Patent Application
20250223329
This application claims priority to Chinese Patent Application No. 202211415261.0, filed on Nov. 11, 2022, which is hereby incorporated by reference in its entirety.
The present disclosure belongs to the technical field of biomedicine, and specifically, relates to a bifunctional fusion protein composed of IL-15 and an antibody against a T cell co-stimulatory molecule.
Interleukin-15 (IL-15) is a 4 alpha-helical glycoprotein composed of 114 amino acids and has a molecular weight of 14-15 kDa [1]. IL-15 is mainly secreted by dendritic cells (DCs), macrophages and monocytes and acts on other cells. Receptors for IL-15 include IL-15Rα (CD215), IL-2Rβ (CD122) and IL-2Rγ (CD132). CD215 is mainly expressed on the surface of DCs, macrophages and monocytes, and IL-15, after secreted by cells, can directly bind to CD215 and be trans-presented to T cells and NK cells expressing IL-2Rβ and IL-2Rγ. After binding to the receptors, IL-15 can phosphorylate downstream STAT3 and STAT5 and activate JAK1 and JAK3 signal pathways [2-4].
IL-15 mainly promotes the proliferation and activation of T cells and NK cells and maintains the homeostasis of lymphocytes. IL-15 can also maintain the survival of memory CD8+T cells and effector T cells and prevent T cells from producing activation-induced cell death (AICD) [5]. IL-15 does not bind to IL-2Rα and does not induce the proliferation of regulatory T cells (Tregs).
In tumor therapy, IL-15 monomer is greatly limited in clinical use due to its short half-life and weak activity. Because IL-15 needs to bind to IL-15Rα for stronger affinity, the complex form of IL-15 and IL-15Rα has become the main use form in the current clinical research. In addition, N72D, an IL-15 mutant, can further enhance the activity of IL-15 [6]. By enhancing IL-15′ half-life and activity, the anti-tumor activity of IL-15 can be improved to a certain extent, but the anti-tumor effect is extremely weak when it is used as monotherapy. Therefore, IL-15 is currently more used in combination therapies with other therapies, such as PD-1 antibody, CTLA-4 antibody, CD20 antibody and CD52 antibody. Because NK cells in peripheral tissues express more IL-2Rβ, IL-15 preferentially binds to peripheral NK cells. These forms of IL-15 are not selective for proliferation and activation of NK cells and T cells, causing significant proliferation of peripheral T cells, especially NK cells, and the enhancement of its activity and half-life also brings an increase in side effects. Many studies have shown that the toxicity caused by IL-15 is mainly caused by peripheral NK cells [7].
To solve the problem of peripheral toxicity caused by IL-15, prior studies mainly focus on fusion of IL-15 with other antibodies or polypeptides, in order to change the targeting of the fusion protein, reduce the peripheral binding of IL-15, increase IL-15's binding in tumor cells, enrich the fusion protein in tumors and allow IL-15 to play an activation role on immune cells. For example, by fusing with IL-15/IL-15Rα, the RGD polypeptide can specifically bind to integrin αvβ3, which is highly expressed in tumor cells and endothelial cells, so that the fusion protein can be enriched in tumors, which allows the drug concentration in tumors to increase and allows IL-15/IL-15Rα to exert the activity [8]. Given that the affinity between IL-15 and IL-15Rα is about 38 pM and the affinity between IL-15 and CD122/CD132 also reaches 1 nM [9, 10], which is close to that between IL-15 and antibody (10−8˜10−10 M), the fusion protein obtained by fusing IL-15 with antibody has targeting, which cannot be determined by antibody, and thus the affinity between IL-15 and its receptor needs to be reduced so as to better target the fusion protein into tumors. Some studies show that IL-15 can directly act on intratumoral T cells and exert its anti-tumor effect by mutating IL-15 to reduce its affinity with the receptors IL-15Rα and CD122/CD132, fusing the IL-15 with a screened PD-1 antibody with high affinity, and targeting the resultant fusion protein into T cells with high PD-1 expression in tumors [11]. In addition to altering the targeting of the fusion protein by mutating IL-15 to reduce affinity, IL-15 can be blocked by linking IL-15 to the extracellular domain of receptor IL-2Rβ through a matrix metalloproteinase-14 (MMP-14)-recognizable substrate sequence, and when the MMP-14-recognizable sequence is cleaved in tumors, IL-15 is released and activates the anti-tumor immune response [12].
With continuous improvement, IL-15 can effectively expand intratumoral CD8+T cells and NK cells, and improve anti-tumor response. However, tumors often exhibit an immunosuppressive state, and thus the expanded immune cells quickly become in an exhausted state or the activity thereof is suppressed by intratumoral immunosuppressive cells, and thus cannot exert the anti-tumor effect. Immunosuppressive cells in the tumor microenvironment (TME) mainly include tumor associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) and Tregs, etc. [13]. How to effectively inhibit immunosuppressive effect of these cells has become a key problem to be solved in the immunotherapy. The intratumoral Tregs inhibit anti-tumor immune response in the following ways: 1) highly expressing CTLA-4 to compete with CD80 and CD86 expressed by DCs; 2) highly expressing CD25 to compete with effector T cells for IL-2; 3) secreting inhibitory cytokines such as TGF-β and IL-10, etc. to inhibit the function of effector T cells; 4) regulating the metabolism level of tryptophan and adenosine to inhibit the function of effector T cells [14]. An increase in the proportion of intratumoral Tregs/CD8+T cells is often associated with poor prognosis. An increase in the number of Tregs is detrimental to the anti-tumor immune response in most cancer types, although it is not the case in some cancer types. Therefore, the effective depletion of intratumoral Tregs without affecting peripheral Tregs can effectively enhance the anti-tumor immune response.
Tregs in the tumor microenvironment often highly express CD25, CTLA-4, ICOS, OX40, CCR4 and CCR8, and antibodies against these molecules are often used to deplete Tregs. Some of these molecules, such as CD25 and CTLA-4, which are highly expressed in intratumoral Tregs, are also transiently highly expressed in effector T cells, and thus when the corresponding antibodies are used to deplete Tregs, effector T cells will be depleted too, which results in a great decrease in anti-tumor effect. Selective depletion of intratumoral Tregs with an antibody against an appropriate marker molecule becomes the key to breaking Tregs-induced immune tolerance.
ICOS, OX40, GITR and 4-1BB, etc. in the tumor necrosis factor receptor superfamily (TNFRSF) members are often constitutively expressed in Tregs and up-regulated in tumors. These molecules are of increasing interest. Initially, antibodies against these targets are often used to activate co-stimulatory molecules to activate T cells. Although some studies have shown that antibodies against these molecules can be used to deplete Tregs, there is no clinical trial for Tregs depletion yet.
4-1BB molecule is constitutively expressed in Tregs and DCs and inducibly expressed in T cells and NK cells. In T cells, TCR stimulation or CD3 signal can induce the upregulation of 4-1BB expression, which, together with TCR signal, synergistically promote the secretion of IL-2 and IFN-γ in T cells and the proliferation of T cells [15]. Further studies have shown that 4-1BB co-stimulation can not only induce effector cells to produce effector factors, but also facilitate the differentiation of memory T cells and effector cells and protect T cells from apoptosis [16]. In tumors, 4-1BB molecule is up-regulated in both T cells and NK cells, especially in Tregs. The function of 4-1BB molecule in Th1 CD4+T cells and Tregs remains unclear. Activation of 4-1BB signal helps to activate NK cells and enhance ADCC and cytotoxic effect thereof [17]. In addition, the high expression of 4-1BB molecule in Tregs makes this molecule an important target for effectively reducing and depleting the intratumoral Tregs, which provides an auxiliary means for immunotherapy. In a mouse model, the use of 4-1BB depletion-type antibody can effectively inhibit tumor growth and deplete intratumoral Tregs, and its effect of depleting Tregs is equivalent to that of CTLA-4 and OX40 antibodies. Meanwhile, after the therapy with 4-1BB antibody, Tregs cells which are not depleted in tumors exhibit a weak inhibitory phenotype [18]. Although some CD8+T cells in tumors also express 4-1BB and the use of 4-1BB antibody may affect the number of this portion of T cells while depleting Tregs, multiple studies have shown that, in a mouse tumor model, the IgG1 isotype 4-1BB antibody has a far less anti-tumor effect than the IgG2a isotype antibody, indicating that the depletion of Tregs is superior to the activation of T cells using 4-1BB antibody in the generation of anti-tumor immune response [19, 20].
On this basis, the present invention is proposed.
The present disclosure first relates to a bifunctional fusion protein composed of IL-15 and an antibody against a T cell co-stimulatory molecule;
Further, the bifunctional fusion protein comprises:
Preferably,
The IL-15 and IL-15R Sushi domain are human or murine proteins,
The present disclosure further relates to a nucleotide fragment encoding the bifunctional fusion protein, or a vector or a host comprising the nucleotide fragment.
The present disclosure further relates to use of the bifunctional fusion protein, or the nucleotide fragment encoding the bifunctional fusion protein, or the vector or the host comprising the nucleotide fragment in:
Preferably, the anti-tumor medicament is a medicament that reduces and depletes intratumoral Treg cells and induces proliferation of CD8+T cells;
The present disclosure has the following beneficial effects:
pEE12.4-IgGκ, containing a signal peptide of mouse IgGκ, for expression of cytokines and antibodies.
The primers used in the experiment were designed by DNAMAN software and synthesized by GENEWIZ Company.
Wild type C57BL/6, BALB/c mice and BALB/c-nude mice were purchased from Beijing Vital River Laboratory Animal Center, China. Unless otherwise specified, all animals used in experiments were female mice aged 8-10 weeks.
The mice were fed in a specific pathogen-free (SPF) barrier environment. The feeding and experimental protocols of animals complied with the relevant regulations of Animal Care and Use Committee of Institute of Biophysics, Chinese Academy of Sciences.
MC38 is a colorectal cancer cell line derived from C57 mice.
CT26 is a colorectal cancer cell line derived from BALB/C mice.
The above cell lines were cultured in DMEM complete medium (containing 10% of inactivated fetal bovine serum, 2 mmol/l of L-glutamine, 0.1 mmol/l of non-essential amino acids, 100 U of penicillin and 100 μg/ml of streptomycin).
TIB-210TM hybridoma cell line (ATCCTIB-210), for expressing the depletion antibody for CD8+T cells (clone: 2.43).
TIB-207TM hybridoma cell line (ATCC-TIB-207), for expressing the depletion antibody for CD4+T cells (clone: GK1.5).
HB-197TM hybridoma cell line (ATCC-HB-197), for expressing an antibody blocking FcγRII/III in mice (clone: 2.4G2).
FreeStyle™ 293F cell line (Invitrogen), a suspension cell line, derived from HEK293 cell strain, cultured in SMM293-TII or CD OptiCHO™ medium, and mainly for transient transfection and expression of the fusion protein.
CTLL-2 cell line, a murine T cell line, for detecting the biological activity of IL-2.
The above cell lines were cultured in RPMI1640 complete medium (containing 10% of inactivated fetal bovine serum, 2 mmol/L of L-glutamine, 0.1 mmol/L of non-essential amino acids, 100 U of penicillin, 100 μg/ml of streptomycin and 100 IU/ml of recombinant IL-2).
The mouse IL-15 has an amino acid sequence as shown in SEQ ID NO.7, and the mouse IL-15R Sushi domain has an amino acid sequence as shown in SEQ ID NO.8;
Light chain variable region+light chain constant region (light chain) of human or murine 4-1BB antibody;
Fc region (CH2+CH3) of human IgG1 or an Fc region variant of human IgG1 with ADCC effect knocked out.
5×105 MC38 single cells were suspended in 100 μL of PBS and inoculated subcutaneously into C57BL/6 mice on the back.
When the mice with regressed tumors were re-challenged with the same type of tumor cells, the number of the inoculated tumor cells was five times that in the initial tumor modeling, and the site of inoculation was subcutaneous on the other side of the back of the mice. The tumor size was monitored twice a week, by using a vernier caliper to measure the long diameter (a), short diameter (b) and height (c) of the tumor, and the mouse tumor volume=a×b×c/2.
The antibody or the antibody fusion protein was injected intraperitoneally, but also intratumorally in some experiments. The specific administration dosage will be described in the specific experiments.
The CD4+ T cell depletion-type antibody GK1.5, the CD8+ T cell depletion-type antibody TIB210 and the FcRII/III blockade antibody used in the experiments were all derived from corresponding hybridoma cells (TIB-210TM, TIB-207TM and HB-197TM), and were produced and purified in our laboratory.
Depletion of CD4+T Cells and CD8+T Cells:
The day before the treatment with the fusion protein, 200 μg of GK1.5 or TIB210 antibody were injected intraperitoneally to deplete CD4+ T cells or CD8+ T cells, and then injected every 4 days. The number of injections was adjusted based on the treatment period. The depletion efficiency was determined by flow cytometry.
FTY720 (purchased from Sigma Company) is an immunosuppressant that can reduce the migration of T cells from lymphoid organs to peripheral blood circulation.
In the present disclosure, FTY720 blockade was performed at different periods of mouse tumor inoculation to change the tumor microenvironment. Blockade was performed during the treatment of tumor in mice: 20 μg of FTY720 was injected intraperitoneally one day before tumor treatment, and then 10 μg was injected intraperitoneally every other day. The blocking time was determined based on the treatment period, which resulted in no T cells newly migrating into the tumor tissue during tumor treatment. With the FTY720 blockade regimen, the importance of infiltrating lymphocytes in tumor tissue could be studied.
Unless otherwise specified, the molecular structure of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein in the following Examples 1-5 is shown in
A human IgG1 isotype Anti-4-1BB antibody was used to verify the antibody function in the MC38 tumor model. Compared with the untreated control group, the antibody treatment group could effectively control the growth of small tumors (
C57BL/6 mice were inoculated with 5×105 MC38 tumor cells subcutaneously, and the tumor-bearing mice were treated in different groups (n=5/group) on Day 7 or 12 by intraperitoneally injecting (ip) 200 μg of the Anti-4-1BB antibody every three days for a total of three times.
The results showed that the treatment by administering the antibody alone had a good anti-tumor effect only on small tumors.
Given the fact that the human IgG1 isotype 4-1BB antibody can effectively deplete the intratumoral Tregs cells, which are a type of intratumoral immunosuppressive cells that have a great inhibitory effect on the function of T cells, while the Anti-4-1BB antibody cannot treat advanced tumors, the function of T cells needs to be further exerted. The depletion of Tregs relieved the inhibition of T cells, but the number of T cells did not change significantly. To effectively expand CD8+T cells, IL-15 was used as a helper to expand T cells. To verify the expansion effect of IL-15, 10 μg and 50 μg of sIL-15, i.e., the fusion protein in the IL-15-IL15Ra-Fc form, was used to treat MC38 tumor-bearing mice every 3 days for 3 consecutive times. Spleens and tumor tissues of mice were collected on Day 2 post the second injection to detect the number and proportion of immune cells. ALT levels in peripheral blood of mice were detected on Day 7 post the last injection.
The results showed that the mice in the 50 μg-dose treatment group had a significant difference in tumor volume compared with the untreated group, while the mice in the 10 μg-dose treatment group had no significant difference in tumor volume compared with the untreated group (
IL-15 has a good expansion effect on T cells, but Tregs cells have a strong inhibitory effect on the proliferation of T cells. To detect whether the expansion of IL-15 on T cells can overcome the inhibitory effect of Tregs in vitro, the anti-CD3 antibody was used to stimulate CFSE-labeled CD8+T cells in vitro, 1 μg/mL of sIL-15 was added, and different proportions of Tregs were added simultaneously. The CFSE intensity change in CD8+T cells and the proliferation proportion of T cells were detected post 3 days of incubation.
The results showed that the addition of IL-15 could not overcome the inhibitory effect of Tregs on the proliferation of CD8+T cells. Compared with the groups with no Tregs added, the proliferation proportion of T cells was reduced post the addition of Tregs (
Given that the 4-1BB antibody and IL-15 per se have a limited therapeutic effect, but are complementary to each other in function, we combined both to test whether they are synergistic in anti-tumor effect. MC38 tumor-bearing mice were given the combination therapy from Day 13 post tumor inoculation, by injecting intraperitoneally 200 μg of the Anti-4-1BB antibody and 15 μg of sIL-15 every 3 days for 3 consecutive times, and then measured for the tumor volume.
The results showed that the combination of the Anti-4-1BB antibody and sIL-15 had a superior effect to that of their respective monotherapy and had a synergistic anti-tumor effect (
Although the combination therapy of the Anti-4-1BB antibody and sIL-15 has a synergistic anti-tumor effect, sIL-15 treatment causes peripheral toxicity. Taking this into consideration, it is aimed to design a fusion protein that can both act as an antibody to deplete the intratumoral Tregs and expand the intratumoral T cells with sIL-15, and make IL-15 exert activity within tumors as much as possible, without expanding immune cells in the periphery so as to avoid peripheral toxicity.
We designed a fusion protein by linking IL-15-IL-15Rα to the antibody at the carboxyl terminal by a short linker, which blocks the activity of IL-15. When the short linker is replaced with a substrate sequence that can be recognized by highly expressed MMP in tumors, the fusion protein can be cleaved within tumors to release IL-15 activity. Each molecule comprises a complete heavy chain of the Anti-4-1BB antibody and the IL-15-IL-15Rα molecule, with an MMP substrate sequence there between (
As the MMP substrate sequence in the fusion protein can be cleaved by MMP2, MMP9 or MMP14, the fusion protein and the activated MMP14 enzyme were co-incubated in vitro, and then subjected to SDS-PAGE analysis to verify in vitro cleavage of the fusion protein. The results showed that the fusion protein could be effectively cleaved in vitro (
The activity of IL-15 in the fusion protein is well blocked. To further verify whether the fusion protein expand immune cells in peripheral tissues of tumor-bearing mice, 100 μg of the fusion protein or an equimolar amount of the Anti-4-1BB antibody and sIL-15 mix was injected intraperitoneally every three days. The numbers of CD4+ T cells, CD8+ T cells and NK cells in peripheral blood were measured on Day 2 post the second treatment. The results showed that, compared with the mix treatment group, the fusion protein did not cause substantial expansion of T cells and NK cells in peripheral blood and had good safety (
C57BL6 mice were inoculated with 5×105 MC38 tumor cells subcutaneously, and were given treatment from D13 post tumor inoculation, by intraperitoneally injecting 10, 50 and 200 μg of the fusion protein, respectively. The treatments were performed on D13, D16 and D19, and the tumor size was measured twice a week.
The results showed that the therapeutic effect of the fusion protein was gradually improved with the increase of therapeutic dose of the fusion protein (
Meanwhile, the weight of the mice was measured every 1-2 days before and after treatment to verify the effect of the fusion protein on the weight of the mice.
The results showed that the weight of the mice did not change significantly with the increase of the used dose (
In addition, we compared the anti-tumor effect of 50 μg of the fusion protein, an equimolar amount of the Anti-4-1BB antibody and sIL-15 mix, and the fusion protein in which the MMP sequence was replaced with a 2×G4S linker. The results showed that the fusion protein had a therapeutic effect superior to that of the mix therapy and the fusion protein without the MMP-recognizable substrate sequence sequence (
Further, in the treatment group with the fusion protein, the tumor re-challenge experiment was performed two months post tumor elimination. In the treatment group with the fusion protein, the tumor re-challenge experiment was performed two months post tumor regression in the mice in which the tumors were completely eliminated. Three times the dose (1.5×106) of MC38 tumor cells were inoculated subcutaneously, and only the tumor cells were inoculated in the re-challenge experiment. After inoculation, all groups were no longer given treatment.
The results showed that, compared to the control group, re-inoculation of three times the dose of MC38 cells did not cause tumor growth, indicating that the treatment with the fusion protein induces the production of memory immune cells in mice (
To explore which population of cells mediates the anti-tumor function of the fusion protein, mice with Rag1 gene knocked out were used. Due to the lack of Rag gene, TCR and BCR cannot rearrange in such mice, and thus the T and B cells cannot develop into mature stage, resulting in lack of T and B lymphocytes.
Rag1−/− mice were inoculated with 5×105 MC38 tumor cells subcutaneously, and 50 μg of the fusion protein was injected intraperitoneally on Days 12, 15 and 18 to verify whether the therapeutic effect of the fusion protein depends on innate immune cells or adaptive immune cells. The fusion protein was found to have no anti-tumor effect in Rag1−/− mice which had received the treatment (
We further verified the role of T cells in the treatment with the fusion protein.
The treatment was initiated on Day 12 post subcutaneous inoculation of 5×105 MC38 tumor cells into C57BL/6 mice on the back, by intraperitoneally injecting 50 μg of Anti-4-1BB-MMP-IL-15-IL15Ra (administration on Days 12, 15 and 18 post tumor inoculation). One day before the treatment, 200 μg of a CD4T cell-depleting antibody (Clone No. GK1.5, prepared in our laboratory) and 200 μg of a CD8T cell-depleting antibody (Clone No. TIB210, prepared in our laboratory) were injected intraperitoneally for a total of four times.
CD8+T cells and CD4+T cells were depleted by the anti-CD8 and anti-CD4 monoclonal antibodies, respectively, and the depletion efficiency was detected. The results showed that the antibodies were effective in depleting T cells in peripheral blood (
To further explore whether the fusion protein-dependent CD8+T cells that perform their roles are the population of T cells existing in tumors per se, or the T cells outside tumors which are activated by the fusion protein and then migrate into tumors, FTY720, an inhibitor that inhibits T cell migration from lymph nodes to the periphery, was used to block the T cell migration from lymph nodes to tumors, thereby maintaining the stability of intratumoral T cells.
The experimental protocols were as follows:
C57BL/6 mice were inoculated with 5×105 MC38 tumor cells subcutaneously on the back, and then were intraperitoneally injected with 50 μg of the fusion protein on Days 12, 15 and 18 post tumor inoculation. In the treatment group with FTY720, FTY720 was injected intraperitoneally at 15 μg on Day 11 post tumor inoculation and then was injected at 10 μg on Days 13, 15, 17, 19 and 21, respectively.
The treatment with the fusion protein remained effective post FTY720 blockade (
4. Protection Effect of the Treatment with Anti-4-1BB-MMP-IL-15-IL15Ra Fusion Protein on Distal Metastatic Tumors
To verify whether the immunoprotection produced from the treatment of tumors in situ also has an inhibitory effect on tumors at other metastatic sites, we conducted an experiment in a double-tumor mouse model. The mice were inoculated subcutaneously with tumor cells on both the left and right sides and only one side was treated in situ to observe whether the distal tumor can also be controlled.
The experimental protocols were as follows:
C57BL6 mice were inoculated with 5×105 MC38 tumor cells on the right side of the back and received intratumoral local treatment with 30 μg of the fusion protein on Days 13, 16 and 19 post tumor inoculation. On Day 13, the mice were inoculated with 1×106 MC38 tumor cells on the left side. The tumor volume on both sides was measured twice a week.
The results showed that the treatment in situ of the right tumor only by intratumoral injection could not only eliminate the tumor in situ, but also control the untreated distal tumor on the opposite side (
In addition, a B16F10 mouse tumor model was used to further verify the therapeutic effect of the treatment with the fusion protein on pulmonary metastatic tumor nodules during melanoma metastasis.
The experimental protocols were as follows:
C57BL6 mice were inoculated with 5×105 B16F10 tumor cells on the right side of the back and received intratumoral local treatment with 30 μg of the fusion protein on Days 9, 12 and 15 post tumor inoculation, and 15 μg of FTY720 was injected intraperitoneally every 2 days from Day 8 post tumor inoculation until the end of experiment. On Day 8 post tumor inoculation, 5×105 B16F10 tumor cells were injected into the tail vein of each mouse intravenously. On Day 10 post the third treatment, the mice were perfused, the lungs of the mice were taken, and the melanoma metastatic tumor nodules in the lung were counted to verify the therapeutic effect of the treatment with the fusion protein on the metastatic tumor nodules.
The results showed that the intratumoral treatment with the fusion protein had a significant anti-tumor effect on the subcutaneous tumors in situ and could partially eliminate the tumors in situ, and the blockade of the T cell migration by FTY720 did not affect the control of the tumors in situ (
The human IgG1 isotype Anti-4-1BB antibody can effectively deplete intratumoral Tregs, and IL-15 can expand CD8+T cells. To verify the function of the fusion protein in vivo, MC38 tumor-bearing mice were treated on Days 13 and 16 post tumor inoculation by intraperitoneally injecting 50 μg of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein. The tumor tissues were taken on Day 18, and subjected to flow cytometry for the proportion of intratumoral Tregs and CD8+T cells. By the flow cytometry analysis, we found that Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein could effectively deplete intratumoral Treg cells and increase the proportion of CD8 T cells, thus having the best therapeutic effect (
The analysis of intratumoral lymphocytes in MC38 tumor-bearing mice showed that Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein has the function of depleting Tregs. To verify whether Treg depletion is the main mechanism by which Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein exerts anti-tumor effect, we constructed Anti-4-1BB-MMP-IL-15-IL15Ra-no ADCC antibody by site mutation in the Fc region. The mutated Fc does not bind to FCγR receptor and thus loses the ADCC or ADCP-mediated depletion function, but still retains the expansion effect of IL-15 on immune cells. A MC38 tumor model was used. C57BL6 mice were inoculated with MC38 tumor cells and received the treatment on Day 13 post inoculation. 50 μg of antibodies with wt Fc or with no ADCC Fc was injected intraperitoneally every three days for a total of three times.
The results showed that, after tumor inoculation, the therapeutic effect of the antibody with wt Fc was compared with that of the antibody with no ADCC Fc, and it was found that the mutated Fc reduced the therapeutic effect of the fusion protein (
1. Up-Regulation of PDL1 in Intratumoral Immune Cells Post the Treatment with the Fusion Protein
When MC38 tumor-bearing mice are treated, the tumor can be effectively controlled, but cannot be eliminated in some mice. Where the tumor is relatively large, the treatment with the fusion protein alone can only control the tumor growth. The treatment with the fusion protein often activates intratumoral immune cells and the improvement of activation degree often results in a negative feedback of the immune system, causing up-regulated expression of immunosuppressive checkpoint molecules, and thus inhibiting the activation of immune cells. To better improve the therapeutic effect, the mice, upon treatment with the fusion protein, were detected for the expression of PD-1 and PD-L1 in intratumoral immune cells, for the purpose of finding an appropriate immune checkpoint blockage antibody, which is used as a combined therapy to observe whether the therapeutic effect can be improved.
Protocols: C57BL/6 mice were inoculated with 5×105 MC38 tumor cells subcutaneously on the back, and received the treatment with 50 μg of the fusion protein on Days 12 and 15. Tumor tissues of the mice were taken on Day 18 to analyze the expression of PD-1 and PD-L1 in intratumoral immune cells.
The results showed that, the mice, upon treatment, had significantly increased PD-L1 expression in intratumoral CD45+ immune cells, but significantly decreased PD-1 expression in CD8+T cells (
Protocols: C57BL/6 mice were inoculated with 1×106 MC38 tumor cells subcutaneously on the back, 200 μg of the aPDL1 antibody was injected intraperitoneally on Days 13 and 16, and 50 μg of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein was injected intraperitoneally on Days 13, 16 and 19.
The results showed that, for advanced tumors, the PD-L1 antibody could further improve the therapeutic effect of the fusion protein and overcome immune tolerance.
Finally, it should be noted that the above examples are intended only to assist those skilled in the art in understanding the essence of the present disclosure and not intended to limit the scope of the present disclosure.
The above studies had proved that the combination therapy of Anti-4-1BB antibody and sIL-15 has a synergistic anti-tumor effect. Given that both OX40 and 4-1BB are members of TNF superfamily and also a T cell activating factor, it was speculated that the Anti-OX40 antibody, upon fusion with sIL-15, might also produce synergistic anti-tumor effect. Here, we once again designed a fusion protein by linking IL-15-IL-15Rα to the immunoglobulin Fc at the carboxyl terminal through a short substrate sequence which can be recognized by MMP highly expressed in tumors between the Fc and IL-15, so that the fusion protein can be cleaved in tumors to release IL-15 activity. Each molecule comprises a complete Fc of the antibody, an IL-15-IL-15Rα molecule, and an MMP substrate sequence between the antibody and the cytokine. The fusion protein and the activated MMP14 enzyme were co-incubated in vitro, and then subjected to SDS-PAGE analysis to verify the in vitro cleavage of the fusion protein. The results showed that the fusion protein could be effectively cleaved in vitro (
CTLL2 cells were cultured in 1640 complete medium containing 100 U/ml of commercial recombinant IL2 cytokine for 24 hours, washed with the complete medium without IL2 for 2˜3 times, and then were diluted to 2×104/ml; samples of sIL15-Fc, Fc-MMP14-sIL15 (not cleaved by MMP14) and Fc-MMP14-sIL15 (cleaved by MMP14) were diluted in the complete culture medium without IL2 at a 5-fold dilution from the initial concentration of 5 μg/ml, with 10 dilution gradients; 100 μl of the cell suspension and 100 μl of the sample were added into a 96-well cell culture plate, and pipetted homogenously by a pipette tip; the cells were cultured for 72 hours, added with 20 μl of CCK8, and then cultured for further 3 hours; OD values at 450 nM and 630 nM were measured by a microplate reader.
The results showed that: (1) Fc-MMP14-sIL-15 (−) had a biological activity which was about 300 times different from that of sIL15Fc, indicating that Fc-MMP14-sIL15 had a very low biological activity when it was not cleaved by MMP14; (2) Fc-MMP14-sIL-15 (+) had a biological activity which was comparable to that of sIL15Fc, indicating that Fc-MMP14-sIL15 had a biological activity which was restored to the level of sIL15-Fc when it was cleaved by MMP14 (
C57BL/6 mice were inoculated with 5×105 MC38 tumor cells subcutaneously until the tumor grew to 100 mm3 and treated with 15 μg of αOX40, 15 μg of sIL15-Fc or 30 μg of αOX40-MMP14-sIL15 every three days for 3 times, and PBS was given for the control group in the same way. The tumor volume was measured (volume=length×width×height/2).
The results showed that the tumor was well controlled by intraperitoneal administration of αOX40-MMP14-sIL15. This indicates that the fusion protein has a better anti-tumor effect than an equimolar mass of αOX40 and sIL15-Fc administered by combination (
To verify that humanized Anti-4-1BB-MMP-IL-15-IL15R fusion protein had a similar anti-tumor effect, the fusion protein was constructed with the heavy chain of the antibody capable of recognizing human 4-1BB molecule and human IL-15-IL-15Rα in the same way. Two fusion proteins comprising linkers of different lengths, G4S-MMP-G4S and GS-MMP-GGS, were constructed, and then subjected to SDS-PAGE analysis (
The MMP substrate sequence in the humanized fusion protein can be cleaved by MMP2, MMP9 or MMP14. The fusion protein and the activated MMP14 enzyme were co-incubated in vitro, and then subjected to SDS-PAGE analysis to verify in vitro cleavage of the fusion protein. The results showed that the fusion proteins with linkers of different length could be effectively cleaved in vitro (
The activity of IL-15 in humanized fusion protein is well blocked. To further verify whether the fusion protein will expand human immune cells in peripheral tissues of tumor-bearing PBMC mice, 100 μg of the fusion protein or an equimolar amount of Anti-4-1BB antibody and sIL-15 mix was injected intraperitoneally every three days, and the numbers of CD4+T cells, CD8+T cells and CD3+T cells in peripheral blood were measured on Day 2 post the second treatment. The fusion protein with GS-MMP-GGS as linker is represented as Human GS, the fusion protein with G4S-MMP-G4S as linker is represented as Human G4S, and the mix treatment group is represented as Abs+sIL-15. The results showed that, compared with the mix treatment group, the fusion protein did not substantially expand T cells in peripheral blood and had good safety (
NSG mice are immunodeficient mice obtained by knockout mutations of Prkdc gene and Il2rg gene, which lack mature T, B and NK cells, do not produce immunoglobulin, and have dendritic cells (DCs) having abnormal function, and are very suitable for transplantation of human cells or tissues. NSG mice aged 6-8 weeks were inoculated with 1×106 CFPAC1 cells subcutaneously on the right side of the back on DO and were inoculated with 5×106 PBMC cells by tail vein injection after 7 days. The mice were grouped based on the tumor size on Day 7 post inoculation of PBMC cells, and the tumor-bearing hu-PBMC mouse model was established.
100 μg of the fusion protein and an equimolar amount of Anti-4-1BB antibody and sIL-15 mix were injected intraperitoneally, respectively. The mice were treated on D14, D17 and D20 for three times, and measured for tumor size and weight twice a week. The results showed that, the fusion protein had a therapeutic effect superior to that of the control group (
| Number | Date | Country | Kind |
|---|---|---|---|
| 202211415261.0 | Nov 2022 | CN | national |
