WO2025129454A1 - Bifunctional fusion protein composed of il-15 and t cell co-stimulatory molecule antibody - Google Patents

Abstract

Provided is a bifunctional fusion protein composed of IL-15 and a T cell co-stimulatory molecule antibody. The T cell co-stimulatory molecule is 4-1BB, ICOS, or OX40, preferably 4-1BB. The fusion protein comprises: (1) a T cell co-stimulatory molecule antibody; (2) a conjugate of IL-15 and an IL-15R Sushi domain, wherein the IL-15 and the IL-15R Sushi domain are linked by means of a second linker fragment; and (3) a first linker fragment, wherein the first linker fragment is used for linking a heavy chain Fc region of the T cell co-stimulatory molecule antibody and the conjugate of IL-15 and the IL-15R Sushi domain, and can be cleaved by matrix metalloproteinases.

Description

Bifunctional fusion protein composed of IL-15 and T cell co-stimulatory molecule antibody Technical Field

The present invention belongs to the field of biomedicine technology, and specifically relates to a bifunctional fusion protein consisting of IL-15 and T cell co-stimulatory molecule antibody.

Background Art

Interleukin-15 (IL-15) is a glycoprotein with four α-helices, composed of 114 amino acids and a molecular weight of 14-15 kDa[1]. IL-15 is mainly secreted by dendritic cells (DC), macrophages and monocytes, and acts on other cells. The receptors of IL-15 include IL-15Rα (CD215), IL-2Rβ (CD122) and IL-2Rγ (CD132). CD215 is mainly expressed on the surface of DC, macrophages and monocytes. After being secreted by cells, IL-15 can directly bind to CD215 and be trans-presented to T cells and NK cells expressing IL-2Rβ and IL-2Rγ. After IL-15 binds to the receptor, it can phosphorylate downstream STAT3 and STAT5, activating the JAK1 and JAK3 signaling 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 (Treg).

In tumor treatment, the 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 to have a strong affinity, the complex form of IL-15 and IL-15Rα has also become the main form used in clinical research. In addition, the IL-15 mutant N72D can further improve the activity of IL-15[6]. By increasing the half-life and activity of IL-15, the anti-tumor activity of IL-15 is improved to a certain extent, but when used as a single drug, the anti-tumor effect is extremely weak . Therefore, IL-15 is currently used more in combination with other treatment methods, such as PD-1 antibodies, CTLA-4 antibodies, CD20 antibodies, and CD52 antibodies. Since NK cells in peripheral tissues express more IL-2Rβ, IL-15 preferentially binds to peripheral NK cells. These forms of IL-15 have no selectivity for the proliferation and activation of NK cells and T cells, causing a large number of peripheral T cells, especially NK cells, to proliferate. The increase in their activity and half-life also leads to an increase in side effects . Many studies have shown that the toxicity caused by IL-15 is mainly caused by peripheral NK cells[7].

In order to solve the problem of peripheral toxicity caused by IL-15, existing studies have mainly fused IL-15 with other antibodies or peptides to change the targeting of the fusion protein, reduce the binding of IL-15 in the periphery, increase its binding to cells in the tumor, and make the fusion protein enriched in the tumor, thereby exerting the activation effect of IL-15 on immune cells. For example, by fusing the RGD peptide sequence with IL-15/IL-15Rα, the RGD peptide can specifically bind to the integrin αvβ3 highly expressed by tumor cells and endothelial cells, so that the fusion protein can be enriched inside the tumor, increase the drug concentration in the tumor, and exert the activity of IL-15/IL-15Rα[8]. Considering that the affinity of IL-15 to IL-15Rα is about 38pM, and the affinity of IL-15 to CD122/CD132 is also 1nM[9,10], which is close to the affinity of antibodies ( ^10-8 to ^10-10M ) , after fusion of IL-15 and antibodies, the targeting of the fusion protein cannot be determined by the antibody. It is necessary to reduce the affinity between IL-15 and the receptor in order to better target the fusion protein to the tumor . Some studies have mutated IL-15 to reduce its affinity to the receptors IL-15Rα and CD122/CD132, and screened for high-affinity PD-1 antibodies. The PD-1 antibodies were fused with IL-15, and the fusion protein was targeted to T cells in the tumor that highly expressed PD-1. IL-15 can directly act on T cells in the tumor and exert anti-tumor effects[11]. In addition to changing the targeting of fusion proteins by reducing the affinity of IL-15 by mutating it, the extracellular domain of the receptor IL-2Rβ can also be used to block IL-15 and connect the extracellular domain to IL-15 through a substrate sequence that can be recognized by matrix metalloproteinase-14 (MMP-14). The MMP-14 recognition sequence is cleaved in the tumor, releasing the activity of IL-15 and activating the anti-tumor immune response [12] .

After continuous improvement, IL-15 can effectively amplify CD8+T cells and NK cells in tumors and enhance anti-tumor responses. However, tumors often show an immunosuppressive state, and proliferating immune cells quickly become exhausted or their activity is suppressed by immunosuppressive cells in the tumor, making them unable to exert anti-tumor effects. Immunosuppressive cells in the tumor microenvironment (TME) mainly include tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC) and Tregs [13]. How to effectively inhibit these cells from exerting immunosuppressive effects has also become a key issue that needs to be urgently addressed in immunotherapy. Tregs in tumors mainly inhibit anti-tumor immune responses in the following ways: 1) High expression of CTLA-4 competes for DC expression 1) CD80 and CD86; 2) High expression of CD25 to compete with effector T cells for IL-2; 3) Secretion of inhibitory cytokines such as TGF-β and IL-10 to inhibit the function of effector T cells; 4) Regulating the metabolic levels of tryptophan and adenosine to inhibit the function of effector T cells [14]. An increase in the ratio of Tregs/CD8+T cells in tumors is often associated with poor prognosis. Although some cancer types are more special, in most cancer types, an increase in the number of Tregs is not conducive to anti-tumor immune response. Therefore, effectively eliminating Tregs in tumors without affecting peripheral Tregs can effectively enhance 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 delete Tregs. Although some of these molecules, such as CD25 and CTLA-4, are highly expressed in Tregs within the tumor, they are also transiently highly expressed on effector T cells. Using the corresponding antibodies to delete Tregs will also delete effector T cells, causing a significant decrease in the anti-tumor effect. Using antibodies against appropriate marker molecules to selectively delete Tregs within the tumor has become the key to breaking the immune tolerance induced by Tregs .

ICOS, OX40, GITR and 4-1BB in the tumor necrosis factor receptor superfamily (TNFRSF) are often constitutively expressed on Tregs and upregulated in tumors. These molecules are gaining increasing attention. Initially, antibodies against these targets were often used to activate co-stimulatory molecules to activate T cells. Although studies have shown that antibodies against these molecules can be used to delete Tregs, there have been no clinical trials for the deletion of Tregs.

4-1BB is constitutively expressed on Tregs and DCs, and inducibly expressed on T cells and NK cells. In T cells, TCR stimulation or CD3 signals can induce upregulation of 4-1BB expression, which synergizes with TCR signals to promote the secretion of IL-2 and IFN-γ by T cells and the proliferation of T cells[15]. Further studies have shown that in addition to inducing effector cells to produce effector factors, 4-1BB co-stimulation is also beneficial for the differentiation of memory T cells and effector cells and protects T cells from apoptosis[16]. In tumors, 4-1BB is upregulated in both T cells and NK cells, especially in Tregs. The function of 4-1BB in Th1CD4 ⁺ T cells and Tregs is still unclear. Activation of 4-1BB signals helps activate NK cells and enhance their ADCC and cytotoxic effects[17]. In addition, the high expression of 4-1BB in Tregs makes it an important target for effectively reducing and eliminating intratumoral Tregs, providing an auxiliary means for immunotherapy . In mouse models, the use of 4-1BB-deleting antibodies can effectively inhibit tumor growth and eliminate Tregs in tumors. The effect of eliminating Tregs is equivalent to that of CTLA-4 and OX40 antibodies. At the same time, after treatment with 4-1BB antibodies, Tregs cells that were not eliminated in the tumor showed a weaker inhibitory phenotype [18]. Although some CD8 ⁺ T cells in tumors also express 4-1BB, the use of 4-1BB antibodies may affect the number of these T cells while deleting Tregs. However, many studies have shown that the anti-tumor effect of IgG1 subtype 4-1BB antibodies in mouse tumor models is far inferior to that of IgG2a subtype antibodies , indicating that the elimination of Tregs is better than the use of 4-1BB antibodies to activate T cells for the generation of anti-tumor immune responses [19,20].

Based on this, the present invention is proposed.

[References]

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[16]SHUFORD W W, KLUSSMAN K, TRITCHLER D D, et al.4-1BB Costimulatory Signals Preferentially Induce CD8+TCell Proliferation and Lead to the Amplification In Vivo of Cytotoxic T Cell Responses[J].Journal of Experimental Medicine,1997,186(1):47-55.

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[18] MAJ T, WANG W, CRESPO J, et al. Oxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumor [J]. Nature Immunology, 2017.

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Summary of the invention

The present invention firstly relates to a bifunctional fusion protein consisting of IL-15 and T cell co-stimulatory molecule antibody;

The T cell co-stimulatory molecules are: 4-1BB, ICOS, OX40; preferably 4-1BB;

The fusion protein comprises:

(1) Antibodies to T cell co-stimulatory molecules;

(2) a conjugate of IL-15 and IL-15R Sushi domain, wherein the IL-15 and IL-15R Sushi domain are connected via a second linker fragment; and

(3) a first connecting fragment; the first connecting fragment is used to connect the heavy chain Fc region of the T cell co-stimulatory molecule antibody and the IL-15 and IL-15R Sushi domain conjugate, and can be cleaved by metal matrix proteases;

Preferably, the T cell co-stimulatory molecule antibody is an IgG1 antibody.

Preferably, the first linking fragment comprises: 2-4 G ₄ S linking units and a short peptide contained therein that can be recognized and cleaved by metal matrix proteases;

More preferably, the amino acid sequence of the short peptide that can be recognized and cleaved by the metal matrix protease is as shown in SEQ ID NO.6;

Most preferably, the amino acid sequence of the first connecting fragment is shown as SEQ ID NO.12 or SEQ ID NO.11;

Preferably, the second connecting fragment comprises: 2-5 GnS connecting units, wherein n is an integer of 1-4; more preferably, the amino acid sequence of the second connecting fragment is as shown in SEQ ID NO.5.

Furthermore, the bifunctional fusion protein comprises:

(1) The first structural unit, from N-terminus to C-terminus, consists of: 4-1BB antibody heavy chain, first linker fragment, IL-15 and IL-15R Sushi domain conjugate;

(2) A second structural unit: a 4-1BB antibody light chain paired with the 4-1BB antibody heavy chain;

Furthermore, the bifunctional fusion protein dimerizes through the Fc region of the 4-1BB antibody heavy chain to form a homodimer.

Preferably,

The 4-1BB antibody is an antibody formed by fusing the Fab region of a human or mouse antibody with the Fc region of IgG1.

The amino acid sequence of the heavy chain (VH+CH1) of the murine 4-1BB antibody is shown in SEQ ID NO.2;

The amino acid sequence of the heavy chain (VH+CH1) of the human 4-1BB antibody is shown in SEQ ID NO.10;

The amino acid sequence of the light chain (VL-CL) of the murine 4-1BB antibody is shown in SEQ ID NO.1;

The amino acid sequence of the light chain (VL-CL) of the human 4-1BB antibody is shown in SEQ ID NO.9;

The IgG1 Fc region is the Fc region of human IgG1, or a human IgG1 Fc region variant with ADCC effect knocked out;

The amino acid sequence of the Fc region of human IgG1 is shown in SEQ ID NO.3, and the amino acid sequence of the Fc region variant of human IgG1 with ADCC effect knocked out is shown in SEQ ID NO.4.

The IL-15 and IL-15R Sushi domains are human or mouse proteins.

The amino acid sequence of the murine IL-15 is shown in SEQ ID NO.7, and the amino acid sequence of the murine IL-15R Sushi domain is shown in SEQ ID NO.8;

The amino acid sequence of the human IL-15 is shown in SEQ ID NO.13, and the amino acid sequence of the human IL-15R Sushi domain is shown in SEQ ID NO.14.

The present invention also relates to a nucleotide fragment encoding the bifunctional fusion protein, a vector containing the nucleotide fragment, and a host.

The present invention also relates to the following applications of the bifunctional fusion protein, or a nucleotide fragment encoding the bifunctional fusion protein, or a vector or host containing the nucleotide fragment:

(1) Preparation of anti-tumor drugs;

(2) Preparation of combined anti-tumor drugs.

Preferably, the anti-tumor drug is a drug that reduces and eliminates intratumoral Treg cells and induces CD8 ⁺ T cell proliferation;

Preferably, the tumor is: a tumor in an immunosuppressive state;

Preferably, the combination anti-tumor drug is: a combination anti-tumor drug used in combination with an immune checkpoint inhibitor; the immune checkpoint inhibitor includes but is not limited to: PD-1/PD-L1 antibody, CTLA4 antibody, TIGIT antibody, LAG-3 antibody, TIM-3 antibody.

The beneficial effects of the present invention are:

(1) IL-15 has a strong ability to expand CD8+ T cells and NK cells, but the expanded T cells do not show high anti-tumor ability. The therapeutic effect of IL-15 can be improved by changing the immunosuppressive state in the tumor;

(2) Based on the ability of 4-1BB antibody to eliminate Tregs in tumors and relieve the inhibitory state of Tregs on effector T cells, the fusion protein bifunctional antibody composed of 4-1BB antibody and IL-15 can simultaneously play the role of eliminating Tregs and expanding CD8+ T cells;

(3) By designing a short substrate sequence between the antibody and IL-15 that can be recognized and cleaved by MMPs that are highly expressed in tumors, the short connecting peptide segment can maintain the activity of IL-15 and avoid peripheral toxicity. At the same time, after the fusion protein enters the tumor, the connecting peptide segment can be cleaved by MMP, releasing IL-15 and restoring its activity, which has great development potential.

(4) The fusion protein can overcome the tolerance of PD-L1 antibody and has a synergistic effect when used in combination with PD-L1 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Anti-4-1BB-hIgG1 antibody can control the growth of small tumors, but has poor therapeutic effect in large tumors. 1A, the inhibitory effect of Anti-4-1BB antibody on the growth of small tumors; 1B, the inhibitory effect of Anti-4-1BB antibody on the growth of large tumors; 1C, the clearing effect of Anti-4-1BB antibody on Tregs in tumors; 1D, flow cytometry diagram of the clearing effect of Anti-4-1BB antibody on Tregs in tumors.

Figure 2. Anti-tumor effects of different doses of IL-15 and their effects on lymphocyte proliferation. 2A. High-dose IL-15 inhibits tumor growth; 2B. IL-15 massively expands peripheral and intratumoral immune cells; 2C. IL-15 treatment causes an increase in ALT levels in the blood.

Figure 3. IL-15 and Anti-4-1BB combined therapy has a synergistic effect. 3A. IL-15 cannot overcome the inhibitory effect of Tregs on CD8+ T cells; 3B. Synergistic anti-tumor effect of Anti-4-1BB antibody and IL-15.

Figure 4. Construction and expression of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein. 4A. Structural model of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein; 4B. SDS-PAGE analysis of the constructed Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein, R is the reducing band, and NR is the non-reducing band.

Figure 5. In vitro and in vivo functional detection of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein. 5A. In vitro cleavage of fusion protein by MMP. Buffer indicates the band after fusion protein was incubated in the cleavage solution without MMP14 for 12 hours (h). MMP 6h or 12h band is the protein band after fusion protein was cleaved by MMP14 for 6h or 12h. 5B. Activation of STAT5 signaling pathway of CTLL-2 cell line by fusion protein and cleaved protein. 5C. The amplification effect of fusion protein on immune cells in peripheral blood.

Figure 6. Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein has a synergistic therapeutic effect. 6A. Tumor inhibitory effect of different doses of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein (MC38 tumor model); 6B. Effect of different doses of fusion protein on mouse body weight after treatment; 6C. Comparison of the therapeutic effects of fusion protein with Mix and fusion protein without MMP (MMP sequence replaced with 2G4S); 6D. Results of re-challenge test in the Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein treatment group (MC38 tumor model).

Figure 7. The therapeutic effect of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein is independent of innate immune cells. The therapeutic effect of the fusion protein on Rag tumor-bearing mice.

Figure 8. The function of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein depends on CD8 T cells. 8A. One day after injection of 200ug of deletion antibody, flow cytometry was used to detect the deletion efficiency of CD4 T cells and CD8 T cells in peripheral blood; 8B. Tumor growth curves of different treatment groups.

Figure 9. Intratumoral T cells play a key role in the treatment of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein, and the therapeutic effect of the fusion protein on FTY720-injected tumor-bearing mice.

Figure 10. Immune responses induced by local treatment of orthotopic tumors can control distant tumor growth. 10A. Tumor treatment effects on the left and right sides (drug administration sides) of different treatment groups in the MC38 model; 10B. Orthotopic tumor treatment effects in the B16F10 tumor model; 10C. Treatment effects on lung metastasis in the B16F10 tumor model

Figure 11. Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein treatment deleted intratumoral Tregs and increased the ratio of CD8T cells to Tregs. 11A, flow cytometry of intratumoral Treg cells in different treatment groups; 11B, the ratio of intratumoral Tregs to CD4+T in different treatment groups; 11C, flow cytometry of intratumoral CD8+T cells in different treatment groups; 11D, the ratio of intratumoral CD8+T to CD45+ cells in different treatment groups.

Figure 12. Antibody function depends in part on the binding of Fc to FcγR.

Figure 13. aPDL1 antibody can synergize with Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein to improve the anti-tumor effect.

Figure 14. Anti-OX40-MMP-IL-15-IL15Ra fusion protein has good anti-tumor effect.

Figure 15. In vitro functional detection of humanized Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein. 15A. SDS-PAGE detection of humanized fusion protein; 15B. In vitro cleavage of fusion protein by MMP, bands after the fusion protein expressed in buffer was incubated in a cleavage solution without MMP14 for a certain period of time, and the MMP 6h or 12h bands were protein bands after the fusion protein was cleaved by MMP14 for 6h or 12h; 15C. Activation of STAT5 signal in HEK-IL2 cell line by fusion protein and cleaved protein and EC50.

Figure 16. Detection of the immune cell proliferation activity of humanized Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein on hu-PBMC mice.

Figure 17. Therapeutic efficacy and safety of humanized Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein in hu-PBMC tumor-bearing mice. 17A. Anti-tumor effect of fusion protein and mixed treatment group; 17B. Body weight change of fusion protein and mixed treatment group.

DETAILED DESCRIPTION

Experimental Materials

1. Bacterial strains and plasmids

Bacteria: Top10E.coli, DH5αE.coli competent cells (Beijing Quanshijin Biotechnology Co., Ltd.)

Plasmids:

pEE12.4-IgGκ, containing the signal peptide of mouse IgGκ, is used for the expression of cytokines and antibodies.

The primers used in the experiment were designed by DNAMAN software and synthesized by Genewise.

2. Experimental Animals

Wild-type C57BL/6, BALB/c mice, and BALB/c-nude mice were purchased from the Weitonglihua Laboratory Animal Center, Beijing, China. Unless otherwise specified, all experiments used female mice aged 8–10 weeks.

All mice were housed in a specific pathogen-free (SPF) barrier environment. Animal husbandry and experimental procedures were in accordance with the relevant regulations of the Animal Management Committee of the Institute of Biophysics, Chinese Academy of Sciences.

3. Cell lines

MC38 is a mouse colorectal cancer cell line with a C57 background.

CT26 is a mouse colorectal cancer cell line on a BALB/C background.

All of the above cell lines were cultured in DMEM complete medium (containing 10% inactivated fetal bovine serum, 2 mmol/l L-glutamine, 0.1 mmol/l non-essential amino acids, 100 U penicillin and 100 μg/ml streptomycin).

TIB-210TM hybridoma cell line (ATCC TIB-210), used to express CD8+ T cell deleting antibody (clone: 2.43).

TIB-207TM hybridoma cell line (ATCC-TIB-207) expressing CD4+ T cell deleting antibody (clone: GK1.5).

HB-197TM hybridoma cell line (ATCC-HB-197), used to express an antibody that blocks mouse FcγRII/III (clone: 2.4G2).

FreeStyleTM 293F cell line (Invitrogen) is a suspension cell line derived from the HEK293 cell line and cultured in SMM293-TII or CD OptiCHOTM medium. It is mainly used for transient transfection to express fusion proteins.

CTLL-2 cell line is a mouse T cell line used to detect the biological activity of IL-2

The above cell lines were cultured in RPMI1640 complete medium (containing 10% inactivated fetal bovine serum, 2 mmol/L L-glutamine, 0.1 mmol/L non-essential amino acids, 100 U penicillin and 100 μg/ml streptomycin, and 100 IU/ml recombinant IL-2).

IL15&IL-15R Sushi

The amino acid sequence of mouse IL-15 is shown in SEQ ID NO.7, and the amino acid sequence of mouse IL-15R Sushi region is shown in SEQ ID NO.8;

The human IL-15 gene sequence is shown in SEQ ID NO.13, and the human IL-15R Sushi region gene sequence is shown in SEQ ID NO.14;

The sIL15 mentioned in the example corresponds to IL15 RA Fc in CN201810420739.6, and its amino acid sequence is shown in SEQ ID NO.15:

Fab region of Anti-4-1BB antibody

Light chain variable region + light chain constant region (light chain) of human or mouse 4-1BB antibody;

Heavy chain variable region + CH1 region (heavy chain) of human or mouse 4-1BB antibody.

Fc region of Anti-4-1BB antibody

The Fc region (CH2+CH3) of human IgG1 or a human IgG1 Fc region variant with ADCC effect knocked out.

Mouse tumor inoculation and treatment

(1) Tumor inoculation and measurement:

Establishment of tumor model,

5 × 10 ⁵ MC38 single cells were suspended in 100 μL PBS and inoculated subcutaneously on the back of C57BL/6 mice;

When the mice with tumor regression were re-challenged with the same tumor cells, the number of tumor cells inoculated was 5 times that of the initial tumor modeling, and the inoculation site was subcutaneous on the other side of the mouse's back. The tumor size was monitored twice a week, and the long diameter (a), short diameter (b) and height (c) of the tumor were measured using a vernier caliper. The mouse tumor volume = a×b×c/2.

(2) Treatment:

The antibody or antibody fusion protein is administered by intraperitoneal injection, and some experiments also use intratumoral administration. The specific dosage will be described in the specific experiment.

Monoclonal antibody preparation (mouse ascites method)

The CD4 ⁺ T cell-deleting antibody GK1.5, CD8 ⁺ T cell-deleting antibody TIB210, and FcRII/III blocking antibody used in the experiment were all derived from the corresponding hybridoma cells (TIB-210TM, TIB-207TM, HB-197TM), produced and purified by our laboratory.

Cell deletion in mice

Deletion of CD4 ⁺ T cells and CD8 ⁺ T cells:

One day before fusion protein treatment, 200 μg of GK1.5 or TIB210 antibody was intraperitoneally injected to delete CD4 ⁺ T cells and CD8 ⁺ T cells, and then injected every 3 days, and the injection frequency was adjusted according to the treatment cycle. The deletion efficiency was detected by flow cytometry.

T cell emigration blockade

FTY720 (purchased from Sigma) is an immunosuppressant that can reduce the migration of T cells from lymphoid organs to peripheral blood circulation.

In the present invention, FTY720 blocking is performed at different stages of mouse tumor inoculation to change the tumor microenvironment. Blocking is performed during mouse tumor treatment: 20 μg FTY720 is injected intraperitoneally one day before tumor treatment, and then 10 μg is injected intraperitoneally every other day. The blocking time depends on the treatment cycle. This will result in no new T cells migrating into the tumor tissue during tumor treatment. With the help of the FTY720 blocking scheme, the importance of lymphocytes infiltrating in tumor tissue can be studied.

Unless otherwise specified, the molecular structures of the Anti-4-1BB-MMP-IL-15-IL15Ra fusion proteins in the following Examples 1-5 are all the molecular structures shown in FIG. 4A .

Example 1: Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein significantly improves the effect of single treatment

1. Anti-4-1BB antibodies are effective against small tumors but not large tumors

We used human IgG1 subtype Anti-4-1BB antibody and verified the function of the antibody in the MC38 tumor model. Compared with the untreated control group, the antibody treatment group was able to effectively control the growth of small tumors (Figure 1A), but the antibody treatment failed to completely eliminate the tumor when the tumor was larger (Figure 1B). Different doses of 4-1BB antibody can eliminate Tregs in the tumor to varying degrees (Figure 1C).

C57BL/6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells. Tumor-bearing mice were divided into groups for treatment starting on the 7th or 12th day (n=5/group): 200 μg Anti-4-1BB antibody was intraperitoneally injected (ip) once every three days for a total of three treatments.

The results showed that antibody treatment alone had a good anti-tumor effect only on small tumors .

2. Anti-tumor effect of different doses of IL-15 and its effect on lymphocyte proliferation

Given that the human IgG1 subtype 4-1BB antibody can effectively delete Tregs in tumors, and Tregs cells are a type of immunosuppressive cells in tumors that have a greater inhibitory effect on T cell function, but the Anti-4-1BB antibody cannot effectively treat advanced tumors, we need to further develop the T Cell function. Considering that the deletion of Tregs relieves the inhibitory effect on T cells, the number of T cells does not change significantly. In order to effectively expand CD8 ⁺ T cells, we chose to use IL-15 as an auxiliary to expand T cells. In order to verify the expansion effect of IL-15, we used two different doses of sIL-15, IL-15-IL15Ra-Fc fusion protein, 10μg and 50μg, to treat MC38 tumor-bearing mice once every 3 days for 3 consecutive times. The spleen and tumor tissues of mice were taken 2 days after the second injection of treatment to detect the number and proportion of immune cells. The level of ALT in the peripheral blood of mice was detected 7 days after the last injection of treatment.

The results showed that the tumor volume of mice in the 50μg treatment group was significantly different from that in the untreated group, while the tumor volume of mice in the 10μg treatment group was not significantly different from that in the untreated group (Figure 2A). Both the low-dose and high-dose groups significantly increased the expansion of CD8 ⁺ T cells in the spleen and tumor (Figure 2B). After IL-15 treatment, the ALT level in the peripheral blood of mice increased significantly (Figure 2C).

3. IL-15 and Anti-4-1BB combined therapy has a synergistic effect

IL-15 has a good proliferation effect on T cells, but Tregs cells have a strong inhibitory effect on T cell proliferation. In vitro, we tested whether IL-15 on T cell proliferation can overcome the inhibitory effect of Tregs. We used anti-CD3 antibodies to stimulate CFSE-labeled CD8 ⁺ T cells in vitro, added 1 μg/mL sIL-15, and added different proportions of Tregs. After incubation for 3 days, we detected the changes in CFSE intensity in CD8 ⁺ T cells and the proliferation ratio of T cells.

The results showed that the addition of IL-15 could not overcome the inhibitory effect of Tregs on the expansion of CD8 ⁺ T cells. Compared with the group without Tregs, the proliferation rate of T cells was reduced after the addition of Tregs (Figure 3A) (the light-colored peak in the figure represents the division generation of CD8 ⁺ T cells. As the number of Treg cells added increased, the division generation of CD8 ⁺ T cells decreased significantly).

Given that 4-1BB antibody and IL-15 have limited therapeutic effects on their own, and the two have complementary functions, we combined the two to test whether they have synergistic anti-tumor effects. Using MC38 tumor-bearing mice, combined treatment began on the 13th day after tumor inoculation, with 200μg Anti-4-1BB antibody and 15μg sIL-15, using intraperitoneal injection, once every 3 days, for 3 consecutive treatments, and the tumor volume was measured.

The results showed that the combined use of Anti-4-1BB antibody and sIL-15 was superior to the monotherapy of either drug and had a synergistic anti-tumor effect ( Figure 3B ).

4. Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein has a synergistic anti-tumor effect

4.1 Construction and expression of fusion protein

The combined treatment of Anti-4-1BB antibody and sIL-15 has a synergistic anti-tumor effect, but considering that sIL-15 treatment can cause peripheral toxicity, how to design a fusion protein that can have both antibody deletion of intratumor Tregs and sIL-15 amplification of intratumor T cells, while trying to maximize the activity of IL-15 inside the tumor without amplifying immune cells in the periphery to avoid peripheral toxicity.

We designed a fusion protein, connecting IL-15-IL-15Ra at the carboxyl end of the antibody, connecting the antibody and IL-15 through a short linker to mask the activity of IL-15. After replacing the short linker with a substrate sequence that can be recognized by MMPs highly expressed in tumors, the fusion protein can be cleaved in the tumor to release IL-15 activity. Each molecule contains a complete Anti-4-1BB antibody heavy chain and IL-15-IL-15Ra molecule, and an MMP substrate sequence is contained between the antibody and the cytokine (Figure 4A) . The light chain of the antibody and the fusion protein plasmid were co-transfected into 293F cells. After seven days, the cell supernatant was collected and the protein was purified with a protein A column after centrifugation. The purified antibody was identified by protein gel. The protein molecular composition was analyzed by SDS-PAGE, which proved that the molecule could express the light chain and fused heavy chain of the Anti-4-1BB antibody (Figure 4B).

4.2. Verification of the function of the fusion protein

(1) In vitro activity detection of fusion protein

The MMP substrate sequence in the fusion protein can be cleaved by MMP2, MMP9 or MMP14. We co-incubated the fusion protein with the activated MMP14 enzyme in vitro, and then performed SDS-PAGE analysis to verify the in vitro cleavage effect of the fusion protein. The results showed that the fusion protein can be effectively cleaved in vitro (Figure 5A). IL-15 often induces the activation of the downstream STAT5 signaling pathway after binding to the receptor. We used the CTLL2-STAT5-Luc cell line and the Anti-4-1BB antibody of the same clone to block the 4-1BB molecules expressed in CTLL-2 cells, and then added different concentrations of fusion protein to stimulate the cells. After 4 hours, the activity of luciferase in the cells was detected. The results showed that the fusion protein had a decreased ability to activate the STAT5 signaling pathway. After in vitro enzyme cleavage, the activation ability was completely restored (Figure 5B), indicating that the activity of IL-15 in the fusion protein was well blocked, and its activity was fully restored after MMP cleavage.

(2) Detection of the proliferation activity of fusion protein on immune cells in mice

The activity of IL-15 in the fusion protein was well blocked. To further verify whether the fusion protein would amplify immune cells in the peripheral tissues of tumor-bearing mice, we intraperitoneally injected 100 μg of fusion protein or a mixture of equimolar amounts of Anti-4-1BB antibody and sIL-15, treated once every three days, and detected the number of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells in the peripheral blood 2 days after the second treatment. The results showed that compared with the mixed treatment group, the fusion protein did not cause a large number of T cells and NK cells in the peripheral blood, and had good safety (Figure 5C).

4.3. Fusion protein has good safety and anti-tumor effect

5×10 ⁵ MC38 tumor cells were subcutaneously inoculated in C57BL6 mice, and treatment was started on D13 after tumor inoculation; 10, 50, and 200 μg of fusion protein were injected intraperitoneally, respectively. Treatment was performed three times on D13, D16, and D19, and tumor size was measured twice a week.

The results showed that as the therapeutic dose of the fusion protein increased, the therapeutic effect of the fusion protein gradually improved ( FIG. 6A ).

At the same time, we measured the body weight of mice every 1-2 days before and after treatment to verify the effect of the fusion protein on the body weight of mice.

The results showed that with the increase of dosage, there was no significant change in the body weight of mice (Figure 6B).

In addition, we compared the anti-tumor effects of 50 μg fusion protein mixed with equimolar amounts of Anti-4-1BB antibody and sIL-15 and fusion protein in which the MMP sequence in the fusion protein was replaced with 2×G ₄ S linker. The results showed that the therapeutic effect of the fusion protein was better than that of the mixed treatment and the fusion protein without MMP sequence (Figure 6C).

Furthermore, in the fusion protein treatment group, a tumor re-challenge experiment was performed two months after tumor clearance. In the fusion protein treatment group, mice with completely cleared tumors were subjected to a tumor re-challenge experiment two months after tumor regression, with five times the dose (1.5×10 ⁶ ) of MC38 tumor cells inoculated subcutaneously, and only tumor cells were inoculated in the re-challenge, and no drug treatment was given to each group after inoculation.

The results showed that compared with the control group, re-inoculation of three times the dose of MC38 cells did not cause tumor growth, indicating that fusion protein treatment induced the production of memory immune cells in mice (Figure 6D).

Example 2: Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein can activate CD8 T cells

1. The therapeutic effect of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein is not dependent on innate immune cells

In order to explore which group of cells mediates the anti-tumor function of the fusion protein, we first used Rag1 knockout mice. Due to the lack of Rag gene, TCR and BCR cannot be rearranged in this mouse, so T and B cells cannot mature and lack T and B lymphocytes.

Rag1-/- mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells, and 50 μg of fusion protein was intraperitoneally injected on days 12, 15, and 18 to verify whether the therapeutic effect of the drug depends on innate immune cells or adaptive immune cells. After treatment, it was found that the fusion protein had no anti-tumor effect in Rag1-/- mice (Figure 7), indicating that the effect of the drug depends on adaptive immune cells.

2. The therapeutic effect of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein depends on CD8 ⁺ T cells

We further verified the role of T cells in fusion protein therapy.

C57BL/6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells on the back and treatment began on the 12th day, with an intraperitoneal injection of 50ug Anti-4-1BB-MMP-IL-15-IL15Ra (administered on the 12th, 15th, and 18th days after tumor inoculation). On the day before treatment, 200μg CD4T cell deleting antibody (clone number: GK1.5, prepared in our laboratory) and 200μg CD8T cell deleting antibody (clone number: TIB210, prepared in our laboratory) were intraperitoneally injected for a total of four times.

We deleted CD8 ⁺ T cells and CD4 ⁺ T cells using anti-CD4 and anti-CD8 monoclonal antibodies, respectively, and tested the deletion efficiency. The results showed that the antibody can effectively eliminate T cells in peripheral blood (Figure 8A). Deleting CD4 ⁺ T cells while using fusion protein treatment does not affect the anti-tumor effect of the drug. After deleting CD8 ⁺ T cells, the anti-tumor effect completely disappeared (Figure 8B).

3. The therapeutic effect of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein depends on CD8 ⁺ T cells in the tumor

In order to further explore whether the CD8+T cells that the drug relies on are the T cells in the tumor itself, or whether the drugs activate T cells outside the tumor and then migrate to the tumor to play a role. We used FTY720, an inhibitor that inhibits the migration of T cells from lymph nodes to the periphery, to block the migration of T cells from lymph nodes to tumors, thereby maintaining the stability of T cells in tumors.

The experimental plan is as follows:

C57BL/6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells on their backs, and 50 μg of fusion protein was injected intraperitoneally on days 12, 15, and 18 after tumor inoculation. In the FTY720-treated group, 15 μg of FTY720 was injected intraperitoneally on day 11 after tumor inoculation, and 10 μg was injected intraperitoneally on days 13, 15, 17, 19, and 21, respectively.

After FTY720 blockade, the treatment with the fusion protein still had an effect (Figure 9). The above results indicate that the anti-tumor effect of the fusion protein depends on the CD8 ⁺ T cells already present in the tumor.

4. The protective effect of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein on distant metastatic tumors

In order to verify whether the immune protection generated by in situ tumor treatment also has an inhibitory effect on tumors at other metastatic sites, we conducted a mouse dual tumor model experiment. We simultaneously inoculated tumor cells on the left and right sides of the mouse subcutaneously and performed in situ treatment on only one side to observe whether the distant tumors could also be controlled.

The experimental plan is as follows:

C57BL6 mice were inoculated with 5×10 ⁵ MC38 tumor cells on the right side of the back, and 30 μg of fusion protein was used for local intratumoral treatment on days 13, 16, and 19 after tumor inoculation. On day 13, 1×10 ⁶ MC38 tumor cells were inoculated on the left side of the mice, and the tumor volumes on both sides were measured twice a week.

The results showed that orthotopic treatment by intratumoral injection of only the right tumor could not only eliminate the orthotopic tumor but also control the untreated tumor in the contralateral distal part ( FIG. 10A ).

In addition, we used the B16F10 mouse tumor model to further validate the therapeutic effect of fusion protein therapy on lung metastases during melanoma metastasis.

The experimental plan is as follows:

C57BL6 mice were inoculated with 5×10 ⁵ B16F10 tumor cells on the right side of the back. 30 μg of fusion protein was used for local intratumoral treatment on days 9, 12, and 15 after tumor inoculation. Starting from day 8 after tumor inoculation, 15 μg of FTY720 was injected intraperitoneally every 2 days until the end of the experiment. On day 8 after tumor inoculation, 5×10 ⁵ B16F10 tumor cells were injected into the tail vein of each mouse. Ten days after the third treatment, the mice were perfused, and the lungs of the mice were taken to count the melanoma metastases in the lungs to verify the therapeutic effect of fusion protein treatment on metastases.

The results showed that the fusion protein had a significant anti-tumor effect on subcutaneous tumors in situ after intratumoral treatment, and was able to partially eliminate the in situ tumors. FTY720 blocking T cell migration did not affect the control of in situ tumors (Figure 10B). After FTY720 blocking, the lung metastases in the fusion protein treatment group were similar to those in the untreated control group, while the number of lung metastases in the fusion protein treatment group was significantly reduced (Figure 10C).

Example 3: Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein can delete intratumoral Tregs and increase the CD8/Treg cell ratio

1. Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein deletes intratumoral Tregs and increases the CD8/Treg cell ratio

Human IgG1 subtype Anti-4-1BB antibody can effectively delete Tregs in tumors, and IL-15 can expand CD8+T cells. In order to verify the function of the fusion protein in vivo, MC38 tumor-bearing mice were treated on the 13th and 16th days after tumor inoculation, and 50μg Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein was intraperitoneally injected. Tumor tissue was taken on the 18th day, and the proportion of Treg cells and CD8+T cells in the tumor were analyzed by flow cytometry. Through flow cytometry analysis, we found that Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein can effectively delete Treg cells in tumors and increase the proportion of CD8T, which has the best therapeutic effect (Figure 11A-D).

2. The therapeutic effect of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein depends on the binding of Fc to FcγR

Through the analysis of intratumoral lymphocytes in MC38 tumor-bearing mice, Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein has the effect of deleting Treg. In order to verify whether Treg deletion is the main mechanism of Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein to exert anti-tumor effect, we constructed Anti-4-1BB-MMP-IL-15-IL15Ra-no ADCC antibody by point mutation of Fc region. The mutated Fc does not bind to FcγR receptor and loses ADCC and ADCP-mediated deletion function, but still retains the amplification effect of IL-15 on immune cells. Using MC38 tumor model, C57BL6 mice were inoculated with MC38 tumor cells and treatment started on the 13th day after inoculation. 50μg wt Fc or no ADCC Fc antibody was injected intraperitoneally, and treatment was once every three days for a total of three times.

The results showed that after tumor inoculation, the therapeutic effects of wt Fc and no ADCC Fc were compared, and it was found that the mutant Fc reduced the therapeutic effect of the fusion protein (Figure 12) . In the Anti-4-1BB-MMP-IL-15-IL15Ra-no ADCC treatment group, the fusion protein still retained some anti-tumor effects, indicating that the function of the Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein depends largely on the deletion of Treg cells.

Example 4. Anti-4-1BB-MMP-IL-15-IL15Ra combined with PDL1 antibody therapy

1. PDL1 is upregulated in tumor immune cells after fusion protein treatment

When treating MC38 tumor-bearing mice, although the tumor can be effectively controlled, the tumor cannot be eliminated in some mice. When the tumor is large, the fusion protein treatment alone can only control the tumor growth; and the fusion protein treatment often activates the immune cells in the tumor, and the increase in the degree of activation often leads to negative feedback of the immune system, causing the expression of immune inhibitory checkpoint molecules to be upregulated, thereby inhibiting the activation of immune cells. In order to better improve the treatment effect, we detected the expression of PD-1 and PD-L1 in the immune cells in the mouse tumor after fusion protein treatment, in order to find a suitable immune checkpoint inhibitory antibody as a combined use scheme to observe whether it can improve the treatment effect.

Scheme: 5×10 ⁵ MC38 tumor cells were subcutaneously inoculated on the back of C57BL/6 mice. 50 μg of fusion protein was used for treatment on days 12 and 15. Tumor tissues of mice were collected on day 18 to analyze the expression of PD-1 and PD-L1 in immune cells in the tumor.

The results showed that after treatment, the expression of PD-L1 in CD45 ⁺ immune cells in the tumor of mice increased significantly, while the expression of PD-1 in CD8 ⁺ T cells decreased significantly (Figure 13). This suggests that we can use the combined use of fusion protein and PD-L1 antibody to further relieve the degree of immunosuppression in the tumor. In order to further improve the therapeutic effect of fusion protein, fusion protein and PD-L1 antibody were used to treat advanced tumor-bearing mice (tumor volume of about ^150-200mm3 ).

Protocol: C57BL/6 mice were subcutaneously inoculated with 1×10 ⁶ MC38 tumor cells on the back and intraperitoneally injected with 200 μg aPDL1 antibody on days 13 and 16, and intraperitoneally injected with 50 μg Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein on days 13, 16, and 19.

The results showed that for advanced tumors, PD-L1 antibodies can further enhance the therapeutic effect of fusion proteins and overcome immune tolerance.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the protection scope of the present invention.

Example 5: Anti-OX40-MMP-IL-15-IL15Ra fusion protein has good anti-tumor effect

1. Construction and expression of fusion protein

The above studies have shown that the combined treatment of Anti-4-1BB antibody and sIL-15 has a synergistic anti-tumor effect. Considering that OX40 and 4-1BB are both members of the TNF superfamily and are also T cell activation factors, it is speculated that the fusion of Anti-4-1BB antibody and sIL-15 may also produce a synergistic anti-tumor effect. Here, we once again verified that IL-15-IL-15Ra was connected to the carboxyl terminus of immunoglobulin Fc, and the Fc and IL-15 were fused and expressed through a short substrate sequence that can be recognized by MMPs highly expressed in tumors, so that the fusion protein can be cleaved in the tumor to release IL-15 activity. Each molecule contains a complete antibody Fc and IL-15-IL-15Ra molecule, and an MMP substrate sequence is contained between the antibody and the cytokine. We co-incubated the fusion protein with the activated MMP14 enzyme in vitro, and then performed SDS-PAGE analysis to verify the in vitro cleavage effect of the fusion protein. The results showed that the fusion protein can be effectively cleaved in vitro (Figure 14A).

2. In vitro activity detection of fusion protein (lymphocyte proliferation assay (CCK8 assay))

CTLL2 cells were cultured with 1640 complete medium containing 100U/ml commercial recombinant IL2 cytokine for 24 hours; then washed 2-3 times with complete medium without IL2, and the cells were diluted to 2×10 ⁴ /ml; the diluted samples sIL15-Fc, Fc-MMP14-sIL15 (without MMP14 cut), and Fc-MMP14-sIL15 (with MMP14 cut) were cultured with complete medium without IL2. The starting concentration was 5μg/ml, and 10 dilutions were made with 5-fold dilutions; 100μl of cell suspension and 100μl of sample were added to a 96-well cell culture plate, and mixed by pipetting with a pipette tip; after 72 hours of culture, 20μl of CCK8 was added, and culture was continued for 3 hours, and the OD values of 450nM and 630nM wavelengths were detected by an enzyme marker.

The test results showed that: (1) the biological activity of Fc-MMP14-sIL-15(-) differed by about 300 times from that of sIL15Fc, indicating that the biological activity of Fc-MMP14-sIL15 was very low when not cleaved by MMP14; (2) the biological activity of Fc-MMP14-sIL-15(+) was equivalent to that of sIL15Fc, indicating that when cleaved by MMP14, the biological activity of Fc-MMP14-sIL15 returned to the level of sIL15-Fc (Figure 14B).

3. Fusion protein has good anti-tumor effect

C57BL6 mice were subcutaneously inoculated with 5×10 ⁵ MC38 tumor cells, and when the tumor grew to 100 mm ³ , αOX40 15 μg, sIL15-Fc 15 μg or αOX40-MMP14-sIL15 30 μg were given for 3 times with an interval of 2 days. The control group was given PBS 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, indicating that the fusion protein had a better anti-tumor effect than the combined administration of αOX40 and sIL15-Fc of equal molar mass ( FIG. 14C ).

Example 6: Humanized Anti-4-1BB-MMP-IL-15-IL15Ra fusion protein has good anti-tumor effect and safety

6.1 Construction and expression of humanized fusion protein

In order to verify that the humanized Anti-4-1BB-MMP-IL-15-IL15R fusion protein has similar anti-tumor effects, we constructed a fusion protein with the antibody heavy chain that can recognize the human 4-1BB molecule and human IL-15-IL-15Ra in the same way. We constructed fusion proteins containing two linker lengths, one is _G4S -MMP- _G4S and the other is GS-MMP-GGS, and performed SDS-PAGE analysis (Figure 15A) (R: reducing gel, NR: non-reducing gel).

6.2. Verification of the function of the fusion protein

(1) In vitro activity detection of fusion protein

The MMP substrate sequence in the humanized fusion protein can be cleaved by MMP2, MMP9 or MMP14. We co-incubated the fusion protein with the activated MMP14 enzyme in vitro, and then performed SDS-PAGE analysis to verify the in vitro cleavage effect of the fusion protein. The results showed that fusion proteins of different Linker lengths can be effectively cleaved in vitro (Figure 15A). We used the HEK-IL2 cell line to detect the activation effect of human IL-15 in the fusion protein on the STAT5 signal of the cell line. The results showed that the fusion protein without enzyme cleavage had a decreased ability to activate the downstream signal of the HEK-IL2 cell line. After in vitro enzyme cleavage, the activation ability was basically restored (Figure 15B), indicating that the activity of IL-15 in the humanized fusion protein was well blocked, and its activity was fully restored after MMP cleavage.

(2) Fusion protein amplifies immune cells in hu-PBMC mice

The activity of IL-15 in the humanized fusion protein is well blocked. In order to further verify whether the fusion protein can amplify human immune cells in the peripheral tissues of tumor-bearing PBMC mice, we intraperitoneally injected 100 μg of fusion protein or a mixture of equimolar amounts of Anti-4-1BB antibody and sIL-15, treated once every three days, and detected the number of CD4 ⁺ T cells, CD8 ⁺ T cells and CD3 ⁺ T cells in peripheral blood 2 days after the second treatment. The fusion protein with linker GS-MMP-GGS is expressed as Human GS, the fusion protein with linker G ₄ S-MMP-G ₄ S is expressed as Human G ₄ S, and the mixed treatment group is expressed as Abs+sIL-15. The results showed that compared with the mixed treatment group, the fusion protein did not cause a large number of T cells in peripheral blood to expand, and had good safety (Figure 16).

6.3. Fusion protein has good safety and anti-tumor effect

NSG mice are immunodeficient mice caused by knockout mutations of the Prkdc gene and the Il2rg gene. They lack mature T, B, and NK cells, do not produce immunoglobulins, and have abnormal dendritic cells (DC) function, making them very suitable for human cell or tissue transplantation. We used 6-8 week old NSG mice, inoculated 1ⅹ10 ⁶ CFPAC1 cells subcutaneously on the right back on D0, and inoculated 5ⅹ10 ⁶ PBMC cells by tail vein injection 7 days later. Seven days after PBMC cell inoculation, the mice were grouped according to tumor size to construct a tumor-bearing hu-PBMC mouse model.

100 μg of fusion protein and an equimolar amount of Anti-4-1BB antibody and sIL-15 were injected intraperitoneally for mixed treatment. The treatment was performed three times on D14, D17, and D20 days, and the tumor size and body weight were measured twice a week. The results showed that the therapeutic effect of the fusion protein was better than that of the control group (Figure 17A). The fusion protein did not cause weight loss. The mixed treatment group lost more than 10% of its weight after the second treatment, causing death, indicating that the safety of the fusion protein was better than that of the mixed treatment group (Figure 17B). The x in Figures 17A and B indicates that all mice in the mixed treatment group died.

Claims (10)

A bifunctional fusion protein consisting of IL-15 and T cell co-stimulatory molecule antibody, characterized in that the fusion protein comprises: (1) Antibodies to T cell co-stimulatory molecules; (2) a conjugate of IL-15 and IL-15R Sushi domain, wherein the IL-15 and IL-15R Sushi domain are connected via a second linker fragment; and (3) a first connecting fragment; the first connecting fragment is used to connect the heavy chain Fc region of the T cell co-stimulatory molecule antibody and the IL-15 and IL-15R Sushi domain conjugate, and can be cleaved by metal matrix proteases; The T cell co-stimulatory molecules are: 4-1BB, ICOS, OX40; preferably 4-1BB; preferably, the T cell co-stimulatory molecule antibody is an IgG1 antibody; Preferably, the first connecting fragment is: 2-4 GnS connecting units and a short peptide contained therein that can be recognized and cleaved by metal matrix proteases, wherein n is an integer of 1-4; The bifunctional fusion protein according to claim 1, characterized in that The amino acid sequence of the short peptide that can be recognized and cleaved by metal matrix proteases is shown in SEQ ID NO.6; The amino acid sequence of the first connecting fragment is shown in SEQ ID NO.12; The bifunctional fusion protein according to claim 1, characterized in that the second connecting segment is: 2-5 G ₄ S connecting units; preferably, the amino acid sequence of the second connecting segment is as shown in SEQ ID NO.5. The bifunctional fusion protein according to any one of claims 1 to 4, characterized in that the bifunctional fusion protein comprises: (1) The first structural unit, from N-terminus to C-terminus, consists of: 4-1BB antibody heavy chain, first linker fragment, IL-15 and IL-15R Sushi domain conjugate; (2) A second structural unit: a 4-1BB antibody light chain paired with the 4-1BB antibody heavy chain; Furthermore, the bifunctional fusion protein dimerizes through the Fc region of the 4-1BB antibody heavy chain to form a homodimer. The bifunctional fusion protein according to claim 4, characterized in that The 4-1BB antibody is an antibody formed by fusing the Fab region of a human or mouse antibody with the Fc region of IgG1. The amino acid sequence of the heavy chain (VH+CH1) of the murine 4-1BB antibody is shown in SEQ ID NO.2; The amino acid sequence of the heavy chain (VH+CH1) of the human 4-1BB antibody is shown in SEQ ID NO.10; The amino acid sequence of the light chain (VL+CL) of the murine 4-1BB antibody is shown in SEQ ID NO.1; The amino acid sequence of the light chain (VL+CL) of the human 4-1BB antibody is shown in SEQ ID NO.9; The IgG1 Fc region is the Fc region of human IgG1, or a human IgG1 Fc region variant with ADCC effect knocked out. The bifunctional fusion protein according to claim 5 is characterized in that the amino acid sequence of the Fc region of human IgG1 is as shown in SEQ ID NO.3, and the amino acid sequence of the Fc region variant of human IgG1 with ADCC effect knocked out is as shown in SEQ ID NO.4. The bifunctional fusion protein according to claim 4, characterized in that the IL-15 and IL-15R Sushi domains are human or mouse proteins; preferably, The amino acid sequence of mouse IL-15 is shown in SEQ ID NO.7, and the amino acid sequence of mouse IL-15R Sushi domain is shown in SEQ ID NO.8; The amino acid sequence of human IL-15 is shown in SEQ ID NO.13, and the amino acid sequence of human IL-15R Sushi domain is shown in SEQ ID NO.14. A nucleotide fragment encoding the bifunctional fusion protein according to any one of claims 1 to 7, a vector containing the nucleotide fragment, and a host. The following uses of the bifunctional fusion protein according to any one of claims 1 to 7, or the nucleotide fragment encoding the bifunctional fusion protein according to claim 8, or a vector or host containing the nucleotide fragment: (1) Preparation of anti-tumor drugs; (2) Preparation of combined anti-tumor drugs. The use according to claim 9, characterized in that The anti-tumor drug is a drug that reduces and eliminates intratumoral Treg cells and induces CD8 ⁺ T cell proliferation; preferably, the tumor is a tumor in an immunosuppressive state; The combination anti-tumor drug is: a combination anti-tumor drug used in combination with an immune checkpoint inhibitor, and the combination anti-tumor drug can overcome tolerance to the immune checkpoint inhibitor; the immune checkpoint inhibitor includes but is not limited to: PD-1/PD-L1 antibody, CTLA4 antibody, TIGIT antibody, LAG-3 antibody, TIM-3 antibody.