Cytokine blocking antibody and use thereof

Abstract

An antibody selectively activated in a target cell or tissue for treating one of symptoms. The antibody comprises a functional antibody which contains at least one light chain and at least one heavy chain and can be used for treating a condition in an activated state; at least one peptide, which can bind to an interferon receptor as required and activate a signaling pathway of the peptide; and at least one cleavable polypeptide linker, wherein each cleavable polypeptide linker contains a substrate peptide that can be cleaved by means of an enzyme, the enzyme is specifically or highly expressed in a target cell or tissue, and the cleavable polypeptide linker links one of the peptides to the N terminal of one of the at least one light chain and the at least one heavy chain of the functional antibody.

Description

Cytokine blocking antibodies and their applications Technical Field

[0001] The present invention relates to a specific selective antibody, in particular an antibody for improving the efficacy and safety of treating metastatic cancer. Background Art

[0002] Immune checkpoint inhibitor therapy is one of the most promising new strategies for treating cancer. It blocks immunosuppressive signaling between human immune cells and tumors, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death protein ligand 1 (PDL-1). This activates the immune system, allowing the body's immune T cells to attack cancer cells, ultimately achieving a therapeutic effect. It is one of the most promising cancer therapies in recent years. In 2014, global sales of cancer drugs reached $41 billion, accounting for nearly half of the market. Science magazine even hailed immune checkpoint blockade as "a major breakthrough in cancer immunotherapy in 2013." Yervoy (α-human CTLA-4 antibody, ipilimumab), the first immune checkpoint drug to be marketed in 2011, had global sales of $960 million by 2018. The US FDA approved Yervoy in March 2011 for the treatment of melanoma, advanced metastatic colorectal cancer, and renal cancer. Clinical results have shown promising results in a wide range of cancers. For example, melanoma patients treated with Yervoy combined with conventional chemotherapy (dacarbazine injection) had a three-year survival rate of 22%, significantly higher than the 12.2% three-year survival rate for chemotherapy alone, demonstrating its significant therapeutic efficacy in melanoma. However, long-term use of Yervoy antibodies can lead to systemic immune overactivation, resulting in systemic autoimmune toxicity. FDA reports and literature indicate that Yervoy antibody administration may cause side effects such as enteritis, hepatitis, endocrine abnormalities, and toxic epidermolysis due to overactivation of the immune system. Furthermore, it may also cause inflammation of the nervous system and lead to paralysis. Therefore, increasing the selectivity of Yervoy antibodies in tumor areas to reduce side effects is urgently needed.

[0003] Immune checkpoint inhibitor therapy is a highly promising strategy for the clinical treatment of cancer. These inhibitors typically target cancer by activating the immune system. However, immune system activation can also cause systemic immune-related adverse reactions and tumor immune escape, limiting the effectiveness of immune checkpoint inhibitor therapy. Summary of the Invention

[0004] The interferon-functional fusion antibody provided by the present invention, taking the interferon-Yervoy fusion antibody (IFN-Yervoy) as an example, has the following advantages:

[0005] (1) Tumor specificity and reduced side effects: Successfully utilize spatial blocking to allow IFNα, IFNβ or IFNγ and Yervoy to block each other's functions. Only in the tumor area where MMPs protease is overexpressed can their activity be restored, so that Yervoy and IFNγ can locally activate the immune response in the tumor area, thereby enhancing the anti-cancer effect of immune checkpoint blocking antibodies and overcoming their serious systemic side effects.

[0006] (2) Overcoming the clinical limitations of Yervoy antibody efficacy: The efficacy and application of current immune checkpoint blockade therapy are still limited by the expression of MHC molecules in the patient's tumor and the lack of IFNγ signal transmission in the tumor area. The present invention increases the expression of tumor MHC molecules through local IFNγ activation, thereby increasing the efficacy of Yervoy antibody, thereby overcoming the problem that the efficacy of Yervoy antibody is limited by insufficient expression of tumor MHC molecules in clinical patients.

[0007] (3) Wide range of antibody applications: Since human antibody structures are highly similar, this strategy can be easily applied to cancer immunotherapy (Immuno-Oncology, IO) drugs through genetic engineering technology, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1) or all clinical antibodies. In addition, MMPs substrate peptides can also be replaced with different protease substrate peptides, increasing the design flexibility of this targeted drug to respond to targeted treatment of tumors with different characteristics.

[0008] (4) Low immune response: Because Yervoy antibody and IFNγ have been humanized and are proteins in the human body, they will not produce an immune response in the human body, which will accelerate the clinical application of this precursor protein drug.

[0009] As used herein, the term "antibody" refers to a protein comprising at least one heavy (H) chain and one light (L) chain interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In certain antibodies (e.g., naturally occurring IgG antibodies), the heavy chain constant region comprises a hinge and three domains: CH1, CH2, and CH3. In certain antibodies (e.g., naturally occurring IgG antibodies), each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. For example, "antibodies" include both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; and fully synthetic antibodies.

[0010] The antibodies or fragments thereof provided by the present invention may be in any form such as monoclonal antibodies, single-chain antibodies, bifunctional antibodies, single-domain antibodies, nanobodies, fully or partially humanized antibodies or chimeric antibodies, or the antibodies or fragments thereof may be half antibodies or antigen-binding fragments of half antibodies, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

[0011] The tumor-selective pro-interferon-Yervoy fusion antibody (IFNγ-Yervoy) provided by this invention will reduce the side effects of Yervoy, an emerging antibody drug in current immunotherapy, and improve the effectiveness of Yervoy antibody treatment in clinical patients. This will significantly enhance the anti-cancer efficacy and clinical safety of current immune checkpoint blockade therapies, accelerate the development of cancer treatment, and provide patients with better medical quality.

[0012] An antibody selectively activated in target cells or tissues to treat one of the symptoms thereof, the antibody comprising: a functional antibody comprising at least one light chain and at least one heavy chain, which can be used to treat the condition when activated; at least one peptide capable of optionally binding to an interferon receptor and activating the signaling pathway of the peptide, wherein the interferon receptor comprises IFNAR1, IFNAR2, IFNGR1, or IFNGR2; and at least two cleavable polypeptide linkers, wherein each cleavable polypeptide linker comprises a substrate peptide that can be cleaved by an enzyme that is specifically or highly expressed in the target cells or tissues, and the cleavable polypeptide linker connects one of the peptides to the N-terminus of one of the at least one light chain and the at least one heavy chain of the functional antibody.

[0013] In one embodiment, the functional antibody is immunoglobulin G1 (IgG1), IgG2, IgG3, or IgG4.

[0014] In one embodiment, the functional antibody is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-RANKL antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-GITR antibody, anti-HER2 antibody, anti-EGFR antibody, anti-VEGF antibody, anti-VEGFR2 antibody, anti-HLA-DR antibody, anti-ICOS antibody, anti-OX40 antibody, anti-IL6R antibody, anti-IL12 / 23 antibody, anti-CD3 antibody, anti-CD11a antibody, anti-CD20 antibody, anti-CD25 antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-CD52 antibody, anti-CD137 antibody and anti-CD138 antibody.

[0015] In a preferred embodiment, the functional antibody is an anti-CTLA-4 antibody or an anti-PD-1 antibody.

[0016] In one embodiment, the peptide is an interferon. In a preferred embodiment, the peptide comprises IFN-α, IFN-β or IFN-γ.

[0017] In one embodiment, the cleavable polypeptide linker comprises a substrate peptide cleavable by a matrix metalloproteinase (MMP).In a preferred embodiment, the polypeptide linker is MMP-2.

[0018] In one embodiment, the substrate peptide can be cleaved by any of the following enzymes: matrix metalloproteinases, such as MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, and MMP-14; autolytic enzymes, such as CTS A, CTS B, CTSD, CTS E, and CTS K); caspases, such as CASP-1, CASP-2, CASP-3, CASP-4, CASP-5, CASP-6, CASP-7, CASP-9, CASP-10, CASP-11, CASP-12, CASP-13, and CASP-14; or disintegrins and metalloproteinases, such as ADAM-10, ADAM-12, ADAM-17, ADAM-TS, and ADAM-TS5.

[0019] The present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises: the antibody as described above; and a pharmaceutically acceptable carrier.

[0020] The present invention further provides a use of a composition in preparing a medicament for treating metastatic cancer, comprising: administering an effective dose of the composition comprising the antibody as described above to an individual suffering from metastatic cancer.

[0021] In one embodiment, the metastatic cancer comprises metastatic melanoma, advanced renal cell carcinoma, microsatellite instability-high (MSI-H) metastatic colorectal cancer, mismatch repair-deficient (dMMR) metastatic colorectal cancer, hepatocellular carcinoma, or metastatic non-small cell lung cancer.

[0022] In a preferred embodiment, the metastatic cancer is microsatellite instability-high metastatic colorectal cancer, mismatch repair-deficient metastatic colorectal cancer, or metastatic melanoma.

[0023] In one embodiment, the effective dose is 0.1 mg / kg-6 mg / kg; in a preferred embodiment, the effective dose is 0.2 mg / kg-4.5 mg / kg; in an optimal embodiment, the effective dose is 0.5 mg / kg-3 mg / kg.

[0024] The present invention further provides a method of treating a subject having metastatic cancer, wherein the method comprises administering to the subject an effective amount of the above-described antibody.

[0025] In one embodiment, the metastatic cancer comprises metastatic melanoma, advanced renal cell carcinoma, microsatellite instability-high metastatic colorectal cancer, mismatch repair-deficient metastatic colorectal cancer, hepatocellular carcinoma, or metastatic non-small cell lung cancer.

[0026] In a preferred embodiment, the metastatic cancer is microsatellite instability-high (MSI-H) metastatic colorectal cancer, mismatch repair-deficient metastatic colorectal cancer, or metastatic melanoma.

[0027] In one embodiment, the antibody treats metastatic cancer by: the aforementioned peptide, taking interferon as an example, which reduces the antibody recognition ability of the functional antibody with its universal shielding properties; and increasing the activity of immune cells in the diseased area against cancer cells, thereby enhancing the efficacy of the functional antibody and reducing the side effects of the functional antibody.

[0028] In one embodiment, the effective dose is 0.1 mg / kg-6 mg / kg; in a preferred embodiment, the effective dose is 0.2 mg / kg-4.5 mg / kg; in an optimal embodiment, the effective dose is 0.5 mg / kg-3 mg / kg.

[0029] In one embodiment, the effective dose is administered once every 3 weeks for 4 weeks. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A is a schematic diagram of the gene construction of IFN-γ-Yervoy. Figure 1B shows the difference in molecular weight of IFN-γ-Yervoy before and after MMP-2 / 9 activation.

[0031] FIG2 is a schematic diagram of the IFN-γ-Yervoy antibody.

[0032] Figure 3 shows the ability of Antibody Lock to inhibit the binding of IFN-γ-Yervoy and its downstream signaling analysis. Figure 3A shows the binding ability of Yervoy, IFN-γ-Yervoy, and MMP-2 / 9-activated IFN-γ-Yervoy using a CTLA4 ELISA. Figure 3B shows the CTLA4 activation ability of IFN-γ-Yervoy and Yervoy in the presence and absence of MMP-2 / 9, as measured by a luciferase CTLA4 / IL-2 reporter assay.

[0033] Figure 4 shows the binding ability analysis of IFN family proteins IFN-α, IFN-β and IFN-γ with therapeutic antibodies Yervoy, Tremelimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Durvalumab or Avelumab, respectively.

[0034] Figure 5 shows the results of cytotoxicity analysis of IFN-γ-Yervoy. Figure 5A shows the cell viability assay of Yervoy, IFN-γ-Yervoy, and Yervoy + IFN-γ-Yervoy after reaction with MMP-2 / 9 at different times. Figure 5B shows the MHC I level assay of SW480 cells treated with Yervoy and IFN-γ-Yervoy with or without MMP-2 / 9. Figure 5C shows the cytotoxicity analysis of PBMC, PBMC + Yervoy, and PBMC + IFN-γ-Yervoy (with MMP-2 / 9) at different effector (E) to target (T) ratios. Values ​​are mean ± SEM, * indicates significant difference, P < 0.05. Error: standard error of triplicate determinations.

[0035] Figure 6 is an evaluation of the efficacy of IFN-γ-Yervoy in colorectal cancer. Figure 6A is a flow chart of the human PBMC animal model. Figure 6B is an analysis of tumor volume of cells treated with PBS, different concentrations of Yervoy, and IFN-γ-Yervoy on day 33. Figure 6C is a graph showing the CD4 + 、CD8 + and MHC I expression analysis. Figure 6D shows the expression of CD4 + 、CD8 + and statistical plots of staining areas for MHC I expression.

[0036] Figure 7 shows the toxicity evaluation of IFN-γ-Yervoy in colorectal cancer experiments. Figure 7A shows the survival rate of mice treated with PBS, different doses of Yervoy, and IFN-γ-Yervoy. Figure 7B shows the body weight analysis of mice under different treatments. Figure 7C shows the CD4 counts in the blood of mice on days 1, 8, and 34. + Figure 7D shows the expression of CD8 in the blood of mice on day 1, day 8 and day 34. + Figure 7E shows the expression analysis of MHC I in the blood of mice on days 1, 8, and 34. Figure 7F shows the staining of organ damage in mice under different treatments. Figure 7G shows the statistical diagram of organ damage or bleeding area in mice under different treatments.

[0037] Figure 8 shows the structural integrity of the IFNα-Nivolumab fusion antibody analyzed by SDS-PAGE. Figure 8A shows that the IFNα-Nivolumab fusion antibody has heavy chain and light chain bands. Figure 8B shows that the IFNα-Nivolumab fusion antibody has a higher molecular weight band.

[0038] Figure 9 shows the binding affinity analysis of IFNα-Nivolumab fusion antibody after protease cleavage. Figures 9A and 9B analyze the molecular weight of the light and heavy chains of IFNα-Nivolumab fusion antibody after protease cleavage by Western blotting. Figure 9C analyzes the antibody binding capacity of IFNα-Nivolumab fusion antibody after protease cleavage by ELISA. Data are shown as mean ± standard deviation, with asterisks ( * ) indicates statistically significant differences (P<0.05).

[0039] Figure 10 shows flow cytometric analysis of MHC class I expression in A375 melanoma cells under different treatments. A375 melanoma cells (A) were treated with 3.5 nM IFNα (Figure 10A), 0.875 nM nivolumab (Figure 10B), 0.875 nM IFNα-nivolumab (Figure 10C), and 0.875 nM MMP2 / 9-activated IFNα-nivolumab (Figure 10D). The control group consisted of untreated cells.

[0040] Figure 11 shows an IFNα-Nivolumab trial conducted in an A375 melanoma cell mouse model. Figure 11A shows the tumor volume monitored every 3 days. Figure 11B shows an immunohistochemical staining image. Figure 11C shows the MHC I expression area in the mouse model under different treatments. Figure 11D shows the CD4 + Figure 11E shows the expression of CD8 T cell infiltration in mouse models under different treatments. + Figure 11F shows the expression of IFNγ in tumor tissues by sandwich ELISA. Figure 11G shows the expression of IFNα in tumor tissues by sandwich ELISA. Data are expressed as mean ± standard deviation, with asterisks ( * ) indicates statistically significant differences (P<0.05).

[0041] Figure 12 is a PBMC-mediated cytotoxicity assay. Data are shown as mean ± standard deviation, with asterisks ( * ) indicates statistically significant differences (P<0.05).

[0042] Figure 13 shows the survival status monitoring and cytotoxicity detection of the IFNα-Nivolumab test using the A375 melanoma cell mouse model. Figure 13A shows the survival rate of the A375 melanoma cell mouse model under different treatments. Figure 13B shows the weight change of the A375 melanoma cell mouse model under different treatments. Figure 13C shows hematoxylin and eosin (H&E) staining of the lungs, liver, kidneys and small intestine of the A375 melanoma cell mouse model. The black arrows indicate significant damage in other groups. Figure 13D shows the quantification of damage to various organs of the mouse model under different treatments. Figure 13E shows the CD3 + 、CD4 + 、CD8 + and FoxP3 + The expression levels of T cells. Data are expressed as mean ± standard deviation, asterisk ( * ) indicates significant difference (P<0.05). DETAILED DESCRIPTION

[0043] The following examples are non-limiting and merely represent various aspects and features of the present invention.

[0044] Example 1: Construction of IFN-γ-Yervoy

[0045] To understand whether IFN-γ-Yervoy can increase the efficacy and reduce the side effects of colorectal cancer, this example constructed Yervoy and IFN-γ-Yervoy, as shown in FIG1A .

[0046] [Corrected 09.05.2025 according to Rule 26] Complementary DNA encoding the Yervoy heavy and light chains was cloned by assembly PCR. The human IgG1 hinge sequence was obtained from the National Center for Biotechnology Information (NCBI). A GGGGS linker and an MMP-2 / 9 substrate coding sequence (GPLGVR) (SEQ ID NO: 1) were introduced upstream of the Yervoy light and heavy chains to generate IFNγ-yervoy. Yervoy or IFN-γ-yervoy was produced using the Expi293 expression system (Thermo Fisher Scientific, Waltham, MA, USA) and purified using Protein A-Sepharose (GE Healthcare, Milwaukee, WI, USA).

[0047] After constructing Yervoy and IFN-γ-Yervoy, the plasmids were transfected and used in CHO cells, respectively. The antibodies were collected and purified from the supernatant, and the molecular weights of Yervoy and IFN-γ-Yervoy were analyzed by Western blotting. The results showed that Yervoy and IFN-γ-Yervoy were successfully produced, as shown in Figure 1B.

[0048] Example 2: Mechanism of IFN-γ-Yervoy

[0049] This embodiment discloses the mechanism by which IFN-γ-Yervoy improves immune checkpoint blockade therapy. As shown in the schematic diagram of the antibody structure in Figure 2, the N-terminus of the IFN-γ-Yervoy antibody structure contains Type I IFN or Type II IFN, such as IFN-γ, and a cleavable polypeptide linker, such as an MMP-2 / 9 substrate. IFN-γ-Yervoy is inactivated in the blood. When IFN-γ-Yervoy enters the tumor microenvironment, it is cleaved by MMP-2 / 9 into Yervoy and IFN-γ. Yervoy promotes CD8 + The proliferation of T cells and IFN-γ increase the expression of MHC molecules in tumors. The synergistic effect of the two enhances CD8 + The ability of T cells to recognize and kill tumors.

[0050] We have confirmed that adding a protease cleavage site and interferon to the N-terminus of an antibody's VL or VH can be used as a precursor antibody design approach to increase the specific activation ability of the antibody and interferon, thereby improving the efficacy and safety of cancer treatment. Therefore, IFN-γ-Yervoy improves the limitations of immune checkpoint blockade therapy.

[0051] Example 3: IFN-γ-Yervoy activation assay

[0052] To confirm that IFN-γ-Yervoy can be activated by MMPs and maintain the biological activity of Yervoy, this example further reacted equal amounts of 5 μg of MMPs and 5 μg of Yervoy at 37°C for 10 minutes, and the reaction was terminated with 10 μg of BSA. The resulting mixture was then serially diluted and added to a CTLA4 ELISA plate for a 1-hour reaction. A secondary antibody was then added for a 1-hour reaction, and ABTS was added for a 30-minute reaction. The absorbance was then analyzed using an ELISA analyzer.

[0053] As shown in Figures 3A and 3B, the results demonstrate that Yervoy does not affect its function in the presence or absence of MMPs. Furthermore, the antibody binding capacity of the IFN-γ-Yervoy group treated with MMPs was significantly improved compared to the IFN-γ-Yervoy group treated without MMPs, with antibody binding restored 457-fold. This phenomenon demonstrates that IFN-γ-Yervoy can activate its antigen-binding capacity through MMPs.

[0054] Example 4: IFN family binding therapeutic antibodies

[0055] In addition to the fact that IFN-γ-Yervoy can activate its antigen binding ability through MMP, the present invention also confirms that the IFN family also has the same effect.

[0056] In this example, IFN-α, IFN-β, and IFN-γ were each combined with various therapeutic antibodies, including Yervoy (Ipilimumab), Tremelimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Durvalumab, and Avelumab, and the antigen-binding ability of the various therapeutic antibodies was tested. As shown in FIG4 , the results showed that the IFN family can reduce the antigen-binding ability of the therapeutic antibodies and restore the antigen-binding ability of the therapeutic antibodies through MMP-2 / 9 activation.

[0057] Example 5: Cytotoxicity Assay of IFN-γ-Yervoy

[0058] To confirm that IFN-γ-Yervoy can enhance the IFN-γ signaling pathway in SW480 cells, this example added Yervoy and IFN-γ-Yervoy to SW480 cells, and then allowed IFN-γ and Yervoy to bind to MMP-2 at different times. As shown in Figure 5A , the binding ability of IFN-γ-Yervoy increased depending on the duration of the reaction with MMP-2, demonstrating that IFN-γ-Yervoy of the present invention can bind to IFN receptors and stimulate downstream signaling.

[0059] To illustrate the effects of different concentrations of IFN-γ-Yervoy on MHC I expression in SW480 cells, different concentrations of IFN-γ-Yervoy were added to SW480 cells in this example. As shown in Figure 5B , the results indicate that activated IFN-γ-Yervoy can increase MHC I expression in SW480 cells.

[0060] In addition, to understand whether IFN-γ in IFN-γ-Yervoy can increase MHC I expression in SW480 cells, this example also added Yervoy, Yervoy + IFNγ, IFN-γ-Yervoy + BSA, and IFN-γ-Yervoy + MMP-2 to SW480 cells, respectively. As shown in Figure 5C, the results showed that compared with the other groups, Yervoy + IFNγ and IFN-γ-Yervoy + MMP-2 had better ability to activate PBMC cytotoxicity in SW480 cells.

[0001] Therefore, IFN-γ-Yervoy can be activated by matrix metalloproteinases, such as MMP-2, thereby promoting the IFNγ signaling pathway and enhancing T cell cytotoxicity.

[0061] Example 6: Efficacy of IFN-γ-Yervoy on Rectal Cancer

[0062] To confirm that IFN-γ-Yervoy can specifically activate CD4+ and CD8+ lymphocytes, as shown in Figure 6A, this example first used ASID mice to establish a human PBMC heterotopic colorectal cancer mouse model, and PBS, Yervoy, and IFN-γ-Yervoy were administered, respectively. Blood was collected on day 1 (Day 1, D1), day 8, and day 34 to analyze the activation of human lymphocytes in the blood. As shown in Figures 6B-6D, compared with day 1, day 8, and day 34, the lymphocyte activation on day 33 was higher, indicating that the present invention successfully established a human PBMC heterogeneous colorectal cancer mouse model.

[0063] After administering IFN-γ-Yervoy and Yervoy to a human PBMC ectopic colorectal cancer mouse model, the differences in the blood levels of the two drugs were compared. As shown in Table 1, all indicators of IFN-γ-Yervoy were similar to those of Yervoy.

[0064] Table 1. Characteristics of IFN-γ-Yervoy and Yervoy in blood

[0065] %ID / g: Percent injected dose per gram. AUC: Area under the influence of the plasma concentration. CI: Interval. Cmax: Mean maximum serum concentration. h: Hours. MRT: Mean residence time.

[0066] To confirm that IFN-γ-Yervoy can enhance the efficacy of Yervoy, this example tracked the tumor volume of different treatment groups from day 1 to day 34. As shown in Figure 6B, the results showed that the tumor volume of the IFN-γ-Yervoy group was smaller than that of the Yervoy group and the PBS-treated group.

[0067] In addition, to explore whether IFN-γ-Yervoy can reduce the levels of CD4+ and CD8 + The activation of lymphocytes is shown in Figures 6C and 6D. The results showed that on day 34, the CD4 + and CD8 + Low activation.

[0068] Therefore, IFN-γ-Yervoy enhances the efficacy of Yervoy in colorectal cancer by increasing the infiltration of CD8+ cells and CD4+ cells and increasing the expression of MHC I in tumors.

[0069] Example 7: Effect of IFN-γ-Yervoy on Survival Rate

[0070] To confirm that high-dose IFN-γ-Yervoy can improve individual survival rates, in this example, animals were treated with PBS, 1 mg / kg or 3 mg / kg of Yervoy, or IFN-γ-Yervoy three times a week for two weeks. The survival rate of the animals was observed on day 34. As shown in Figure 7A, the results showed that the survival rate of the 1 mg / kg or 3 mg / kg IFN-γ-Yervoy-treated group was higher than that of the Yervoy-treated group, demonstrating that IFN-γ-Yervoy is less toxic than the Yervoy-treated group.

[0071] Example 8: Effects of IFN-γ-Yervoy on Organ Damage

[0072] In order to understand the relationship between the efficacy of different groups and the expression of CD4 + 、CD8 + The relationship between CD4 and MHC I was analyzed by IHC staining on day 34 (D34) in the PBS, Yervoy and IFN-γ-Yervoy treatment groups. + 、CD8 + 7B-7E, the results showed that the CD4 + 、CD8 + and MHC I expression were the highest, indicating that IFN-γ-Yervoy can increase CD4 + and CD8 + The infiltration of lymphocytes and activation of MHC I expression in SW480 cells increased the cytotoxicity of Yervoy against colorectal cancer cells.

[0073] To demonstrate that the higher toxicity in the Yervoy-treated group was caused by greater organ damage, this example analyzed lung, liver, kidney, and intestinal damage in the PBS, IFN-γ-Yervoy, and Yervoy groups on day 34. As shown in Figures 7F-7G , compared with the Yervoy-treated group, the organs in the IFN-γ-Yervoy-treated group showed no significant differences from those in the PBS-treated group, whereas the organ tissues in the Yervoy-treated group were significantly damaged. This suggests that IFN-γ-Yervoy can mitigate the side effects of Yervoy by reducing lymphocyte infiltration, normal tissue bleeding, and organ damage.

[0074] Example 9: Binding and efficacy testing of IFN family and Nivolumab

[0075] In addition to binding to various therapeutic antibodies, including Yervoy, Tremelimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Durvalumab, and Avelumab, to mitigate their side effects, the IFN family can also bind to Nivolumab via MMPs. In this example, an IFNα-Nivolumab fusion antibody was used as a representative for efficacy testing and analysis.

[0076] As shown in Figures 8A and 8B, purified nivolumab and IFNα-nivolumab fusion antibody were analyzed by SDS-PAGE. The results showed that nivolumab had the expected heavy and light chain bands, while the fusion antibody showed a higher molecular weight band, confirming its structural integrity.

[0077] Western blotting and ELISA were also used to analyze the antibody binding capacity of IFNα-Nivolumab after MMP-2 / 9-mediated protease cleavage, as shown in Figures 9A and 9B , confirming that MMP-2 / 9 can effectively cleave the fusion antibody, releasing nivolumab. As shown in Figure 9C , ELISA experiments confirmed that the antibody binding capacity of the IFNα-Nivolumab fusion antibody after MMP-2 / 9 cleavage was restored to a level comparable to that of nivolumab alone.

[0078] Example 10: Effect of IFNα-Nivolumab on Cellular MHC I Expression

[0079] A375 melanoma cells were cultured and treated with IFNα, Nivolumab, IFNα-Nivolumab, and IFNα-Nivolumab+MMP-2 / 9, respectively. The MHC I expression levels of the treated A375 melanoma cells were analyzed.

[0080] As shown in Figures 10A-10D, flow cytometry was used to detect the expression of MHC I on the cell surface, indicating that compared with the control group, each drug treatment group could increase the expression of MHC I in A375 cells, among which the MMP-2 / 9-activated IFNα-Nivolumab fusion antibody had the most significant effect.

[0081] Example 10: Efficacy of IFNα-Nivolumab in a mouse model

[0082] A mouse A375 cell tumor model was established, and different treatments were given, and the tumor volume was measured every three days.

[0083] After 33 days of treatment, tumor tissues were removed and subjected to immunohistochemistry (IHC) staining to detect CD4+ and CD8+ T cell infiltration and the expression of MHC I in the tumor tissues. Sandwich ELISA was used to measure the levels of IFNγ and TNFα in the tumor tissues.

[0084] As shown in FIG11A , the tumor growth was significantly inhibited in the IFNα-Nivolumab group compared with the other groups.

[0085] As shown in Figures 11B-11E, immunohistochemical staining showed that CD4+ and CD8+ T cell infiltration and MHC I expression were significantly increased in the tumor tissues of the IFNα-Nivolumab group.

[0086] As shown in Figures 11F and 11G, the levels of IFNγ and TNFα were also significantly increased in the tumor tissues of the IFNα-Nivolumab group.

[0087] Example 11: Cytotoxicity Test and Safety Assessment of IFNα-Nivolumab

[0088] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and mixed with A375 melanoma cells at varying effector to target cell ratios (E:T ratios). Interferon-α-nivolumab fusion antibodies, either activated or not by MMP-2 / 9, were then added. After culture, A375 cell survival was measured to assess PBMC-mediated cytotoxicity.

[0089] As shown in FIG12 , IFNα-Nivolumab fusion antibody can enhance the killing effect of PBMC on tumor cells at an increased effector to target ratio.

[0090] During the treatment of Example 10, the survival rate and individual body weight changes of each treatment group were monitored. After the treatment, the lungs, liver, kidneys, small intestine and other organs of the mice were removed and stained with hematoxylin and eosin (H&E) to observe the pathological changes in the organ tissues.

[0091] As shown in Figures 13A and 13B, mice in the IFNα-Nivolumab group exhibited a higher survival rate and less change in body weight.

[0092] As shown in the H&E staining results of Figures 13C and 13D , the degree of organ damage in the IFNα-Nivolumab group was significantly reduced compared with the other treatment groups.

[0093] Peripheral blood was drawn from mice, and the activation status of T cell populations (CD3+, CD4+, CD8+ T cells) and the proportion of FoxP3+ regulatory T cells were analyzed using flow cytometry.

[0094] As shown in Figure 13E , the degree of T cell activation in each group was similar, and the proportion of FoxP3+ regulatory T cells remained balanced, indicating that IFNα-Nivolumab did not cause excessive immune activation or immunosuppression.

[0095] In summary, this example demonstrates that the IFNα-Nivolumab fusion protein can be cleaved by MMP-2 / 9, restoring the activity of Nivolumab, enhancing MHC-I expression, and improving the cytotoxicity of T cells. It also demonstrated good tumor suppression effects and safety in animal experiments.

[0096] While the invention has been described and illustrated in sufficient detail for those skilled in the art to make and use it, it will be apparent that various alternatives, modifications and improvements will occur without departing from the spirit and scope of the invention.

[0097] Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the foregoing objects and obtain the aforementioned purposes and advantages, as well as those inherent therein. The processes and methods for producing the same represent preferred embodiments and are exemplary and do not limit the scope of the invention. Modifications and other uses thereof will occur to those skilled in the art. Such modifications are within the spirit of the invention and are defined by the scope of the claims.

Claims

1. An antibody that selectively activates in target cells or tissues to treat a symptom thereof, the antibody comprising: Functional antibodies comprising at least one light chain and at least one heavy chain, which, when activated, can be used to treat a disease condition; At least one peptide capable of optionally binding to an interferon receptor and activating a signaling pathway of the peptide, wherein the interferon receptor comprises IFNAR1, IFNAR2, IFNGR1 or IFNGR2; and At least two cleavable polypeptide linkers, wherein each cleavable polypeptide linker comprises a substrate peptide that can be cleaved by an enzyme that is specifically or highly expressed in target cells or tissues, and the cleavable polypeptide linker connects one of the peptides to the N-terminus of one of the at least one light chain and the at least one heavy chain of the functional antibody.

2. The antibody of claim 1, wherein the functional antibody is immunoglobulin G1 (IgG1), IgG2, IgG3 or IgG4.

3. The antibody of claim 1 , wherein the functional antibody is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-RANKL antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-GITR antibody, anti-HER2 antibody, anti-EGFR antibody, anti-VEGF antibody, anti-VEGFR2 antibody, anti-HLA-DR antibody, anti-ICOS antibody, anti-OX40 antibody, anti-IL6R antibody, anti-IL12 / 23 antibody, anti-CD3 antibody, anti-CD11a antibody, anti-CD20 antibody, anti-CD25 antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-CD52 antibody, anti-CD137 antibody, and anti-CD138 antibody. The antibody of claim 1 , wherein the functional antibody is an anti-CTLA-4 antibody or an anti-PD-1 antibody.

5. The antibody of claim 1, wherein the cleavable polypeptide linker comprises a substrate peptide cleavable by a matrix metalloproteinase (MMP).

6. A pharmaceutical composition, comprising: the antibody according to any one of claims 1 to 6; and a pharmaceutically acceptable carrier.

7. Use of a composition in preparing a medicament for treating metastatic cancer, comprising: administering an effective dose of the composition comprising the functional antibody according to claim 3 to an individual suffering from metastatic cancer.

8. The method of claim 7, wherein the metastatic cancer comprises metastatic melanoma, advanced renal cell carcinoma, metastatic colorectal cancer with high microsatellite instability (MSI-H), metastatic colorectal cancer with gene mismatch repair deficiency (dMMR), hepatocellular carcinoma, or metastatic non-small cell lung cancer.

9. The use according to claim 7, wherein the metastatic cancer is metastatic colorectal cancer with high microsatellite instability, metastatic colorectal cancer with gene mismatch repair function deficiency, or metastatic melanoma.

10. The use according to claim 7, wherein the effective dose is 0.1 mg / kg-3 mg / kg.

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