. AL72-FSY expands the number of Treg cells more than WT-IL-2, and the percentage of the total cells occupied by the Treg cells is not obvious to CD8+ T cells. B L72-FSY activates expression of Treg upper surface markers, including CD25 and Foxp3, more than WT-IL-2, and only to an equivalent extent activates expression of CD26 and CD49d on CD8+ T.
Figure 5 shows that L72-FSY has a durable and potent Treg-biased activation in vivo. Human PBMCs were intravenously injected into immunodeficient murine NSG to prepare a humanized mouse model. WT-IL-2, L72-FSY and PBS were injected subcutaneously once a day for a total of 10 days. Spleen cells from mice were isolated on the fifth day after the last injection and examined by flow cytometry. The proportion of Treg cells after treatment in each group A to CD4+ T cells is counted. B Treg, cd8+t, tconv cell count statistics and Treg/cd8+t and Treg/Tconv calculations. The results indicate that the L72-FSY bias significantly expanded cd4+ T cells in mice with less impact on cd8+ T and Tconv cells. C activating marker detection. Including CD25 on cd8+t and Foxp3 in Treg.
FIG. 6 shows the long lasting and potent in vivo Treg-biased activation effect of L72-FSY was demonstrated using CD25 humanized B-hIL-2Rα mice. B-hIL-2Rα mice are humanized from the transgenic level, the extracellular segment of CD25 is still murine, and L72-FSY can be evaluated in mice. B-hIL-2Rα mice were subcutaneously injected with WT-IL-2, L72-FSY and PBS, respectively, once a day for a total of 10 days. Spleen cells from mice were isolated on the fifth day after the last injection and examined by flow cytometry. Statistics of the proportion of Treg cells in splenocytes to CD4+ T cells after treatment of each group A, and statistics of the numbers of Treg, CD8+ T, tconv, NK, th1 and Th17 cells in splenocytes. The ratio of B Treg/CD8+T, treg/Tconv, treg/Th1, treg/Th17 was calculated.
FIG. 7 shows site-directed PEG coupling to L72-FSY. A scheme for coupling L72-FSY was performed using 20kDa propanal-PEG (ALD-20K-PEG). SDS-PAGE analysis of the B-coupled products. It can be seen that ALD-20K-PEG successfully coupled to L72-FSY, exhibiting a single uniform band. And the coupled product still has the covalent crosslinking activity of CD 25.
FIG. 8 shows in vitro activity characterization of 20K-PEG modified L72-FSY (PEG-L72 FSY). Different concentrations of PEG-L72FSY were stimulated with human PBMC and PEG-L72FSY was tested for activation of Treg cells and CD8+ T cells pSTAT5 therein. The PEG modification at the AN end is slight, reduces the influence of IL-2 on Treg cells and CD8+ T cells to the same extent, and has no obvious influence on the deviation and activity of the Treg cells and the CD8+ T cells. B shows the detection of the affinity of each sample for IL-2Rα and IL-2Rβ.
Fig. 9 shows pharmacokinetic evaluations. Each sample was injected subcutaneously 5ug each at a time into C57BL/6 mice and B-hIL-2RA mice. A shows the drug concentration and time profile. B shows calculated in vivo clearance half-life and average retention time.
Figure 10 shows that PEG-L72FSY has a durable and potent Treg-biased activation effect in vivo using CD25 humanized B-hIL-2ra mice. An equal amount of the sample was subcutaneously injected into B-hIL-2Rα, once daily for unmodified PEG, and once daily for modified PEG. Spleen cells from mice were isolated on the fifth day after the last injection and examined by flow cytometry. The ratio of the number of tregs to effector cells expanded in each treatment group was counted and calculated.
FIG. 11 is a graph showing the therapeutic effect of IL-2 containing covalent crosslinking activity in autoimmune disease models, including pristane-induced systemic lupus erythematosus and graft-versus-host rejection disease models.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1 selection of sites for covalent crosslinking and preparation of IL-2 containing FSY amino acids
Depending on the nature of the FSY amino acid reaction, it remains inert under natural conditions and covalent crosslinking reactions can only occur when it is sufficiently close to the reacting amino acid. Based on the structure of the binding crystal of IL-2 to its receptor, several potential sites were selected for insertion of FSY amino acids, specifically including R38, F42 and L72, which are spatially close enough to the 120 th histidine on CD25 to initiate a cross-linking reaction when IL-2 binds to CD25 (FIG. 1A). For each site, the inventor designs a primer capable of mutating the codon encoding the amino acid into an amber codon, and then uses a site-directed mutagenesis kit [ ]Lighting Site-Directed Mutagenesis Kits, catalyst # 210518), operating according to instructions, and mutating the corresponding position of IL-2 to an amber stop codon using the wild type IL-2 expression vector pET21a-IL-2 (WT) as a template, to obtain a mutated IL-2 expression plasmid. The expression plasmid with ampicillin resistance and the auxiliary plasmid with spectinomycin resistance are simultaneously transformed into escherichia coli OrigamiB (DE 3), and the co-transformed positive strain is screened out by a chloramphenicol and spectinomycin double-resistance flat plate, so that the expression strain with two plasmids is obtained. Culturing the obtained expression strain in 2 XYT culture medium at 37deg.C for 12-16 hr, performing secondary amplification until the OD value of the bacterial liquid reaches 0.6-1.0, adding FSY to final concentration of 0.5mM, continuing amplification at 37deg.C for 30 min, adding IPTG to final concentration of 0.5mM, and inducing expression at 24deg.C for 12 hr, and collecting thallus. Suspending the collected thallus with Ni-NTA-Bind-Buffer balance weight, circularly crushing with ultra-high pressure homogenizing crusher 1200bar, centrifuging at high speed to remove cell debris, subjecting to Ni-NTA metal chelate affinity chromatography, washing with Ni-NTA-Wash-Buffer, and washing with Ni-NTA-Wash-BufferThe Elute-Buffer eluted to give a primarily purified interleukin-2 sample with a purity of about 90%. The product was checked by SDS-PAGE, coomassie blue and mass spectrometry to confirm that the unnatural amino acid was inserted at the specific site.
Example 2 in vitro validation of covalent crosslinking Activity of IL-2 mutants
The IL-2 mutant with different sites inserted into FSY is incubated with CD25 extracellular segment in equal amount under physiological condition, the incubated sample is added into the loading buffer solution with reductant, boiled at 100 deg.c for 10 min and the reduced sample is analyzed by SDS-PAGE. Coomassie brilliant blue staining and Western Blot results show that in the reduced samples, the IL-2 mutants with FSY inserted at the R38 and L72 sites, namely R38-FSY and L72-FSY, produced distinct covalent cross-linked bands with molecular weights equal to the sum of the molecular weights of the two proteins, wherein the cross-linking efficiency of L72-FSY was higher than that of R38-FSY (FIG. 1B). Mass spectrometry analysis of the crosslinked band of L72-FSY with CD25 revealed that the FSY amino acid at position 72 did covalently crosslink with the H120 position of CD25, and a covalently crosslinked fragment was retrieved (fig. 1C). On the other hand, L72-FSY was incubated with YT cells expressing CD25, and Western blot was performed on the denatured cell lysates. Consistent with the results of molecular level detection, it was found that L72-FSY successfully produced covalent cross-links with CD25 at the cellular level, with the cross-linking activity exhibiting a pronounced dose and time dependence (FIG. 1D).
Example 3 characterization of in vitro Activity of L72-FSY
To evaluate the bias and effectiveness of L72-FSY on activation of Treg cells, we first evaluated the effect of this mutation on IL-2 activity. We assessed the effect of 72 site insertion of FSY on IL-2 activity by affinity detection and the phosphorylation 5 of YT-CD25 (pSTAT 5) index. The results show that L72-FSY slightly reduced the binding to CD25 (approximately 3-fold) compared to WT-IL-2. This result is consistent with literature reports, since the L72 moiety is involved in binding of IL-2 to CD 25. However, this decrease did not affect the activation of CD25-YT cells by L72-FSY, which was shown to have the same degree of pSTAT5 activation as WT-IL-2, with affinity and EC50 for cell detection as shown in Table 1 and FIG. 2.
TABLE 1 WT-IL-2 vs L72-FSY affinity for CD25 and CD25-YT cell in vitro Activity
| Sample of | WT-IL-2 | L72-FSY |
| kon(1/Ms) | 3.89E+06 | 1.39E+06 |
| kdis(1/s) | 1.30E-02 | 1.42E-02 |
| KD(M) | 3.33E-09 | 1.03E-08 |
| EC50(μg/ml) | 4.82E-05 | 9.47E-05 |
Example 4 covalent crosslinking enhances the time of presentation and intensity of action of IL-2 on Treg cells
Based on the covalent crosslinking activity shown in example 2, we then verify whether covalent crosslinking with CD25 can enhance the recycling process of IL-2 with CD25 and its specificity for Treg action. Based on the existing literature studies (Suat al., sci Transl Med.2015Oct 28;7 (311): 311ra 170), we selected a pulse incubation cell model to verify this. We incubated WT-IL-2 or L72-FSY with isolated pure Treg cells and cd8+ T cells, respectively, for 1.5 hours, followed by washing the cells with an acidic buffer to remove cytokines from the cell surface and medium, and continued culturing the cells to detect the residual amount of IL-2 above. The results showed that during continued culture, the L72-FSY treated group had higher cell surface IL-2 than the WT-IL-2 group, an effect that was not exhibited in CD8+ T cells lacking CD25 expression, suggesting that more efficient IL-2 recirculation occurred in the L72-FSY treated group, enhancing the time for which IL-2 was present on Treg cells (FIG. 3A). Fluorescence confocal analysis consistent with this result, fluorescence labeled WT-IL-2 or L72-FSY was incubated with CD25-YT cells, and intracellular and surface IL-2 co-localization with CD25 and lysosomes was detected. The results show that after co-incubation, the L72-FSY group has obvious IL-2 residues on the cell surface and co-localizes with CD 25; whereas the WT-IL-2 group labeled fluorescent molecules were more present in the cell and co-localized with lysosomes (fig. 3B). Further, we examined pSTAT5 signal intensity of Treg cells and cd8+ T cells after prolonged incubation. The results showed that L72-FSY treated Treg cells retained a stronger pSTAT5 signal intensity during incubation than the WT-IL-2 group, which exhibited CD25 dependence (FIG. 3C). These results indicate that the crosslinking activity does enhance the recycling of IL-2, which in turn enhances its time of presence and action activity on cells. More importantly, this effect has Treg cell selectivity.
Example 5 cell number and activation marker detection of L72-FSY in vitro targeted expanded Treg cells
L72-FSY or WT-IL-2 was incubated overnight with isolated pure Treg cells or CD8+ T cells, acid washed, incubated for 3 days in the absence of cytokines, and expansion of Treg and CD8+ T cells and expression of cell surface markers were detected by flow cytometry, as a result of which complementation with pSTAT5 was achieved. The results show that during cytokine-depleted co-culture, the L72-FSY treated group expanded Treg cells more than WT-IL-2, in terms of an increase in the proportion of CD4+ T cells occupied by Treg and the absolute number of Treg cells, while having no significant effect on the proportion of CD8+ T cells to CD3+ T cells and the absolute number of CD8+ T cells (FIG. 4A). The results of activation markers on the cell surface and cell expansion were consistent: the L72-FSY treated group enhanced CD25 and Foxp3 expression on Treg over WT-IL-2, while there was no significant effect on CD26 and CD49d expression on CD8+ T cells (FIG. 4B). This result suggests that L72-FSY can activate Treg cells more effectively than WT-IL-2, and that the activation effect is selective.
EXAMPLE 6L72-FSY in vivo Targeted expansion of Treg cells
We further evaluated the targeted activation of Treg cells by L72-FSY using in vivo models. According to literature reports, we have established an evaluation model of human cells in mice (Trotta et al, nat Med.2018Jul;24 (7): 1005-1014). Briefly, we intravenously injected freshly isolated human PBMC cells into NSG-deficient mice, while subcutaneously injecting either WT-IL-2 or L72-FSY once a day for 10 days. The spleen of the mouse was taken the fifth day after the last injection, and the number of each immunocyte therein was examined. The results indicate that the L72-FSY bias expanded Treg cells in mice compared to WT-IL-2 combination and PBS control, which was manifested by an increase in the proportion of Treg cells to cd4+ T cells, an increase in numbers, while the numbers of cd8+ T cells and Tconv cells were instead lower than WT-IL-2, resulting in a significant increase in Treg/cd8+ T and Treg/Tconv in the L72-FSY treated group (fig. 5A and 5B). Consistent with the in vivo cell activation markers and expansion results, L72-FSY significantly activated Foxp3 expression in Treg cells, while less affecting CD25 expression on cd8+ T cells (fig. 5C).
On the other hand, we used CD25 humanized mice (B-hIL-2Rα), i.e., CD25 extracellular domain was transgenic to humanize, CD25 intracellular domain was still murine, and the activation of L72-FSY on Treg cells in vivo was evaluated. As described above, B-hIL-2Rα mice were subcutaneously injected with WT-IL-2, L72-FSY and PBS, respectively, once a day for a total of 10 days. Spleen cells from mice were isolated on the fifth day after the last injection and examined by flow cytometry. The results indicate that L72-FSY significantly targets expanded Treg cells in B-hIL-2Rα, while affecting other cells less, and is characterized by an increase in the proportion and number of mouse Treg cells in the L72-FSY treated group, CD8+T, tconv and NK, lower than in the WT-IL-2 group, and a decrease in the number of Th1 and Th17 cells, higher than in the WT-IL-2 group, than in the PBS and WT-IL-2 treated groups. These results resulted in an increase in the ratio of Treg to these cells in the L72-FSY treated mice (fig. 6A and 6B). Overall, consistent with in vitro results, L72-FSY had a higher bias for Treg activation in vivo than WT-IL-2 in vivo model assays of PBMC-established humanized mice and CD25 humanized mice.
EXAMPLE 7 polyethylene glycol modified L72-FSY
The above examples demonstrate that covalent cross-linking CD25 activity, exemplified by L72-FSY, is effective in preferentially activating Treg cells. In view of the low molecular weight of IL-2 and rapid clearance in vivo, to enhance the timing of covalent cross-linking of IL-2 mutants with CD25 in vivo, we modified IL-2 with biomacromolecules to increase its half-life and stability in vivo. Using polyethylene glycol modification as an example (PEG), we performed 20kDa N-terminal PEG modification on L72-FSY. As shown in FIG. 7, the results demonstrate that 20kDa PEG was successfully coupled to L72-FSY, which resulted in a single modified band, while modified PEGylated L72-FSY (PEG-L72 FS 7) still retained CD25 covalent crosslinking activity (FIG. 7).
We further evaluated the activity and selectivity of this modified product. Different concentrations of PEG-L72FSY were stimulated with human PBMC and PEG-L72FSY was tested for activation of Treg cells and CD8+ T cells pSTAT5 therein. The results show that the PEG modification was mild and reduced to the same extent the activation of both Treg cells and CD8+ T cells by L72-FSY, affecting the selectivity to a lesser extent than WT-IL-2 (FIG. 8A). The results of the receptor affinity detection and the cell activity detection are consistent, the binding force of the modified product to IL-2 Ralpha and IL-2 Rbeta is reduced to the same extent by PEG modification, and the influence on the deviation is not obvious (figure 8B). The data sets associated with the affinity assays are shown in the following table.
TABLE 2 Effect of 20kDa-PEG modification at N end on affinity of L72-FSY for IL-2Rα and IL-2Rβ
Example 8 Effect of covalent Cross-linking and/or PEG modification on the in vivo half-life of IL-2
The modification of PEG can improve the molecular volume of protein, reduce glomerular filtration, shield antigen sites, further improve the stability of modified IL-2 in vivo and prolong half-life. On the other hand, the half-life period is prolonged, the reaction time of the IL-2 derivative with covalent binding capacity with CD25 in vivo can be prolonged, and the covalent crosslinking effect is further enhanced. To this end, we evaluated the pharmacokinetics of WT-IL-2, L72-FSY,20kDa-PEG modified WT-IL-2 (PEG-WT), and 20kDa-PEG modified L72-FSY (PEG-L72 FSY) in C57BL/6 and B-hIL-2RA mice. The results indicate that 20kDa-PEG modification can significantly improve the pharmacokinetics of IL-2 in mice. In particular, samples containing FSY have stronger pharmacokinetic properties than WT-IL-2 and PEG-WT, which do not possess crosslinking activity. This difference was not shown in the control group of C57BL/6, demonstrating that it was indeed caused by covalent cross-linking (FIG. 9). Pharmacokinetic data are summarized in table 3.
Table 3 summary of pharmacokinetic parameters detected in two mice
1.AUC:Area under drug concentration versus time curve；2.Tmax：time of maximal drug concentration；3.MRT:mean residence time；4.T 1/2 :half-life of clearance；5.Cl/f:apparent total plasma clearance.
Example 9PEG-L72FSY in vivo Targeted expansion of Treg cells
We used an in vivo model to evaluate the targeted activation of PEG-L72FSY on Treg cells. Briefly, we used B-hIL-2Rα mice subcutaneously injected with either PEG-WT-IL-2 or PEG-L72FSY once a day, 5 total. WT-IL-2 and L72-FSY served as controls. The results indicate that PEG-L72FSY can activate Treg cells more efficiently and preferentially than L72-FSY, reacting to higher Treg/cd8+t, treg/Tconv and Treg/NK (fig. 10).
EXAMPLE 10 covalent Cross-linking active IL-2 has greater therapeutic efficacy and safety in autoimmune disease models
We used pristane-induced lupus model to evaluate the therapeutic effect of IL-2 with covalent cross-linking activity in lupus in B-hIL-2rα mice. The results show that the L72-FSY and PEG-L72FSY treated groups significantly reduced disease progression in lupus, as demonstrated by reduced antinuclear, anti-rNP and anti-Sm antibodies, relative to the wild-type IL-2 and PBS treated groups; and the inflammatory action of the kidney and the spleen is obviously improved, the deposition of IgG and IgM of the kidney is reduced, and the formation of germinal centers of the spleen is reduced. On the other hand, inflammatory lesions of the lung were one of the important disease progression of pristane-induced lupus, and we found that the L72-FSY and PEG-L72FSY treated groups significantly improved pulmonary inflammatory responses in lupus mice, suggesting that IL-2 with covalent crosslinking activity had a significant immunosuppressive effect in improving autoimmune diseases typified by lupus (FIG. 11A).
On the other hand, we examined the use of IL-2 with covalent crosslinking activity in another inflammatory disease, graft versus host disease. We found that the survival time and weight retention of mice can be significantly prolonged at both high and low doses in L72-FSY treated mice compared to PBS groups; whereas the wild-type IL-2 group only has a weaker protective effect at low doses. These results further demonstrate that IL-2 with covalent crosslinking activity has a more desirable efficacy and stability than wild-type IL-2 in ameliorating the progression of inflammatory disease (fig. 11B).
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Sequence listing
<110> Beijing synergetic Hospital at the national academy of medical science
BEIJING Hospital
Beijing University
<120> Interleukin-2 of covalently crosslinkable CD25 and its use in the treatment of autoimmune diseases
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<170> SIPOSequenceListing 1.0
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<211> 133
<212> PRT
<213> Homo sapiens
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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
Claims (5)
1. Human interleukin-2 covalently crosslinkable with CD25, characterized in that one or both of the amino acids in position L72 and R38 shown in SEQ ID No.1 are mutated to unnatural amino acids, which are
(I) The unnatural amino acid Fluoosulfate-L-tyrosine is shown.
2. The human interleukin-2 covalently cross-linkable to CD25 of claim 1, wherein said human interleukin-2 covalently cross-linkable to CD25 is modified by a PEG modifier of different molecular weight.
3. The human interleukin-2 covalently cross-linkable with CD25 of claim 2, wherein the PEG modifier is acetaldehyde-methoxy PEG, propionaldehyde-methoxy PEG, succinimidyl ester-methoxy PEG, maleimide-methoxy PEG; the molecular weight of the PEG is 5kDa,10kDa,20kDa,30kDa, or 40kDa.
4. A pharmaceutical composition comprising a therapeutically effective amount of human interleukin-2 covalently cross-linkable to CD25 according to any one of claims 1-3 and a pharmaceutically acceptable carrier.
5. Use of human interleukin-2 covalently crosslinkable with the CD25 receptor according to any one of claims 1-3 for the preparation of a medicament for the biased and long-acting activation of regulatory T cells (Treg), the treatment of autoimmune diseases caused by loss of Treg function or number, said autoimmune diseases being graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus, autoimmune disease diabetes mellitus, dermatomyositis, scleroderma, multiple sclerosis, myasthenia gravis, demyelinating diseases, primary adrenocortical atrophy, chronic thyroiditis, chronic non-specific ulcerative colitis, chronic active hepatitis, cachexia and atrophic gastritis, autoimmune glomerulonephritis, pulmonary hemorrhagic syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia and/or autoimmune alopecia.
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