A sequential dosing cytokine-targeted therapy system and uses thereof

Abstract

This invention discloses a pharmaceutical composition comprising a first pharmaceutical component and a second pharmaceutical component. The first pharmaceutical component comprises a fusion protein molecule containing a fragment capable of binding IL-15 or IL-15 mutants, such as the IL-15Rα or IL-15Rα sushi domain; the second pharmaceutical component comprises an IL-15 drug molecule. The first and second pharmaceutical components are used for sequential administration, preferably the first pharmaceutical component is administered first, followed by the second pharmaceutical component. This composition achieves precise targeting through spatiotemporal reconstruction. After the fusion protein accumulates at the tumor site, it captures IL-15, forming a local high-concentration immune activation center, thereby significantly enhancing the anti-tumor efficacy while reducing systemic toxicity. This invention also relates to the fusion protein molecule, the IL-15 drug molecule, the method of administration, and its use in the prevention or treatment of interleukin-15 related tumors (such as liver cancer, breast cancer, lymphoma, etc.).

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a sequential drug delivery IL-15 targeted therapy system and its application. Background Technology

[0002] Interleukin (IL)-15 is a cytokine belonging to the four-alpha helical bundle family and is structurally related to IL-2. Because IL-2 and IL-15 share the IL-2 / IL-15Rβ and γc receptor chains, they exhibit similar biological properties in vitro. Furthermore, IL-2 and IL-15 each use proprietary alpha chains (IL-2Rα and IL-15Rα), which confer cytokine specificity and enhance cytokine binding affinity. IL-2 promotes rapid T cell proliferation during immune responses and facilitates activation-induced cell death (AICD) to eliminate self-reactive T cells, while also being crucial for the development and maintenance of regulatory T cells (Tregs). IL-2 primarily affects activated T cells and regulatory T cells (Tregs). IL-15 supports memory CD8+. + It contributes to the long-term survival and maintenance of T cells and natural killer (NK) cells. It typically has anti-apoptotic effects, primarily affecting NK cells and memory CD8+ cells. + T cells.

[0003] IL-15 primarily promotes anti-tumor responses by enhancing the cytotoxicity and survival rate of NK cells, thereby directly killing tumor cells. It also expands and maintains CD8+. + Memory T cells, which aid in long-term tumor surveillance. IL-15 can bypass Tregs and target CD8. + Memory T cells play a role in reducing immunosuppression in the tumor microenvironment.

[0004] IL-15 is an essential signaling pathway for NK cell development and homeostasis. After acquiring the IL-15 receptor, NK progenitor cells respond to IL-15 signaling and differentiate into mature NK cells. IL-15 regulates NK cell survival by preventing apoptosis (programmed cell death). This is achieved by activating survival pathways, leading to the upregulation of anti-apoptotic proteins such as Mcl-1. Simultaneously, IL-15 inhibits the expression and activation of pro-apoptotic proteins such as Bim, shifting cellular homeostasis towards survival. IL-15 mediates proliferation and expansion; it is a potent driver of NK cell proliferation and expansion, both in laboratory culture and in vivo. It primarily signals through the PI3K-AKT-mTOR and JAK-STAT5 pathways to drive cell growth and division. IL-15 promotes NK cell activation and cytotoxicity, enhancing NK cell cytotoxicity against cancer cells, a effect more pronounced when NK cells are co-stimulated with other cytokines such as IL-12. It significantly upregulates the expression of NK cell surface activation receptors, including NKp30, NKp46, and NKG2D, which helps NK cells recognize and kill cancer cells. In addition to directly killing cells, IL-15 can also promote the secretion of other pro-inflammatory cytokines by NK cells, including interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), thereby further enhancing the immune response.

[0005] IL-15 against CD8 + Regarding the role of T cells, IL-15 maintains the memory cell population and drives memory CD8 cells. + Homeostatic proliferation of T cells helps maintain a constant pool of these cells in the absence of neoantigen exposure. IL-15 promotes memory CD8. + T cells can survive and their programmed cell death (apoptosis) can be suppressed.

[0006] IL-15 can reduce CD8 + The activation threshold of T cells makes them more responsive to antigen stimulation. It can also independently activate some memory CD8+ cells. + T cells. Upon activation by IL-15, they can induce CD8... + T cells proliferate, produce cytotoxicity, and generate effector molecules. It can also enhance tumor-responsive CD8+. + In vivo antitumor activity of T cells. IL-15 promotes the depletion of progenitor CD8 cells. + Self-renewal of T cells (Tpex) is crucial for maintaining antigen-specific CD8 during continuous stimulation. + T cell repositories are crucial. In some cases, even in the absence of CD4... + Helper T cells, IL-15 can also be used for CD8+ T cells provide the assistance needed for longevity.

[0007] Although IL-15 has been reported to enhance the proliferation and function of regulatory T cells (Tregs), in the presence of both effector T cells and Tregs, IL-15 preferentially supports the proliferation and survival of antigen-specific cytotoxic T lymphocytes (CTLs) and other effector T cells. It can reduce the inhibitory activity of Tregs on effector tumor antigen-specific CTLs, making them more resistant to the inhibitory effects of Tregs. This allows effector T cells to better target tumor cells, thus benefiting cancer treatment.

[0008] IL-15 functions in vivo through a unique trans-presentation mechanism, a distinctive cell-contact-dependent mechanism in which antigen-presenting cells deliver the cytokine IL-15, which binds to its high-affinity receptor IL-15Rα, to neighboring target cells via IL-2 / 15Rα. and together The receptor complex is composed of the gC chain. This highly regulated process is crucial for key immune cells, including natural killer (NK) cells and memory CD8+ cells. + The development, survival, and function of T cells are crucial.

[0009] The IL-15 receptor IL-15Rα contains a sushi domain, which is the high-affinity binding region of the IL-15Rα protein. The unique structure and properties of the sushi domain enable it to form an exceptionally strong and stable binding to IL-15, which is crucial for IL-15 function. In many basic, preclinical, and clinical studies, the IL-15Rα sushi domain is frequently used to replace the extracellular region of the full-length IL-15Rα protein, performing its trans-presentation function.

[0010] The antitumor effects of IL-15 have been validated in numerous animal models, including solid tumors and hematologic malignancies. IL-15 can exert its antitumor activity not only on its own, but it can also be combined with immune checkpoint inhibitors, monoclonal antibodies, chemotherapy, or radiotherapy to achieve better therapeutic effects.

[0011] Clinically, several IL-15 drugs are currently in different stages of clinical trials. Apart from the earliest recombinant IL-15, other IL-15s are fusion proteins combining IL-15 with various proteins, including IL-15Rα, antibodies, etc. The following is a brief description of several IL-15 drugs.

[0012] NIZ985 (hetIL-15), developed by Novartis, has completed a Phase 1 / 1b clinical trial for metastatic and advanced solid tumors. The drug is also currently recruiting for a Phase 1 / 1b clinical trial for recurrent advanced solid tumors and lymphomas. Its mechanism of action involves the recombination of a non-covalent heterodimer of the full-length extracellular region of one molecule of IL-15 and one molecule of IL-15Rα.

[0013] SOT101 (SO-C101), developed by SOTIO Biotech, has completed a Phase 1 / 1b clinical trial and has entered a Phase 2 clinical trial in combination with pembrolizumab for the treatment of advanced / refractory solid tumors. Its structure is very similar to NIZ985, but there is a covalent linker peptide between the IL-15 and IL-15Rα sushi domains.

[0014] SAR445877, developed by Sanofi, is in early-stage clinical research for cancer. It is composed of a human anti-PD-1 antibody and a fusion protein of mutant human IL-15 and human IL-15Rα sushi domain.

[0015] NKTR-255, developed by Nektar Therapeutics, has completed a Phase 1 clinical trial in relapsed or refractory multiple myeloma and non-Hodgkin's lymphoma. A Phase 2 clinical trial in metastatic urothelial carcinoma is underway. Its structure is a polyethylene glycol conjugate of recombinant human IL-15.

[0016] SHR-1501 (P22339), developed by Hengrui Medicine Co., Ltd., is currently undergoing Phase 1 and Phase 2 clinical trials for advanced solid tumors and non-muscle-invasive bladder cancer (NMIBC). It is a fusion protein composed of an IL-15 / IL-15Rα complex and an IgG1-Fc fragment.

[0017] HCW9218, developed by HCW Biologics, is currently undergoing phase 1 / 1b and phase 2 clinical trials for advanced / metastatic solid tumors and advanced pancreatic cancer. It is a bifunctional molecule containing an IL-15 agonist and a TGF-β antagonist.

[0018] SIM0237, developed by Simcere, is currently undergoing a Phase 1 clinical trial for the treatment of various cancers, including non-muscle-invasive bladder cancer (NMIBC). Its molecule comprises a bifunctional molecule consisting of a mutated IL-15Rα sushi domain fused with IL-15, acting as both an agonist and a PD-L1 antagonist. The contained IL-15 is a modified molecule with low activity.

[0019] PF-07209960, developed by Pfizer, contains a PD-1 antibody and a modified, lower-activity IL-15.

[0020] SAR445710 / KD033, developed by Sanofi / Kadmon, had its Phase 1 clinical trial terminated in January 2024. One part of its molecule is an anti-PD-L1 antibody, and the other part is an IL-15 and IL-15Rα complex.

[0021] Xmab24306, developed by Xencor / Genentech, is currently undergoing or has completed Phase 1 studies. This molecule is a fusion product of an IL-15 / IL-15Rα complex and Fc.

[0022] N-803, also known as Anktiva, was developed by ImmunityBio. Anktiva contains a mutated human IL-15 protein called IL-15N72D (nogapendekin alfa), with the asparagine (N) at position 72 mutated to aspartic acid (D). This modification increases its biological activity and binding affinity to IL-15 receptor signaling components. Another component is a dimer fusion protein, where the high-affinity sushi domain of IL-15Rα binds to the Fc portion of a human IgG1 antibody (inbakicept). IL-15N72D forms a non-covalent complex with the IL-15Rα sushi in inbakicept. Anktiva is approved for the treatment of adult patients with non-MIBC who are unresponsive to BCG and is currently the only approved IL-15 drug. It should be noted that Anktiva is administered via intratumoral injection for this clinical application. In addition, several other clinical trials of Anktiva are underway.

[0023] The aforementioned IL-15 drug molecules, whether alone or in complex form with IL-15Rα, are expressed in a fixed ratio when forming fusion proteins with target molecules or peptides such as Fc. They are usually divalent, meaning that a complete drug molecule contains two IL-15 molecules, such as N-803 and SIM0237, or monovalent, meaning that a complete drug molecule contains one IL-15 molecule, such as PF-07209960 and Xmab24306.

[0024] IL-15 itself has a molecular weight of only 14 kDa, is rapidly excreted by the kidneys, and has a very short half-life. In vivo, peripheral immune cells such as those in the blood and mucous membranes contain a large number of IL-15 receptors, which consume the drug, a phenomenon known as TMDD (Target-Mediated Drug Deposition). TMDD not only accelerates IL-15 consumption and reduces its blood concentration but also causes side effects. In mice, the half-life of intravenous, subcutaneous, or abdominal administration is approximately 40 minutes. In humans, the half-life is approximately 2.5 hours for intravenous administration and about 4 hours for subcutaneous administration. These studies suggest that IL-15 monotherapy has limited efficacy and is difficult to develop into a viable drug; therefore, the mechanisms limiting its therapeutic efficacy and further improvement strategies need to be considered. Summary of the Invention

[0025] The present invention aims to solve the existing technical problems and provides a sequential drug delivery IL-15 targeted therapy system and its application.

[0026] According to one aspect of the present invention, a pharmaceutical composition is provided comprising a separately packaged first pharmaceutical component and a second pharmaceutical component, wherein: the first pharmaceutical component comprises a fusion protein molecule, the fusion protein molecule comprising a fragment capable of binding IL-15 or an IL-15 mutant; the second pharmaceutical component comprises an IL-15 pharmaceutical molecule; and the first and second pharmaceutical components are for sequential administration.

[0027] Preferably, the fragment is an IL-15Rα or IL-15Rα sushi domain.

[0028] Preferably, the IL-15Rα or IL-15Rα sushi domain is not bound to IL-15 or an IL-15 mutant.

[0029] Preferably, the IL-15Rα or IL-15Rα sushi domain is wild-type.

[0030] Preferably, the IL-15Rα or IL-15Rα sushi domain is abruptly modified.

[0031] Preferably, the dissociation constant KD of the fusion protein molecule binding to IL-15 or the IL-15 mutant is less than 1 / 2. M.

[0032] Preferably, the dissociation constant KD is less than M.

[0033] Preferably, the dissociation constant KD is less than M.

[0034] Preferably, the fusion protein molecule further comprises an antibody or antigen-binding fragment or a receptor fragment.

[0035] Preferably, the antibody or antigen-binding fragment is selected from Fab, nanobodies, scFv antibodies, or bispecific antibodies.

[0036] Preferably, the fusion protein molecule further comprises an Fc fragment or human serum albumin HSA or a fragment thereof.

[0037] Preferably, the antibody, antigen-binding fragment, or receptor fragment is capable of binding to tumor antigens or tumor targets, and its dissociation constant KD is less than [value missing]. M.

[0038] Preferably, the tumor antigens or tumor targets that the fusion protein molecule can bind to are selected from the group consisting of: PD-1, PD-L1, CTLA4, CD20, CD19, CD30, Her2, TROP-2, BCMA, CD33, CD22, Nectin-4, c-MET, Tissuefactor, FRα, CD38, gp100, Her3, CEACAM5, EpCAM, MUC16, DLL3, CLDN18.2, CLDN6, B7-H3, B7-H4, ROR1, ITGB6, CDH6, CDH17, MUC1, GPC-3, CSF1R, LAG3, TIM3, PSMA, Mesothelin, CD79b, CAIX, DDR1, FAP, OSMR, VEGF, EGF, and TGFβ-1.

[0039] Preferably, the IL-15Rα or IL-15Rα sushi domain is located at the amino terminus, carboxyl terminus, or intermediate position of the fusion protein molecule.

[0040] Preferably, the IL-15 drug molecule is wild-type IL-15, an IL-15 mutant, or a protein containing wild-type and mutant IL-15, or chemically modified wild-type IL-15, or chemically modified IL-15 mutant.

[0041] Preferably, the dissociation constant KD of the IL-15 drug molecule binding to the fusion protein molecule is less than 1 / 2. M.

[0042] Preferably, the time intervals for sequential administration are 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, and 14 days.

[0043] Preferably, the time interval is from 1 hour to 7 days.

[0044] Preferably, the molar ratio of the fusion protein molecule in the first drug component to the IL-15 drug molecule in the second drug component is greater than 4:1.

[0045] According to another aspect of the invention, a fusion protein molecule is provided comprising a fragment capable of binding IL-15 or an IL-15 mutant, said fragment being an IL-15Rα or IL-15Rα sushi domain.

[0046] Preferably, the IL-15Rα or IL-15Rα sushi domain is not bound to IL-15 or an IL-15 mutant.

[0047] Preferably, it further comprises an antibody or antigen-binding fragment or receptor fragment, which is capable of binding to tumor antigens or tumor targets.

[0048] According to another aspect of the invention, an IL-15 drug molecule is provided for use in combination with the previously described fusion protein molecule in sequential administration.

[0049] According to another aspect of the invention, a method of administration is provided, comprising sequentially administering to a subject in need of: (a) a first pharmaceutical component comprising the previously described fusion protein molecule; and (b) a second pharmaceutical component comprising an IL-15 pharmaceutical molecule.

[0050] Preferably, the first drug component is administered first, followed by the second drug component.

[0051] Preferably, the time interval for sequential administration is 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0052] Preferably, the molar ratio of the fusion protein molecule to the IL-15 drug molecule is greater than 4:1.

[0053] Preferably, the administration is performed via intravenous or subcutaneous injection.

[0054] According to another aspect of the invention, an isolated nucleic acid molecule is provided that encodes the previously described fusion protein molecule or IL-15 drug molecule.

[0055] According to another aspect of the invention, an expression vector is provided comprising the previously described isolated nucleic acid molecules.

[0056] According to another aspect of the invention, a host cell is provided comprising the previously described isolated nucleic acid molecule or the previously described expression vector.

[0057] According to another aspect of the invention, a method for producing a fusion protein molecule or IL-15 is provided, comprising culturing the aforementioned host cells and isolating the fusion protein molecule or IL-15 from the culture.

[0058] According to another aspect of the invention, the use of the previously described pharmaceutical composition or the previously described method of administration is provided in the preparation of a medicament for the prevention or treatment of interleukin-15-related tumors.

[0059] Preferably, the interleukin-15-related tumor is one that can be activated or proliferated by IL-15. Tumor cells and / or NK cells have a response.

[0060] Preferably, the interleukin-15 related tumors are selected from the group consisting of: liver cancer, stomach cancer, esophageal cancer, skin cancer, prostate cancer, pancreatic cancer, colorectal cancer, ovarian cancer, head and neck cancer, bladder cancer, urothelial carcinoma, cervical cancer, breast cancer, kidney cancer, glioblastoma, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, and non-Hodgkin lymphoma.

[0061] Improving the metabolism and toxicity of IL-15 drugs can be achieved through attenuation, targeting tumor tissue, and forming complexes with other peptides. Attenuation aims to reduce binding to IL-15 receptors in the peripheral system, decrease and delay drug consumption by TMDD (tumor-mediated diuresis), prolong the half-life, and allow more time for the drug to enter the lesion. Complexation can increase the molecular weight of IL-15-containing drugs, altering their renal metabolic pathway. Complex formation methods include covalent or non-covalent binding with IL-15Rα, Fc fusion, fusion with other antibodies, fusion with peptides or other proteins, PEGylation, etc. Once the molecular weight of the complex exceeds the glomerular filtration threshold, the primary metabolic pathway becomes hepatic metabolism. Binding drugs to molecules targeting tumors or immune cells can increase the drug's effect on the lesion or immune cells, alter drug distribution, and reduce side effects. Targeting methods typically involve fusing with antibodies or peptides targeting tumor antigens. Targeting targets for drugs in clinical and preclinical investigations include PD-1, PD-L1, LAG-3, CD20, GD2, MUC1, and integrins. Compared to IL-15 itself, both complexes and attenuation can significantly improve the pharmacokinetic (PK) properties of IL-15 drugs. Adding a targeting molecule can also increase targeted binding to tumor antigens, leading to drug accumulation in tumor tissue. However, due to the widespread presence of the IL-15 receptor, the overall PK properties of drugs containing IL-15 remain quite limited. In particular, compared to conventional antibodies, antibodies linked to IL-15 exhibit faster clearance efficiency and shorter in vivo residence time. However, this limitation cannot be compensated for by increasing the dosage and dosing frequency, as the increased IL-15 content would increase toxicity and reduce tolerability. Similarly, although attenuation can reduce toxicity to some extent, selecting an attenuated IL-15 mutant that possesses sufficient IL-15 activity, tolerable IL-15 toxicity, and allows the antibody molecule in the fusion protein to utilize a sufficient dose to achieve ideal receptor occupancy is extremely challenging in early development. Furthermore, the ideal antibody dose varies for different antigens; therefore, the same mutated IL-15 cannot be matched with all antibodies.

[0062] In summary, the pharmacokinetic (PK) properties and toxicity of drugs bound to IL-15 by target molecules (such as antibodies) are affected by the TMDD of IL-15. This means that the target molecule in the drug can only perform its targeting function, while its own tumor-suppressive function cannot be utilized due to dose and drug concentration limitations. For example, Kadmon's KD033 (PD-L1 fusion IL-15 complex) was administered at two doses in phase I clinical trials: 3 μg / kg and 25 μg / kg every two weeks. In contrast, clinically used monotherapy PD-L1 antibodies such as durvalumab are administered at doses of 10 mg / kg every two weeks or 20 mg / kg every three weeks, avelumab at a single dose of 800 mg, and atezolizumab at doses of 840 mg every two weeks, 1200 mg every three weeks, or 1680 mg every four weeks. The combination of a target molecule with mutation-induced attenuation of IL-15 offers a possibility of both reducing drug toxicity and improving drug pharmacokinetics, thereby increasing the dose of the target molecule. However, in practice, it is very difficult to achieve a balance where both the target molecules and IL-15 are used at ideal drug doses. Moreover, due to differences in antigen expression levels, the doses of target molecules targeting different antigens are also different. Therefore, a mutation-reduced IL-15 cannot be applied to all target molecules.

[0063] In existing technologies, SIM0237 is an anti-PD-L1 monoclonal antibody and an inactivated IL-15 / IL-15Rα sushi fusion protein. It can block the PD-1 / PD-L1 immunosuppressive pathway by binding to PD-L1, while simultaneously activating the immune system through IL-15, thus achieving a synergistic effect of relieving immunosuppression and activating immune anti-tumor activity through a dual pathway. Because it binds to the IL-15 molecule, its half-life after administration is relatively short. SIM0237 aims to prolong the half-life of the fusion protein by inactivating IL-15. However, even with reduced IL-15 activity, the half-life of the fusion protein cannot reach the level of the naked antibody (e.g., ...). Figure 1 (As shown).

[0064] Rituximab (RTX) is a chimeric monoclonal antibody (mAb) targeting the CD20 antigen, currently used in combination with chemotherapy to treat follicular non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma, and chronic lymphocytic leukemia (CLL). IL-15 has been shown to increase the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of RTX against lymphoma B-cell lines and lymphoma cells from CLL patients. Recently, in vitro demonstrations of IL-15 delivery to B-cell leukemia cells from CLL patients have shown its ability to stimulate and expand autologous NK cells, leading to the clearance of B-cell leukemia cells; this process is significantly enhanced in the presence of monoclonal antibodies such as RTX or GA101 (an optimized anti-CD20 antibody). Anti-CD20-RLI is a protein that combines RTX and RLI (which binds the human IL-15 rα-sushi+ domain to human IL-15). It exhibits antibody-dependent cytotoxicity, classical complement-dependent cytotoxicity, and the cytokine potential of RLI. However, the half-life of RTX antibodies is significantly reduced after incorporation into the IL-15 complex (e.g., ...). Figure 2 (As shown).

[0065] Therefore, while the binding complex can significantly improve the pharmacokinetic (PK) properties of IL-15 drugs and increase their targeted binding to tumor antigens compared to IL-15 itself, the overall PK properties of the drug are not significantly altered due to the widespread presence of the IL-15 receptor, particularly in terms of significantly reducing the PK of the contained antibody. Furthermore, although attenuated IL-15 can reduce its toxicity to some extent, there are still dose limitations; the high doses used with conventional antibodies cannot be employed. Additionally, antigen-positive cells in non-tumor tissues can also bind to the drug and produce side effects. At limited doses, the amount of drug that can actually reach tumor tissue is also limited. In conclusion, the PK properties and toxicity of drugs bound to IL-15 by target molecules (such as antibodies) are affected by the TMDD of IL-15, meaning that the target molecule in the drug can only perform its targeting function, while its own tumor-suppressive function cannot be utilized due to dose and drug concentration limitations.

[0066] In developing IL-15-based targeted immunotherapies, the industry faces a long-standing core technological paradox: within the same molecule, the three key attributes of a drug—long-acting circulation, potent activation, and controllable safety—are mutually exclusive, forming an irreconcilable triangle. Specifically, pursuing long-acting circulation requires the drug to remain stable in the body for an extended period, like an antibody, necessitating the avoidance of rapid clearance. However, due to the widespread expression of its receptor, IL-15 triggers a strong target-mediated drug disposition effect (TMDD), drastically shortening the half-life of any target molecule (such as an antibody) it fuses with, thus preventing long-term efficacy. Pursuing potent activation requires IL-15 to maintain high activity to fully activate immune cells. However, this exacerbates its non-specific stimulation of the systemic immune system, leading to severe dose-limiting toxicity, preventing the use of effective high doses, and resulting in uncontrollable safety. If controllable safety is prioritized, toxicity can be reduced by inactivating IL-15 mutations. While this improves tolerability, it directly weakens the core therapeutic function of potent activation, resulting in compromised efficacy.

[0067] Therefore, existing technologies have reached a stalemate. Any optimization of a single fusion protein molecule is like a seesaw, where improving one property inevitably comes at the expense of another. This patent aims to solve this deep-seated structural contradiction at the molecular design level: how to break the mutually exclusive shackles between efficacy, pharmacokinetics, and safety in IL-15 targeted therapy.

[0068] To address the problems existing in the prior art, firstly, this invention decouples the system into two independent molecules. The pharmacokinetic properties of the IL-15Rα-containing fusion protein are no longer interfered with by IL-15, enabling it to achieve long-lasting circulation and high accumulation within tumors, similar to a natural antibody. Simultaneously, the subsequent administration of IL-15 is captured, improving and prolonging its pharmacokinetic profile and effective duration of action. Thus, all components within the system achieve optimal pharmacokinetic properties. Secondly, this invention does not sacrifice potency for safety, but rather achieves precise and potent efficacy through spatiotemporal reconstruction. The lead fusion protein forms a trapping network in vivo, rapidly capturing and enriching subsequently injected highly active natural IL-15 in the target region, thereby creating a locally high-concentration immune activation center within the tumor microenvironment, achieving more efficient and precise stimulation than systemic administration. Simultaneously, this strategy can also utilize mutated or modified IL-15, such as the IL-15N72D (nogapendekin alfa) mutant, making it more potent than the wild type, or attenuated IL-15 mutants, making it safer than the wild type. Furthermore, this strategy achieves spatiotemporal control of function through physical separation. The fusion protein, lacking cytokine activity, circulates for the vast majority of the time within the system, fundamentally eliminating systemic toxicity caused by continuous activation. Low-dose IL-15 is anchored immediately after administration, its activity directed and restricted to the tumor site, significantly reducing the risk of off-target toxicity and making the safe administration of high-dose fusion proteins possible.

[0069] This invention breaks the traditional dilemma of trade-offs, simultaneously optimizing three originally contradictory goals: targeted delivery of fusion proteins, long half-life, complete activity of IL-15, and overall high safety. It opens up a new path for the clinical application of IL-15 that combines excellent efficacy and outstanding safety.

[0070] One aspect of this invention is the use of a fusion protein, the core of which is IL-15Rα. As part of the fusion protein, IL-15Rα typically refers to its extracellular portion, also known as soluble IL-15Rα. The amino acid sequence of the extracellular region of human IL-15Rα is shown in Seq ID NO. 02.

[0071] In practical applications, usually only the sushi domain is needed, as in the sequences used in preferred embodiments 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17. Specific amino acid sequences are shown in Seq ID NO. 04, Seq ID NO. 05, Seq ID NO. 06, and Seq ID NO. 07.

[0072] In other embodiments, such as Examples 1, 2, 3, 4, 5, and 6, the mouse-derived IL-15Rα sushi domain was used to construct the fusion protein. The full-length sequence of the mouse-derived IL-15Rα extracellular region is shown in Seq ID NO. 03, and the sequence of the mouse-derived IL-15Rα sushi is shown in Seq ID NO. 29.

[0073] In some specific embodiments, the IL-15Rα sushi domain forms a fusion protein with immunoglobulin, as in Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 16, 17.

[0074] In some specific embodiments, the IL-15Rα sushi domain may be located at the C-terminus of the fusion protein, as in Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 16, and 17. In another specific embodiment, the IL-15Rα sushi domain may be located at the N-terminus of the fusion protein, as in Example 14. In yet another embodiment, the IL-15Rα sushi domain may be located in the middle of the fusion protein, as in Example 10.

[0075] In some embodiments, the IL-15Rα sushi domain can form fusion proteins with other antibody forms via the Fc region of immunoglobulin. In one specific embodiment, the IL-15Rα sushi domain forms a fusion protein with a heavy-chain single-domain antibody (VHH, also known as a single-domain antibody or nanobody) via the Fc region of immunoglobulin, as in Example 10. In another specific embodiment, the IL-15Rα sushi domain forms a fusion protein with a single-chain variable fragment antibody via the Fc region of immunoglobulin, as in Example 14. The human IgG1 Fc sequence is shown in Seq ID NO. 08.

[0076] In some embodiments, the fusion protein containing the IL-15Rα sushi domain may also contain extracellular portions of other receptors such as VEGFR1-D2 (vascular endothelial growth factor receptor 2-D2 domain), as in Examples 11, 13, 16, and 17. The VEGFR1-D2 sequence is available in Seq ID NO. 09.

[0077] In some embodiments, the IL-15Rα sushi domain can also form fusion proteins with bifunctional antibodies, as in Examples 11, 13, 16, and 17.

[0078] In the embodiments of this invention, the preferred IL-15Rα sushi domain is derived from wild-type human IL-15Rα and wild-type mouse IL-15Rα. This invention is also applicable to IL-15Rα from other mammals or non-mammals.

[0079] In the embodiments of this invention, the IL-15Rα sushi domain preferably used is derived from wild-type human IL-15Rα and wild-type mouse IL-15Rα. This invention is also applicable to mutants developed based on preferred wild-type sequences, as well as mutants of other mammalian or non-mammal IL-15Rα.

[0080] In some embodiments, the IL-15Rα sushi domain forms a fusion protein with other proteins without a linker, while in another specific embodiment, the IL-15Rα sushi domain forms a fusion protein with other proteins through a linker, as in Examples 10 and 14.

[0081] In the embodiments described in this invention, the half-life of the IL-15Rα-containing fusion protein is primarily extended through the Fc region of the immunoglobulin. In clinical and research practice, human serum albumin (HSA) has also been shown to extend the half-life; therefore, the Fc region of the fusion protein in this invention can be replaced by human serum albumin (HSA). The Fc region can also enhance the immunological function of the drug, such as antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

[0082] In the embodiments described in this invention, the half-life of the IL-15Rα-containing fusion protein is extended primarily by utilizing the Fc region of human immunoglobulin IgG1 and the Fc region of mouse IgG2a. Mouse IgG2a is generally considered equivalent to human IgG1. The Fc region can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. The Fc region sequence of mouse IgG2a is shown in Seq ID No. 10.

[0083] In the embodiments of the present invention, the extracellular portion of different forms of antibodies or receptors targets multiple tumor antigens, including cell membrane surface antigens such as PD-L1 (Examples 1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17), Claudin18.2 (Example 9), as well as soluble antigens such as VEGF-A (Examples 11, 13, 16, 17) and EGF (Example 14). Similarly, the antigens (targets) to which this invention can be applied include, but are not limited to: PD-1, PD-L1, CTLA4, CD20, CD19, CD30, Her2, TROP-2, BCMA, CD33, CD22, Nectin-4, c-MET, Tissuefactor, FRa, CD38, gp100, Her3, CEACAM5, EpCAM, MUC16, DLL3, CLDN18.2, CLDN6, B7-H3, B7-H4, ROR1, ITGB6, CDH6, CDH17, MUC1, GPC-3, CSF1R, LAG3, TIM3, PSMA, Mesothelin, CD79b, CAIX, DDR1, FAP, OSMR, VEGF, EGF, and TGFb-1.

[0084] A second aspect of the invention is the use of interleukin-15 (IL-15). Preferably, embodiments 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17 of the invention all use wild-type human IL-15, the sequence of which is shown in Seq ID NO. 01.

[0085] Preferably, the present invention is also applicable to IL-15 in other mammalian or non-mammal species, such as mice, monkeys, pigs, rabbits, and chickens. The IL-15 sequence of mice is shown in Seq ID NO.11.

[0086] In the embodiments of the present invention, human wild-type IL-15 is preferred. The present invention can also be applied to mutants developed based on preferred wild-type sequences, as well as mutants of other mammalian or non-mammal IL-15.

[0087] The third aspect of this invention is a fusion protein of IL-15Rα and sequential administration of IL-15, wherein the fusion protein is injected first, followed by IL-15 after a period of time. The first-injected fusion protein captures the subsequently injected IL-15 in vivo and trans-presents it to effector cells; this process can be termed "capture".

[0088] In embodiments of the present invention, the preferred time interval between the injection of the fusion protein and IL-15 is 30 minutes, as in Examples 3, 4, 6, 7, 8, 15, 16, and 17. In actual clinical use, IL-15 can be injected at intervals of 5 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, up to 1 day, 7 days, and 14 days after the fusion protein injection. The time interval depends on the PK behavior and serum concentration of the fusion protein, as well as the treatment requirements, and IL-15 can even be injected more than once within a single fusion protein injection cycle. This is precisely one of the advantages of the present invention.

[0089] Another advantage of the sequential dosing of the present invention is that the dosage of the fusion protein and the dosage of IL-15 depend on the balance of their respective efficacy and toxicity, and do not limit each other.

[0090] This invention preferably uses intravenous injection to administer the fusion protein and IL-15. In practical applications, antibodies or fusion proteins can also be administered subcutaneously, which is a trend in the administration of large molecule drugs. IL-15 is also administered clinically via both intravenous and subcutaneous injection, and practice has shown that the bioavailability of IL-15 is the same under both methods. Therefore, IL-15 can also be administered subcutaneously in this invention.

[0091] In the embodiments of this invention, the tumor types that have been tested and demonstrated therapeutic efficacy include skin cancer (melanoma) (Example 4), colorectal cancer (Examples 7, 8, 15, 16, 17), and gastric cancer (Example 9). Similarly, the types of cancer to which this invention is applicable include, but are not limited to: liver cancer, gastric cancer, esophageal cancer, skin cancer, prostate cancer, pancreatic cancer, colorectal cancer, ovarian cancer, head and neck cancer, bladder cancer, urothelial carcinoma, cervical cancer, breast cancer, kidney cancer, glioblastoma, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, and non-Hodgkin lymphoma.

[0092] A fourth aspect of the present invention is to provide a method for preventing and / or treating diseases in an individual, said diseases including, but not limited to, liver cancer, stomach cancer, esophageal cancer, skin cancer, prostate cancer, pancreatic cancer, colorectal cancer, ovarian cancer, head and neck cancer, bladder cancer, urothelial carcinoma, cervical cancer, breast cancer, kidney cancer, glioblastoma, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, etc.

[0093] Terminology Definitions and Explanations Unless otherwise specified, the terms used herein have the meanings as understood by one of ordinary skill in the art.

[0094] The terms "interleukin-15" or "IL-15" refer to a cytokine with a specific amino acid sequence. IL-15 is a cytokine belonging to the four-alpha helical bundle family and is structurally related to IL-2. They share the IL-2 / IL-15Rβ and γ(c) receptor chains and exhibit similar biological properties in vitro. However, IL-2 and IL-15 each use their proprietary alpha chains (IL-2Rα and IL-15Rα), which confer cytokine specificity and enhance cytokine binding affinity. IL-15 supports memory. It promotes the long-term survival and maintenance of cells and natural killer (NK) cells. It typically has anti-apoptotic effects. It primarily affects NK cells and memory cells. Cells. IL-15 can be IL-15 from common mammals such as humans, mice, and monkeys, or from other mammals or non-mammals. IL-15 also refers to "wild-type IL-15" or "IL-15 mutants." "Wild-type IL-15" refers to naturally occurring IL-15 from common mammals such as humans, mice, and monkeys, or IL-15 from non-mammals. The sequence of human wild-type IL-15 can be found in Seq ID NO.01, or in the UniProtKB database at accession number P40933. "IL-15 mutants" refer to IL-15 molecules with different activities or specificities obtained through gene recombination by substituting, deleting, or inserting amino acid sequences into the IL-15 sequence.

[0095] The term "IL-15Rα" refers to the proprietary receptor for IL-15. The IL-15 receptor has three subunits, only the α subunit is specific to IL-15; the other two subunits are shared by IL-15 and IL-2. IL-15Rα has extremely high affinity for IL-15. "IL-15Rα" can refer to IL-15Rα from common mammals such as humans, mice, and monkeys, or from other mammals or non-mammals. "IL-15Rα" can also refer to "wild-type IL-15Rα" or "IL-15Rα mutants." Wild-type IL-15Rα refers to naturally occurring IL-15Rα from common mammals such as humans, mice, and monkeys, or IL-15Rα from non-mammals. The sequence of human wild-type IL-15Rα can be found in Seq ID NO.02, or in the UniProtKB database at accession number Q13261. The sequence of wild-type IL-15Rα in mice is referenced in Seq ID NO.03, or see the sequence with accession number Q60819 in the UniProtKB database. "IL-15Rα mutant" refers to IL-15Rα molecules with different affinities obtained by substituting, deleting, or inserting amino acid sequences of IL-15Rα through gene recombination.

[0096] The term "soluble IL-15Rα (sIL-5Rα)" usually refers to the extracellular portion of IL-15Rα, and human sIL-15Rα contains amino acid sequence 31-205.

[0097] The term "IL-5Rα sushi domain" refers to a segment of the IL-5Rα protein, a key protein sequence for the high-affinity binding of IL-15Rα to the cytokine IL-15. The IL-15Rα sushi domain generally refers to a 65-amino acid-long extracellular region of the IL-15Rα protein, which is the shortest segment capable of binding IL-15. Sequences of slightly different lengths are also considered as IL-15Rα sushi domains. These sequences are listed in Seq ID NO. 04, Seq ID NO. 05, Seq ID NO. 06, and Seq ID NO. 07. The IL-15Rα sushi domain is preferred as a component of fusion proteins.

[0098] The terms "tumor target" and "tumor antigen" are used interchangeably. A tumor target is a specific molecule, protein, or genetic feature present in cancer cells that drives their growth, division, and survival. These are specific molecules or pathways in cancer cells that targeted therapeutic drugs attack. A tumor antigen, on the other hand, is a molecule expressed by tumor cells, typically on the cell membrane surface. Tumor-specific antigens are expressed only on tumor cells, while tumor-associated antigens, although also present in some normal cells, are usually present in much lower concentrations in tumor cells than in cancer cells, making them potential targets for immunotherapy.

[0099] The term "targeting molecule" refers to a drug molecule that acts on specific biomolecules that cause disease; it can also be called a targeted drug, and there are macromolecular and small molecule targeted drugs. Macromolecular targeted drugs usually refer to the extracellular region of antibodies or receptors. Unless otherwise specified, the targeting molecules in this article refer to macromolecular targeting molecules. These molecules can enter the tumor microenvironment and accumulate in the tumor environment.

[0100] The term "antibody" refers to an immunoglobulin molecule secreted by B cells that recognizes specific antigens. Antibody molecules are typically tetramers composed of two heavy chains and two light chains. The amino terminus (N-terminus) of both the light and heavy chains is the variable region, followed by the constant region. The carboxyl terminus (C-terminus) of the heavy chain's constant region is the Fc region. Antibodies can be classified into IgG, IgM, IgA, IgD, IgE, etc. In addition, the term "antibody" also includes antibody fragments such as Fab, recombinant antibodies such as scFv (single-chain variable fragment antibody), nanobodies, bispecific antibodies, etc.

[0101] The term "nanobody" (Nb), also known as a single-domain antibody (sdAb) with a variable heavy chain domain (VHH), refers to a small, recombinant antigen-binding fragment derived from unique heavy chain antibodies found in camelidae animals (such as camels, llamas, and alpacas) and cartilaginous fish (such as sharks).

[0102] The term "bispecific antibody" or "bispecific monoclonal antibody" (BsAb, BsMAb) refers to an artificial protein capable of simultaneously binding to two different types of antigens or two different epitopes on the same antigen. Its antigen-binding site is typically derived from two monoclonal antibodies, expressed through genetic engineering.

[0103] The term "fusion protein" refers to a hybrid protein formed by the linking and expression of two or more genes that originally encode different proteins. The resulting polypeptide chain retains the functional properties of each original component. Fusion proteins can occur naturally, such as in certain cancers, or can be artificially synthesized using recombinant DNA technology for specific applications in research and medicine. A common example is the Fc fusion protein, which binds a therapeutic protein to the Fc (crystallizable fragment) of an antibody. The Fc region prolongs the drug's half-life in the bloodstream, thus reducing the frequency of drug administration to patients. Other common fusion proteins include immunotoxins, which are used to target cancer therapy by linking the targeting portion (e.g., an antibody) to a cytotoxic drug or toxin. This allows the toxin to be specifically delivered to cancer cells. Bispecific fusion proteins contain multiple binding domains, allowing them to target two different molecules simultaneously. For example, they can link cancer cells to T cells, triggering an immune response against the tumor. Multidomain fusion proteins bind multiple functional domains, resulting in a synergistic effect. A key aspect of this invention is the use of a fusion protein, the core of which is IL-15Rα, preferably the IL-15Rα sushi domain. Other portions of the fusion protein include targeting regions such as antibodies, nanobodies, and scFv antibodies, and may also include other functional domains such as receptors. The fusion protein of this invention preferably uses an Fc fragment to extend its half-life. Similarly, human serum albumin (HSA) can also be used as the functional portion for extending the half-life.

[0104] The term "linker" refers to a short polypeptide used during the construction of a fusion protein to connect two proteins from different functional regions, ensuring proper folding and stability. Linkers are classified as rigid or flexible. This invention preferably uses the flexible linker (GGS)n or (GGGGS)n, where n can be 1, 2, 3, 4, 5, 6, etc., preferably 2, 3, 4, or 5. Attached Figure Description

[0105] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 This study compares the pharmacokinetics of Simcere's anti-PD-L1 monoclonal antibody and its attenuated IL-15 / IL-15Rα sushi fusion protein in cynomolgus monkeys with those of a conventional anti-PD-L1 antibody in the same monkeys. Figure 1 This demonstrates the comparison of three doses of the Similac fusion protein in cynomolgus monkeys. Figure 1b shows the pharmacokinetic (PK) of MPDL3280A (i.e., Atezolizumab) in cynomolgus monkeys. It is evident that although the IL-15 activity in SIM0237 was reduced by more than 600-fold, the circulation time of SIM0237 in vivo was still significantly shortened compared to the IL-15-free MPDL3280A molecule.

[0106] Figure 2 The half-life of the anti-CD20-RLI fusion protein and rituximab in animals was compared.

[0107] Figure 3 To identify the expression, purification, and in vitro function of Ab1.

[0108] Figure 4 This is a comparison of the in vivo efficacy and toxicity of Ab1 with that of Ab2.

[0109] Figure 5 The effect of Ab1 on the PK of IL-15 in vivo.

[0110] Figure 6 The purpose of this study was to identify Ab3 expression, purification, and in vitro function, and to detect the in vivo efficacy of Ab3 combined with sequential administration of IL-15.

[0111] Figure 7 To identify the expression, purification, and in vitro function of Ab4 and Ab5.

[0112] Figure 8 This study compares the in vivo PK behavior of Ab4 and avelumab.

[0113] Figure 9 The in vivo efficacy of Ab5 and its combination with IL-15 in CT-26 colorectal cancer was investigated.

[0114] Figure 10 The in vivo efficacy of Ab5 and its combination with IL-15 in sequential administration against colorectal cancer MC38 was investigated.

[0115] Figure 11 To identify the expression, purification, and in vitro function of Ab7.

[0116] Figure 12 To identify the expression, purification, and in vitro function of Ab9.

[0117] Figure 13 To identify the expression, purification, and in vitro function of Ab10.

[0118] Figure 14 To identify the expression, purification, and in vitro function of Ab12.

[0119] Figure 15To identify the expression, purification, and in vitro function of Ab13.

[0120] Figure 16 To identify the expression, purification, and in vitro function of Ab14.

[0121] Figure 17 The in vivo efficacy of Ab12 and its combination with IL-15 in CT-26 colorectal cancer was investigated.

[0122] Figure 18 The in vivo efficacy of Ab5 combined with IL-15 sequential administration and Ab10 monotherapy against colorectal cancer MC38 was compared.

[0123] Figure 19 Comparison of the efficacy of Ab12 and Ab13 and their combination with IL-15 in CT-26. Detailed Implementation

[0124] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that the following examples are not intended to limit the scope of protection claimed by the present invention, but are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

[0125] 1. Example 1: Ab1 Expression and Functional Identification a) Ab1 is a mouse anti-mouse PD-L1 antibody clone 80 (Schoffeld et al., MABS 2021, VOL. 13, NO. 1, e1857100) that fused to the C-terminus of its heavy chain to express the sushi domain of mouse IL-15Rα, but without the D265A mutation. The sequence of Ab1 is shown in Seq ID NO. 12 (light chain) and Seq ID NO. 13 (heavy chain). The DNA vector sequence encoding the Ab1 protein was synthesized and the expression sequence was constructed into the pcDNA3.4 vector, and confirmed by sequencing to obtain the protein expression plasmid. The constructed plasmid of the protein to be expressed was transfected into Expi CHO cells for transient expression, with antibody expression time of 7 days. One day before transfection, Expi CHO cells were pre-treated with... cells / mL Cells / mL, cultured overnight. On the day of transfection, cell counting was performed; the Expi CHO cell density was approximately [missing information]. cells / mL cells / mL, viability > 98%; adjust Expi CHO cell density to Cells / mL. Determine the cell quantity based on the required expression volume. Before transfection, incubate the transfection reagent / plasmid DNA mixture at room temperature for 1-5 minutes, then add it dropwise to the prepared Expi CHO cell suspension while gently shaking the suspension. After transfection, incubate the cells at 37°C in a shaker with 7% CO2. On the first day after transfection (18-22 hours), add feed while gently shaking the cell suspension, then transfer the flask to a shaker with 7% CO2 for continued culture. On the fifth day after transfection, add feed to Expi CHO cells while gently shaking the suspension, then return the flask to the shaker with 7% CO2 for continued culture. On days 2, 5, and 7 after transfection, randomly sample different volumes of transfected Expi CHO cells to measure cell density and viability. Collect samples when the Expi CHO viable cell density is not less than 50% of the highest cell density and the cell viability is not less than 40%. Collect the cell supernatant after expression is complete.

[0126] b) Protein A affinity column chromatography purification: First, wash the chromatography system and affinity column with double-distilled water, then wash the column with 0.1M NaOH for 15 min; equilibrate with PBS buffer until pH and conductivity are stable; filter the expression supernatant and load the sample, retaining it for 4-6 min; after loading, wash the column with PBS until the A280 UV absorbance drops to baseline; then wash away impurities with 20mM phosphate buffer (pH 7.0) containing 0.1M NaCl. After washing away impurities, wash the column with PBS until the A280 UV absorbance and conductivity reach baseline. Finally, wash the column with 20mM citrate elution buffer (pH 3.4), and collect the elution sample according to the A280 UV absorption peak, neutralizing it to neutral with 1M Tris HCl (pH 9.0). The collected components were analyzed by SDS-PAGE electrophoresis, and necessary pooling was performed. Purity was analyzed, concentration and endotoxin content were measured, and endotoxin removal and storage buffer replacement were performed as needed. Figure 3 As shown, after one round of Protein A purification, the target protein Ab1 with a purity of 99% was obtained.

[0127] c) Direct ELISA method to identify the function of Ab1 in binding to PD-L1 or IL-15. Dilute the antibody to 2 μg / mL with 1× phosphate-buffered saline (PBS), coat the microplate with this solution, add 30 μL to each well, and incubate overnight at 4°C. Wash the microplate three times with phosphate-buffered saline (PBST) containing Tween 20, then block the microplate with 5% phosphate-buffered saline (5% PBSM) containing skim milk powder at room temperature for 2 hours. Wash the microplate three times again with PBST, add 30 µL of biotin-labeled mouse PD-L1 (Mouse-PD-L1-Biotin, Sanyou Biotechnology, catalog number P00632B5.93) diluted with 1% PBSM to each well, and incubate at room temperature for 60 minutes. Wash the microplate three times with PBST, then add streptavidin-horseradish peroxidase (NeutrAvidin-HRP, Thermo Fisher Scientific, catalog number 31001, diluted 1:2000 with 1×PBS), or anti-6×His-horseradish peroxidase (HRP). The sample was purchased from Pujian Biotechnology, catalog number HRP-66005, diluted 1% PBSM at a ratio of 1:4000. 30 µL of sample was added to each well and incubated at room temperature for 50 minutes. The plate was washed six times with PBST, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added. After the reaction, the reaction was terminated with 2 mol / L (2M) stop solution. Finally, the absorbance (OD450) was measured at 450 nm. Figure 3 b and Figure 3 As shown in c, Ab1 retains the ability to bind mouse-derived PD-L1, while the mIL-15Rαsushi domain at the C-terminus of its heavy chain can bind hIL-15.

[0128] d) Sandwich ELISA method to identify the function of Ab1 in simultaneously binding to IL-15 and PD-L1. The microplate was coated with 1×PBS solution (8 μg / mL) containing SY01-musPD-L1-ECD-huFC (Sanyou Bio, catalog number P00632), 30 μL per well, and incubated overnight at 4°C. The microplate was washed three times with PBST, then blocked with 5% PBSM and incubated at room temperature (RT) for 2 hours. The microplate was washed three times with PBST, and 30 μL of antibody diluted with 1% PBSM was added to each well, and incubated at room temperature for 60 minutes. The microplate was washed three times with PBST, and 30 μL of WW001-GSV0-IL-15-P40933-chis (Sanyou Bio, catalog number COAC0285) solution (4 μg / mL) prepared with 1% PBSM was added to each well, and incubated at room temperature for 60 minutes. Wash the ELISA plate three times with PBST, then add anti-6×histidine-tagged horseradish peroxidase conjugate antibody (Anti-6) diluted 1:4000 with 1% PBSM to the plate. his-HRP (Brand: Proteintech, Catalog No.: HRP-66005) was incubated at room temperature for 50 minutes. The plate was washed six times with PBST, TMB chromogenic buffer was added, and the reaction was terminated with 2M sulfuric acid (H2SO4) stop solution after color development. The absorbance (OD450) was measured at 450 nm. Figure 3 As shown in c, Ab1 can bind to both mouse-derived PD-L1 and hIL-15 simultaneously.

[0129] 2. Example 2: Efficacy and Toxicity of Ab1 a) Expand the tumor cell line MC38 to a certain quantity in vitro, and resuspend the cells at a density of 2×10⁶. 7 Tumor models were established by subcutaneous inoculation of 0.1 mL cells / mL into the back of each C57 mouse; on day 8 post-tumor implantation, the tumor volume was approximately 110 g / mL. The subjects were then randomly assigned to three groups (N=8). On the day of group assignment, they were given intraperitoneal injections of the solvent control and the corresponding drug according to the dosing regimen. Group G1 received PBS, Group G2 received 7.5 mg / kg Ab1, and Group G3 received 7.5 mg / kg Ab2. The injection volume for all three groups was 10 ml / kg, and the injection frequency was twice a week for three weeks. Ab2 was a mouse anti-mouse PD-L1 antibody clone 80 (Schoffeld et al., MABS 2021, VOL. 13, NO. 1, e1857100), but without the D265A mutation. The sequence is shown in Seq ID NO. 12 (light chain) and Seq ID NO. 14 (heavy chain). Ab1 was formed by fusing the sushi domain of mouse IL-15Rα to the C-terminus of the heavy chain of Ab2 (see Seq ID NO. 12 and Seq ID NO. 13). Tumor size, body weight, and other parameters were measured to observe the efficacy and toxicity. Figure 4 As shown in figure a, groups G1 and G2 have similar efficacy at the same mass dose. For example... Figure 4 As shown in b, none of the eight animals in each of the three groups showed significant weight loss, thus concluding that the addition of IL-15Rα to the PD-L1 antibody did not introduce additional toxicity.

[0130] 3. Example 3 Pre-addition of Ab1 to mice can bind to subsequently added IL-15 and alter its PK behavior (sequential administration).

[0131] a) Prepare Balb / c mice, SPF grade, 18-20g, male, 3 mice / group, intraperitoneally injected with the drug. Group 1 was injected with 500ug Ab1 first, followed by 2.5ug hIL-15 (Bepsys, IL-5-H4117) 0.5 hours later; Group 2 was injected with 2.5ug hIL-15 (Bepsys, IL-5-H4117) alone; Group 3 was injected with a pre-mixed mixture of 500ug Ab1 and 2.5ug hIL-15. Blood was collected from the orbital venous plexus of the mice using the canthal vein plexus method, at the following time points: 0h before drug administration, and 15min, 30min, 1h, 2h, 4h, 8h, 24h, 48h, 72h, and 96h after drug administration. The blood volume collected from each mouse was 40-60 μL.

[0132] b) Gently transfer the collected blood into a centrifuge tube without anticoagulant. Incubate the tube at room temperature (approximately 22-25°C) for 30-60 minutes to allow the blood to clot naturally. The blood will coagulate into a gel-like consistency, and a clear, pale yellow liquid will separate out; this is serum. Place the coagulated blood sample into a centrifuge and centrifuge at 4°C and 2000×g for 15 minutes. Using a micropipette, carefully aspirate the top layer of clear or pale yellow serum and transfer it to a new, pre-chilled, sterile centrifuge tube. Store under appropriate conditions.

[0133] c) After appropriate dilution, serum samples were analyzed using an ELISA method to determine the hIL-15 content. The ELISA kit was purchased from ACRO Biosystems (catalog number CRS-A024), and the experimental procedure was performed according to the kit's instructions. Figure 5 As shown in Figure a, compared with hIL-15 injection alone, pre-addition of Ab1 in vivo significantly altered the PK behavior of subsequently added hIL-15, with Cmax increasing from 2 ng / ul to nearly 30 ng / ul and half-life increasing from less than one hour to 20 hours. We also investigated whether pre-mixing in vitro and forming a complex in vivo had different efficiencies on IL-15 retention. Two animals were selected from the third group, which received pre-mixed 500 μg Ab1 and 2.5 μg hIL-15, and their serum IL-15 concentrations at 4 hours were measured and compared with the serum IL-15 concentrations at the same time point of two animals selected from the first group, which received 500 μg Ab1 first, followed by 2.5 μg hIL-15 0.5 hours later. Figure 5 As shown in b, although the serum IL-15 concentrations of the two animals in the third group (in vitro) injected with the premixed in vitro drug were comparable to those of the two animals in the first group injected with the sequential drug, it is still evident that the effect of the premixed in vitro drug tends to be inferior to that of the sequentially injected drug. Furthermore, experiments confirmed that in the sequential dosing regimen, the pharmacokinetic curve of Ab1 (which does not carry IL-15), as a fusion protein, was essentially consistent with that of the control group injected with Ab1 alone. This result demonstrates that the dispensing strategy of this invention successfully decouples the antibody from the TMDD effect of IL-15, allowing the targeting antibody to maintain its inherent long half-life and high exposure advantage even in the presence of IL-15—a feat unmatched by any existing fusion protein technology.

[0134] d) To further demonstrate that IL-15 in vivo forms a complex with IL-15Rα in the antibody fusion protein Ab1, we used ELISA to verify whether hIL-15 exists in vivo in the form of a complex. It is known that the anti-IL-15 monoclonal antibody Ordesekimab does not interfere with the binding of IL-15 to IL-15Rα. If IL-15 exists in the form of an antibody complex, the IL-15 complex captured from serum by Ordesekimab can be detected by a secondary antibody against a mouse antibody. Therefore, 1× phosphate buffer (1... Ordesekimab (catalog number P100708, Sanyou Biotechnology) was diluted to 2 μg / mL and used to coat an enzyme-linked immunosorbent assay (ELISA) plate (microplate). 30 µL of sample was added to each well, and the plate was incubated overnight at 4°C. The plate was washed three times with phosphate-buffered saline (PBST) containing Tween 20, followed by blocking with 5% phosphate-buffered saline (5% PBSM) at room temperature (RT) for 2 hours. The plate was washed three times again with PBST, and 30 µL of sample diluted with 1% PBSM was added to each well. The plate was incubated at room temperature for 60 minutes. Wash the microplate three times with PBST, add anti-mouse IgG Fc-horseradish peroxidase (Anti-mouse-IgG-Fc-HRP, purchased from Abcam, catalog number ab97265, diluted 1:8000 with 1% PBSM), 30 µL per well, and incubate at room temperature for 50 minutes. Wash the microplate six times with PBST, add 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution, and after the reaction, rinse with 2 mol / L (2M) sulfuric acid. The reaction was terminated with a stop solution, and the absorbance (OD450) at 450 nm was measured. Test samples were selected from the serum of animals G1-1 and G1-3 in the first group of sequentially administered drugs, as well as serum at 0.5 hours and 2 hours, and were diluted several times. Figure 5 As shown in c, the ELISA results for these samples were all positive, and the OD450 value decreased with sample dilution, exhibiting a dose-dependent effect. Based on this, it can be inferred that serum IL-15 exists in the form of a complex formed by binding the IL-15Rαsushi domain to Ab1.

[0135] 4. Example 4: Identification of Ab3 expression and in vivo efficacy experiment.

[0136] a) Based on the results of Examples 1 and 3, we expressed the Ab3 antibody fusion protein, sequenced as Seq ID NO. 15 (light chain) and Seq ID NO. 16 (heavy chain), which is a mouse IgG fused with a mouse IL-15Rαsushi fragment at the C-terminus of its heavy chain. Its IgG portion is derived from antibody A13-d6.3 (GenBank: CBI70317.1), an antibody against pseudorabies virus glycoprotein L. Normal laboratory mice do not carry the pseudorabies virus unless inoculated with it; therefore, A13-d6.3 and Ab3 did not have antigenic targets in this experiment. The experimental procedures for expression, purification, and identification of Ab3 were the same as those for Ab1 in Example 1. Figure 6 As shown in a, after one round of Protein A affinity chromatography purification, a protein with a purity of 98.5% was obtained.

[0137] b) ELISA method to identify the in vitro function of Ab3. First, coat the ELISA plate with antibody, and then use 1× phosphate buffer (…). Dilute the antibodies (Ab3 and Ordesekimab) to 2 μg / mL and add 30 µL per well to the microplate. Incubate overnight at 4°C. Wash the microplate three times with phosphate-buffered saline (PBST) containing Tween 20, then add 5% PBSM containing skim milk powder and block the microplate at room temperature (RT) for 2 hours. Wash the microplate three times again with PBST, add 30 µL of humanized interleukin-15 (IL-15-P40933-chis, Sanyou Biotechnology, catalog number CoAC0285) diluted with 1% PBSM to each well, and incubate at room temperature for 60 minutes. Wash the microplate three times with PBST, then add anti-6×histidine tag-horseradish peroxidase (Anti-6... his-HRP (purchased from Pujian Biotechnology, catalog number HRP-66005) antibody was diluted 1:4000 with 1% PBSM and incubated at room temperature for 50 minutes. The plate was washed six times with PBST, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added. After the reaction reached a suitable extent, 2 mol / L (2M) stop solution was added to terminate the reaction. Finally, the absorbance value (OD450) was measured at 450 nm. Figure 6 As shown in b, Ab3's ability to bind to hIL-15 is similar to or slightly stronger than that of Ordesekimab.

[0138] c) Ab3 antibody antitumor efficacy experiment: The tumor cell line B16-F10 was expanded in vitro to a certain quantity, and the cells were resuspended at a certain density. Tumor models were established by subcutaneous inoculation of 0.1 mL per cell / mL into the back of 6-8 week old female C57 mice; on day 7 post-tumor implantation, tumors approximately 90 mm in size were examined. 3 The subjects were then randomly assigned to 5 groups (N=8). On the day of group assignment, they received intraperitoneal injections of the solvent control and the corresponding drugs according to the dosing regimen. Group G1 received PBS, Group G2 received 455ug of A13-d6.3, Group G3 received 500ug of Ab3, Group G4 received 2.5ug of hIL-15 (Bepsys, IL-5-H4117), and Group G5 received sequential injections of 500ug of Ab3 and 2.5ug of hIL-15, with a 30-minute interval between the sequential injections. The injection frequency was once a week for two weeks. Tumor size, body weight, and other parameters were measured to observe drug efficacy and toxicity. Figure 6 As shown in Figure c, group G5 exhibited a significant anti-tumor effect compared to all other groups. At the end of the experiment, 70% of the mice in group G5 survived, while all mice in the other groups either died (e.g., group G1) or had only a 15% survival rate (e.g., groups G2, G3, and G4). Figure 6 As shown in d, it can be inferred that the Ab3 antibody captures hIL-15 in vivo, forming an active complex, thereby exerting its antitumor activity. When Ab3 is used alone, since no hIL-15 complex is formed, it, like A13-d6.3, does not have antitumor activity. When hIL-15 is used alone, it is rapidly metabolized and cleared, so it also lacks significant antitumor activity.

[0139] 5. Example 5: Expression and in vitro functional identification of Ab4 and Ab5 a) Both Ab4 and Ab5 are fusion proteins derived from Avelumab. Ab4 has a mouse IL-15Rα sushi domain attached to the C-terminus of its heavy chain, while Ab5 has a human IL-15Rα sushi domain fused to the C-terminus of the Avelumab heavy chain. The light chain sequences of Ab4 and Ab5 are shown in Seq ID NO. 17, the heavy chain sequence of Ab4 is shown in Seq ID NO. 18, and the heavy chain sequence of Ab5 is shown in Seq ID NO. 19. The specific expression and purification methods and procedures are the same as those for Ab1 in Example 1. Figure 7 As shown in a and 7b, after one round of Protein A affinity chromatography purification, Ab4 and Ab5 proteins with purities of 91% and 96%, respectively, were obtained.

[0140] b) ELISA method to identify the in vitro function of Ab4 and Ab5. Direct ELISA was used to identify the binding function of Ab4 and Ab5 to hIL-15. The antibodies to be tested included Ab4, Ab5, and Ordesekimab. The antibodies were diluted to 2 μg / mL with 1× phosphate-buffered saline (PBS) and coated onto the microplate. 30 μL of the solution was added to each well, and the plate was incubated overnight at 4°C. The microplate was washed three times with phosphate-buffered saline (PBST) containing Tween 20, and then blocked with 5% phosphate-buffered saline (5% PBSM) containing skim milk powder for 2 hours at room temperature. The microplate was washed three times again with PBST. 30 µL of His-tagged human interleukin-15 (IL-15, Sanyou Biotechnology, catalog number P40933-chis) diluted with 1% PBSM was added to each well, and the plate was incubated for 60 minutes at room temperature. Wash the ELISA plate three times with PBST, then add anti-6×His-horseradish peroxidase (Anti-6). his-HRP, purchased from Pujian Biotechnology (catalog number HRP-66005), was diluted 1% PBSM at a ratio of 1:4000. 30 µL was added to each well and incubated at room temperature for 50 minutes. The plate was washed six times with PBST, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added. After the reaction, the reaction was terminated with 2 mol / L (2M) stop solution. Finally, the absorbance (OD450) was measured at 450 nm. Figure 7 As shown in c, Ab4 and Ab5 have similar binding abilities to hIL-15 as Ordesekimab. Ab4 contains the mouse IL-15Rα sushi domain, while Ab5 contains the human IL-15Rα sushi domain. Figure 7 The results of c indicate that both have equivalent IL-15 capture capabilities.

[0141] c) The sandwich ELISA method was used to identify the simultaneous binding of IL-15 and PD-L1 by Ab4 and Ab5. The ELISA method was the same as in Experiment d of Example 1. Figure 7 As shown, both Ab4 and Ab5 can bind to both mouse PD-L1 and human IL-15 simultaneously. 6. Example 6: The PK behavior of Ab4 is similar to that of Avelumab.

[0142] a) Prepare C57BL / 6N mice, SPF grade, 18-20g, male, 3 mice / group, intraperitoneally injected with the drug. Group 1 was injected with 500ug Ab4; Group 2 was injected with 454ug Avelumab. Blood was collected from the orbital venous plexus of the mice at the following time points: 0h before drug administration, 15min, 30min, 1h, 2h, 4h, 8h, 24h, 48h, 72h, 96h, 192h, 264h, and 336h after drug administration. The blood volume collected from each mouse was 40-60 μL.

[0143] b) Gently transfer the collected blood into a centrifuge tube without anticoagulant. Incubate the tube at room temperature (approximately 22-25°C) for 30-60 minutes to allow the blood to clot naturally. The blood will coagulate into a gel-like consistency, and a clear, pale yellow liquid will separate out; this is serum. Place the coagulated blood sample into a centrifuge and centrifuge at 4°C and 2000×g for 15 minutes. Using a micropipette, carefully aspirate the top layer of clear or pale yellow serum and transfer it to a new, pre-chilled, sterile centrifuge tube. Store under appropriate conditions.

[0144] c) After appropriate dilution, serum samples were analyzed using ELISA to determine the levels of Ab4 and Avelumab in the serum. For coating the microplate: mus-PD-L1-C-His (Sanyou Bio, catalog number P268386) was diluted to 2 μg / mL with 1× phosphate-buffered saline (1×PBS), and 30 µL was added to each well of the microplate. The plate was incubated overnight at 4°C. The microplate was washed three times with phosphate-buffered saline (PBST) containing Tween 20, followed by the addition of 2% bovine serum albumin (2% BSA) solution. The plate was blocked at room temperature (RT) for 2 hours. The plate was washed three times again with PBST, and 30 µL of appropriately diluted serum was added to each well. The plate was incubated at room temperature for 60 minutes. Wash the ELISA plate three times with PBST, add the secondary antibody Goat-Anti-Human-IgG-Fc-HRP (Jackson Immuno, 109-035-008) containing goat anti-human IgG Fc at a ratio of 1:8000 (diluted with 1% BSA solution), add 30 µl to each well, and incubate at room temperature for 60 minutes. Wash the ELISA plate six times with PBST, add 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution, and after the reaction reaches the expected extent, add 2 mol / L (2M) sulfuric acid (… The reaction was terminated with a stop solution, and the absorbance (OD450) at 450 nm was measured using a microplate reader. The contents of Ab4 and Avelumab were calculated based on the measured OD450 values ​​using a pre-established standard curve. Figure 8As shown, at the concentrations used, Ab4 and Avelumab exhibited similar PK behaviors, indicating that the fusion of the antibody molecule with the IL-15Rαsushi fragment had no significant effect on its PK.

[0145] 7. Example 7: In vivo pharmacodynamic studies of sequential administration of Ab5 and hIL-15 demonstrated improved efficacy that was dose-dependent on IL-15.

[0146] a) Expand the tumor cell line CT-26 to a certain quantity in vitro, and resuspend the cells at a density of 2×10⁶. 7 Tumor models were established by subcutaneous inoculation of 0.1 mL per 6-8 week old female Balb / c mice with cells / mL; on day 7 post-tumor implantation, tumors of approximately 90 mm were randomly selected for examination. 3 The subjects were then randomly assigned to groups of six. On the day of grouping, they received intraperitoneal injections of the solvent control and the corresponding drugs according to the dosing regimen. Group G1 received PBS, Group G2 received sequential injections of 500 μg and 4 μg hIL-15 (Bepsys, IL-5-H4117), Group G3 received sequential injections of 500 μg Ab5 and 8 μg hIL-15, and Group G4 received sequential injections of 500 μg Ab5 and 10 μg hIL-15, with a 30-minute interval between each injection. The injection frequency was once a week for two weeks. Tumor size, body weight, and other parameters were measured to observe drug efficacy and toxicity. Figure 9 As shown in Figure a, all groups receiving sequential injections of Ab5 and IL-15 exhibited significant tumor-suppressing effects, and the tumor-suppressing effect increased with increasing IL-15 dose from 4 μg to 8 μg and then to 10 μg, demonstrating a dose-dependent effect. Furthermore, as... Figure 9 As shown in b, the changes in body weight of the animals in each group were basically similar, with no significant differences, indicating that IL-15 at 10ug and below had no significant toxicity to mice.

[0147] b) Following the above methods and steps, two additional experimental groups were set up. One group was injected with 454ug of Avelumab, and the other group was injected with 500ug of Ab5, ensuring that the two groups were at equimolar dose levels. In the two groups without IL-15, both groups showed almost identical tumor-suppressing effects, such as... Figure 9 As shown in c. Figure 9 The results showed that neither Ab5 alone nor Avelumab alone had a significant effect on body weight. Therefore, it can be inferred that the introduction of the IL-15Rα sushi fragment into Avelumab did not affect the antibody's toxicity or efficacy.

[0148] 8. Example 8: Ab5 plus hIL-15 exhibited long-lasting antitumor activity. a) Expand the tumor cell line MC38 to a certain quantity in vitro, and resuspend the cells at a density of 2×10⁶. 7 Tumor models were established by subcutaneous inoculation of 0.1 mL per 6-8 week old female C57 mice with cells / mL; approximately 120 tumors were randomly selected for examination. Mice were then randomly assigned to groups. On the day of grouping, mice were administered intraperitoneally with the solvent control and the corresponding drug according to the dosing regimen. Group G1 (n=6) received PBS, Group G2 (n=7) received only 500 μg Ab5, and Group G3 (n=7) received sequential injections of 500 μg and hIL-15 (Bepsys, IL-5-H4117). The sequential injections in Group G3 were administered 30 minutes apart. Dosing was performed weekly. Tumor size, body weight, and other parameters were measured to observe efficacy and toxicity.

[0149] b) The first three doses of IL-15 are 2.5 μg, such as Figure 10 As shown in Figure a, although group G3 exhibited tumor-suppressive activity, the effect was not significant. Referring to the IL-15 dosage relationship in Example 7, group G3 received an additional dose on day 17, sequentially administering 500 μg Ab5 and 10 μg hIL-15. Group G2 received an additional dose of 500 μg Ab5. After this administration, all groups discontinued their medication. Figure 10 As shown in b, the tumor-suppressing effect was significantly improved in group G3. After drug withdrawal, the tumor volume in mice in group G2 recovered rapidly, almost paralleling that of the control group. However, the tumor volume in mice in group G3 recovered slowly, only recovering at the end of the experiment on day 30, demonstrating sustained tumor-suppressing activity. Figure 10 d represents the survival time comparison of the three groups of mice. The survival time of mice in the G3 group was better than that of the PBS control group and also better than that of Ab5 single drug.

[0150] 9. Example 9: Expression and in vitro functional identification of Ab7.

[0151] a) Ab7 is a mouse IgG1 antibody fusion protein containing the Fab of Zolbetuximab and the Fc of mIgG1, with an hIL-15Rα sushi domain linked to the C-terminus of its heavy chain. The sequences are shown in Seq ID NO. 20 (light chain) and Seq ID NO. 21 (heavy chain). The expression and purification procedures for Ab7 were the same as in experiments a and b of Example 1. Figure 11 As shown in Figure a, after one round of Protein A chromatography purification, a protein with 100% purity was obtained as shown by SEC-HPLC.

[0152] b) ELISA to identify Ab7's in vitro binding function to hClaudin18.2. Human Claudin 18.2 virus-like particles (hu-CLDN18.2-VLP, Sanyou Bio, catalog number P104156) were diluted to 16 μg / mL with 1× phosphate-buffered saline (1×PBS) and added to each well in a volume of 30 µL. The plates were incubated overnight at 4°C. The plates were washed three times with phosphate-buffered saline (PBST) containing Tween 20, followed by adding 180 µL of 5% phosphate-buffered saline (PBSM) containing skim milk powder to each well. The plates were then blocked at room temperature (RT) for 2 hours. The plates were washed three times again with PBST, and 30 µL of antibody diluted with 1% PBSM was added to each well. The plates were incubated at room temperature for 60 minutes. Wash the microplate three times with PBST, add horseradish peroxidase-labeled goat anti-mouse IgG-Fc secondary antibody (Goat-Anti-mouse-IgG-Fc-HRP, purchased from AbCam, catalog number ab97265), which needs to be diluted with 1% PBSM at a ratio of 1:8000. Add 30 µL to each well and incubate at room temperature for 60 minutes. Wash the microplate six times with PBST, add 30 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution to each well, and after the reaction reaches the appropriate level, add 2 mol / L (2M) sulfuric acid. The reaction was terminated with a stop solution, and the absorbance (OD450) at a wavelength of 450 nm was measured using an ELISA reader. Figure 11 As shown in b, Ab7 can bind to hClaudin18.2 VLP.

[0153] c) ELISA was used to identify the ability of Ab7 to simultaneously bind hClaudin18.2 and hIL-15 in vitro. The ELISA method is basically the same as described above. However, instead of Goat-Anti-mouse-IgG-Fc-HRP, the secondary antibody used was Human IL-15, His Tag (Sanyou Bio, P211120), followed by Mouse-Anti-6×His-HRP (Proteintech, HRP-66005) and subsequent TMB staining. Figure 11 As shown in c, Ab7 can bind to both hClaudin18.2 VLP and hIL-15 simultaneously. d) FACS method was used to identify Ab7 binding to cell membrane-expressed hClaudin18.2. Per well Cells were plated at a density of 1E5 cells / well; then centrifuged and washed with flow cytometry buffer (FACS buffer). 100 μL of different dilutions of Ab7 were added to the cells, and the cells were incubated at 4°C for 60 minutes. After incubation, the cells were centrifuged and washed twice with FACS buffer. 100 μL of fluorescently labeled secondary antibody, PE (phycoerythrin)-labeled goat F(ab')2 fragment of anti-mouse IgG (purchased from Abcam, catalog number ab98742, diluted 1:300 with FACS buffer), was added to the cells; the cells were incubated at 4°C for 30 minutes after adding the secondary antibody. The cells were resuspended in FACS buffer and then analyzed by flow cytometry. Two types of engineered cells were used: MDA-MB-231 (hu18.2-MDA-MB-231) and CT-26 (hu18.2-CT-26), which highly express human Claudin18.2. Figure 11 d and Figure 11 As shown in e, both types of cells can be bound by Ab7.

[0154] e) The FACS method identified that Ab7 could simultaneously bind to cells expressing hClaudin18.2 and hIL-15. This detection was performed using hu18.2-CT-26 cells, and the FACS method was basically the same as above. The secondary antibody used was Human IL-15, His Tag (Sanyou Bio, P281964), and finally, PE-labeled mouse anti-His Tag antibody (BioLegend, catalog number 362603, 1:150 dilution) was used for labeling and analysis. Figure 11 As shown in f, Ab7 can bind to hIL-15 while binding to hClaudin18.2-positive CT-26 cells.

[0155] 10. Example 10: Expression and in vitro functional identification of Ab9.

[0156] a) Ab9 is a fusion protein of an anti-PD-L1 nanobody (heavy chain single-domain antibody) with the hIL-15Rα sushi domain and the VEGFR1 D2 domain. The nanobody sequence is derived from KN035, located at the N-terminus of the fusion protein, with the hIL-15Rα sushi domain linked to its C-terminus; an hIgG1 Fc fragment is linked to the C-terminus of the hIL-15Rα sushi domain, and VEGFR1-D2 is linked to the C-terminus of the Fc fragment. The Ab9 sequence is shown in Seq ID NO. 22. The expression and purification methods and procedures for Ab9 are the same as those in experiments a and b of Example 1. Figure 12 As shown in a, a single round of Protein A chromatography purification yielded a protein with approximately 90% purity.

[0157] b) ELISA method to verify that Ab9 can bind to VEGF-A. The ELISA method is basically the same as Experiment b in Example 9, but with the following modifications. Coating was performed with 2 μg / mL Human VEGFA, His Tag (Sanyou Bio, P233340), and the secondary antibody was 1:8000 diluted HRP-labeled goat anti-human IgG-Fc (Goat-Anti-Human-IgG-Fc-HRP, Jackson Immuno, 109-035-008). Figure 12 As shown in b, Ab9 can bind to human VEGF-A.

[0158] c) ELISA method to verify that Ab9 can simultaneously bind to huPD-L1 and VEGF-A. The ELISA method is similar to experiment b in this example, but with the following modifications: the coating is 8 μg / mL PD-L1-huFc (Sanyou Biotech, COAA1477), the secondary antibody is human VEGFA, His Tag (Sanyou Biotech, P233340), and finally, mouse anti-His tag antibody (Mouse-Anti-6×His-HRP (Proteintech, HRP-66005) diluted 1:4000 is used for color development. Figure 12 As shown in c, Ab9 can bind to both human VEGF-A and human PD-L1 simultaneously.

[0159] d) ELISA method to verify that Ab9 can simultaneously bind huPD-L1 and hIL-15. The ELISA method is similar to experiment c in this embodiment, but with the following modifications: the secondary antibody used is Human IL-15, His Tag (Sanyou Bio, P211120), followed by Mouse-Anti-6×His-HRP (Proteintech, HRP-66005) for color development. Figure 12 As shown in d, Ab9 can bind to both human IL-15 and human PD-L1 simultaneously.

[0160] 11. Example 11: Expression and in vitro functional identification of Ab10 a) Ab10 is based on Ab5 with the addition of VEGFR1-D2, which is linked to the N-terminus of the heavy chain of Ab5 via a linker peptide (GGGGS)3. The sequence of Ab10 is shown in Seq ID NO. 17 (light chain) and Seq ID NO. 23 (heavy chain). The expression and purification methods and procedures for Ab10 are the same as those in experiments a and b of Example 1. Figure 13 As shown in figure a, a protein with a purity of approximately 95% was obtained after one round of Protein A chromatography purification.

[0161] b) ELISA method to verify that Ab10 can bind to VEGF-A. The ELISA method is the same as in Experiment b of Example 10. Figure 13 As shown in b, Ab10 can bind to human VEGF-A.

[0162] c) ELISA method verification shows that Ab10 can simultaneously bind to huPD-L1 and hIL-15. The ELISA method is the same as in Experiment d of Example 10. Figure 13 As shown in c, Ab10 can bind to both human IL-15 and human PD-L1 simultaneously.

[0163] d) ELISA method verification that Ab10 can simultaneously bind to hVEGF-A, huPD-L1, and musPD-L1. The ELISA method for verifying Ab10 binding to hVEGF-A and huPD-L1 is the same as in Experiment c of Example 10. For verifying the binding of hVEGF-A and musPD-L1, the coating was 8 μg / mL of musPD-L1-ECD-huFC (Sanyou Bio, P00632), and the rest was the same as in Experiment c of Example 10. Figure 12 As shown in d, Ab10 can bind to both human VEGF-A and human PD-L1 or mouse PD-L1 simultaneously.

[0164] 12. Example 12: Expression and in vitro functional testing of Ab12 a) Ab12 is a fusion protein based on a rat anti-mouse PD-L1 antibody clone, 10F.9G2, a widely used model molecule (Bu et al., Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, Vol. 41, 202-209, 2022). Ab12 is derived from the murine-derived 10F.9G2, with its Fab sequence derived from 10F.9G2 and its Fc being the Fc of mIgG2a, with an hIL-15Rαsushi domain attached to the C-terminus of its Fc. The murine-derived 10F.9G2 was named Ab11. The light chain sequences of Ab11 and Ab12 are shown in Seq ID NO. 24, the heavy chain sequence of Ab11 is shown in Seq ID NO. 25, and the heavy chain sequence of Ab12 is shown in Seq ID NO. 26. The expression and purification methods and procedures of Ab12 are the same as those in experiments a and b of Example 1. Figure 14 As shown in a, a single round of Protein A chromatography purification yielded a protein with approximately 90% purity.

[0165] b) ELISA method verification that Ab12 can simultaneously bind to musPD-L1 and hIL-15. In the ELISA method, except that the secondary antibody was changed to Human IL-15, His Tag (Sanyou Bio, P211120), the rest was the same as experiment d in Example 1. Figure 14 As shown in b, Ab12 can bind to both musPD-L1 and hIL-15 simultaneously.

[0166] 13. Example 13: Expression and in vitro functional testing of Ab13 a) Ab13 is a fusion protein formed by attaching a VEGFR1-D2 domain to the N-terminus of the heavy chain of Ab12. The light chain sequence of Ab13 is shown in Seq ID NO. 24, and the heavy chain sequence is shown in Seq ID NO. 27. The expression and purification methods and procedures for Ab13 are the same as those in experiments a and b of Example 1. Figure 15 As shown in Figure a, after one round of Protein A chromatography purification, approximately 100% pure protein was obtained as shown by SEC-HPLC.

[0167] b) ELISA method to verify that Ab13 can bind to VEGF-A. The ELISA method is basically the same as Experiment b in Example 9, but with the following modifications. Coating was performed with 2 μg / mL Human VEGFA, His Tag (Sanyou Bio, P233340), and the secondary antibody was a 1:8000 diluted HRP-labeled goat anti-mouse IgG-Fc (Goat-Anti-mouse-IgG-Fc-HRP (Abcam, ab97265) antibody. Figure 15 As shown in b, Ab13 can bind to human VEGF-A.

[0168] c) ELISA method to verify that Ab13 can simultaneously bind to hVEGF-A and musPD-L1. The ELISA method is the same as in Experiment c of Example 10. When verifying simultaneous binding to hVEGF-A and musPD-L1, the coating was 8 μg / mL of musPD-L1-ECD-huFC (Sanyou Bio, P00632), and the rest was the same as in Experiment c of Example 10. Figure 15 As shown in c, Ab13 can bind to both human VEGF-A and mouse PD-L1 simultaneously.

[0169] d) ELISA method verification that Ab13 can simultaneously bind to musPD-L1 and hIL-15. In the ELISA method, except that the secondary antibody was changed to Human IL-15, His Tag (Sanyou Bio, P211120), the rest was the same as experiment d in Example 1. Figure 15As shown in d, Ab13 can bind to both musPD-L1 and hIL-15 simultaneously.

[0170] e) ELISA method verification: Ab13 can simultaneously bind to huVEGF-A and hIL-15, and binding to VEGF-A does not interfere with the binding of Ab13 to hIL-15, and vice versa. WW001-GSV0-IL-15-P40933-TEV-hFc 32-4 (IL-15 fusion protein, Sanyou Bio, catalog number P274741) was diluted to 8 μg / mL with 1× phosphate-buffered saline (1×PBS), and 30 µL was added to each well of the microplate. The plate was incubated overnight at 4°C. The microplate was washed three times with phosphate-buffered saline (PBST) containing Tween 20, and then 180 µL of 5% phosphate-buffered saline (PBSM) containing skim milk powder was added to each well. The microplate was blocked at room temperature (RT) for 2 hours. Wash the microplate three times with PBST. Add a 1:3 premixed mixture of Ab13 and VEGF-A (Human VEGFA, His Tag, Sanyou Biotechnology, catalog number P233340) to each well, 30 µL, and incubate at room temperature for 60 minutes. Alternatively, add Ab13 and VEGF-A sequentially. First, add 30 µL of Ab13 diluted with 1% PBSM to each well and incubate at room temperature for 60 minutes. After washing the microplate three times with PBST, add 30 µL of human vascular endothelial growth factor A-histidine tag protein (Human VEGFA, His Tag, Sanyou Biotechnology, catalog number P233340) diluted with 1% PBSM to a concentration of 4 μg / mL, and incubate at room temperature for 60 minutes. After adding Ab13 and VEGF-A using a premixed or sequential method and incubating, the microplate was washed three times with PBST. A 1:4000 dilution of mouse anti-6×histidine-tagged horseradish peroxidase (Mouse-Anti-6×His-HRP, purchased from Pujian Biotechnology, catalog number HRP-66005) was added to each well, at 30 µL, and incubated at room temperature for 60 minutes. The microplate was then washed six times with PBST. 30 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added to each well. Once the reaction reached the appropriate level, 2 mol / L (2M) sulfuric acid was added. The reaction was terminated with a stop solution, and the absorbance (OD450) at a wavelength of 450 nm was measured using an ELISA reader. Figure 15 e and Figure 15As shown in f, whether Ab13 and VEGF-A are added premixed or sequentially, Ab13 can bind to both hIL-15 and VEGF-A simultaneously, and the EC50 values ​​are very close (0.2477 for sequential addition and 0.2280 for premixing). This proves that Ab13 can not only bind to both VEGF-A and hIL-15 simultaneously, but also that the binding of Ab13 to VEGF-A does not interfere with the binding of Ab13 to hIL-15, and vice versa.

[0171] 14. Example 14: Expression of Ab14 and detection of its in vitro function.

[0172] a) Ab14 is a fusion protein composed of an IL-15Rα sushi domain, an hIgG1 Fc, and an anti-EGFR extracellular domain scFv antibody. The scFv sequence is derived from the light and heavy chain variable regions of panitumumab. The scFv is linked to the C-terminus of the Fc with a 3xGGGGS structure, while the IL-15Rα sushi domain is directly linked to the N-terminus of the IgG1 Fc. The sequence of Ab14 is shown in Seq ID NO. 28. The expression and purification methods and procedures of Ab14 are the same as those in experiments a and b of Example 1. Figure 16 As shown in Figure a, after one round of Protein A chromatography purification, a protein with a purity of 75% was obtained as shown by SEC-HPLC.

[0173] b) ELISA method to verify that Ab14 can bind to the extracellular domain of EGFR. The ELISA method is basically the same as Experiment b in Example 9, but with the following modifications. The coating was 2 μg / mL Human EGFR, his Tag (Sanyou Bio, P305971), and the secondary antibody was 1:8000 diluted HRP-labeled goat anti-human IgG-Fc (Goat-Anti-Human-IgG-Fc-HRP, Jackson Immuno, 109-035-008). Figure 16 As shown in b, Ab14 can bind to human EGFR-ECD.

[0174] c) ELISA method to verify that Ab14 can simultaneously bind to huEGFR-ECD and hIL-15. Except for coating the microplate with 8 μg / mL Human EGFR, Fc tag (Sanyou Bio, P303610), the ELISA method was the same as in Example 9, Experiment c. Figure 16 As shown in c, Ab14 can bind to both huEGFR-ECD and hIL-15 simultaneously.

[0175] 15. Efficacy of sequential administration of Ab12 and hIL-15 in Example 15 a) Expand the tumor cell line CT-26 in vitro to a certain quantity, and resuspend the cells at a density of 5 × 10⁻⁶. 6 Tumor models were established by subcutaneous inoculation of 0.1 mL per 5-week-old female Balb / c mice with cells / mL; tumors of approximately 60 mm were randomly selected for examination. 3 Subjects were then randomly assigned to groups. On the day of grouping, intraperitoneal injection of the solvent control and the corresponding drug was administered according to the dosing regimen. The control group received PBS, the Ab11 group received 10 mg / kg antibody Ab11, the Ab12 group received 11 mg / kg antibody Ab12, and the Ab11+IL-15 group received sequential injections of 10 mg / kg antibody Ab11 and 10 μg hIL-15 (Bepsys, IL-5-H4117). The two drugs were injected sequentially 30 minutes apart. The Ab12+IL-15 group received sequential injections of 11 mg / kg antibody Ab12 and 10 μg hIL-15 (Bepsys, IL-5-H4117), also with a 30-minute interval. Dosing was administered every 5 days, up to a maximum of 3 times. Tumor size, body weight, and other parameters were measured to observe efficacy and toxicity.

[0176] b) such as Figure 17 As shown in Figure a, at equimolar doses and without the addition of IL-15, Ab11 and Ab12 exhibited almost identical tumor inhibition rates, further validating the conclusions of Examples 2 and 7, namely that the introduction of the IL-15Rα sushi fragment onto the antibody did not affect the antibody's therapeutic efficacy. Figure 17 Furthermore, the study showed that in a 5-day dosing regimen (days 0 and 5), Ab11, lacking IL-15 receptor antagonists, could not capture IL-15, leading to its rapid excretion. Therefore, sequential IL-15 injection did not enhance the tumor-suppressive effect of Ab11. Conversely, because Ab12 could capture IL-15, even with a 5-day dosing frequency, IL-15 significantly increased the efficacy of Ab12. Further experiments demonstrated that increasing the dosing frequency, adding an extra dose on day 8, resulted in a slight increase in efficacy in the Ab11+IL-15 group. Figure 17 As shown in b. However, the Ab12+IL-15 group showed weight loss, indicating toxicity (data not shown). Figure 17 c showed that IL-15 could provide a durable therapeutic effect. After the third dose, the drug was discontinued. In the Ab12 monotherapy group, the tumor volume doubled starting from day 14, while the Ab12+IL-15 group maintained a tumor inhibition rate of more than 90%.

[0177] 16. Example 16: Comparison of the efficacy of Ab5+hIL-15 and Ab10 a) Expand the tumor cell line MC38 in vitro to a certain quantity, and resuspend the cells at a density of 2×10⁻⁶. 7 Tumor models were established by subcutaneous inoculation of 0.1 mL per 6-8 week old female C57 mice with cells / mL; approximately 90 tumor cells / mL were randomly selected for examination. Mice were then randomly assigned to groups. On the day of grouping, mice were administered intraperitoneally with the solvent control and the corresponding drug according to the dosing regimen. Group 1 (6 mice) received PBS, Group 2 (6 mice) received only 347 μg of Ab10, and Group 3 (6 mice) received sequential injections of 300 μg and 9 μg of hIL-15 (Bepsys, IL-5-H4117), with a 30-minute interval between the injections. Dosing was administered weekly for 3 weeks. Tumor size, body weight, and other parameters were measured to observe efficacy and toxicity.

[0178] b) As described in Examples 5 and 11, Ab5 is a fusion protein of the PD-L1 antibody Avelumab and hIL-15Rα sushi, while Ab10 is a VEGFR1-D2 domain linked to the N-terminus of the heavy chain of Ab5. Figure 18 As shown, the efficacy of Ab5 sequential injection of hIL-15 (i.e., IL-15 and anti-PD-L1) is similar to that of Ab10 monotherapy without IL-15 (i.e., anti-VEGF and anti-PD-L1).

[0179] 17. Efficacy of Ab12 and Ab13 in Example 17 a) Expand the tumor cell line CT-26 to a certain quantity in vitro, and resuspend the cells at a density of 2×10⁶. 7 Tumor models were established by subcutaneous inoculation of 0.1 mL per 6-8 week old female Balb / c mice with cells / mL; approximately 90 tumor cells / mL were randomly selected for examination. Mice were then randomly assigned to groups. On the day of grouping, mice were administered intraperitoneally with the solvent control and the corresponding drug according to the dosing regimen. Group 1 (6 mice) received PBS; Group 2 (6 mice) received only 490 μg Ab12; Group 3 (6 mice) received sequential injections of 490 μg Ab12 and 8 μg hIL-15 (Bepsys, IL-5-H4117); Group 4 (6 mice) received only 566 μg Ab13; Group 5 (6 mice) received sequential injections of 566 μg Ab13 and 4 μg hIL-15; and Group 6 (6 mice) received sequential injections of 566 μg Ab13 and 8 μg hIL-15 (Bepsys, IL-5-H4117). The interval between sequential injections was 30 minutes. Dosing was administered once a week for two weeks. Tumor size, body weight, and other parameters were measured to observe efficacy and toxicity.

[0180] b) As described in Examples 12 and 13, Ab12 is a fusion protein of murine PD-L1 antibody 10F.9G2 and hIL-15Rαsushi, while Ab13 is a VEGFR1-D2 domain linked to the N-terminus of the heavy chain of Ab12. Without IL-15, Ab12 is equivalent to PD-L1 antibody 10F.9G2, while Ab13 is equivalent to a bispecific antibody against PD-L1 and anti-VEGF. Figure 19 As shown in Figure a, the therapeutic effect of Ab13 was enhanced as expected after sequential injection of hIL-15, and a dose-response effect was observed. Figure 19 b compared the efficacy of Ab12 and Ab13 with and without hIL-15. As expected, Ab13 showed stronger antitumor activity than Ab12 without hIL-15; the efficacy of Ab12 was enhanced by sequential addition of 8 μg hIL-15, which was better than Ab-13 monotherapy. The efficacy of Ab13 was also enhanced by sequential addition of 8 μg hIL-15, with a tumor inhibition rate similar to that of Ab12 with sequential addition of 8 μg hIL-15.

Claims

1. A pharmaceutical composition, characterized in that, It comprises a first drug component and a second drug component packaged separately, wherein: The first pharmaceutical component comprises a fusion protein molecule, said fusion protein molecule comprising a fragment capable of binding IL-15 or an IL-15 mutant, an antibody or antigen-binding fragment or a receptor fragment, and an Fc fragment or human serum albumin HSA or a fragment thereof. The fragment capable of binding IL-15 or IL-15 mutants is an IL-15Rα or IL-15Rα sushi domain, and the IL-15Rα or IL-15Rα sushi domain does not bind IL-15 or IL-15 mutants. The antibody, antigen-binding fragment, or receptor fragment described herein can bind to tumor antigens or tumor targets; The second drug component contains IL-15 drug molecules; The first drug component and the second drug component are used for sequential administration, wherein the sequential administration is to first administer the first drug component and then administer the second drug component, wherein the first drug component captures the subsequently injected second drug component in the body; The molar ratio of the fusion protein molecule in the first drug component to the IL-15 drug molecule in the second drug component is greater than 4:

1.

2. The pharmaceutical composition according to claim 1, characterized in that, The IL-15Rα or IL-15Rα sushi domain is wild-type.

3. The pharmaceutical composition according to claim 1, characterized in that, The IL-15Rα or IL-15Rα sushi domain is mutated, and the dissociation constant KD of the fusion protein molecule binding to IL-15 or the IL-15 mutant is less than 1 × 10⁻⁶. -8 M.

4. The pharmaceutical composition according to claim 3, characterized in that, The dissociation constant KD is less than 1 × 10⁻⁶. -9 M.

5. The pharmaceutical composition according to claim 3, characterized in that, The dissociation constant KD is less than 1 × 10⁻⁶. -10 M.

6. The pharmaceutical composition according to claim 1, characterized in that, The antibody or antigen-binding fragment is selected from Fab, nanobodies, scFv antibodies, or bispecific antibodies.

7. The pharmaceutical composition according to claim 1, characterized in that, The antibody, antigen-binding fragment, or receptor fragment is capable of binding to tumor antigens or tumor targets, and its dissociation constant KD is less than 1 × 10⁻⁶. -8 M.

8. The pharmaceutical composition according to claim 1, characterized in that, The tumor antigens or tumor targets that the fusion protein molecule can bind to are selected from the following group: PD-1, PD-L1, CTLA4, CD20, CD19, CD30, Her2, TROP-2, BCMA, CD33, CD22, Nectin-4, c-MET, Tissue factor, FRα, CD38, gp100, Her3, CEACAM5, EpCAM, MUC16, DLL3, CLDN18.2, CLDN6, B7-H3, B7-H4, ROR1, ITGB6, CDH6, CDH17, MUC1, GPC-3, CSF1R, LAG3, TIM3, PSMA, Mesothelin, CD79b, CAIX, DDR1, FAP, OSMR, VEGF, EGF, TGFβ-1.

9. The pharmaceutical composition according to claim 1, characterized in that, The IL-15Rα or IL-15Rα sushi domain is located at the amino terminus, carboxyl terminus, or intermediate position of the fusion protein molecule.

10. The pharmaceutical composition according to claim 1, characterized in that, The IL-15 drug molecule is wild-type IL-15, an IL-15 mutant, or chemically modified IL-15, or a mutant of chemically modified IL-15, or a protein containing IL-15, wherein the dissociation constant KD of the IL-15 drug molecule binding to the fusion protein molecule is less than 1 × 10⁻⁶. -8 M.

11. The pharmaceutical composition according to claim 1, characterized in that, The sequential dosing intervals are 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, and 14 days.

12. The pharmaceutical composition according to claim 1, characterized in that, The time interval for sequential administration is from 1 hour to 7 days.

13. Use of the pharmaceutical composition of any one of claims 1-12 in the preparation of a medicament for the prevention or treatment of interleukin-15-related tumors, wherein the interleukin-15-related tumors are CD8 cells that can be activated or proliferated by IL-15. + Tumors that respond to T cells and / or NK cells.

14. The use according to claim 13, characterized in that, The interleukin-15 related tumors are selected from the following group: liver cancer, stomach cancer, esophageal cancer, skin cancer, prostate cancer, pancreatic cancer, colorectal cancer, ovarian cancer, head and neck cancer, bladder cancer, urothelial carcinoma, cervical cancer, breast cancer, kidney cancer, glioblastoma, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, and non-Hodgkin lymphoma.

15. The use according to claim 13, characterized in that, The pharmaceutical composition is administered via intravenous or subcutaneous injection.

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