Bifunctional composite molecule of Anti-tumor antibody and interleukin-15 precursor, and use of bifunctional composite molecule

Abstract

The present invention provides a composite molecule, a nucleic acid, a vector, a host cell, and a pharmaceutical composition, and uses of the composite molecule, the nucleic acid, the vector, the host cell, and the pharmaceutical composition in preparation of drugs for treating cancers. The composite molecule comprises an anti-tumor antibody domain, a linker, and pro-IL-15; the anti-tumor antibody domain is linked to the pro-IL-15 by means of the linker; the anti-tumor antibody domain is a complete antibody against an immune checkpoint molecule, a tumor antigen molecule or an immune activation molecule, or a nano antibody or an antigen binding fragment thereof; the linker is a polypeptide linker or a non-peptide linker; the pro-IL-15 is a fusion protein comprising IL-15, an IL-15Rα sushi domain, and a linker peptide, and optionally comprising an IL-15Rβ extracellular domain; and the IL-15Rβ extracellular domain, the IL-15, and the IL-15Rα sushi domain are linked by means of the linker peptide.

Description

Bifunctional complex molecule of anti-tumor antibody and interleukin-15 precursor and its application Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a bifunctional molecule composed of an IL-15 precursor connected to an anti-tumor antibody or an antigen-binding fragment thereof, and its application in treating tumors. Background Art

[0002] Cancer immunotherapy, particularly immune checkpoint blockade (ICB), has been a revolutionary advance in cancer treatment. Immune checkpoints, such as CTLA-4, PD-1, and PD-L1, are present on the surface of activated immune cells and serve as signaling molecules that prevent overactivation of the immune system. When an immune response becomes excessive, they silence these cells, effectively acting as brakes on the immune response. The tumor microenvironment exploits these molecules to evade immune attack. Immune checkpoint blockade drugs can relieve the inhibitory effects of the tumor microenvironment on tumor-infiltrating lymphocytes (TILs), thereby activating anti-tumor immune responses. Compared to CTLA-4 blocking antibodies, PD-L1 / PD-1 (PD) blocking antibodies have attracted considerable attention due to their reduced side effects and greater efficacy. Clinically, 10-20% of patients with malignant tumors achieve remarkable therapeutic responses with PD-blocking drugs. However, a significant proportion of patients remain unresponsive to these drugs or, despite an early response, relapse later in life. While PD-blocking drugs can partially restore the function of T lymphocytes in tumors, these cells can still differentiate into an exhausted state, reducing or even eliminating their anti-tumor function.

[0003] On the other hand, T cell growth factor has become an important regulatory molecule in tumor immunotherapy because of its ability to amplify T lymphocytes. Among them, IL-2 is a cytokine drug that was earlier approved by the U.S. FDA for use in malignant melanoma and renal cancer, but IL-2 has not been widely used in clinical practice mainly due to the following three reasons: ① Activation of regulatory T cells (Treg) with immunosuppressive function; ② Activated effector T cells will be cleared through activation-induced cell death (AICD); ③ Because IL-2 receptors are widely expressed on vascular endothelial cells, IL-2 will produce serious toxic side effects during treatment. IL-15 is another important T cell growth factor and a multi-effect cytokine that can induce and maintain innate immunity and adaptive immune responses. IL-15 can promote CD8 +Activation, proliferation and survival of T cells; inducing the production of long-lived memory T cells and maintaining steady-state proliferation; inducing and amplifying NK and NKT cells; and promoting the activation, proliferation and differentiation of DC cells in an autocrine manner, promoting the expression of MHC-II and CD80 / CD86, and improving the ability of DC cells to cross-present antigens. Compared with IL-2, IL-15 has the following advantages: ① It does not induce the production of AICD; ② It does not amplify Treg cells. Studies have shown that IL-15 is a component of the inflammatory environment in tumor tissues and is necessary for tumors to establish a normal number of CD8 + Required for T cell infiltration. For example, loss of IL-15 in colorectal cancer patients is closely associated with reduced T cell proliferation, tumor recurrence, and decreased patient survival.

[0004] However, the clinical application of IL-15 also has bottlenecks. This is because most cytokine receptors are widely expressed in peripheral tissue cells. On the one hand, systemic injection of cytokine drugs will cause serious toxic side effects due to the effects on peripheral cells; on the other hand, the drugs are consumed or cleared in peripheral tissues, so that the drug concentration entering the tumor is insufficient to activate intratumoral immune cells, resulting in unsatisfactory therapeutic effects. The above are common difficulties and bottlenecks encountered by cytokine drugs in clinical applications. At present, clinical and preclinical research is still mainly focused on developing IL-15 fusion proteins such as IL-15 / IL-15Rα complexes or screening IL-15 mutants to extend the half-life of IL-15 and improve the biological activity of the drug, ultimately hoping to improve the therapeutic effect of tumors. However, IL-15R is mainly expressed in NK cells, NKT cells and T cells. The increase in the affinity of IL-15 to the receptor will cause IL-15 to be consumed in large quantities before entering the tumor due to high-affinity binding to peripheral immune cells, and will also increase the toxic side effects induced by IL-15. It has been found that the serious toxic side effects caused by IL-15 treatment are mainly mediated by a large number of expanded peripheral NK cells. 1,2 .

[0005] To ensure that IL-15 releases its activity only in tumors to reduce toxic side effects and improve tumor treatment efficacy, the inventors previously designed an IL-15 precursor (pro-IL-15) that specifically releases active IL-15 in tumor tissues. The pro-IL-15 contains sIL-15 (a fusion protein of IL-15 and IL-15Rαsushi domain), an MMP-cleavable peptide, and an IL-15Rβ extracellular domain. 2Among them, because MMP (matrix metalloproteinase) is highly expressed in tumor tissue but is expressed at a low level in normal tissue, Pro-IL-15 is in a closed state in the periphery to avoid IL-15 release and toxicity. When it enters the tumor tissue, MMP-14, which is highly expressed in the tumor, cuts the connecting peptide, releasing IL-15 to activate the anti-tumor immune response. Previous research results have shown that tumor-responsive pro-IL-15 maintains the same anti-tumor effect as IL-15 while significantly reducing toxic side effects. It can also amplify T lymphocytes in tumor tissue, especially stem cell-like lymphocytes (TCF1) with self-renewal and differentiation potential. + Tim3 - CD8 + T cells). Summary of the Invention

[0006] Although pro-IL-15 has been found to effectively amplify CD8 + How to deliver IL-15 specifically to antigen-specific CD8 T cells in tumors + T cells to allow IL-15 to more effectively expand CD8 + CTL, thereby restoring, reversing or enhancing CD8 + The effector function of CTLs remains an unresolved issue.

[0007] Some current clinical and preclinical studies mainly use tumor antigen-targeted antibodies or peptides to deliver IL-15 to tumors. However, these strategies only consider delivering cytokines to tumor sites rather than effector CD8 + In addition, the affinity of most cytokines to receptors is much higher than the affinity of antibodies to antigens. Therefore, these strategies will lead to a cytokine-dominated driving model, thereby affecting the targeting effect of antibodies.

[0008] After in-depth research, the inventors unexpectedly discovered that by combining tumor-specific responsive pro-IL-15 with an anti-tumor antibody domain (e.g., a complete antibody or antigen-binding fragment thereof against an immune checkpoint molecule, a tumor antigen molecule, or an immune activation molecule, preferably a PD-blocking antibody, more preferably a PD-1 antibody), it is possible to achieve targeted delivery of pro-IL-15 and to synergistically exert the anti-tumor function of the anti-tumor antibody and IL-15. For example, when the anti-tumor antibody is a PD-1 antibody, a tumor PD-1 antibody can be constructed. + The CTL-responsive anti-PD-1 and pro-IL-15 complex molecule achieves the following effects: (1) The activity of the complex molecule is blocked before entering the tumor, so it will not cause serious toxic side effects; (2) The complex molecule specifically targets and acts on tumor-infiltrating PD1 + CD8 +CTL cells; (3) After reaching the target site, PD-1 antibodies relieve the tumor microenvironment from CD8 + Suppression of CTL cells, restoration of CD8 + T effector function; (4) IL-15 expands CD8 in situ after being released + CTL, enhanced CD8 + The effector function and number of CTLs can be increased, and long-lived memory immune cells can be induced to produce, thereby breaking the immune tolerance of the tumor microenvironment, improving the tumor cure rate and preventing recurrence.

[0009] Therefore, the first aspect of the present invention provides a composite molecule comprising an anti-tumor antibody domain, a linker and pro-IL-15, wherein the anti-tumor antibody domain and pro-IL-15 are connected by the linker, the anti-tumor antibody domain is a complete antibody, nanobody or antigen-binding fragment thereof against an immune checkpoint molecule, a tumor antigen molecule or an immune activation molecule, and the linker is a polypeptide or a non-peptide linker; wherein the pro-IL-15 is a fusion protein comprising an IL-15Rβ extracellular domain, IL-15, an IL-15Rαsushi domain, a first connecting peptide and a second connecting peptide, or a fusion protein consisting of them, wherein the IL-15Rβ extracellular domain, IL-15 and the IL-15Rαsushi domain are connected by the first connecting peptide and the second connecting peptide; or the pro-IL-15 is a fusion protein comprising an IL-15, an IL-15Rαsushi domain, a first connecting peptide or a fusion protein consisting of them, wherein IL-15 and the IL-15Rαsushi domain are optionally connected by the first connecting peptide.

[0010] In the composite molecule (e.g., anti-PD-1-pro-IL-15), the pro-IL-15 can be (i) a fusion protein consisting of, from N-terminus to C-terminus, the IL-15Rβ extracellular domain (RβD1), a first connecting peptide (e.g., an MMP-cleavable peptide), IL-15, a second connecting peptide, and an IL-15Rαsushi domain; (ii) a fusion protein consisting of, from N-terminus to C-terminus, the IL-15Rαsushi domain, a second connecting peptide, IL-15, a first connecting peptide (e.g., an MMP-cleavable peptide), and an IL-15Rβ extracellular domain (RβD1); (iii) a fusion protein consisting of, from N-terminus to C-terminus, IL-15, a first connecting peptide (e.g., an MMP-cleavable peptide), and an IL-15Rαsushi domain; or (iv) a fusion protein consisting of, from N-terminus to C-terminus, a first connecting peptide (MMP-cleavable peptide), IL-15, and an IL-15Rαsushi domain. In case (iv), the linker can be the first connecting peptide (e.g., an MMP-cleavable peptide), i.e., the anti-tumor antibody domain and pro-IL-15 are connected together via the first connecting peptide (e.g., an MMP-cleavable peptide). The first connecting peptide can be an MMP-cleavable peptide or an enzymatically inoperable peptide, and the second connecting peptide can be an enzymatically inoperable peptide.

[0011] In an embodiment of the composite molecule of the present invention, the immune checkpoint molecule may include PD-1, PD-L1, CTLA4, TIGIT, Tim3 or Lag3; the tumor antigen molecule may include CLDN18.2, EGFR, Her2 or mesothelin; the immune activation molecule may include OX40, 4-1BB, CD28 or CD3; but are not limited thereto.

[0012] In an embodiment of the composite molecule of the present invention, the anti-tumor antibody domain may be a PD-1 antibody or an antigen-binding fragment thereof; the antigen-binding fragment may include ScFv, Fab or F(ab')2.

[0013] In an embodiment of the composite molecule of the present invention, the composite molecule may be a fusion protein, the connector may be a polypeptide, and the composite molecule may be selected from the following forms:

[0014] (a) the C-terminus of at least one heavy chain of the intact antibody or Nanobody is linked to the N-terminus of pro-IL-15 via a linker;

[0015] (b) the C-terminus of the antigen-binding fragment is linked to the N-terminus of pro-IL-15 via a linker;

[0016] (c) the N-terminus of one Fc chain is linked to the C-terminus of the fusion protein of the form of (b) via a linker, and the N-terminus of the other Fc chain is not linked to a peptide or is linked to the C-terminus of the fusion protein of the form of (b), the C-terminus of the antigen-binding fragment, or the C-terminus of pro-IL-15 via a linker;

[0017] Wherein, the C-terminus of the antigen-binding fragment described in (b) and (c) refers to the C-terminus of the heavy chain or light chain of Fab, the C-terminus of scFv, or the C-terminus of one heavy chain or light chain of F(ab')2; wherein, when multiple linkers appear in the composite molecule, they are the same or different from each other; and when two pro-IL-15s appear in the composite molecule, they are the same or different from each other.

[0018] In an embodiment of the composite molecule of the invention, the whole antibody or Nanobody may be a bispecific antibody.

[0019] In an embodiment of the composite molecule of the present invention, the amino acid sequence of the connector is preferably SEQ ID No: 8 or SEQ ID No: 10.

[0020] In an embodiment of the composite molecule of the present invention, the composite molecule may be a conjugate, and the linker may be a non-peptide linker.

[0021] In an embodiment of the composite molecule of the present invention, the linker may be an enzymatically cleavable linker.

[0022] In an embodiment of the composite molecule of the present invention, the linker may be a non-enzymatically cleavable linker.

[0023] In an embodiment of the composite molecule of the present invention, the two chains of the Fc segment of the anti-tumor antibody can be respectively linked to pro-IL-15 with different sequences, wherein the two linkers can be the same or different.

[0024] In an embodiment of the composite molecule of the present invention, the composite molecule may be in the form of the above-mentioned (a) C-terminus of at least one heavy chain of the complete antibody or nanobody connected to the N-terminus of pro-IL-15 via a linker, wherein the sequence of at least one heavy chain of the composite molecule (i.e., the peptide chain formed by the connection of at least one heavy chain of the complete antibody or nanobody and pro-IL-15) from N-terminus to C-terminus may be at least one of the following: (1) anti-tumor antibody Fab heavy chain, Fc, linker, Rαsushi domain, second connecting peptide, IL-15, first connecting peptide and RβD1; (2) anti-tumor antibody Fab heavy chain, Fc, linker, RβD1, A first connecting peptide, IL-15, a second connecting peptide and an Rαsushi domain; (3) an anti-tumor antibody Fab heavy chain, Fc, a linker, IL-15, a first connecting peptide and an Rαsushi domain; and (4) an anti-tumor antibody Fab heavy chain, Fc, a first connecting peptide, IL-15 and an Rαsushi domain; wherein the anti-tumor antibody is an anti-PD-1 antibody, an anti-Lag3 antibody or an anti-Claudin18.2 antibody; wherein each Fab heavy chain is associated with a corresponding antibody light chain; wherein the linker is a non-enzymatically cleavable linker, the first connecting peptide is an MMP-cleavable peptide or a non-enzymatically cleavable peptide, and the second connecting peptide is an non-enzymatically cleavable peptide.

[0025] In an embodiment of the composite molecule of the present invention, when an Fc is included, the Fc may be a wild-type Fc, or preferably an Fc that has been modified (e.g., mutated) to reduce or eliminate its binding affinity to FcγR, or an Fc that has been modified (e.g., mutated) to enhance its binding affinity to FcγR. Preferably, the Fc segment may lack ADCC function (mFc), for example, the amino acid sequence of the Fc segment may be SEQ ID No: 3, and the Fc segment may further include an L235E mutation.

[0026] In an embodiment of the composite molecule of the present invention, the IL-15 in the pro-IL-15 can be a mammalian wild-type IL-15 (e.g., SEQ ID No: 6 or 16), or can be one of its N71Q, N72D, N79Q or N112Q mutant proteins.

[0027] In an embodiment of the composite molecule of the present invention, the anti-tumor antibody may be a complete anti-tumor antibody or a Fab fragment thereof, and the Fab may be at least one selected from the following: (i) the Fab heavy chain sequence is SEQ ID No: 2 or SEQ ID No: 12, and the light chain sequence is SEQ ID No: 1 or SEQ ID No: 11; (ii) the Fab heavy chain sequence is SEQ ID No: 19, and the light chain sequence is SEQ ID No: 18; or (iii) the Fab heavy chain sequence is SEQ ID No: 21, and the light chain sequence is SEQ ID No: 20.

[0028] In an embodiment of the composite molecule of the present invention, in the pro-IL-15, the sequence of IL-15 may be SEQ ID No: 6 or SEQ ID No: 16, the sequence of the IL-15Rαsushi domain may be SEQ ID No: 7 or SEQ ID No: 17, the sequence of the IL-15Rβ extracellular domain may be SEQ ID No: 4, SEQ ID No: 14, SEQ ID No: 5 or SEQ ID No: 15, the sequence of the first connecting peptide may be SEQ ID No: 8, 9 or 10, and the sequence of the second connecting peptide may be SEQ ID No: 8 or SEQ ID No: 9.

[0029] The present invention also provides a composite molecule obtained by inserting pro-IL-15 or IL-15 between the CH1 region and the CH2 region of at least one heavy chain of a complete anti-tumor antibody, wherein a linker is optionally connected to the N-terminus and C-terminus of the pro-IL-15 or IL-15; wherein the pro-IL-15 is a fusion protein, which comprises, from the N-terminus to the C-terminus: IL-15, a connecting peptide and an IL-15Rαsushi domain, or an IL-15Rαsushi domain, a connecting peptide and IL-15; the anti-tumor antibody, the linker, and the connecting peptide may be the same as those described in the above embodiments.

[0030] In an embodiment of the composite molecule of the present invention, the composite molecule is a fusion protein in the following form: one heavy chain is, from N-terminus to C-terminus, an anti-tumor antibody Fab heavy chain, a linker, pro-IL-15 / IL-15, a linker, and one chain of Fc; the other heavy chain is, from N-terminus to C-terminus, an anti-tumor antibody Fab heavy chain, a linker, pro-IL-15 / IL-15, a linker, and another chain of Fc, wherein the two Fab heavy chains are identical or different and each associates with the corresponding antibody light chain through, for example, a disulfide bond to form Fab, the two chains of Fc are associated with each other through a disulfide bond, the linkers are identical or different, and the two pro-IL-15s are identical or different; preferably, the Fab is selected from the Fab of anti-PD-1, Lag3 or Claudin18.2 antibody; the linker is selected from SEQ ID No: 8, SEQ ID No: 9 and SEQ ID No: 10; the IL-15 is selected from SEQ ID No: 6 and SEQ ID No: 16; the IL-15Rαsushi domain is selected from SEQ ID No:7 and SEQ ID No:17; the connecting peptide is selected from SEQ ID No:8 and SEQ ID No:9; the Fc can be a wild-type Fc, or preferably an Fc whose binding ability to FcγR is reduced or eliminated after modification (e.g., mutation), or an Fc whose binding ability to FcγR is enhanced after modification (e.g., mutation), for example, the amino acid sequence of the Fc segment can be SEQ ID No:3, and the Fc segment can further include an L235E mutation; more preferably, the connector connected to the Fab heavy chain is SEQ ID No:8 or SEQ ID No:9, and the connector connected to the Fc is SEQ ID No:10.

[0031] When the composite molecule of the present invention is a fusion protein, the present invention also provides a nucleic acid encoding any of the above fusion proteins, a vector comprising the nucleic acid, and a host cell comprising the vector.

[0032] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising the composite molecule, nucleic acid, vector, or host cell of the first aspect of the present invention, and a pharmaceutically acceptable carrier or excipient. In an embodiment of the second aspect, the anti-tumor antibody domain may be an intact antibody, nanobody, or antigen-binding fragment thereof against PD-1, and the pharmaceutical composition may further comprise an anti-PD-L1 antibody.

[0033] In the third aspect of the present invention, there is provided use of the composite molecule, nucleic acid, vector or host cell of the first aspect of the present invention or the pharmaceutical composition of the second aspect in the preparation of a drug for treating cancer, preferably solid tumors.

[0034] In a fourth aspect of the present invention, a method for treating cancer is provided, comprising administering an effective amount of the composite molecule of the first aspect or the pharmaceutical composition of the second aspect of the present invention to a subject; the subject is preferably a mammal, more preferably a human.

[0035] The cancers described in the third and fourth aspects include, but are not limited to, lung cancer, liver cancer, gastric cancer, cervical cancer, ovarian cancer, breast cancer, colorectal cancer, melanoma, and lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic diagram of the structures of two examples of the anti-PD-1-pro-IL-15 complex molecule of the present invention. The left figure shows an inseparable anti-PD-1-pro-IL-15 fusion protein, where "Rα-IL-15" indicates a fusion molecule consisting of the Rα sushi domain (Rα) from the N-terminus to the C-terminus, an enzymatically inaccessible linker, and IL-15; the right figure shows a separable anti-PD-1-pro-IL-15 fusion protein, where "IL-15-Rα" indicates a fusion molecule consisting of IL-15, an enzymatically inaccessible linker, and the Rα sushi domain from the N-terminus to the C-terminus.

[0037] Figure 2 shows the binding ability of each protein used in Example 2 to cell surface PD-1: IL-15-Rα only connected to the Fc segment (IL-15-Rα-Fc), PD-1 antibody only (α-PD-1-mFc), inseparable anti-PD-1-pro-IL-15 fusion protein (α-PD-1-mFc-Rα-IL-15-RβD1), and separable anti-PD-1-pro-IL-15 fusion protein (α-PD-1-mFc-RβD1-IL-15-Rα).

[0038] Figure 3 shows the IL-15 biological activity of two IL-15-Rα fusion proteins linked only to the Fc segment (Fc-Rα-IL-15 and IL-15-Rα-Fc) and the two anti-PD-1-pro-IL-15 fusion proteins used in Example 2.

[0039] FIG4 shows gel electrophoresis of two anti-PD-1-pro-IL-15 fusion proteins before and after treatment with MMP14 enzyme.

[0040] FIG5 shows the IL-15 biological activities of two anti-PD-1-pro-IL-15 fusion proteins before and after MMP14 enzyme treatment.

[0041] Figure 6 shows the therapeutic effects of two anti-PD-1-pro-IL-15 fusion proteins, a blank control (CTR), and a non-masked anti-PD-1-mFc-Rα-IL-15 fusion protein (without RβD1) on the mouse MC38 tumor model.

[0042] Figure 7 shows the therapeutic effects of a blank control (CTR), an inseparable anti-PD-1-pro-IL-15 fusion protein (α-PD-1-mFc-Rα-IL-15-RβD1), and a physical mixture of PD-1 antibody and pro-IL-15 (α-PD-1-mFc+pro-IL-15) on the mouse MC38 tumor model. *** indicates p < 0.001.

[0043] Figure 8 shows that after blocking IFN-γ and deleting CD8 + Antitumor effect of anti-PD-1-pro-IL-15 fusion protein (α-PD-1-mFc-Rα-IL-15-RβD1) after T cell or NK cell depletion.

[0044] Figure 9 shows the expression of antigen-specific CD8 in tumors after treatment with anti-PD-1-pro-IL-15. + Changes in T cells and stem-like T cells.

[0045] FIG10 shows the inhibitory effect of anti-PD-1-pro-IL-15 in combination with PD-L1 antibody on tumors.

[0046] FIG11 shows the size exclusion chromatography and static light scattering (SEC-MALS) detection results of different protein molecules prepared in Example 8.

[0047] Figure 12 shows the binding ability of the fusion protein prepared in Example 8 to human PD-1 (hPD-1) on the cell surface: human IgG1 (hIgG1); anti-PD-1 antibody only (αPD-1), anti-PD-1 antibody only linked to sIL-15 (αPD-1-sIL-15), human fusion protein αPD-1-proIL-15 (RβD1), and human fusion protein αPD-1-proIL-15 (Rβ).

[0048] Figure 13 shows gel electrophoresis of the two human fusion proteins prepared in Example 8 and their products after treatment with MMP14. Lanes 1 and 2 in (A) are PD-1 antibody-Rα-IL-15-Rβ, and lane 3 is PD-1 antibody-Rα-IL-15; lanes 1 and 2 in (B) are PD-1 antibody-Rα-IL-15-RβD1, lane 3 is PD-1 antibody-Rα-IL-15-RβD1 after treatment with MMP14, and lane 4 is PD-1 antibody-Rα-IL-15.

[0049] Figure 14 shows the effects of two human fusion proteins prepared in Example 8 and their products after treatment with MMP14 enzyme on (A) NK cells and (B) CD8 + Biological activity of IL-15 in T cells.

[0050] Figure 15 shows the therapeutic effects of the two human fusion proteins prepared in Example 8, a blank control (CTR), and a physical mixture of PD-1 antibody and pro-IL-15 (proIL-15 + α-PD-1) on CFPAC1 tumors in a CBMC humanized mouse model. * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

[0051] FIG16 shows a schematic diagram of the structure of the physically shielded fusion protein anti-PD-1-Fab-IL-15-Rα-mFc.

[0052] Figure 17 shows the mass spectrophotometer detection results of the physically shielded fusion proteins anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc prepared in Example 9 after separation and purification.

[0053] Figure 18 shows the binding ability of the physically shielded fusion proteins prepared in Example 9 to PD-1: anti-PD-1 antibody only (αPD-1), anti-PD-1-Fab-IL-15-Rα-mFc, anti-PD-1-Fab-IL-15-mFc, and human IgG1 (hIgG1).

[0054] FIG19 shows the binding ability of IL-15 or sIL-15 in the physically shielded fusion protein prepared in Example 9 to the IL-15β receptor CD122.

[0055] Figure 20 shows the biological activity of the physically shielded fusion proteins anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc prepared in Example 9 in the CTLL2 proliferation assay.

[0056] Figure 21 shows the anti-tumor effects and side effects of the physically shielded fusion proteins anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc prepared in Example 9 in a mouse MC38 tumor model. *** indicates p < 0.001; **** indicates p < 0.0001.

[0057] Figure 22 shows the anti-tumor effect of the fusion protein anti-Lag3-pro-IL-15 prepared in Example 10 in the mouse MC38 tumor model, where **** indicates p<0.0001.

[0058] FIG23 shows the IL-15 biological activities of the two anti-Claudin18.2-pro-IL-15 fusion proteins prepared in Example 11.

[0059] Figure 24 shows the anti-tumor effect of the fusion protein anti-Claudin18.2-Fc-RβD1-IL-15-Rα prepared in Example 11 in the mouse MC38 tumor model, where **** indicates p<0.0001. DETAILED DESCRIPTION

[0060] The exemplary embodiments of the present invention will now be described in detail. However, it should be understood that the exemplary embodiments described below are only used to illustrate the subject matter of the present invention and are not intended to limit the scope of the present invention.

[0061] Unless otherwise specified, the terms "Rα sushi domain," "Rα," and "sushi" are used interchangeably herein to refer to the sushi domain of the IL-15 Rα receptor, which may be SEQ ID No: 7 or SEQ ID No: 17, or a functionally equivalent mutant thereof.

[0062] Unless otherwise specified, the term "IL-15Rβ extracellular domain" in this application refers to the extracellular domain of the Rβ receptor of IL-15 or a truncated functional fragment thereof (for example, "IL-15Rβ extracellular domain 1" or "RβD1"), the sequence of which can be SEQ ID No: 4, SEQ ID No: 14, SEQ ID No: 5 or SEQ ID No: 15, or a mutant sequence having equivalent function thereto.

[0063] Unless otherwise specified, the term "anti-tumor antibody domain" in this application refers to a complete antibody, nanobody or antigen-binding fragment thereof that can specifically bind to tumor-associated molecules and exert anti-tumor effects (including but not limited to inhibiting the occurrence, development and metastasis of tumors). The tumor-associated molecules may include immune checkpoint molecules (such as PD-1, PD-L1, CTLA4, TIGIT, Tim3 or Lag3), tumor antigen molecules (CLDN18.2, EGFR, Her2 or mesothelin) or immune activation molecules (OX40, 4-1BB, CD28 or CD3), but are not limited thereto. The "complete antibody" refers to a complete structural antibody with two heavy chains and two light chains in the usual sense; the "nanobody" refers to an antibody that has only two heavy chains and no light chain compared to a complete antibody, and the heavy chain lacks the constant region CH1; the antigen-binding fragment may include ScFv, Fab or F(ab')2 fragments of a complete antibody, and may also include Fab or F(ab')2 fragments of a nanobody.

[0064] The term "treat" as used herein generally refers to obtaining a desired pharmacological and / or physiological effect that partially or completely stabilizes or cures a disease and / or its adverse effects, or inhibits or alleviates the symptoms of a disease.

[0065] The term "effective amount" in this application refers to the amount of the compound molecule or pharmaceutical composition of the present invention that achieves a therapeutic effect."Therapeutically effective amount" can vary depending on, for example, the disease state, age, sex, and weight of the individual, as well as the ability of the therapeutic agent to elicit a desired response in the subject.

[0066] The term "IL-15" herein refers to interleukin-15, a cytokine of the interleukin family, including IL-15 of mammalian origin, such as IL-15 derived from mouse or human.

[0067] The present invention provides a composite molecule comprising an anti-tumor antibody domain, a linker and pro-IL-15, wherein the anti-tumor antibody domain and pro-IL-15 are connected by the linker, the anti-tumor antibody domain is a complete antibody, nanobody or antigen-binding fragment thereof targeting an immune checkpoint molecule, a tumor antigen molecule or an immune activation molecule, and the linker is a polypeptide or a non-peptide linker; wherein the pro-IL-15 is a fusion protein comprising an IL-15Rβ extracellular domain, IL-15, an IL-15Rαsushi domain, a first connecting peptide and a second connecting peptide, wherein the IL-15Rβ extracellular domain, IL-15 and the IL-15Rαsushi domain are connected by the first connecting peptide and the second connecting peptide; or the pro-IL-15 is a fusion protein comprising IL-15, an IL-15Rαsushi domain and a first connecting peptide, wherein IL-15 and the IL-15Rαsushi domain are optionally connected by the first connecting peptide. Unless otherwise specified, the order in which the functional units of pro-IL-15, namely the IL-15Rβ extracellular domain, IL-15 and IL-15Rα sushi domain, are linked is not limited.

[0068] In a specific embodiment, the present invention provides a complex molecule formed by connecting an anti-tumor antibody domain (such as a PD-blocking antibody) and pro-IL-15 via a linker, which specifically targets and acts on tumor-infiltrating PD-1. + CD8 + After CTL cells reach the target site, PD-1 antibodies relieve the tumor microenvironment from CD8 + Inhibition of CTL cells and restoration of CD8 + T effector function, IL-15 is released, and CD8 + CTL, enhanced CD8 + The composite molecule of the present invention can break the immune tolerance of the tumor microenvironment, improve the cure rate of tumors and prevent recurrence.

[0069] In an embodiment of the composite molecule of the present invention, the PD-blocking antibody may be a PD-1 antibody or a PD-L1 antibody, and may be a monoclonal antibody known in the art. For example, the heavy chain sequence of the Fab of the PD-1 antibody is SEQ ID No: 2 or SEQ ID No: 12, and the light chain sequence is SEQ ID No: 1 or SEQ ID No: 11, but is not limited thereto.

[0070] In an embodiment of the composite molecule of the present invention, the Lag3 antibody may be a monoclonal antibody known in the art. For example, the heavy chain sequence of the Fab of the Lag3 antibody is preferably SEQ ID No: 19, and the light chain sequence is preferably SEQ ID No: 18, but are not limited thereto.

[0071] In an embodiment of the composite molecule of the present invention, the Claudin18.2 (CLDN18.2) antibody can be a monoclonal antibody known in the art. For example, the heavy chain sequence of the Fab of the Claudin18.2 antibody is preferably SEQ ID No: 21, and the light chain sequence is preferably SEQ ID No: 20, but are not limited thereto.

[0072] In an embodiment of the composite molecule of the present invention, pro-IL-15 is a fusion protein and can be (i) a fusion protein comprising, from N-terminus to C-terminus, the IL-15Rβ extracellular domain (e.g., RβD1), a first connecting peptide (e.g., an MMP-cleavable peptide), IL-15, a second connecting peptide, and an IL-15Rαsushi domain, (ii) a fusion protein comprising, from N-terminus to C-terminus, the IL-15Rαsushi domain, a second connecting peptide, IL-15, a first connecting peptide (e.g., an MMP-cleavable peptide), and an IL-15Rβ extracellular domain (e.g., RβD1), (iii) a fusion protein comprising, from N-terminus to C-terminus, IL-15, a first connecting peptide (e.g., an MMP-cleavable peptide), and an IL-15Rαsushi domain, or (iv) a fusion protein comprising, from N-terminus to C-terminus, a first connecting peptide (e.g., an MMP-cleavable peptide), IL-15, and an IL-15Rαsushi domain. In case of (iv), the connector may be the first connecting peptide (eg, an MMP-cleavable peptide). The first connecting peptide may be an MMP-cleavable peptide or an enzymatically inaccessible peptide, and the second connecting peptide may be an enzymatically inaccessible peptide.

[0073] The activity of IL-15 in the complex molecule can be blocked before entering the tumor, thereby preventing serious toxic side effects. After the complex molecule enters the tumor, the highly expressed MMP-14 in the tumor can cleave the MMP-cleavable peptide serving as the first connecting peptide, releasing IL-15 to exert its function. The second connecting peptide (if present) in the pro-IL-15, which connects IL-15 and the IL-15Rα sushi domain, can be enzymatically inaccessible.

[0074] In an embodiment of the composite molecule of the present invention, the linker connecting the anti-tumor antibody domain (e.g., PD-blocking antibody) and pro-IL-15 can be a polypeptide or non-peptide linker. Examples of polypeptide linkers include SEQ ID Nos: 8, 9, and 10.

[0075] In an embodiment of the composite molecule of the present invention, the composite molecule may be a fusion protein, wherein the connector may be a polypeptide, and the composite molecule may be selected from the following forms:

[0076] (a) the C-terminus of at least one heavy chain of the intact antibody or Nanobody is linked to the N-terminus of pro-IL-15 via a linker;

[0077] (b) the C-terminus of the antigen-binding fragment is linked to the N-terminus of pro-IL-15 via a linker;

[0078] (c) the N-terminus of one Fc chain is linked to the C-terminus of the fusion protein of the form of (b) via a linker, and the N-terminus of the other Fc chain is not linked to a peptide or is linked to the C-terminus of the fusion protein of the form of (b), the C-terminus of the antigen-binding fragment, or the C-terminus of pro-IL-15 via a linker;

[0079] Wherein, the C-terminus of the antigen-binding fragment described in (b) and (c) refers to the C-terminus of the heavy chain or light chain of Fab, the C-terminus of scFv, or the C-terminus of one heavy chain or light chain of F(ab')2; wherein, when multiple linkers appear in the composite molecule, they are the same or different from each other; and when two pro-IL-15s appear in the composite molecule, they are the same or different from each other.

[0080] In an embodiment of the composite molecule of the present invention, the composite molecule is obtained by inserting pro-IL-15 or IL-15 between the CH1 region and the CH2 region of at least one heavy chain of a complete anti-tumor antibody, wherein a linker is optionally connected to the N-terminus and C-terminus of the pro-IL-15 or IL-15; wherein the pro-IL-15 is a fusion protein, which comprises, from the N-terminus to the C-terminus: IL-15, a connecting peptide and an IL-15Rαsushi domain, or an IL-15Rαsushi domain, a connecting peptide and IL-15; the anti-tumor antibody, the linker, and the connecting peptide may be the same as those described above.

[0081] In an embodiment of the composite molecule of the present invention, the composite molecule is a fusion protein in the following form: one heavy chain is, from N-terminus to C-terminus, an anti-tumor antibody Fab heavy chain, a linker, pro-IL-15 / IL-15, a linker, and one chain of Fc; the other heavy chain is, from N-terminus to C-terminus, an anti-tumor antibody Fab heavy chain, a linker, pro-IL-15 / IL-15, a linker, and another chain of Fc, wherein the two Fab heavy chains are identical or different and each associates with the corresponding antibody light chain through, for example, a disulfide bond to form Fab, the two chains of Fc are associated with each other through a disulfide bond, the linkers are identical or different, and the two pro-IL-15s are identical or different; preferably, the Fab is selected from the Fab of anti-PD-1, Lag3 or Claudin18.2 antibody; the linker is selected from SEQ ID No: 8, SEQ ID No: 9 and SEQ ID No: 10; the IL-15 is selected from SEQ ID No: 6 and SEQ ID No: 16; the IL-15Rαsushi domain is selected from SEQ ID No: 7 and SEQ ID No: 17; the connecting peptide is selected from SEQ ID No: 8 and SEQ ID No: 9; the Fc may be a wild-type Fc, or an Fc whose binding ability to FcγR is reduced or eliminated after modification (e.g., mutation), or an Fc whose binding ability to FcγR is enhanced after modification (e.g., mutation). Preferably, the Fc segment may not have ADCC function (mFc), for example, the amino acid sequence of the Fc segment may be SEQ ID No: 3, and the Fc segment may further include an L235E mutation; more preferably, the connector connected to the Fab heavy chain is SEQ ID No: 8 or SEQ ID No: 9, and the connector connected to the Fc is SEQ ID No: 10.

[0082] In embodiments of the composite molecules of the present invention, the linker may be enzymatically cleavable or non-enzymatically cleavable, but is preferably non-enzymatically cleavable. The inventors have discovered that when the linker is non-enzymatically cleavable, the anti-tumor antibody domain (e.g., PD-blocking antibody) and pro-IL-15 are essentially inseparable in the periphery and within the tumor, unexpectedly exhibiting superior anti-tumor efficacy.

[0083] In addition, the composite molecule of the present invention can use a composite molecule that does not contain the IL-15Rβ extracellular domain (e.g., RβD1) in pro-IL-15, for example, it can contain the above-mentioned pro-IL-15 (iii) and (iv). The inventors found that when RβD1 is not present, the activity of the composite molecule "(anti-PD-1-Fc)-linker-(IL-15)-(IL-15Rαsushi)" is reduced compared to sIL-15; however, when (iii) or (iv) is used as the composite molecule, for example, "(anti-PD-1-Fc)-linker-(IL-15)-MMP cleavable peptide-(IL-15Rαsushi)" or "(anti-PD-1-Fc)-linker-MMP cleavable peptide-(IL-15)-(IL-15Rαsushi)", the activity is restored.

[0084] Furthermore, both the first and second connecting peptides in the pro-IL-15 complex molecule of the present invention can be non-enzymatically cleavable peptides. Typically, the first connecting peptide in a single pro-IL-15 molecule is a peptide cleavable by MMPs, thereby ensuring efficient release of IL-15 in the tumor environment to exert its function. However, the inventors unexpectedly discovered that when linked to an antibody (e.g., the Fc of a PD-1 antibody), even if both the first and second connecting peptides of pro-IL-15 are non-enzymatically cleavable peptides, the complex molecule can still exert significantly higher IL-15 activity than pro-IL-15 not linked to the antibody (when cells express both the PD-1 molecule and the IL-15 receptor).

[0085] In a specific embodiment, the complex molecule of the present invention is a fusion protein as shown in the left figure of Figure 1 , wherein the functional units thereof, from N-terminus to C-terminus, are: anti-PD-1 Fab, mFc (with reduced binding affinity to FcγR), linker, Rαsushi domain, connecting peptide, IL-15, MMP-cleavable peptide, and RβD1, wherein the linker is a non-enzymatically cleavable polypeptide linker, and thus the antibody and pro-IL-15 in this complex molecule are substantially inseparable in vivo (i.e., an "inseparable" complex molecule). It should be understood that the "inseparable" described herein is not limited to the complex molecule of this embodiment; as long as the antigen-binding portion of the antibody is linked to pro-IL-15 via a non-enzymatically cleavable linker, it can be referred to as "inseparable."

[0086] In a specific embodiment, the complex molecule of the present invention is a fusion protein as shown in the right figure of Figure 1, wherein the functional units thereof are, from N-terminus to C-terminus, the following: anti-PD-1 Fab, mFc (reduced binding to FcγR), linker, RβD1, MMP-cleavable peptide, IL-15, linker peptide, and Rαsushi domain, wherein the linker is an enzymatically cleavable polypeptide linker, for example, an MMP-cleavable peptide. Therefore, the antibody portion and pro-IL-15 of the complex molecule are separated from each other after entering the tumor due to the disconnection of the linker (i.e., a "separable" complex molecule). It should be understood that the "separable" described herein is not limited to the complex molecule of this embodiment; as long as the antigen-binding portion of the antibody is connected to pro-IL-15 via an enzymatically cleavable linker, it can be referred to as a "separable" complex molecule.

[0087] The structure of the composite molecule of the present invention is not limited to the above-described embodiments. For example, different types of pro-IL-15 molecules can be linked to the two Fc segments of an antibody. In addition, the non-enzymatically cleavable linker in the left figure of FIG1 can be replaced with an enzymatically cleavable linker to form a separable composite molecule. The enzymatically cleavable linker in the right figure of FIG1 can be replaced with a non-enzymatically cleavable linker to form an inseparable composite molecule.

[0088] When the composite molecule of the present invention is a fusion protein, the present invention also provides a nucleic acid encoding any of the above fusion proteins, a vector comprising the nucleic acid, and a host cell comprising the vector.

[0089] The present invention also provides a pharmaceutical composition comprising the composite molecule, nucleic acid, vector or host cell of the present invention, and a pharmaceutically acceptable carrier or excipient. In one embodiment, the pharmaceutical composition may further comprise a second active agent, such as another anticancer agent (e.g., a small anticancer agent or an antibody anticancer agent). In one embodiment, the pharmaceutical composition is used to treat a tumor or cancer.

[0090] The present invention also provides the use of the composite molecule, nucleic acid, vector, host cell or pharmaceutical composition of the present invention in the preparation of a drug for treating cancer, preferably solid tumors.

[0091] The present invention also provides a method for treating cancer, preferably solid tumors, comprising administering an effective amount of the composite molecule or pharmaceutical composition of the present invention to a subject; the subject is preferably a mammal, more preferably a human.

[0092] The above-mentioned tumors or cancers include but are not limited to: lung cancer, liver cancer, gastric cancer, cervical cancer, ovarian cancer, breast cancer, colorectal cancer, melanoma and lymphoma.

[0093] Example

[0094] The following experiments provide examples that represent the gist of the present invention or demonstrate the effects of the present invention, but it should be understood that the present invention is not limited to these examples, but various equivalent replacements or changes can be made based on the gist of the present invention.

[0095] Materials and methods

[0096] Construction of fusion proteins

[0097] The pEE12.4-IgGκ plasmid was obtained from our laboratory and contains the signal peptide of mouse IgGκ. The Fab sequence of the PD-1 antibody is a J43 clone that can block PD-L1 / PD-1 signaling. The Fc sequence is a human IgG1 sequence lacking ADCC function (SEQ ID No: 3). IL-15-related genes (including IL-15 (SEQ ID No: 6), IL-15Rα (SEQ ID No: 7), IL-15Rβ extracellular domain (SEQ ID No: 5), and IL-15Rβ extracellular domain 1 (SEQ ID No: 4)) were synthesized by Shanghai Bio-Technology Co., Ltd. and fused to the PD-1 antibody into the pEE12.4 expression vector. Plasmids were extracted using a Tiangen plasmid extraction kit and stored at -80°C.

[0098] experimental animals

[0099] Wild-type C57BL / 6 mice were purchased from the Weitonglihua Laboratory Animal Center in Beijing, China. All experiments used female mice aged 8–10 weeks, unless otherwise noted. Mice were maintained in a specific pathogen-free (SPF) barrier environment. Animal husbandry and experimental procedures were in accordance with the regulations of the National Laboratory Animal Care Committee.

[0100] cell lines

[0101] The FreeStyle™293F cell line (Invitrogen) is a suspension cell line derived from the HEK293 cell line. It is cultured in SMM293-TII or CD OptiCHO™ medium and is primarily used for transient transfection to express fusion proteins.

[0102] CTLL-2 is a mouse T cell line used to detect the biological activity of IL-15. It was cultured in RPMI1640 complete medium (containing 10% inactivated fetal bovine serum, 2 mmol / L L-glutamine, 0.1 mmol / L non-essential amino acids, 100 U penicillin, 100 μg / ml streptomycin, 100 IU / ml recombinant IL-2, and 55 μM β-mercaptoethanol).

[0103] CTLL-2-mPD-1 is a cell line that stably expresses the mouse PD-1 molecule and is cultured in RPMI1640 complete medium (containing 10% inactivated fetal bovine serum, 2 mmol / L L-glutamine, 0.1 mmol / L non-essential amino acids, 100 U penicillin, 100 μg / ml streptomycin, 400 IU / ml recombinant IL-2, 55 μM β-mercaptoethanol, and 4 μg / ml puromycin).

[0104] MC38 is a mouse colorectal cancer cell line with a C57 background and was cultured in DMEM complete medium (containing 10% inactivated fetal bovine serum, 2 mmol / l L-glutamine, 0.1 mmol / l non-essential amino acids, 100 U penicillin and 100 μg / ml streptomycin).

[0105] Protein binding capacity assay

[0106] Cells expressing the specific antigen were harvested and resuspended in FACS buffer (PBS + 2% FBS + 0.05% sodium azide). 0.2 μg of 2.4G2 antibody was added to each sample and incubated on ice for 15 minutes to block FcγR. The fusion protein was diluted serially in FACS buffer. The protein was incubated with the cell suspension and incubated on ice for 30 minutes. After washing once with FACS buffer, fluorescently labeled anti-human IgG1 antibody was added and incubated on ice for 30 minutes. After washing twice with FACS buffer, the cells were analyzed.

[0107] CTLL-2 proliferation assay

[0108] CTLL-2 cells are a mouse T cell line. The cell proliferation assay can be used to detect the biological activity of IL-15. Dilute the fusion protein or the product cleaved by MMP-14 in complete culture medium in series, and add 100 μl to a 96-well plate. Collect CTLL-2 cells or CTLL-2-mPD-1 cells and wash away the IL-2 in the culture medium. Adjust the cell concentration to 3x10 4 / ml, take 100 μl of cell suspension and add it to each well. After 72 hours of incubation at 37°C, 5% CO2, add 20 μl of CCK8 reagent to each well and continue incubation at 37°C, 5% CO2 for 3-4 hours. OD450 is measured using a microplate reader, and the curve is plotted and analyzed.

[0109] Verification of the in vitro cleavage and activity recovery effects of anti-PD-1-pro-IL-15

[0110] Activation buffer (50 mM Tris, 1 mM CaCl2, 0.5% Brij-35, pH 9.0) and assay buffer (50 mM Tris, 3 mM CaCl2, 1 μM ZnCl2, pH 7.5) were prepared separately and sterile filtered. In the activation buffer, rhMMP-14 (R&D Company) was activated with rhFurin at 37°C. Anti-PD-1-pro-IL-15 was diluted with assay buffer, and activated MMP-14 was added at a molar ratio of 1:5 between the enzyme and the protein to be cleaved. The cells were incubated at 37°C overnight. SDS-PAGE was used to determine whether anti-PD-1-pro-IL-15 could be specifically cleaved by MMP-14 and the cleavage efficiency was determined. A CTLL-2 cell proliferation assay was used to determine the activity recovery of anti-PD-1-pro-IL-15 after cleavage by MMP-14.

[0111] Establishment of mouse tumor model

[0112] We selected MC38 as the research model. 5 Single MC38 cells were suspended in 100 μl of PBS and subcutaneously inoculated on the right side of the back of C57BL / 6 mice. Tumor length (a), length (b), and height (c) were measured using a vernier caliper. Tumor volume = a × b × c / 2. Tumor size was monitored twice weekly, and tumor growth curves were recorded.

[0113] Peripheral blood immune cell detection

[0114] Blood was collected from the orbital cavity into an anticoagulant tube containing K2 EDTA. 50 μL was transferred to a flow cytometer, and a fluorescent antibody mixture was prepared according to the specific staining protocol. The mixture was added to the flow cytometer, mixed, and incubated at room temperature in the dark for 15 minutes. 450 μL of red blood cell lysis buffer (eBioscience 1XRBC Lysis Buffer) was added dropwise to the blood sample while shaking and incubated at room temperature in the dark for 15 minutes. 15 μL of microspheres (Invitrogen Absolute Counting Beads) were added before loading the flow cytometer. Data were collected using a BD FACS Fortessa flow cytometer. The absolute number of each immune cell population in the peripheral blood was calculated based on the ratio and number of microspheres added.

[0115] Preparation of single-cell suspension of mouse tumor tissue

[0116] Tumor tissue was placed in a 6-well plate pre-filled with 100 μl of RPMI1640 medium. The tumor tissue was thoroughly minced with ophthalmic scissors and resuspended in 2 ml of digestion solution (RPMI1640 medium containing 1 mg / ml collagenase and 500 U / ml DNase I). The tissue suspension was placed in a 37°C shaker at 145 rpm until no obvious particulate matter was present (the entire process was approximately 40 min), with pipette aspiration 20 times every 20 minutes. The digested tissue suspension was filtered through a 70-mesh sieve and the cells were washed twice with RPMI1640 medium containing 2% FBS. The cells were resuspended in 1 ml of RPMI1640 complete medium and counted for later use.

[0117] Flow cytometry analysis of immune cells in tumors

[0118] Tumor single cell suspension was transferred to a flow cytometry tube, 1×10 6 cells / tube; Fc receptor blocking antibody (2.4G2 clone) was added to block Fc receptors on the cell surface and the cells were placed on ice for 20 minutes; according to the specific staining protocol, a fluorescent antibody mixture was prepared and added to the flow cytometry tube and placed on ice in the dark for 30 minutes. Single-stained tubes, isotype control tubes, and blank tubes were also prepared simultaneously; after washing twice with FACS buffer, the cells were resuspended and data were collected on a BD FACS Fortessa flow cytometer.

[0119] Intracellular protein staining was performed as follows: resuspend cells in 300 μl of pre-chilled Foxp3 Fixation / Permeabilization buffer. Incubate on ice in the dark for 30-60 minutes. Resuspend in 1 ml of permeabilization buffer, centrifuge at 500 g for 3 minutes, and discard the supernatant. Resuspend cells in 100 μl of permeabilization buffer, add fluorescent antibody, and incubate on ice in the dark for 30 minutes. Wash twice with permeabilization buffer, resuspend cells, and collect data using a BD FACS Fortessa flow cytometer.

[0120] Biostatistical analysis

[0121] All data were analyzed using GraphPad Prism statistical software. Mouse survival curves were analyzed using one-way ANOVA, and all other data were analyzed using two-tailed t-tests. P values ​​less than 0.05 were considered statistically significant (*), less than 0.01 were considered significantly significant (**), and less than 0.001 (***) or 0.0001 (****) were considered extremely significant.

[0122] Sequence Description

[0123] SEQ ID No: 1: Anti-mouse PD-1 antibody light chain

[0124] SEQ ID No: 11: Anti-human PD-1 antibody light chain

[0125] SEQ ID No: 2: Anti-mouse PD-1 antibody Fab heavy chain

[0126] SEQ ID No: 12: Anti-human PD-1 antibody Fab heavy chain

[0127] SEQ ID No: 3: Mutated human Fc (L19A, L20A, P114G)

[0128] SEQ ID No: 13: Human Fc (WT)

[0129] SEQ ID No: 4: Mouse IL-15Rβ extracellular domain 1

[0130] SEQ ID No: 14: Human IL-15Rβ extracellular domain 1

[0131] SEQ ID No: 5: Mouse IL-15Rβ extracellular domain

[0132] SEQ ID No: 15: Human IL-15Rβ extracellular domain

[0133] SEQ ID No: 6: Mouse IL-15

[0134] SEQ ID No: 16: Human IL-15

[0135] SEQ ID No: 7: Mouse IL-15Rα-sushi domain

[0136] SEQ ID No:17: Human IL-15Rα-sushi domain

[0137] SEQ ID No: 8: Connecting peptide

[0138] SEQ ID No: 9: Connecting peptide

[0139] SEQ ID No: 10: MMP-cleavable linker peptide

[0140] SEQ ID No: 18: Anti-human / mouse Lag3 antibody light chain:

[0141] SEQ ID No: 19: Anti-human / mouse Lag3 antibody Fab heavy chain:

[0142] SEQ ID No: 20: Anti-human / mouse Claudin 18.2 antibody light chain:

[0143] SEQ ID No: 21: Anti-human / mouse Claudin 18.2 antibody Fab heavy chain:

[0144] Example 1. Construction of Anti-PD-1-pro-IL-15 Fusion Protein

[0145] In this example, two anti-PD-1-pro-IL-15 fusion proteins were designed and constructed. The Fab sequence of the PD-1 antibody (anti-PD-1) was the J43 clone, which can block PD-L1 / PD-1 signals; the Fc sequence was a human IgG1 Fc sequence with three amino acid mutations (L19A, L20A, P114G), which lacks ADCC function (designated mFc).

[0146] The first is an inseparable anti-PD-1-pro-IL-15 fusion protein, the structural diagram of which can be seen in the left figure of Figure 1. Its functional units are as follows from N-terminus to C-terminus: anti-PD-1 Fab (SEQ ID No: 1 and SEQ ID No: 2), mFc (SEQ ID No: 3), connector (SEQ ID No: 8), Rαsushi domain (SEQ ID No: 7), connecting peptide (SEQ ID No: 9), IL-15 (SEQ ID No: 6), MMP cleavable peptide (SEQ ID No: 10) and RβD1 (SEQ ID No: 4), wherein the connector is an enzymatically non-cleavable polypeptide connector, so that the antibody and pro-IL-15 of the complex molecule are basically inseparable in vivo.

[0147] The second type is a separable anti-PD-1-pro-IL-15 fusion protein. Its structural diagram can be seen in the right figure of Figure 1. Its functional units from N-terminus to C-terminus are: anti-PD-1 Fab (SEQ ID No: 1 and SEQ ID No: 2), mFc (SEQ ID No: 3), connector (SEQ ID No: 10), RβD1 (SEQ ID No: 4), MMP-cleavable peptide (SEQ ID No: 10), IL-15 (SEQ ID No: 6), connector peptide (SEQ ID No: 9) and Rαsushi domain (SEQ ID No: 7), wherein the connector is an MMP-cleavable polypeptide. Therefore, the antibody part of the complex molecule and pro-IL-15 are separated from each other under the action of MMP after entering the tumor.

[0148] The corresponding anti-PD-1-Fab heavy chain-pro-IL-15 expression plasmid and anti-PD-1 light chain (SEQ ID No: 1) expression plasmid were constructed using the PEE12.4 eukaryotic expression vector system. The anti-PD-1-Fab heavy chain-pro-IL-15 expression plasmid and the anti-PD-1 light chain expression plasmid were co-transfected into 293F cells to obtain expression supernatant. The Fc-tagged protein was purified by Protein A affinity chromatography, and the protein size was verified by SDS-PAGE.

[0149] Example 2. Study on the biological activity of Anti-PD-1-pro-IL-15 in vitro

[0150] The biological activity of each active unit in the fusion protein was tested in vitro.

[0151] First, flow cytometry was used to examine the ability of the fusion protein prepared in Example 1 to bind to its antigen. Antibodies without pro-IL-15 (anti-PD-1) and pro-IL-15-Fc without antibody Fab were used as controls. The results showed that anti-PD-1-pro-IL-15 and anti-PD-1 had similar binding abilities to PD-1 molecules on the surface of EG7 cells (Figure 2).

[0152] The biological activity of IL-15 in the fusion protein was tested using a CTLL-2 proliferation assay. The results showed that compared to unshielded IL-15-Rα-Fc or Fc-Rα-IL-15, the IL-15 activity of the two anti-PD-1-pro-IL-15 proteins of the present invention was significantly reduced in a non-MMP catalytic environment, indicating that the IL-15 activity in the fusion protein can be blocked by the receptor (Figure 3).

[0153] The fusion protein was co-incubated with the activated MMP14 enzyme in vitro, and then SDS-PAGE analysis was performed to verify the in vitro cleavage effect of the fusion protein. The results showed that the fusion protein could be effectively cleaved in vitro (Figure 4). The recovery of the biological activity of the fusion protein after enzyme cleavage was detected by a CTLL-2 proliferation experiment. The results showed that after in vitro enzyme cleavage, the biological activity of IL-15 in the fusion protein could be well restored (Figure 5). The above results indicate that the activity of IL-15 in the fusion protein can be well blocked by the IL-15 receptor, and its activity can be effectively restored after MMP cleavage.

[0154] Example 3. Non-separable Anti-PD-1-pro-IL-15 exhibits better anti-tumor effects than separable Anti-PD-1-pro-IL-15

[0155] After verifying the function of the anti-PD-1-pro-IL-15 fusion protein in vitro, the anti-tumor efficacy and toxicity of these proteins in the mouse MC38 tumor model were evaluated. PBS was used as a blank control (CTR); non-masked anti-PD-1-mFc-Rα-IL-15 (without RβD1) was used as an isotype control. 5×10 5 MC38 tumor cells were injected intravenously with 80 μg of either anti-PD-1-pro-IL-15 fusion protein or 70 μg of the control protein, anti-PD-1-mFc-Rα-IL-15, on days 8 and 11, respectively. Tumor size and body weight were measured regularly, and mouse survival was recorded. Tumor growth and body weight curves were plotted.

[0156] The results, as shown in Figure 6, show that upon systemic administration, mice in the non-masked anti-PD-1-mFc-Rα-IL-15 treatment group experienced severe weight loss, with all mice dying from toxicity the day after the second injection. In contrast, no mice died in the groups receiving either form of the anti-PD-1-pro-IL-15 fusion protein, and no significant weight loss was observed. Furthermore, the non-masked anti-PD-1-pro-IL-15 fusion protein exhibited superior anti-tumor efficacy compared to the detachable form.

[0157] The following examples all used the inseparable anti-PD-1-pro-IL-15 fusion protein prepared in Example 1.

[0158] Example 4. Anti-PD-1-pro-IL-15 has a better anti-tumor effect than a physical mixture of PD-1 antibody and pro-IL-15

[0159] In a mouse tumor model, we evaluated whether the anti-tumor efficacy of the tumor-targeting anti-PD-1-pro-IL-15 fusion protein was superior to that of a non-targeting PD-1 antibody mixed with pro-IL-15. PBS was used as a blank control (CTR). 5×10 5 MC38 tumor cells were injected intravenously with 80 μg of inseparable anti-PD-1-pro-IL-15 fusion protein or a physical mixture of 53 μg of PD-1 antibody and 45 μg of pro-IL-15 on days 8 and 11. The mice were monitored for tumor growth at regular intervals.

[0160] The results are shown in Figure 7. When administered systemically, anti-PD-1-pro-IL-15 demonstrated significantly greater anti-tumor efficacy compared to the physical mixture of PD-1 antibody and pro-IL-15. At day 31, the physical mixture reduced tumor volume by approximately 17% compared to the control, which was not statistically significant, while the fusion protein reduced tumor volume by over 90%, which was statistically significant. This result demonstrates that the anti-tumor efficacy of the fusion protein of the present invention is not solely due to the additive effects of the PD-1 antibody and pro-IL-15, but rather that delivering the PD-1 antibody and pro-IL-15 to the same target site is crucial for the tumor inhibitory effect.

[0161] Example 5. The in vivo anti-tumor effect of Anti-PD-1-pro-IL-15 depends on T cells and IFN-γ

[0162] To explore the source of the anti-tumor effect of the fusion protein, deleting antibodies were used to systematically delete NK cells and CD8 + T cells were treated with blocking antibodies to block the activity of IFN-γ. The efficiency of T cell and NK cell depletion was tested by FACS. The results showed that the depletion antibodies could effectively eliminate CD8+ T cells and NK cells in peripheral blood. 5×10 5 After MC38 tumor cells were inoculated, 80 μg of anti-PD-1-pro-IL-15 was injected intravenously on days 9 and 12. On days 9, 12, 15, and 18, 200 μg of a CD8 T cell-depleting antibody (clone: ​​TIB210, prepared in our laboratory), 400 μg of a NK cell-depleting antibody (clone: ​​PK136, prepared in our laboratory), or 500 μg of an IFN-γ blocking antibody (clone: ​​R4-6A2) were injected intraperitoneally. Tumor growth in mice was monitored regularly.

[0163] The results are shown in Figure 8. Deleting NK cells while using fusion protein treatment does not affect the anti-tumor effect of the drug; however, deleting CD8+ After the induction of T cells, the anti-tumor effect completely disappeared. Blocking IFN-γ signaling with a blocking antibody yielded the same experimental results, with the fusion protein completely failing to control tumor growth. These results suggest that the in vivo anti-tumor effect of Anti-PD-1-pro-IL-15 is dependent on both T cells and IFN-γ.

[0164] Example 6. Different CD8 subsets after Anti-PD-1-pro-IL-15 treatment + T cell differentiation and transformation

[0165] C57BL / 6 mice were inoculated subcutaneously on the back with 5 × 10 5 After MC38-OVA tumor cells were transfected, 80 μg of anti-PD-1-pro-IL-15 was injected on days 8 and 11. Tetramer-positive antigen-specific CD8 + T and TCF + Tim3 - CD8 + The number of T.

[0166] The results showed that after anti-PD-1-pro-IL-15 treatment, antigen-specific CD8 + T cells (OT-I Tetramer + CD8 + T) and stem-like cells (OT-I Tetramer + Tim3 - PD1 + TCF-1 + CD8 + The above results prove that the anti-PD-1-pro-IL-15 fusion protein of the present invention can make the antigen-specific CD8 + T cells and stem cell-like CD8 + The increase in T can lead to a good prognosis after immunotherapy.

[0167] Example 7. Anti-PD-1-pro-IL-15 and PD-L1 antibodies have synergistic anti-tumor effects

[0168] Previous experiments have shown that the therapeutic effect of the fusion protein of the present invention is dependent on IFN-γ, which can upregulate the expression of PD-L1 molecules, thereby further limiting the tumor therapeutic effect of the fusion protein. 5×10 5After MC38 tumor cells were injected, mice were intraperitoneally injected with 100 μg of PD-L1 antibody on days 10 and 13, and intravenously injected with 80 μg of anti-PD-1-pro-IL-15 on the same day. Tumor growth in mice was monitored regularly.

[0169] The results are shown in Figure 10. The effect of the mixture of PD-L1 antibody and fusion protein is significantly better than that of either alone, demonstrating that PD-L1 antibody can further enhance the therapeutic effect of the fusion protein of the present invention and overcome immune tolerance.

[0170] The above results show that the anti-PD-1-pro-IL-15 fusion protein of the present invention can mask the activity of IL-15 in a non-tumor environment, and can release IL-15 and restore the activity of IL-15 under the action of MMP enzymes after entering the tumor. When the PD-1 antibody is connected to pro-IL-15 with an enzyme-insoluble peptide, the anti-tumor effect is better. The anti-PD-1-pro-IL-15 fusion protein of the present invention can activate the antigen-specific CD8 + T cells and stem cell-like CD8 + Its anti-tumor effect is better than the combined effect of administering PD-1 antibody and pro-IL-15 alone, and its anti-tumor effect depends on T cells and IFN-γ. When used in combination with PD-L1 antibody, the anti-tumor effect can be further enhanced.

[0171] Example 8. Construction of human anti-PD-1-pro-IL-15 fusion protein

[0172] 8.1 In this example, two human anti-PD-1-pro-IL-15 (RβD1 or Rβ) proteins were designed and constructed. The Fab sequence of the PD-1 antibody (anti-PD-1) is that of pembrolizumab, which can block PD-L1 / PD-1 signaling. The Fc sequence is a human IgG1 Fc sequence with three amino acid mutations (L19A, L20A, and P114G), which lacks ADCC function (designated mFc). From the N-terminus to the C-terminus, pro-IL-15 consists of the Rα-connector peptide-IL-15-MMP cleavable peptide-RβD1 / Rβ. A schematic diagram of its structure can be found in the left panel of Figure 1. Specifically, the functional units of the fusion protein are, from N-terminus to C-terminus, anti-PD-1 Fab (SEQ ID No: 11 and SEQ ID No: 12), mFc (SEQ ID No: 3), connector (SEQ ID No: 8), Rαsushi domain (SEQ ID No: 17), connecting peptide (SEQ ID No: 9), IL-15 (SEQ ID No: 16), MMP cleavable peptide (SEQ ID No: 10), and IL-15Rβ extracellular domain Rβ (SEQ ID No: 15) or IL-15Rβ extracellular domain truncated RβD1 (SEQ ID No: 14), wherein the connector (SEQ ID No: 8) is a non-enzymatically cleavable polypeptide connector, making the antibody and pro-IL-15 of the complex molecule essentially inseparable in vivo. Therefore, the two human fusion proteins of this example are inseparable fusion proteins.

[0173] The PEE12.4 eukaryotic expression vector system was used to construct the corresponding anti-PD-1-Fab heavy chain-pro-IL-15 expression plasmid and anti-PD-1 light chain (SEQ ID No: 11) expression plasmid. The anti-PD-1-Fab heavy chain-pro-IL-15 expression plasmid and the anti-PD-1 light chain expression plasmid were co-transfected into 293F cells to obtain the expression supernatant. The protein containing the Fc tag was purified by Protein A affinity chromatography, and the protein size was verified by SDS-PAGE. The quality of the protein was detected by SEC-MALS (molecular exclusion chromatography and static light scattering) (Figure 11). As shown in Figure 11, in the SEC-MALS spectrum, the fusion protein with a molecular weight of 263.8kD is anti-PD-1-pro-IL-15 (RβD1), and the fusion protein with a molecular weight of 297.5kD is anti-PD-1-pro-IL-15 (Rβ).

[0174] 8.2 In vitro biological activity studies of human anti-PD-1-pro-IL-15

[0175] Flow cytometry was used to examine the binding ability of the PD-1 antibody portion of the two human fusion proteins prepared in 8.1 to human PD-1 on the surface of CTLL2 (CTLL2-hPD1 cells). Anti-PD-1 antibody (αPD-1) without pro-IL-15 (its sequence is the same as in the above fusion protein), anti-PD-1 antibody (αPD-1-sIL-15) linked only to sIL-15 (i.e., IL-15-Rαsushi) (its sequence is the same as in the above fusion protein), and human IgG1 (hIgG1) were used as controls. The results showed that the two human anti-PD-1-pro-IL-15 fusion proteins had similar binding abilities to the PD-1 molecules of the anti-PD-1 antibody (Figure 12).

[0176] The two human fusion proteins were co-incubated with the activated MMP14 enzyme in vitro, and then SDS-PAGE analysis was performed to verify the in vitro cleavage effect of the fusion proteins. The results showed that the two human fusion proteins can be effectively cleaved in vitro (Figure 13). In Figure 13, lanes 1 and 2 in (A) are PD-1 antibody-Rα-IL-15-Rβ, and lane 3 is PD-1 antibody-Rα-IL-15; lanes 1 and 2 in (B) are PD-1 antibody-Rα-IL-15-RβD1, lane 3 is PD-1 antibody-Rα-IL-15-RβD1 after treatment with MMP14 enzyme, and lane 4 is PD-1 antibody-Rα-IL-15

[0177] NK cells and CD8 + T cell STAT5 phosphorylation assays were used to assess the recovery of the fusion protein's biological activity after enzymatic cleavage. The results showed that the IL-15 biological activity of the fusion protein was significantly restored after in vitro enzymatic cleavage (Figure 14). These results demonstrate that the IL-15 activity of the fusion protein is effectively blocked by the IL-15 receptor and that its activity is effectively restored after MMP cleavage.

[0178] 8.3 Humanized Anti-PD-1-pro-IL-15 Effectively Controls Tumor Growth in Humanized Mouse Models

[0179] The anti-tumor effects of the two humanized anti-PD-1-pro-IL-15 fusion proteins prepared in 8.1 were tested in a humanized mouse model. NSG mice were subcutaneously inoculated with 1×10 6 CFPAC1 cells were injected intravenously on the 10th day after tumor cell inoculation at 7×10 6Human CBMC cells were intraperitoneally injected with 80 μg of anti-PD-1-pro-IL-15 (RβD1), 90 μg of anti-PD-1-pro-IL-15 (Rβ), or a mixture of 40 μg of anti-PD-1 antibody and 40 μg of pro-IL-15 (their sequences are identical to those in the above-mentioned fusion proteins) on days 12, 15, 18, and 21. Tumor size was then recorded. The results showed that both humanized anti-PD-1-pro-IL-15 fusion proteins effectively controlled tumors in the humanized CBMC mouse model (Figure 15).

[0180] In summary, human anti-PD-1-pro-IL-15 fusion protein can significantly control the growth of tumors in humanized mice, which has guiding significance for cytokine application, bioengineering and translational medicine.

[0181] Example 9. anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc fusion proteins

[0182] 9.1 This example designs a physically shielded IL-15. By utilizing steric hindrance, IL-15 or super IL-15 (sIL-15, i.e., IL-15-Rα or Rα-IL-15) is constructed between the Fab fragment and Fc segment of an antibody, thereby reducing the binding of IL-15 to receptor β and avoiding peripheral toxic side effects. Two physically shielded fusion proteins were designed and prepared: anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc.

[0183] The functional units of each arm of anti-PD-1-Fab-IL-15-Rα-mFc are as follows from N-terminus to C-terminus: anti-PD-1 Fab (SEQ ID No: 1 and SEQ ID No: 2), connector (SEQ ID No: 8), IL-15 (SEQ ID No: 16), connecting peptide (SEQ ID No: 9), Rαsushi domain (SEQ ID No: 17), connector (SEQ ID No: 8), and mFc (SEQ ID No: 3), wherein the N-terminus of IL-15 is connected to the C-terminus of the heavy chain in Fab via a connector. Its structural schematic can be seen in Figure 16.

[0184] The functional units of each arm of anti-PD-1-Fab-IL-15-mFc are, from N-terminus to C-terminus: anti-PD-1 Fab (SEQ ID No: 1 and SEQ ID No: 2), linker (SEQ ID No: 8), IL-15 (SEQ ID No: 16), linker (SEQ ID No: 8), and mFc (SEQ ID No: 3), wherein the N-terminus of IL-15 is connected to the C-terminus of the heavy chain in Fab via a linker.

[0185] The PEE12.4 eukaryotic expression vector system was used to construct the corresponding anti-PD-1-Fab-IL-15-Rα-mFc, anti-PD-1-Fab-IL-15-mFc expression plasmids, and anti-PD-1 light chain (SEQ ID No: 1) expression plasmids. The anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc expression plasmids were co-transfected with the anti-PD-1 light chain expression plasmid to obtain expression supernatants. The Fc-tagged proteins were purified by Protein A affinity chromatography, and the protein size was verified by SDS-PAGE. The protein quality was tested by MASS (mass spectrophotometer), and the results showed that the protein quality was good and there was almost no protein aggregation (Figure 17).

[0186] 9.2 In vitro biological activity studies of anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc

[0187] Flow cytometry was used to examine the ability of the PD-1 antibody Fab in the prepared fusion proteins to bind to their antigens, with hIgG1 used as a control. The results showed that anti-PD-1-Fab-IL-15-Rα-mFc, anti-PD-1-Fab-IL-15-mFc, and anti-PD-1 had similar binding abilities to PD-1 molecules on the surface of EL4 cells (Figure 18).

[0188] The binding ability of the prepared fusion proteins, IL-15 or sIL-15, to the IL-15β receptor, CD122, was assessed by ELISA, with human IL-15-Rα-mFc and human IL-15-Fc used as controls. The results showed that the binding ability of anti-PD-1-Fab-IL-15-Rα-mFc to CD122 decreased approximately 5-fold compared to IL-15-Rα-mFc, and the binding ability of anti-PD-1-Fab-IL-15-mFc to CD122 decreased even more significantly compared to IL-15-Rα-mFc (Figure 19).

[0189] Using a CTLL2 proliferation assay, we tested the biological activity of IL-15 in anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc. The results showed that the activity of anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc was significantly reduced (Figure 20). This suggests that placing IL-15 or sIL-15 within the PD-1 antibody can significantly block the activity of IL-15.

[0190] 9.3 In vivo efficacy and toxicity studies of anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc

[0191] The anti-tumor effects and side effects of anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc fusion proteins were evaluated in the mouse MC38 tumor model. 5×10 5 MC38 tumor cells were injected intravenously on days 12, 15, and 19 with (i) a blank control (CTR), (ii) a physical mixture of 45 μg anti-PD-1 (whose Fab and Fc sequences are identical to those in anti-PD-1-Fab-IL-15-Rα-mFc) and 30 μg IL-15-Rα-mFc (whose sequence is identical to that in anti-PD-1-Fab-IL-15-Rα-mFc), (iii) 60 μg anti-PD-1-Fab-IL-15-Rα-mFc, or (iv) 50 μg anti-PD-1-Fab-IL-15-mFc. Tumor size and body weight of mice were measured regularly. Tumor growth curves and body weight change curves were plotted.

[0192] The results, as shown in Figure 21, show that when administered systemically, mice in the physical mixture of anti-PD-1 and IL-15-Rα-mFc group experienced severe weight loss, and 60% of the mice died from toxicity after two injections. In contrast, no mice died or experienced significant weight loss in the anti-PD-1-Fab-IL-15-Rα-mFc and anti-PD-1-Fab-IL-15-mFc groups. Both groups were able to control tumor growth.

[0193] Example 10. Design of anti-Lag3-pro-IL-15 fusion protein and in vivo anti-tumor effect

[0194] In this example, an antibody targeting Lag3 was designed and linked to pro-IL-15. Lag3 molecules are similar to PD-1 molecules and are also highly expressed in tumor tissues, especially CD8 + An anti-Lag3-pro-IL-15 fusion protein targeting Lag3 was constructed. Its structure is similar to the left figure in Figure 1, except that the PD-1 antibody was replaced with the Lag 3 antibody. The design goals are: (1) to target pro-IL-15 to CD8 T cells that highly express Lag3 in tumors through the Lag3 antibody. + T cells; (2) Lag3 blocks immunosuppressive signals; (3) pro-IL-15 activates CD8 in tumors while maintaining low peripheral toxicity + T cells, which ultimately control tumor growth.

[0195] In this example, an anti-Lag3-pro-IL-15 fusion protein was designed and constructed. The Fab of the Lag3 antibody (anti-Lag3) is the S119 clone, which can block Lag3 signaling; the Fc sequence is a human IgG1 Fc sequence with three amino acid mutations (L19A, L20A, and P114G) that lacks ADCC function (designated mFc). The functional units of this fusion protein, from N-terminus to C-terminus, are: anti-Lag3 Fab (SEQ ID Nos. 18 and 19), mFc (SEQ ID No. 3), linker (SEQ ID No. 8), Rαsushi domain (SEQ ID No. 7), connecting peptide (SEQ ID No. 9), IL-15 (SEQ ID No. 6), MMP-cleavable peptide (SEQ ID No. 10), and RβD1 (SEQ ID No. 4). The linker is an enzymatically non-cleavable polypeptide linker, rendering the antibody and pro-IL-15 complex essentially inseparable in vivo.

[0196] Next, the anti-tumor effect of anti-Lag3-pro-IL-15 was tested in a mouse tumor model. 5×10 5MC38 tumor cells were intravenously injected on days 11 and 14 with (i) a PBS blank control (CTR), (ii) 60 μg of anti-Lag3 antibody (whose sequence is the same as in the fusion protein), (iii) 80 μg of anti-Lag3-pro-IL-15 fusion protein, (iv) a mixture of 52 μg of anti-PD-1 (whose sequence is the same as in the anti-PD-1-pro-IL-15 fusion protein in Example 1) and 45 μg of pro-IL-15 (whose sequence is the same as in the fusion protein), or (v) 80 μg of anti-PD-1-pro-IL-15 fusion protein (the inseparable anti-PD-1-pro-IL-15 fusion protein in Example 1). The results showed that the anti-Lag3-pro-IL-15 fusion protein significantly enhanced the anti-tumor effect of anti-Lag3 ( Figure 22 ).

[0197] Example 11. Design of anti-Claudin18.2-pro-IL-15 fusion protein and its anti-tumor effect in vivo

[0198] 11.1 designed a fusion protein anti-Claudin18.2-pro-IL-15 that targets tumor cells. Claudin18.2 is only expressed in differentiated epithelial cells of the gastric mucosa and is not normally expressed in any other healthy tissues. However, it is highly expressed in primary malignant tumors such as gastric cancer, breast cancer, colon cancer, and liver cancer. The design goals of this fusion protein are: (1) to target pro-IL-15 to tumors that highly express Claudin18.2 through the Claudin18.2 antibody; (2) pro-IL-15 maintains low toxicity and side effects in the periphery. Once it reaches the tumor, the enzyme highly expressed in the tumor cleaves and releases the activity of IL-15, activating the immune response in the tumor and controlling tumor growth.

[0199] In this example, the following two releasable anti-Claudin18.2-pro-IL-15 fusion proteins were designed and constructed:

[0200] Anti-Claudin18.2-Fc-RβD1-IL-15-Rα: Its functional units, from N-terminus to C-terminus, are: anti-Claudin18.2 Fab (SEQ ID No: 20 and SEQ ID No: 21), WT Fc (SEQ ID No: 13), linker (SEQ ID No: 10), RβD1 (SEQ ID No: 4), MMP-cleavable peptide (SEQ ID No: 10), IL-15 (SEQ ID No: 6), connecting peptide (SEQ ID No: 9), and Rα sushi domain (SEQ ID No: 7). A schematic diagram of its structure can be seen in the right figure of Figure 1, in which the PD-1 antibody is replaced with the above-mentioned Claudin18.2 antibody, and there is no mutation or modification in the Fc region.

[0201] Anti-Claudin18.2-Fc-RβD1-Rα-IL-15: Its functional units, from N-terminus to C-terminus, consist of: anti-Claudin18.2 Fab (SEQ ID Nos. 20 and 21), WT Fc (SEQ ID No. 13), linker (SEQ ID No. 10), RβD1 (SEQ ID No. 4), MMP-cleavable peptide (SEQ ID No. 10), Rα sushi domain (SEQ ID No. 7), connecting peptide (SEQ ID No. 9), and IL-15 (SEQ ID No. 6). The linker is an MMP-cleavable polypeptide. Therefore, the antibody portion and pro-IL-15 of this complex molecule are separated from each other by MMP after entering the tumor.

[0202] The corresponding anti-Claudin18.2-Fab heavy chain-pro-IL-15 expression plasmids and anti-Claudin18.2 light chain expression plasmids were constructed using the PEE12.4 eukaryotic expression vector system. The anti-Claudin18.2-Fab heavy chain-pro-IL-15 expression plasmid and the anti-Claudin18.2 light chain expression plasmid were co-transfected into 293F cells to obtain expression supernatants. The Fc-tagged proteins were purified by Protein A affinity chromatography, and protein size was verified by SDS-PAGE. The results confirmed the successful construction of the two fusion proteins.

[0203] 11.2 The biological activity of IL-15 in the above fusion protein was tested by a CTLL-2 proliferation assay. The results showed that compared with the unmasked IL-15-Rα-Fc (whose sequence from N-terminus to C-terminus is: IL-15 (SEQ ID No: 6), connecting peptide (SEQ ID No: 9), Rα sushi domain (SEQ ID No: 7), WT Fc (SEQ ID No: 13)), anti-Claudin18.2-Fc-RβD1-IL-15-Rα could effectively block the biological activity of IL-15, indicating that the IL-15 activity in the fusion protein can be blocked by the receptor. The activity of anti-Claudin18.2-Fc-RβD1-Rα-IL-15 was similar to that of the unmasked IL-15-Rα-Fc, indicating that the IL-15 activity in this RβD1-Rα-IL-15 structure cannot be blocked by the receptor ( Figure 23 ).

[0204] 11.3 Testing the anti-tumor effect of the maskable anti-Claudin18.2-Fc-RβD1-IL-15-Rα in the mouse MC38 tumor model. 5×10 5 MC38 tumor cells were intravenously injected with (i) a PBS blank control (CTR), (ii) 60 μg of anti-Claudin18.2 antibody (whose sequence is identical to that in the fusion protein), or (iii) 80 μg of anti-Claudin18.2-pro-IL-15 fusion protein on days 8, 11, and 14. The results showed that the anti-Claudin18.2-pro-IL-15 fusion protein significantly inhibited tumor growth ( Figure 24 ).

[0205] This specification has described specific implementation methods in detail. Those skilled in the art should recognize that the above implementation methods are merely exemplary and cannot be understood as limitations of the present invention. For those skilled in the art, without departing from the principles of the present invention, the technical solutions obtained after making several improvements and modifications to the present invention also fall within the scope of the present invention.

[0206] References

[0207] 1. Guo, Y. et al. IL-15 Superagonist-Mediated Immunotoxicity: Role of NK Cells and IFN-gamma. Journal of immunology (Baltimore, Md.: 1950) 195, 2353-2364 (2015).

[0208] 2.Guo,J.et al.Tumor-conditional IL-15pro-cytokine reactivates anti-tumor immunity with limited toxicity.Cell Res 31,1190-1198,(2021).

Claims

1. A complex molecule, comprising an antitumor antibody domain, a linker, and pro-IL-15, wherein the antitumor antibody domain and the pro-IL-15 are linked by the linker,wherein the antitumor antibody domain is a complete antibody, a nanobody, or an antigenbinding fragment thereof against an immune checkpoint molecule, a tumor antigen molecule, or an immune activation molecule,wherein the linker is a polypeptide linker or a non-peptide linker;wherein the pro-IL-15 is a fusion protein comprising, or consisting of, an IL-15R0 extracellular domain, IL-15, an IL-15Ra sushi domain, a first linking peptide, and a second linking peptide, wherein the IL-15R0 extracellular domain, IL-15, and the IL-15Ra sushi domain are linked by the first linking peptide and the second linking peptide; orthe pro-IL-15 is a fusion protein comprising, or consisting of, IL-15, an IL-15Ra sushi domain, and a first linking peptide, wherein IL-15 and the IL-15Ra sushi domain are optionally linked by the first linking peptide.

2. The complex molecule according to claim 1, wherein the pro-IL-15 is(i) a fusion protein consisting of, from the N-terminus to the C-terminus, an IL-15R0 extracellular domain, a first linking peptide, IL-15, a second linking peptide, and an IL-15Ra sushi domain,(ii) a fusion protein consisting of, from the N-terminus to the C-terminus, an IL-15Ra sushi domain, a second linking peptide, IL-15, a first linking peptide, and an IL-15R0 extracellular domain,(iii) a fusion protein consisting of, from the N-terminus to the C-terminus, IL-15, a first linking peptide, and an IL-15Ra sushi domain, or(iv) a fusion protein consisting of, from the N-terminus to the C-terminus, a first linking peptide, IL-15, and an IL-15Ra sushi domain.

3. The complex molecule according to claim 2, wherein the first linking peptide is an MMP-cleavable peptide or a non-enzymatically cleavable peptide, and the second linking peptide is a non-enzymatically cleavable peptide.

4. The complex molecule according to any one of claims 1 to 3, wherein the immune checkpoint molecule comprises PD-1, PD-L1, CTLA4, TIGIT, Tim3, or Lag3; the tumor antigen molecule comprises CLDN18.2, EGFR, Her2, or mesothelin; and the immune activation molecule comprises OX40, 4-1BB, CD28, or CD3.

5. The complex molecule according to any one of claims 1 to 4, wherein the antitumor39 antibody domain is a complete antibody, a nanobody, or an antigen-binding fragment thereof against PD-1, Lag3, and / or Claudin18.2.

6. The complex molecule according to any one of claims 1 to 4, wherein the antigen-binding fragment comprises ScFv, Fab, or F(ab’)2.

7. The complex molecule according to any one of claims 1 to 6, wherein the complex molecule is a fusion protein, the linker is a polypeptide, and the complex molecule is in a form selected from:form (a) wherein the C-terminus of at least one heavy chain of the complete antibody or nanobody is linked to the N-terminus of a pro-IL-15 via a linker;form (b) wherein the C-terminus of an antigen-binding fragment is linked to the N-terminus of a pro-IL-15 via a linker; orform (c) wherein the N-terminus of one chain of an Fc region is linked, via a linker, to the C-terminus of a fusion protein in form (b); and the N-terminus of the other chain of the Fc region is not linked to a peptide, or is linked, via a linker, to the C-terminus of a fusion protein in form (b), to the C-terminus of an antigen-binding fragment, or to the C-terminus of a pro-IL-15;wherein in forms (b) and (c) the C-terminus of an antigen-binding fragment refers to the C-terminus of the heavy chain or light chain of a Fab, the C-terminus of an scFv, or the C-terminus of one heavy chain or light chain of an F(ab’)2;wherein, when multiple linkers are present in the complex molecule, the linkers are the same as or different from each other; and when two pro-IL-15 are present in the complex molecule, the two pro-IL-15 are the same as or different from each other.

8. The complex molecule according to any one of claims 1 to 6, wherein the complete antibody or nanobody is a bispecific antibody.

9. The complex molecule according to any one of claims 1 to 8, wherein the linker is an enzymatically cleavable peptide linker or a non-enzymatically cleavable linker.

10. The complex molecule according to any one of claims 1 to 6 and 8, wherein the complex molecule is a conjugate, and the linker is a non-peptide linker.

11. The complex molecule according to any one of claims 1 to 9, wherein the linker is a peptide having an amino acid sequence of SEQ ID No: 8 or SEQ ID No: 10.

12. The complex molecule according to claim 7, wherein the complex molecule is in form (a), and the sequence of at least one heavy chain thereof, from the N-terminus to the C-terminus, consists of at least one of the following:a heavy chain of a Fab of the antitumor antibody, an Fc, the linker, the Ra sushi domain, the second linking peptide, IL-15, the first linking peptide, and the R0D1;a heavy chain of a Fab of the antitumor antibody, an Fc, the linker, the R0D1, the first linkingpeptide, IL-15, the second linking peptide, and the Ra sushi domain;a heavy chain of a Fab of the antitumor antibody, an Fc, the linker, IL-15, the first linking peptide, and the Ra sushi domain; anda heavy chain of a Fab of the antitumor antibody, an Fc, the first linking peptide, IL-15, and the Ra sushi domain;wherein the antitumor antibody is an anti-PD-1 antibody, an anti-Lag3 antibody, or an anti-Claudin18.2 antibody;wherein each heavy chain of a Fab is associated with a corresponding antibody light chain;wherein the linker is a non-enzymatically cleavable linker, the first linking peptide is an MMP-cleavable peptide or a non-enzymatically cleavable peptide, and the second linking peptide is a non-enzymatically cleavable peptide.

13. The complex molecule according to claim 12, wherein the Fc is a wild-type Fc, or an Fc mutated or modified to have reduced or enhanced binding to FcyR.

14. The complex molecule according to claim 12, wherein the Fc has a normal ADCC function, has no ADCC function, or has an enhanced ADCC function.

15. The complex molecule according to claim 12, wherein the Fc having no ADCC function has a sequence of SEQ ID No: 3, and the Fc having a normal ADCC function has a sequence of SEQ ID No: 13.

16. The complex molecule according to claim 6 or 12, wherein the Fab is at least one selected from:(i) a Fab having a heavy chain sequence of SEQ ID No: 2 or SEQ ID No: 12, and a light chain sequence of SEQ ID No: 1 or SEQ ID No: 11;(ii) a Fab having a heavy chain sequence of SEQ ID No: 19, and a light chain sequence of SEQ ID No: 18;(iii) a Fab having a heavy chain sequence of SEQ ID No: 21, and a light chain sequence of SEQ ID No: 20.

17. The complex molecule according to any one of claims 1 to 16, wherein in the pro-IL-15, IL-15 has a sequence of SEQ ID No: 6 or SEQ ID No: 16, the IL-15Ra sushi domain has a sequence of SEQ ID No: 7 or SEQ ID No: 17, the IL-15R0 extracellular domain has a sequence of SEQ ID No: 4, SEQ ID No: 14, SEQ ID No: 5, or SEQ ID No: 15, the first linking peptide has a sequence of SEQ ID No: 8, SEQ ID No: 9, or SEQ ID No: 10, and the second linking peptide has a sequence of SEQ ID No: 8 or SEQ ID No: 9.

18. A complex molecule obtained by inserting pro-IL-15 or IL-15 between the CH1 and CH2 of at least one heavy chain of a complete antitumor antibody,wherein linkers are optionally linked to the N-terminus and C-terminus of the pro-IL-15 orIL-15;wherein the pro-IL-15 is a fusion protein consisting of, from the N-terminus to the C-terminus, IL-15, a linking peptide, and an IL-15Ra sushi domain, or an IL-15Ra sushi domain, a linking peptide, and IL-15;5 wherein the antitumor antibody is an antibody against an immune checkpoint molecule, a tumor antigen molecule, or an immune activation molecule,wherein the linkers are each a polypeptide linker or a non-peptide linker; andwherein the linking peptide is an enzymatically cleavable or non-enzymatically cleavable peptide.10       19. The complex molecule according to claim 18, wherein the complex molecule is a fusionprotein in a form in which one heavy chain consists of, from the N-terminus to the C-terminus, a heavy chain of a Fab of the antitumor antibody, a linker, pro-IL-15 / IL-15, a linker, and one chain of an Fc; and the other heavy chain consists of, from the N-terminus to the C-terminus, a heavy chain of a Fab of an antitumor antibody, a linker, pro-IL-15 / IL-15, a linker, and the other chain of15 the Fc;wherein the two heavy chains of the Fabs are the same as or different from each other and are each associated with a corresponding antibody light chain to form a Fab, the two chains of the Fc are associated with each other through a disulfide bond, the linkers are the same as or different from one another, and the two pro-IL-15 are the same as or different from each other.20       20. The complex molecule according to claim 19, wherein the Fab is selected from the Fabsas defined in claim 16; the linker is selected from SEQ ID No: 8, SEQ ID No: 9, or SEQ ID No: 10; the IL-15 is selected from SEQ ID No: 6 or SEQ ID No: 16; the IL-15Ra sushi domain is selected from SEQ ID No: 7 or SEQ ID No: 17; the linking peptide is selected from SEQ ID No: 8 or SEQ ID No: 9; and the Fc is the Fc as defined in any one of claims 13 to 15.25       21. The complex molecule according to claim 20, wherein the linker linked to the heavy chainof a Fab is SEQ ID No: 8 or SEQ ID No: 9, and the linker linked to the Fc is SEQ ID No: 10.

22. A nucleic acid encoding the complex molecule according to any one of claims 7, 12-15, and 17-21.

23. A vector comprising the nucleic acid according to claim 22.30       24. A host cell comprising the vector according to claim 23.

25. A pharmaceutical composition, comprising:the complex molecule according to any one of claims 1 to 21, the nucleic acid according to claim 22, the vector according to claim 23, or the host cell according to claim 24, anda pharmaceutically acceptable carrier or excipient.35       26. The pharmaceutical composition according to claim 25, wherein the antitumor antibodydomain is a complete antibody, a nanobody, or an antigen-binding fragment thereof against PD-1;preferably, wherein the pharmaceutical composition further comprises an anti PD-L1 antibody.

27. Use of the complex molecule according to any one of claims 1 to 21, the nucleic acid according to claim 22, the vector according to claim 23, the host cell according to claim 24, or the 5 pharmaceutical composition according to claim 25 or 26 in the manufacture of a medicament for treating cancer, preferably a solid tumor.

28. The use according to claim 27, wherein the cancer is selected from the group consisting of lung cancer, liver cancer, gastric cancer, cervical cancer, ovarian cancer, breast cancer, colorectal cancer, melanoma, and lymphoma.10

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