Tumor combination therapy targeting CTLA-4, PD-l1 and VEGF

Abstract

Provided is a composition, which contains: i) a CTLA-4 antibody or an antigen-binding portion thereof capable of specifically binding to CLTA-4 and having an FcR-binding affinity, and ii) a recombinant fusion protein, wherein the recombinant fusion protein contains a PD-L1 antibody or an antigen-binding portion thereof capable of specifically binding to PD-L1 and having an FcR binding affinity, and a VEGF binding peptide, wherein the VEGF binding peptide is linked to the N-terminus of the heavy chain or light chain of the PD-L1 antibody or antigen-binding portion thereof. Further provided is the use of the composition in the preparation of a drug for treating tumors.

Description

Combination therapy for tumors targeting CTLA-4, PD-L1, and VEGF Invention Field

[0001] This application relates to a combination therapy for tumors targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death-ligand 1 (PD-L1), and vascular endothelial growth factor (VEGF), as well as corresponding pharmaceutical compositions. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, with the most common cancers including colorectal cancer and breast cancer.

[0003] As research deepens, it has become increasingly clear that cancer is a complex ecosystem. Tumor cells recruit and reprogram surrounding normal tissue cells and immune cells, and remodel the vascular system and extracellular matrix, creating a unique environment called the tumor microenvironment (TME). The tumor microenvironment can be categorized into immune desert, immune rejection, and immune-inflammatory types. Immune desert TMEs do not contain lymphocytes, while immune rejection TMEs contain a large number of immune cells, but these cells only exist in the tumor stroma and do not infiltrate the tumor parenchyma. Immune-inflammatory TMEs, on the other hand, infiltrate the tumor parenchyma, but these immune cells are usually in a state of "exhaustion" (Tiwari A et al., (2023) Towards a consensus definition of immune exclusion in cancer. Front Immunol. 14:1084887). Tumors in the first two types of TMEs are considered "cold" tumors, with CD8+... + T cells cannot approach tumor cells, and CD8 + T cell activation may also be suppressed. For this type of tumor, it is necessary to remodel the TME and alter its immunosuppressive properties in order to enable immune effector cells to kill tumor cells. Tumors in an immune-inflammatory TME are considered "hot" tumors, and combating these tumors requires activating "exhausted" immune cells, especially CD8+ cells. + T effector cells reactivate, and these tumors are often more sensitive to immune checkpoint (ICI) inhibitors that are difficult to work in "cold" tumors.

[0004] CTLA-4 and Treg cells

[0005] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is one of the earliest studied immune checkpoints. Its expression increases dramatically in activated effector T cells, inhibiting effector T cell activation or causing effector T cell exhaustion by competing with the co-stimulatory molecule CD28 for binding to CD80 / 86 on antigen-presenting cells (APCs), thus leading to insufficient killing power against tumor cells. CTLA-4 antibodies can block this effect and promote T cell activation. The first CTLA-4 antibody, ipilimumab, is currently approved for the treatment of melanoma, renal cell carcinoma (RCC), colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), pleural mesothelioma, and esophageal cancer, significantly prolonging patient survival; however, a large proportion of patients do not respond to this antibody. For example, the response rates of ipilimumab monotherapy and combination therapy in melanoma are approximately 20% and 46% (Long GV, et al., (2018) Combination nivolumab and ipilimumab or nivolumab alone in melanoma brain metastases: A multicentre randomised phase 2 study. Lancet Oncol. 19:672–681).

[0006] Besides blocking the binding of CTLA-4 to CD80 / 86, another strategy targeting CTLA-4 may include eliminating regulatory T cells (Tregs) in the TME (Romano E, et al., (2015). Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients. Proc Natl Acad Sci US A. 112(19):6140-5). Treg cells are immunosuppressive cells that constitutively express CTLA-4. They can inhibit APCs by eliminating immunostimulatory cytokines and producing immunosuppressive cytokines such as TGF-β, IL-10, and IL-35. They can also inhibit the activation of effector T cells and their killing of tumors by expressing the IL-2 receptor to bind to IL-2 required by effector T cells. Higher Treg cell levels in TME are associated with poorer prognosis in various cancers (Sobhani N et al., (2021) CTLA-4 in Regulatory T Cells for Cancer Immunotherapy. Cancers (Basel) 13(6):1440). When tumor-bearing mice with knock-in human CTLA-4 gene were administered CTLA-4 antibodies that enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), Treg cells in the mouse tumors decreased and tumor growth was more thoroughly inhibited (Gan X et al., (2022) An anti-CTLA-4 heavy chain-only antibody with enhanced Treg depletion shows excellent preclinical efficacy and safety profile. Proc Natl Acad Sci US A.119(32):e2200879119).

[0007] PD-1 and PD-L1

[0008] Like CTLA-4, programmed death receptor 1 (PD-1) is one of the earliest studied immune checkpoints. It is expressed on macrophages, dendritic cells, monocytes, and activated T, natural killer, and B lymphocytes, and is particularly highly expressed on tumor-specific T cells, mainly playing a role in downregulating immune function and maintaining immune tolerance. One of its ligands is programmed death ligand 1 (PD-L1), which is usually expressed on macrophages, activated T and B cells, and epithelial cells, and is also expressed in a large number of tumor cells and antigen-presenting cells in the TME (Han Y et al., (2020) PD-1 / PD-L1 pathway: current researches in cancer. Am J Cancer Res. 10(3):727-742; Jiang Y et al., (2019) PD-1 and PD-L1 in cancer immunotherapy: clinical implications and future considerations. Hum Vaccin Immunother. 15(5):1111-1122).

[0009] In TME, CD4 + Th1 helper cells and CD8 + T cells produce interferon-γ (IFN-γ), which on the one hand activates macrophages to kill tumors, and on the other hand induces PD-L1 expression in both macrophages and tumor cells. Tumor-specific CD8 + PD-1 on T cells binds to PD-L1 expressed in various cells within the TME, stimulating CD8+. + T-cell dysfunction (Topalian, S. et al., (2016) Nat Rev Cancer 16(5):275–287). PD-1 can also bind to another ligand, PD-L2, downregulating the immune system. An analysis of 196 clinical tumor samples from patients with renal cell carcinoma showed that patients with high expression of PD-L1 in their tumors and / or lymphocytes had a 4.5-fold higher risk of death compared to patients with low PD-L1 expression (Thompson RH et al., (2004) Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target. Proc Natl Acad Sci US A.101(49):17174-17179).

[0010] Antibodies targeting PD-1 or PD-L1 have been approved for the treatment of various cancers. For example, the PD-L1 antibody atezolizumab is clinically used to treat urothelial carcinoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, and melanoma.

[0011] VEGF and its receptor

[0012] As tumors grow larger, they experience a degree of hypoxia, which limits their further growth. Therefore, solid tumors rely on angiogenesis to facilitate their expansion and metastasis. Angiogenesis is determined by the interaction between pro-angiogenic factors and angiogenesis inhibitors.

[0013] Vascular endothelial growth factor (VEGF or VEGF-A) is one of the most important pro-angiogenic factors. Different gene splicing can produce six different transcripts, and VEGF consists of 165 amino acids. 165It is the main molecular form. VEGF family ligands also include VEGF-B, VEGF-C, VEGF-D, and PlGF. Among them, VEGF-B can mediate tumor metastasis independently of VEGF-A, while PlGF is more critical for the survival of some cancer cells (such as medulloblastoma) (Yang X et al., (2015) VEGF-B promotes cancer metastasis through a VEGF-A-independent mechanism and serves as a marker of poor prognosis for cancer patients. Proc Natl Acad Sci USA 112(22):E2900–9; Snuderl M et al., (2013) Targeting placental growth factor / neuropilin 1 pathway inhibits growth and spread of medulloblastoma. Cell 152(5):1065–76). VEGF family ligands function by binding to the VEGF receptor (VEGFR) on the cell surface. VEGF-A, VEGF-B, and PlGF bind to VEGFR1, which is mainly distributed on vascular endothelial cells. This receptor contains seven extracellular immunoglobulin (Ig)-like domains, and ligand binding mainly occurs in the second and third Ig-like domains (Keyt BA et al., (1996) Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. J Biol Chem 271:5638-5646; Tanaka K et al., (1997) Characterization of the extracellular domain in the Vascular Endothelial Growth Factor Receptor-1 (Flt-1tyrosine kinase). Jpn J Cancer Res 88:867–876). VEGFR2 is mainly expressed in blood vessels and growing lymphatic vessels, and VEGF-A, VEGF-C, and VEGF-D are its ligands. VEGF-A mainly functions by binding to VEGFR-2 in the tumor inner epithelium, but its binding affinity to VEGFR-1 is higher than that to VEGFR-2.Ligand binding and receptor dimerization lead to autophosphorylation of VEGFR-2, activating downstream signaling pathways such as PI3K and MAPK, promoting cell growth and migration, as well as vascular pathways (Matsumoto T. and Claesson-Welsh L, (2001) VEGF receptor signal transduction. Sci STKE 2001(112):RE21).

[0014] In tumors, VEGF-A is upregulated through both hypoxia-dependent and hypoxia-independent mechanisms. It not only directly promotes tumor migration and invasiveness but also induces the stem cell nature of tumor cells (Beck B et al., (2011) A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumors. Nature 478(7369):399–403). VEGF-A can also promote the immunosuppressive nature of the tumor microenvironment (TME) by disrupting the function of immune cells. For example, VEGF-A can disrupt the maturation and antigen presentation of dendritic cells and further inhibit T cell-mediated cytotoxic killing (Gabrilovich DI et al., (1996) Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat Med, 2(10): 1096-1103; Dikov MM et al., (2005) Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation. J Immunol 174(1): 215-222). Tumor-associated M2 macrophages exhibiting immunosuppressive activity secrete VEGF-A, thereby promoting tumor angiogenesis and remodeling (Lai YS, et al., (2019) Autocrine VEGF signalling on M2 macrophages regulates PD-L1 expression for immunomodulation of T cells. J Cell Mol Med 23(2):1257-1267). Furthermore, VEGF-A can also migrate myeloid-derived suppressor cells from the bone marrow to the peripheral blood.

[0015] The earliest drug targeting the VEGF / VEGFR pathway was a VEGF-A antibody, which reduced tumor angiogenesis and improved the effects of concurrently administered chemotherapy drugs. This suggests that the drug can temporarily normalize the abnormal structure and function of tumor blood vessels to better deliver oxygen and drugs. The combination of VEGF-A inhibition and chemotherapy has shown promising results in clinical trials for renal cell carcinoma, colorectal cancer, and non-small cell lung cancer (NSCLC), but primary and secondary resistance issues remain.

[0016] Combination therapy and bispecific or multispecific fusion proteins

[0017] As mentioned above, in cancer treatment, in addition to directly targeting and killing tumor cells, it is also necessary to reduce the immunosuppressive effects of the tumor microenvironment (TME), allowing more effector cells to infiltrate the tumor parenchyma and maintain their viability. Simultaneously, issues such as primary and secondary drug resistance may arise in cancer treatment, leading to delays in treatment.

[0018] Combination therapies, or the use of bispecific or multispecific fusion proteins, may be able to target multiple different signaling pathways, thereby remodeling the tumor microenvironment and targeting the tumor parenchyma, and killing cancer cells more quickly and thoroughly.

[0019] Reference to any document in this application is not an admission that such document is prior art. Summary of the Invention

[0020] The inventors of this application have achieved effective inhibition of solid tumors, including colorectal cancer, by targeting the CTLA-4, PD-L1, and VEGF pathways and rationally arranging the timing of targeting each pathway. The effect is better than existing treatment options on the market, such as CTLA-4 monotherapy and PD-L1 / PD-1-VEGF dual-target therapy, including higher tumor suppression efficiency or longer survival.

[0021] Unwilling to be bound by theory, the inventors of this application believe that ADCC or ADCP-enhanced CTLA-4 antibodies can remodel the TME by killing Treg cells, making the TME more conducive to the killing of tumor cells by immune cells, especially effector cells, and can block the binding of CTLA-4 to CD80 / 86 to a certain extent, thus preventing the exhaustion of effector cells; targeting PD-L1 can block the PD-1-PD-L1 interaction, avoiding the exhaustion or apoptosis of immune cells, especially effector cells, and can directly kill PD-L1 through ADCC or antibody-dependent phagocytosis (ADCP). +Tumor cells; targeting VEGF can block tumor angiogenesis and reduce the immunosuppressive effect of the tumor microenvironment (TME), thereby indirectly enhancing the tumor-killing function of effector cells. The combined targeting of these three pathways works synergistically on the tumor TME and tumor itself from several aspects. Theoretically, this should be more effective than single-pathway targeting in controlling tumor growth, and even completely eliminating the tumor. Furthermore, the targeting of PD-L1 and VEGF utilizes a dual / multi-specific recombinant fusion protein, improving ease of administration, patient compliance, and potentially achieving better efficacy than the combination of the two single drugs.

[0022] However, in the experiments, the inventors were surprised to find that when CTLA-4 antibody and PD-L1 / VEGF dual-targeting protein were administered simultaneously, the combined effect was not as good as CTLA-4 single-targeting. The inventors believe this may be because the Fc region of the CTLA-4 antibody and the Fc region of the PD-L1 / VEGF dual-targeting protein competitively bind to immune effector cells, preventing CTLA-4 from fully exerting its function of killing Tregs and improving TME. Subsequently, the inventors administered CTLA-4 antibody first, followed by PD-L1 / VEGF dual-targeting protein 7 days later, and found that the combined effect of the three targets was still lower than that of CTLA-4 antibody alone. The inventors further extended the time interval between the first administration of CTLA-4 antibody and PD-L1 / VEGF dual-targeting protein to 11 days, and found that the combined effect of the three targets exceeded that of CTLA-4 antibody alone or PD-L1 and VEGF dual-targeting protein alone. Due to ethical considerations for experimental animals, the dosing interval was not further extended in animal studies, as this could lead to excessive tumor growth.

[0023] Based on the above experimental results, the inventors have reason to believe that after administering the CTLA-4 antibody and fully utilizing its TME improvement (Treg killing or effector cell activation), subsequent PD-1 / PD-L1 targeting and VEGF blockade can better exert the efficacy of each protein, activate immune cells, and kill tumor cells. The CTLA-4 antibody used in the experiment had a half-life of about 8-12 days as measured in Phase I clinical trials, while the half-life of the IgG1 antibody in mice was 6-8 days (Vieira P, Rajewsky K. (1988) The half-lives of serum immunoglobulins in adult mice. Eur J Immunol. 18(2):313-6). The inventors infer that PD-1 / PD-L1 targeting and VEGF blockade should be initiated at least after about 1.5 half-lives of CTLA-4. For critically ill cancer patients, effective treatment should not be delayed for too long. Therefore, PD-1 / PD-L1 targeting and VEGF blockade can be initiated after about 1.5-2.5 half-lives of CTLA-4.

[0024] Specifically, in a first aspect, this application provides a composition that may comprise:

[0025] i) CTLA-4 antibody or its antigen-binding moiety, and

[0026] ii) Recombinant fusion protein comprising a PD-L1 antibody or its antigen-binding portion and a VEGF-binding peptide.

[0027] CTLA-4 antibodies or their antigen-binding moieties may include a heavy chain variable region, a heavy chain constant region, a light chain variable region, and optionally a light chain constant region. The heavy chain constant region may contain Fc receptor (FcR, e.g., FcγR) and / or complement system protein binding activity, particularly high FcR (e.g., FcγR) and / or complement system protein binding activity. In some embodiments, the heavy chain constant region may contain binding activity for FcγRIIa and / or FcγRIIIa, particularly high binding activity. The heavy chain constant region may be a natural or modified heavy chain constant region having FcR (e.g., FcγR) and / or complement system protein binding activity, particularly high FcR (e.g., FcγR) and / or complement system protein binding activity, or a functional fragment thereof, such as a hinge region, CH2 and CH3 fragments containing the heavy chain constant region, such as the Fc region. The heavy chain constant region may be an IgG1 constant region, such as the human IgG1 constant region, or a functional fragment thereof. The heavy chain constant region, in some embodiments, may contain the amino acid sequence shown in SEQ ID NO:15. The light chain constant region may be a κ or λ light chain constant region, such as a human κ or λ light chain constant region. In some embodiments, the light chain constant region may contain the amino acid sequence shown in SEQ ID NO:16.

[0028] The heavy chain variable region of the CTLA-4 antibody or its antigen-binding moiety may include VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region may include VL-CDR1, VL-CDR2, and VL-CDR3. The VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 may respectively contain the amino acid sequences shown in GFTFSSYT (SEQ ID NO:1), ISYDGNNK (SEQ ID NO:2), ARTGWLGPFDY (SEQ ID NO:3), QSVGSSY (SEQ ID NO:4), GAF, and QQYGSSPWT (SEQ ID NO:5). In some embodiments, the heavy chain variable region and the light chain variable region may contain amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:6 and 7.

[0029] PD-L1 antibodies or their antigen-binding moieties may include a heavy chain variable region, a heavy chain constant region, a light chain variable region, and optionally a light chain constant region. The heavy chain constant region may contain Fc receptor (FcR, e.g., FcγR) and / or complement system protein binding capacity, particularly a high FcR (e.g., FcγR) and / or complement system protein binding capacity. In some embodiments, the heavy chain constant region may contain binding capacity for FcR (e.g., FcγR), such as FcγRIIa and / or FcγRIIIa, particularly a high binding capacity. The heavy chain constant region may be a natural or modified heavy chain constant region having FcR (e.g., FcγR) and / or complement system protein binding capacity, particularly a high FcR (e.g., FcγR) and / or complement system protein binding capacity, or a functional fragment thereof, such as a hinge region, CH2 and CH3 fragments containing the heavy chain constant region, such as the Fc region. The heavy chain constant region may be an IgG1 constant region, such as the human IgG1 constant region, or a functional fragment thereof. The heavy chain constant region, in some embodiments, may contain the amino acid sequence shown in SEQ ID NO:15. The light chain constant region may be a κ or λ light chain constant region, such as a human κ or λ light chain constant region. In some embodiments, the light chain constant region may contain the amino acid sequence shown in SEQ ID NO:16.

[0030] The heavy chain variable region of the PD-L1 antibody or its antigen-binding moiety may include VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region may include VL-CDR1, VL-CDR2, and VL-CDR3. The VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 may respectively contain the amino acid sequences shown in GFTFSDSW (SEQ ID NO:8), ISPHYGGST (SEQ ID NO:9), ARRHWPGGFDY (SEQ ID NO:10), QDVSTA (SEQ ID NO:11), SAS, and QQYLYHPAT (SEQ ID NO:12), or respectively contain the amino acid sequences shown in GFTFSDSW (SEQ ID NO:21), IHPNSGSS (SEQ ID NO:22), ARSYYGSSPYYFDY (SEQ ID NO:23), and QDIINY (SEQ ID NO:12). (SEQ ID NO:24), YTS, and QQGDTLPWT (SEQ ID NO:25). In some embodiments, the heavy chain variable region and the light chain variable region may respectively contain amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:13 and 14, or SEQ ID NO:26 and 27.

[0031] The antibodies of this application, including CTLA-4 antibodies and PD-L1 antibodies, in some embodiments comprise two heavy chains and two light chains, or are composed of two heavy chains and two light chains, wherein each heavy chain comprises the aforementioned heavy chain constant region sequence, heavy chain variable region sequence, and / or CDR sequence, and each light chain comprises the aforementioned light chain constant region sequence, light chain variable region sequence, and / or CDR sequence. In some embodiments, the antibodies of this application may be single-chain antibodies (scFv), or may be composed of antibody fragments, such as Fab or F(ab')2 fragments.

[0032] The VEGF-binding peptide may contain a second extracellular Ig-like domain (VEGFR1D2) of vascular endothelial growth factor receptor 1 (VEGFR1), particularly human VEGFR1D2. VEGFR1D2 may contain a deglycosylation mutation at position 68 corresponding to SEQ ID NO:17, for example, the asparagine (Asn, N) at this position may be replaced by alanine (Ala, A). In some embodiments, VEGFR1D2 may contain alanine (Ala, A) at position 68 corresponding to SEQ ID NO:17. In some embodiments, the VEGF-binding peptide may contain an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:17. In some embodiments, the VEGF-binding peptide may contain the amino acid sequence shown in SEQ ID NO:17.

[0033] The VEGF-binding peptide can be linked to the N-terminus of the heavy chain variable region or the light chain variable region of the PD-L1 antibody or its antigen-binding moiety. In some embodiments, the VEGF-binding peptide can be linked to the N-terminus of the heavy chain variable region of the PD-L1 antibody or its antigen-binding moiety.

[0034] VEGF-binding peptides can be linked to PD-L1 antibodies or their antigen-binding moieties via a linker. The linker can be a peptide of 5-30 amino acids in length, particularly a peptide of 10-30 amino acids or 10-20 amino acids in length. The linker can be a GS linker, such as a GS linker containing the amino acid sequence shown in SEQ ID NO:18.

[0035] The recombinant fusion protein of this application may comprise i) a VEGFR1D2-linker-PD-L1 antibody heavy chain variable region-heavy chain constant region chain, and ii) a PD-L1 antibody light chain variable region-light chain constant region chain. In some embodiments, i) and ii) respectively comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:19 and 20, or SEQ ID NO:28 and 29.

[0036] In some embodiments, the recombinant fusion protein of this application may comprise:

[0037] i) The first polypeptide chain, from the N-terminus to the C-terminus, includes the VEGF-binding peptide, the linker, the PD-L1 antibody heavy chain variable region, and the heavy chain constant region.

[0038] ii) The second polypeptide chain, from the N-terminus to the C-terminus, contains the variable region of the PD-L1 antibody light chain and an optional constant region of the light chain.

[0039] iii) The third polypeptide chain, from the N-terminus to the C-terminus, includes the VEGF-binding peptide, the linker, the PD-L1 antibody heavy chain variable region, and the heavy chain constant region, as well as

[0040] iv) The fourth polypeptide chain, from the N-terminus to the C-terminus, contains the variable region of the PD-L1 antibody light chain and an optional constant region of the light chain.

[0041] The variable regions of the PD-L1 antibody heavy chain in the first polypeptide chain and the variable regions of the PD-L1 antibody light chain in the second polypeptide chain form antigen-binding sites that can specifically bind to PD-L1. The variable regions of the PD-L1 antibody heavy chain in the third polypeptide chain and the variable regions of the PD-L1 antibody light chain in the fourth polypeptide chain form antigen-binding sites that can specifically bind to PD-L1. The constant regions of the heavy chain in the first polypeptide chain and the constant regions of the heavy chain in the third polypeptide chain can be linked together by, for example, a pestle-and-mortar structure or a disulfide bond.

[0042] In some embodiments, the first, second, third, and fourth polypeptide chains may contain the amino acid sequences shown in SEQ ID NO: 28, 29, 28, and 29, respectively.

[0043] The recombinant fusion protein of this application may comprise i) a PD-L1 antibody heavy chain variable region-heavy chain constant region chain, and ii) a VEGFR1D2-linker-PD-L1 antibody light chain variable region-light chain constant region chain.

[0044] The composition of this application may also contain i) a nucleic acid molecule encoding a CTLA-4 antibody or its antigen-binding portion, and ii) a nucleic acid molecule encoding the recombinant fusion protein of this application, or a vector or host cell containing the above nucleic acid molecules.

[0045] The compositions of this application may be pharmaceutical compositions, and may also contain pharmaceutically acceptable carriers.

[0046] The pharmaceutical composition of this application may further include instructions for use specifying how to use the pharmaceutical composition of this application. These instructions may specify that the CTLA-4 antibody or its antigen-binding portion is administered first, and that the recombinant fusion protein is administered only after the blood concentration of the CTLA-4 antibody or its antigen-binding portion has decreased to a specified value. The specified value may be the maximum blood concentration (C0). max Less than 1 / 2 of ), for example, C maxLess than 1 / 2, less than 1 / 4, less than 1 / 8, or less than 1 / 16, etc. The instructions for use may specify that the CTLA-4 antibody or its antigen-binding portion is administered first, and the recombinant fusion protein is administered after at least about one half-life (e.g., about 1, 1.5, 2, 2.5, 3, or 4 half-lives) of the CTLA-4 antibody or its antigen-binding portion has elapsed. In some embodiments, the instructions for use may specify that the CTLA-4 antibody or its antigen-binding portion is administered first, and the recombinant fusion protein is administered after at least about 1.5 half-lives of the CTLA-4 antibody or its antigen-binding portion has elapsed. In some embodiments, the instructions for use may specify that the CTLA-4 antibody or its antigen-binding portion is administered first, and the recombinant fusion protein is administered after about 1.5 to 2.5 half-lives (e.g., 1.5, 2, or 2.5 half-lives) of the CTLA-4 antibody or its antigen-binding portion has elapsed. In some embodiments, the instructions for use may specify that the CTLA-4 antibody or its antigen-binding portion is administered first, followed by the recombinant fusion protein after approximately 1.5 half-lives of the CTLA-4 antibody or its antigen-binding portion. In other embodiments, the instructions for use may specify that the CTLA-4 antibody or its antigen-binding portion is administered first, followed by the recombinant fusion protein after approximately 7, 8, 9, 10, 11, 12, 13, or 14 days. In one treatment course, the CTLA-4 antibody or its antigen-binding portion may be administered once, while the recombinant fusion protein may be administered once or multiple times. For example, the recombinant fusion protein may be administered in four doses as one treatment course, with intervals of approximately 1 week, 2 weeks, or 3 weeks between each dose.

[0047] In a second aspect, this application provides a method for treating tumors in a subject in need, comprising i) administering the CTLA-4 antibody of this application or its antigen-binding portion to the subject, and ii) administering the recombinant fusion protein of this application after a number of days equivalent to at least about 1.5 half-lives of the CTLA-4 antibody or its antigen-binding portion.

[0048] Step i) may include administering the CTLA-4 antibody or its antigen-binding portion only once (e.g., once per treatment course).

[0049] Step ii) may include administering the recombinant fusion protein of this application after a number of days equivalent to at least 1.5 half-lives (e.g., 1.5, 2, 2.5, 3, or 4 half-lives) of the CTLA4-antibody or its antigen-binding portion. In some embodiments, step ii) may include administering the recombinant fusion protein of this application after a number of days equivalent to 1.5 to 2.5 half-lives (e.g., 1.5, 2, or 2.5 half-lives) of the CTLA4-antibody or its antigen-binding portion. In some embodiments, step ii) may include administering the recombinant fusion protein of this application approximately 7, 8, 9, 10, 11, 12, 13, or 14 days after implementing step i).

[0050] The application of the recombinant fusion protein of this application in step ii) may include one or more applications of the recombinant fusion protein. In some embodiments, step ii) may include four applications (e.g., four applications in one treatment course) of the recombinant fusion protein, wherein each application is spaced approximately one week, two weeks, or three weeks apart.

[0051] Tumors can be solid tumors, especially those with Tregs or CTLA-4 in the tumor microenvironment. + Solid tumors that act as effector T cells. In some embodiments, the tumor may be a solid tumor sensitive to Treg regulation or sensitive to CTLA-4 antibody administration, including but not limited to lung cancer (including non-small cell lung cancer), breast cancer (including triple-negative breast cancer, HER2-positive breast cancer), and other similar tumors. - The tumor can be any of the following: breast cancer, melanoma, hepatocellular carcinoma, renal cell carcinoma (including renal cell carcinoma), sarcoma, ovarian cancer, thymic carcinoma, pancreatic cancer, head and neck cancer, pleural mesothelioma, and colorectal cancer (especially colon cancer such as microsatellite instability-high (MSI-H) or mismatch modification-deficient colon cancer). In some embodiments, the tumor may be colorectal cancer, particularly colon cancer such as microsatellite instability-high (MSI-H) colon cancer. In some embodiments, the tumor may be a tumor sensitive to Treg regulation or sensitive to CTLA-4 antibody administration and sensitive to administration of the recombinant fusion protein of this application.

[0052] Subjects can be mammals, especially humans.

[0053] This application also provides the use of the compositions of this application, particularly pharmaceutical compositions, in the preparation of a medicament for treating tumors. The tumor can be a solid tumor, particularly a solid tumor sensitive to Treg regulation or sensitive to CTLA-4 antibody administration, including but not limited to colorectal cancer.

[0054] Other features and advantages disclosed herein will become readily apparent from the following detailed description and embodiments, which should not be construed as limiting. All references, Genbank registration numbers, patents, and published patent applications cited in this specification are incorporated herein by reference. Attached Figure Description

[0055] The following detailed description, given by way of example but not intended to limit the invention to the specific embodiments described, can be better understood in conjunction with the accompanying drawings.

[0056] Figure 1 shows the tumor volume changes in mice in each group under the regimen of simultaneous administration of CTLA-4 antibody and recombinant fusion protein.

[0057] Figure 2 shows the changes in tumor volume in mice in each group following the administration of the recombinant fusion protein 7 days after administration of the CTLA-4 antibody.

[0058] Figure 3 shows the tumor volume changes in mice in each group following the administration of the recombinant fusion protein 11 days after administration of the CTLA-4 antibody. Detailed Implementation

[0059] Unless otherwise specified, the terms used herein have their common meanings as found in dictionaries, textbooks, and technical reference books, or as commonly understood by those skilled in the art. The following descriptions of some terms are for the purpose of understanding this application only and are not intended to impose any particular limitations on these terms, unless otherwise specified.

[0060] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form of the object referred to, unless the context clearly specifies otherwise.

[0061] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0062] The terms “comprising” or “including” mean that the stated elements, integers, or steps are included, but do not exclude the inclusion of any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps.

[0063] The terms “optional” or “optionally” in this document mean that a component or step is not mandatory or necessary to include, that is, a component or step may be included in some cases and may not be included in others.

[0064] In this article, "antibody" is intended to include full-length antibodies of IgG, IgA, IgD, IgE, and IgM, as well as any antigen-binding fragments (i.e., antigen-binding portions). Full-length antibodies are typically glycoproteins containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain is separated by a heavy chain variable region (V). H It consists of a VH domain and a heavy chain constant region. The heavy chain constant region consists of three structural domains, namely C... H1 C H2 and C H3 Each light chain consists of a light chain variable region (V for short). L It consists of a light chain constant region (or VL) and a light chain constant region. The light chain constant region consists of a structural domain C. L Composition. V H and V L The region can also be divided into highly variable regions called complementarity-determining regions (CDRs), which are separated by more conservative framework regions (FRs). Each V H and V L The antibody heavy chain constant region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The antibody heavy chain constant region can mediate the binding of immunoglobulins to host tissues or factors, including binding to various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system. The "functional fragment" of the antibody heavy chain constant region refers to a segment within the constant region that retains certain desired functions; for example, a fragment in the heavy chain constant region that retains FcR / complement system component binding activity, such as an Fc fragment. The antibody light chain constant region is typically used to stabilize the antibody structure.

[0065] As used herein, the “antigen-binding portion” (or simply antigen-binding fragment) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the EGFR protein). It has been demonstrated that the antigen-binding function of an antibody can be performed using fragments of the full-length antibody. Examples of binding fragments contained in the “antigen-binding portion” of an antibody include (i) the Fab fragment, which is composed of V… L V H C L and C H1 (ii) F(ab')2 segment, a divalent segment containing two Fab segments connected by a disulfide bridge in the hinge region; (iii) composed of V H and C H1 (iv) Fd fragments composed of antibody single-arm V L and V H The Fv segment is composed of (v) and V. HThe dAb fragment composed of H (Ward et al., (1989) Nature 341:544-546); (vi) separated complementarity-determining regions (CDRs); and (vii) dAb-V L A fragment containing a single variable structural domain and a heavy-chain constant structural domain. Furthermore, although the two structural domains V of the Fv fragment... L and V H Encoded by different genes, they can be linked via a synthetic linker that makes them single-protein chains through recombination, where V L and V H Regions pair to form monovalent molecules. These single-chain antibodies are also intended to be included in the terminology. These antibody fragments can be obtained using common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0066] In this article, "antigen binding site" refers to the part of an antibody that recognizes and binds to an antigen. It is usually located at the top of the antigen-binding fragment of the antibody and consists of a portion of the heavy chain variable region and the light chain variable region.

[0067] The crystallizable region (Fc region) is the tail region of an antibody and is the domain that determines the effector function of the antibody (i.e., how the antibody interacts with specific cellular receptors or other defense proteins). Fc receptors (FcRs) are proteins expressed on the surface of some cells, such as B lymphocytes, natural killer cells, and macrophages. They can be bound by the Fc portion of an antibody, triggering phagocytosis and cytotoxicity against target cells, playing a crucial role in the immune system. FcRs include Fcα receptors, Fcε receptors, and Fcγ receptors. Among them, Fcγ receptors belong to the immunoglobulin superfamily and are the most important FcRs in initiating phagocytosis of microorganisms, including FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), and FcγRIIIA (CD16A).

[0068] "Antibody-dependent cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refers to cell-mediated immune defense in which immune system effector cells actively bind cell membrane surface antigens (such as CTLA-4, PD-L1, or VEGF) to the antibody or recombinant fusion protein of this application, resulting in the lysis of target cells.

[0069] "Antibody-dependent phagocytosis" or "ADCP" refers to an immune elimination mechanism in which the antibody or recombinant fusion protein of this application binds to target cells and recruits immune effector cells, such as phagocytes, via a constant region, such as Fc, thereby facilitating the phagocytosis of target cells by immune effector cells.

[0070] "Effective cells" or "immune effector cells" generally refer to immune cells that participate in clearing foreign antigens and performing effector functions in the immune response, such as plasma cells, cytotoxic T cells, NK cells, APSC pluripotent cells, mast cells, etc.

[0071] "Plasma drug concentration" refers to the total concentration of a drug in plasma, including drugs bound to plasma proteins or free in the plasma. Sometimes it can also refer to the concentration of a drug in whole blood. The intensity of a drug's effect is generally proportional to its concentration in plasma, and the concentration of a drug in the body varies over time. Drug concentrations in blood samples can be measured using methods such as high-performance liquid chromatography (HPLC). "Maximum plasma concentration" or "C60 concentration" is also referred to as "C60 concentration." max "Refers to the maximum (or peak) serum drug concentration reached in a compartment or specific test area of ​​the body after a single drug administration and before the next drug administration."

[0072] "Half-life" refers to the period during which the blood concentration of a drug in the body decreases by half (e.g., from its highest value (i.e., C50)). max The half-life is the time required for the drug's concentration to decrease by half. Different drugs have different half-lives; the longer the half-life, the longer the drug remains in the body, and the longer its effect typically lasts. Those skilled in the art can determine a drug's half-life by periodically taking blood samples and measuring the drug concentration in the blood.

[0073] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, are preferred.

[0074] The term "therapeutic effective amount" refers to the amount of the recombinant fusion protein of this application sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). Therapeutic effective amount is related to the disease being treated and / or the actual condition of the patient, and those skilled in the art can readily determine the actual effective amount.

[0075] In this article, "sequence identity" refers to the percentage of nucleotides / amino acids in a sequence that are identical to those in a reference sequence after sequence alignment. If necessary, spaces are introduced in the sequence alignment to achieve the maximum percentage of sequence similarity between the two sequences. Those skilled in the art can use various methods, such as computer software, to perform pairwise or multiple sequence alignments to determine the percentage of sequence similarity between two or more nucleic acid or amino acid sequences. Such computer software includes, for example, ClustalOmega, T-coffee, Kalign, and MAFFT.

[0076] In cancer treatment, it may be necessary to remodel the immunosuppressive properties of the tumor microenvironment (TME) to make it more friendly to effector immune cells, enabling these cells to activate normally and attack and kill tumor cells. After TME remodeling, immune checkpoints of immune cells can be manipulated to keep them in a more sustained activated state, thereby better killing tumor cells. Alternatively, tumor cells can be directly eliminated through mechanisms such as ADCC, ADCP, or CDC.

[0077] Reports indicate that CTLA-4 antibodies with ADCC function can kill Treg cells and remodel the TME. Furthermore, CTLA-4 antibodies can, to some extent, block the binding of CTLA-4 to CD80 / 86 on APC cells, thus preventing effector cell exhaustion. Antibodies targeting PD-L1 can block PD-1-PD-L1 interaction, preventing the exhaustion or apoptosis of immune cells, especially effector cells, and can directly kill PD-L1 through ADCC or antibody-dependent phagocytosis (ADCP). + Tumor cells. Targeting VEGF can block the process of tumor angiogenesis and reduce the immunosuppressive effect of TME, thereby indirectly enhancing the tumor-killing function of effector cells.

[0078] The inventors of this application achieved better tumor inhibition by targeting the above three pathways and rationally arranging the timing of targeting each pathway. In particular, the targeting of PD-L1 and VEGF utilizes a dual / multi-specific recombinant fusion protein, which may simplify the operation for medical staff and patients, potentially improve patient compliance, and possibly achieve better efficacy than a combination of two single drugs.

[0079] In early studies, the inventors simultaneously administered ADCC-enhanced CTLA-4 antibody and ADCC-enhanced PD-L1 and VEGF bispecific recombinant fusion protein to tumor-bearing mice. However, unexpectedly, the tumor suppression effect was lower than that of single-drug administration of ADCC-enhanced CTLA-4 antibody (Example 1). The inventors conducted further research on the CTLA-4 antibody and the recombinant fusion protein, summarizing the reasons for the failure of the combination as follows: Both the CTLA-4 antibody and the recombinant fusion protein possess FcR (especially FcγRIIa / IIIa) binding affinity. When administered simultaneously, they compete for binding to FcR on the surface of immune cells. Since the number of immune cells is limited, a portion of the CTLA-4 antibody or recombinant fusion protein, especially a large portion of the CTLA-4 antibody targeting Tregs in the TME, cannot exert its intended ADCC or ADCP effect immediately; or, there is spatial competition between the two administered proteins, preventing them from fully functioning. Therefore, the inventors modified the drug administration protocol later. After administering the CTLA-4 antibody, and after a period of time until the blood concentration of the CTLA-4 antibody decreased, the recombinant fusion protein was then administered. This significantly reduced, or even eliminated, the competition for FcR between the CTLA-4 antibody and the recombinant fusion protein. However, when the inventors administered the PD-L1 / VEGF dual-targeting protein approximately 7 days after administering the CTLA-4 antibody, the combined effect of the three targets was still lower than that of CTLA-4 antibody alone (Example 2). The inventors further extended the time interval between the first administration of the CTLA-4 antibody and the PD-L1 / VEGF dual-targeting protein to 11 days, and found that the combined effect of the three targets exceeded that of CTLA-4 antibody alone, or PD-L1 and VEGF dual-targeting protein alone (Example 3).

[0080] Pre-administration of CTLA-4 antibody can remodel the tumor microenvironment (TME) into a form friendly to immune cells, especially effector cells. It can also activate effector cells by blocking the binding of CTLA-4 to CD80 / 86. Subsequently, administration of the recombinant fusion protein can further reduce the immunosuppressive nature of the TME by blocking the VEGF pathway, allowing immune cells, especially effector cells, to infiltrate the tumor parenchyma and exert their tumor-killing function. The recombinant fusion protein can also block tumor angiogenesis, maintain a more sustained activated state of immune cells by blocking the PD-1 pathway, or directly target PD-L1. + The tumor cells are killed. Through a multi-pronged approach, effective inhibition of solid tumors, including colorectal cancer, is achieved. In principle, this strategy is suitable for tumors with Treg or CTLA4 present in the tumor microenvironment (TME). +Effector T cells, and solid tumors that are sensitive to Treg regulation or CTLA-4 antibody administration, including but not limited to lung cancer (including non-small cell lung cancer), breast cancer (including triple-negative breast cancer, HER2-positive breast cancer). - Solid tumors that are "sensitive to Treg regulation" are defined as tumors that rapidly cease growth, shrink, or disappear after administration of drugs that regulate (kill) Tregs, or respond to other anticancer drugs; or tumors that rapidly cease growth, shrink, or disappear after administration of low doses of drugs that regulate (kill) Tregs, or respond to other anticancer drugs. Similarly, solid tumors that are "sensitive to CTLA-4 antibody administration" are defined as tumors that rapidly cease growth, shrink, or disappear after administration of CTLA-4 antibodies, or respond to other anticancer drugs; or tumors that rapidly cease growth, shrink, or disappear after administration of low doses of CTLA-4 antibodies, or respond to other anticancer drugs.

[0081] Melanoma, kidney cancer (including renal cell carcinoma), lung cancer (including non-small cell lung cancer), breast cancer (including triple-negative breast cancer, HER2-positive breast cancer). -Breast cancer, hepatocellular carcinoma, sarcoma, ovarian cancer, thymic carcinoma, head and neck cancer, pancreatic cancer, pleural mesothelioma, and colon cancer (especially MSI-H colon cancer or mismatch modification deficiency) are relatively sensitive to CTLA-4 antibodies such as ipilimumab. (Tomasini P, et al., (2012) Ipilimumab: its potential in non-small cell lung cancer. Ther Adv Med Oncol. 4(2):43-50; Nederlof, I., et al. (2024) Neoadjuvant nivolumab or nivolumab plus ipilimumab in early-stage triple-negative breast cancer: a phase 2 adaptive trial. Nat Med 30:3223–3235; Barroso-Sousa R, et al. (2025) Nivolumab plus low-dose ipilimumab in hypermutated HER2-negative metastatic breast cancer: a phase II) trial(NIMBUS).Nat Commun.16(1):4430;D'Angelo SP,et al.(2018).Nivolumab with or without ipilimumab treatment for metastatic sarcoma(Alliance A091401):two open-label,non-comparative,randomised,phase 2trials.Lancet Oncol.19(3):416-426;Marques C,et al. (2023) Chemotherapy-free treatment of recurrent advanced ovarian cancer: myth or reality? Int J Gynecol Cancer.33(4):607-618; Nicolas Girard., et al.(2025)Efficacy and safety of nivolumab plus ipilimumab for patients with pre-treated type B3 thymoma and thymic carcinoma:Results from the EORTC-ETOP NIVOTHYM phase II trial.Journal of Clinical Oncology 43(16)_suppl; Terrero G,Datta J,Dennison J,et al.(2022)Ipilimumab / Nivolumab Therapy in Patients With Metastatic Pancreatic or Biliary Cancer With Homologous Recombination Deficiency Pathogenic Germline Variants. JAMA Oncol. 8(6):938-940). Among them, melanoma is the approved therapeutic indication for ipilimumab, while renal cell carcinoma, colorectal cancer (MSI-H or mismatch modification deficiency), hepatocellular carcinoma, non-small cell lung cancer, and pleural mesothelioma are the approved therapeutic indications for the combination of ipilimumab and nivolumab (www.drugs.com / history / yervoy). Meanwhile, studies have shown that ipilimumab contains the IgG1 constant region and has a killing / clearing effect on Tregs in the TME of melanoma, head and neck squamous small cell carcinoma, etc. (Romano E, et al., (2015) ibid.; Sasidharan Nair V, Elkord E. (2018) Immune checkpoint inhibitors in cancer therapy: a focus on T-regulatory cells. Immunol Cell Biol. 96(1):21-33).

[0082] The tumor combination therapy of this application can be achieved by administering the composition of this application.

[0083] The compositions of this application, particularly pharmaceutical compositions, may comprise i) a CTLA-4 antibody or its antigen-binding portion thereof, and ii) a recombinant fusion protein comprising a PD-L1 antibody or its antigen-binding portion thereof, and a VEGF-binding peptide.

[0084] The CTLA-4 antibody or its antigen-binding moiety may include a heavy chain variable region, a heavy chain constant region, a light chain variable region, and optionally a light chain constant region, wherein the heavy chain constant region may contain Fc receptor (FcR) and / or complement system protein binding capacity, particularly a high FcR and / or complement system protein binding capacity, to achieve ADCC or ADCP for Tregs. In some embodiments, the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the CTLA-4 antibody or its antigen-binding moiety may respectively contain the amino acid sequences shown in GFTFSSYT (SEQ ID NO:1), ISYDGNNK (SEQ ID NO:2), ARTGWLGPFDY (SEQ ID NO:3), QSVGSSY (SEQ ID NO:4), GAF, and QQYGSSPWT (SEQ ID NO:5). In some embodiments, the heavy chain variable region and light chain variable region of the CTLA-4 antibody or its antigen-binding portion may contain an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:6 and 7.

[0085] PD-L1 antibodies or their antigen-binding moieties may include a heavy chain variable region, a heavy chain constant region, a light chain variable region, and optionally a light chain constant region. The heavy chain constant region may contain Fc receptor (FcR) and / or complement system protein binding capacity, particularly a high FcR and / or complement system protein binding capacity, to achieve both PD-L1 and PD-L1 inhibition simultaneously. + The tumor cells' ADCC, ADCP, or CDC, etc. This application uses two PD-L1 antibodies, both of which achieve good tumor suppression effects. In some embodiments, the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the PD-L1 antibody or its antigen-binding portion may respectively contain the amino acid sequences shown in GFTFSDSW (SEQ ID NO:8), ISPHYGGST (SEQ ID NO:9), ARRHWPGGFDY (SEQ ID NO:10), QDVSTA (SEQ ID NO:11), SAS, and QQYLYHPAT (SEQ ID NO:12), or its heavy chain variable region and light chain variable region may respectively contain amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:13 and 14.

[0086] In another embodiment, the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of the PD-L1 antibody or its antigen-binding portion may respectively contain the amino acid sequences shown in GYTFTSNW (SEQ ID NO:21), IHPNSGSS (SEQ ID NO:22), ARSYYGSSPYYFDY (SEQ ID NO:23), QDIINY (SEQ ID NO:24), YTS, and QQGDTLPWT (SEQ ID NO:25), or its heavy chain variable region and light chain variable region may respectively contain amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:26 and 27.

[0087] The heavy chain variable region CDR and light chain variable region CDR of the antibody or its antigen-binding moiety in this application are determined using the IMGT numbering system. As is well known in the art, the heavy chain variable region CDR and light chain variable region CDR of the antibody or its antigen-binding moiety can also be determined based on the full-length sequence of the variable region using numbering systems such as Chothia, Kabat, AbM, or Contact.

[0088] The antibody or its antigen-binding moiety of this application may contain one or more conserved modifications in the variable region sequences of the heavy chain and / or light chain, or in the CDR1, CDR2, and CDR3 sequences. It is known in the art that some conserved sequence modifications do not result in the loss of antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8. As used herein, the term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter the antibody-binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or its antigen-binding moiety of this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibody or its antigen-binding portion of this application can be replaced with other amino acid residues from the same side chain group, and the resulting antibody can be tested for retained function (i.e., the function described above) using the functional assays described herein.

[0089] The VEGF-binding peptide may be an extracellular Ig-like domain of VEGFR1. The extracellular Ig-like domain of VEGFR1 may be a second extracellular Ig-like domain or a third extracellular IgG-like domain of VEGFR1. In some embodiments, the extracellular Ig-like domain of VEGFR1 may be the second extracellular Ig-like domain of VEGFR1 (VEGFR1D2). VEGFR1D2 may contain a deglycosylation mutation at position 68 of SEQ ID NO:17, for example, asparagine (Asn, N) at this position may be replaced by alanine (Ala, A). In some embodiments, VEGFR1D2 may contain alanine (Ala, A) at position 68 of SEQ ID NO:17. In some embodiments, VEGFR1D2 may contain the amino acid sequence shown in SEQ ID NO:17. Glycosylation sites are Asn(N)-X-Ser(S) or Asn(N)-X-Thr(T) sequences on proteins that allow oligosaccharides to attach to asparagine (Asn, N), where X is any amino acid other than proline (Pro, P). Glycosylation can decrease certain binding functions of a protein. For example, glycosylation may decrease the affinity of an antibody for an antigen. Deglycosylation may improve certain binding forces of the protein. Furthermore, the presence of glycosylation may also reduce the uniformity of protein products during recombinant expression; this problem can be addressed through deglycosylation mutations. Using VEGFR1D2 as a VEGF-binding peptide allows for binding to VEGF-A, VEGF-B, and PlGF. VEGF-binding peptides can contain one or more conserved modifications without altering their VEGF-binding properties.

[0090] The VEGF-binding peptide can be linked to the N-terminus of the heavy chain variable region or the light chain variable region of the PD-L1 antibody or its antigen-binding moiety. In some embodiments, the VEGF-binding peptide can be linked to the N-terminus of the heavy chain variable region of the PD-L1 antibody or its antigen-binding moiety.

[0091] VEGF-binding peptides can be linked to PD-L1 antibodies or their antigen-binding moieties via a linker. The linker primarily functions as a spacer between the extracellular Ig-like domain of VEGFR1 and the N-terminus of the heavy or light chain of the PD-L1 antibody. The linker can be composed of amino acids linked by peptide bonds, for example, 5-30, 10-30, or 10-20 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. One or more of these amino acids can be glycosylated or deglycosylated, as understood by those skilled in the art. In one embodiment, the 5-30, 10-30, 10-20, or 15 amino acids can be selected from glycine, alanine, proline, asparagine, glutamine, serine, and lysine. In one embodiment, the linker is composed mostly of sterically hindered amino acids with empty bonds, such as glycine and alanine. Exemplary linkers are polyglycine, poly(Gly-Ala), or polyalanine. The exemplary suitable linkers shown in the following examples are GS linkers, such as GS linkers comprising the amino acid sequence shown in SEQ ID NO:18. Linkers can also be non-peptide linkers. For example, alkyl linkers, such as -NH-, -(CH2)sC(O)-, where s = 2-20, can be used. These alkyl linkers can also be via any non-sterically hindered group, such as lower alkyl groups (e.g., C). 1-4 Substitution can be achieved by lower acyl groups, halogens (e.g., Cl, Br), CN, NH2, phenyl, etc.

[0092] The compositions of this application may also comprise i) nucleic acid molecules encoding CTLA-4 antibodies or their antigen-binding moieties, and ii) nucleic acid molecules encoding the recombinant fusion protein of this application, or vectors or host cells comprising the aforementioned nucleic acid molecules. Examples of vectors include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), transformable artificial chromosomes (TAC), mammalian artificial chromosomes (MAC), and artificially attached chromosomes (HAEC). Host cells may be transformed or transfected using expression vectors. Suitable host cells include *Escherichia coli*, yeast, and other eukaryotes. In particular, *Escherichia coli*, yeast, or mammalian cell lines (e.g., COS or CHO) may be used.

[0093] The compositions of this application may be pharmaceutical compositions, and may also include pharmaceutically acceptable carriers, such as excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, stabilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0094] The primary medium or carrier in a pharmaceutical composition can be aqueous or non-aqueous in nature. For example, suitable mediums or carriers may be water for injection, physiological saline, or artificial cerebrospinal fluid, supplemented with other materials commonly used in injection. For example, a medium or carrier may be a neutral buffered saline solution or a saline solution mixed with serum albumin. Other exemplary pharmaceutical compositions contain Tris buffer or acetate buffer, which may also contain sorbitol or suitable alternatives thereof. In one embodiment of this application, the composition can be prepared for storage by mixing selected components of desired purity with any formulation (Remington's Pharmaceutical Sciences, as above) in lyophilized or aqueous form. Furthermore, therapeutic compositions can be formulated as lyophilized preparations using suitable excipients such as sucrose.

[0095] Specifically, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, enteric, spinal, or epidermal administration (e.g., by injection or bolus). Depending on the route of administration, the active molecule may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate it. As used herein, the term "enteric administration" refers to a mode of administration other than the usual enteric and local administration via injection, including, but not limited to, intravenous, intramuscular, intraarterial, intramembranous, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, dura mater, and intrasternal injection and infusion. Alternatively, the antibody of this application may be administered via non-injectable routes, such as local, epidermal, or mucosal administration, for example, intranasal, oral, vaginal, rectal, sublingual, or topical administration.

[0096] Pharmaceutical compositions can be in the form of sterile aqueous solutions or suspensions. They can also be formulated as microemulsions, liposomes, or other ordered structures suitable for high concentrations of the drug.

[0097] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation varies depending on the subject to be treated and the specific route of administration, and is generally the amount of the composition that produces therapeutic efficacy. Typically, this amount, in percentage terms, is about 0.01% to about 99% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0098] Dosing regimens can be adjusted to achieve the optimal desired response (e.g., therapeutic response). For example, multiple fractions can be administered over time, or the dose can be reduced or increased proportionally according to the severity of the treatment condition. Particularly advantageous are parenteral compositions formulated in dose units for ease of administration and to promote dose uniformity. The dose unit type used herein refers to physically separated units suitable for a single dose to the treated subject; each unit contains a pre-calculated amount of active compound, together with the drug carrier, to produce the desired therapeutic effect. Alternatively, fusion proteins can be administered in a sustained-release formulation, in which case the frequency of administration is reduced.

[0099] Therapeutic amounts of the antibodies or recombinant fusion proteins of this application preferably cause a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of damage or disability caused by the disease. For example, in the treatment of tumor-bearing subjects, a "therapeutic amount" means that, relative to untreated subjects, tumor growth is inhibited by at least about 40%, at least about 60%, at least about 80%, or even at least about 99%. Therapeutic amounts of the antibodies or recombinant fusion proteins of this application can reduce tumor volume or alleviate symptoms in subjects (typically humans, or possibly another mammal).

[0100] The pharmaceutical composition can be a controlled sustained-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The pharmaceutical composition can be administered via medical devices such as (1) needle-free subcutaneous injection devices (e.g., U.S. Patents 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556); (2) microinfusion pumps (U.S. Patent 4,487,603); (3) transdermal devices (U.S. Patent 4,486,194); (4) infusion devices (U.S. Patents 4,447,233 and 4,447,224); and (5) permeation devices (U.S. Patents 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0101] In some embodiments, the antibodies or recombinant fusion proteins of this application can be formulated to ensure suitable in vivo distribution. For example, to ensure that the therapeutic fusion protein of this application crosses the blood-brain barrier, the fusion protein is formulated in liposomes and may additionally contain targeting groups to enhance selective delivery to specific cells or organs.

[0102] This application also relates to in vivo gene therapy in which nucleic acid molecules encoding the antibody or recombinant fusion protein of this application are directly introduced into a subject. For example, the nucleic acid sequence encoding the antibody or recombinant fusion protein of this application is introduced into target cells via local injection of a nucleic acid construct with or without a suitable delivery vector, such as an adeno-associated virus vector. Other alternative viral vectors include, but are not limited to, retroviruses, adenoviruses, herpes simplex viruses, and papillomavirus vectors. In vivo physical transfer of the viral vector can be achieved by local injection of the desired nucleic acid construct or other suitable delivery vector containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or particle bombardment (gene gun).

[0103] As mentioned above, to avoid competition for FcR binding on immune cells, the administration of the CTLA-4 antibody and the administration of the recombinant protein of this application need to be spaced out for a certain period of time, for example, approximately 1.5 half-lives or at least 11 days, so that the blood concentration of the CTLA-4 antibody drops to a certain level during this period, such as the peak blood concentration (C). max The dosage can be 1 / 2, 1 / 4, 1 / 8, or 1 / 16 of the original dose. Specifically, this allows the blood concentration of the CLTA-4 antibody to drop to baseline levels or near 0 nM during this period. However, this criterion is not applicable to cases with long drug half-lives and rapid tumor growth. If the patient's condition permits, it is permissible to wait, for example, approximately 1.5–2.5 half-lives, until the blood concentration of the previously administered CLTA-4 antibody drops to a low level before administering the recombinant fusion protein. The instructions for use included in the composition can be used to explain the above dosing regimen.

[0104] The compositions of this application can be used to treat a variety of solid tumors, such as solid tumors that can be treated by CTLA-1 antibody, PD-L1 antibody, VEGF blocker, or the recombinant fusion protein of this application, including but not limited to colorectal cancer, such as colon cancer.

[0105] On the other hand, this application provides a method for treating tumors in a subject in need, comprising i) administering the CTLA-4 antibody of this application or its antigen-binding portion to the subject, and ii) administering the recombinant fusion protein of this application after a number of days equivalent to about 1.5 half-lives of the CTLA-4 antibody or its antigen-binding portion.

[0106] This application will be further described with reference to the following non-limiting embodiments.

[0107] Example

[0108] The exemplary CTLA-4 antibody used in the following examples is an IgG full-body antibody comprising two heavy chains and two light chains. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region, and each light chain consists of a light chain variable region and a light chain constant region. The heavy chain variable region, light chain variable region, heavy chain constant region, and light chain constant region contain the amino acid sequences shown in SEQ ID NO: 6, 7, 15, and 16, respectively. The half-life of this antibody, as measured in Phase I clinical trials, is approximately 8-12 days.

[0109] In the following embodiments, two exemplary recombinant fusion proteins are used, both comprising two VEGFR1D2 (SEQ ID NO: 17) linked to the N-terminus of the two heavy chains of a PD-L1 antibody via a linker (SEQ ID NO: 18). In exemplary recombinant fusion protein 1, the PD-L1 is a full-length IgG antibody comprising two heavy chains and two light chains. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region, and each light chain consists of a light chain variable region and a light chain constant region. The heavy chain variable region, light chain variable region, heavy chain constant region, and light chain constant region comprise the amino acid sequences shown in SEQ ID NO: 13, 14, 15, and 16, respectively. Exemplary recombinant fusion protein 2 uses a different PD-L1 antibody, which is a full-length IgG antibody comprising two heavy chains and two light chains. The heavy chain variable region, light chain variable region, heavy chain constant region, and light chain constant region comprise the amino acid sequences shown in SEQ ID NO: 26, 27, 15, and 16, respectively.

[0110] Example 1. Antitumor efficacy of combination therapy in C57BL / 6-hPD1 / hCTLA4 mice subcutaneously transplanted with MC38-hPDL1 tumor cells

[0111] The in vivo antitumor activity of the composition of this application was evaluated by subcutaneously injecting mouse colon cancer MC-38 cells (MC38-hPDL1, Jicui Yaokang) overexpressing human PD-L1 into C57BL / 6 mice (C57BL / 6-hPD1 / hCTLA4, Jicui Yaokang, T003826) with knocked-in human PD-1 and CTLA-4 genes.

[0112] Specifically, MC38-hPDL1 cells were placed in RPMI-1640 medium containing 10% fetal bovine serum and cultured at 37°C in a 5% CO2 incubator. MC38-hPDL1 cells in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with DPBS buffer. The cells were then resuspended in DPBS, and the cell suspension concentration was adjusted to 1×10⁻⁶. 7 per ml.

[0113] According to 1×10 6 Tumor cells were seeded subcutaneously into the right back of mice using a 1ml syringe (4-gauge needle) at a rate of 100μL per mouse. The cells were introduced when the average tumor volume reached 90mm². 3 Animals were randomly divided into groups of six, with the tumor size difference between groups less than 10% of the mean. The grouping date was recorded as D0. Intraperitoneal (ip) administration was initiated according to the average body weight, as shown in Table 1. During the experiment, the animals' body weight and tumor size were measured twice a week, and clinical symptoms were observed and recorded daily.

[0114] Tumor volume (TV) calculation: TV = a × b 2 / 2, where a and b represent the length and width of the tumor, respectively. The relative tumor proliferation rate T / C (%) is calculated as T / C (%) = TRTV / CRTV * 100%, where TRTV is the average RTV of the treatment group and CRTV is the average RTV of the negative control group. The relative tumor volume (RTV) is calculated as RTV = V t / V0. Where V0 is the tumor volume measured at D0, V t Let Dt be the tumor volume measured on day t. TGI is calculated as TGI(%) = (1 - T / C) × 100%.

[0115] In addition, the complete remission rate (CR) of each group was calculated after the experiment.

[0116] Table 1. Dosing regimen for colon cancer model

[0117] The experimental results showed that, as of day 28, the CR and TGI of group 2 were 2 / 6 and 78.1%, respectively, while the CR of groups 3 and 4 were both 0 / 6, and the TGI of groups 3 and 4 were 57.5% and 57.4%, respectively.

[0118] Figure 1 shows the changes in tumor volume in each group of mice during the experiment.

[0119] It can be seen that the combined use of CTLA-4 antibody and recombinant fusion protein is less effective than CTLA-4 alone.

[0120] Example 2. Optimization of Dosing Regimen

[0121] Given that both the CTLA-4 antibody and the recombinant fusion protein contain heavy chain constant regions capable of binding FcRs, such as FcγR, there may be competition for binding to immune effector cells. Therefore, based on Example 1, the dosing regimen was adjusted by administering the recombinant fusion protein 7 days after CTLA-4 administration to test the antitumor activity of the adjusted combination therapy.

[0122] Specifically, according to 1×10 6MC38-hPDL1 cells were seeded subcutaneously into the right back of mice using a 1ml syringe (4-gauge needle) at a rate of 100μL per mouse. The cells were introduced when the average tumor volume reached 90mm². 3 Animals were randomly divided into groups of 6, with the tumor difference between groups being less than 10% of the mean. The grouping day was recorded as D0, and intraperitoneal (ip) administration was started according to the average body weight as per Table 2.

[0123] During the experiment, the animals' body weight and tumor size were measured twice a week, and clinical symptoms were observed and recorded daily. When the tumor volume in the mice reached 3000 mm², [further details needed]. 3 At that time, euthanasia was performed.

[0124] Table 2. Dosing regimen for the colon cancer model

[0125] The experimental results showed that, as of day 32, the CRs of groups 2, 3 and 4 were 4 / 6, 0 / 6 and 2 / 6, respectively, and the TGIs were 89.4%, 48.8% and 79.7%, respectively.

[0126] Figure 2 shows the changes in tumor volume in each group of mice during the experiment.

[0127] With increasing the CTLA-4 dose and setting a 7-day interval between the administration of CTLA-4 and the recombinant fusion protein, the combined effect improved accordingly, but was still lower than that of CTLA-4 alone. Furthermore, the antitumor effect of the recombinant fusion protein alone decreased compared to Example 1 as the initial administration time was delayed.

[0128] Example 3. Further optimization of the dosing regimen

[0129] Building upon Example 2, the interval between the administration of CTLA-4 and the recombinant fusion protein was further extended to 11 days to test the efficacy of the combined treatment, and another structurally similar recombinant fusion protein 2 was added. Furthermore, the dose of the recombinant fusion protein was increased to compensate for the further delay in its initial administration.

[0130] Specifically, according to 1×10 6 MC38-hPDL1 cells were seeded subcutaneously into the right back of mice using a 1ml syringe (4-gauge needle) at a rate of 100μL per mouse. The cells were introduced when the average tumor volume reached 90mm². 3Animals were randomly divided into groups of six, with the tumor size difference between groups less than 10% of the mean. The grouping day was designated D0, and intraperitoneal (ip) administration was initiated according to average body weight, as shown in Table 3. On D3, groups 2, 4, and 6 (all previously administered CTLA-4) were combined and re-divided into three groups based on tumor volume and body weight. Mice from groups 3 and 5 were also combined and re-divided into two groups based on tumor volume and body weight. During the experiment, animal body weight and tumor size were measured twice weekly, and clinical symptoms were observed and recorded daily. When the tumor volume in the mice reached 3000 mm², the tumor was considered closed. 3 At that time, euthanasia was performed.

[0131] Table 3. Dosing regimen for the colon cancer model

[0132] Figure 3 shows the changes in tumor volume in each group of mice during the experiment, and Table 4 shows the mean survival and median survival of each group of mice.

[0133] Table 4. Mean and median survival time of mice in each group

[0134] By day 25, the TGIs for groups 2 through 6 were 79.4%, 40.40%, 28.72%, 82.94%, and 80.37%, respectively. The data for groups 5 and 6 were similar, slightly higher than those for group 2, and much higher than those for groups 3 and 4.

[0135] As the observation period lengthened, it became apparent that, compared to groups 2 and 3, mice in group 5 exhibited slower overall tumor growth and longer survival. By the end of the experiment, two mice from group 2 had survived and achieved complete remission (CR), while three mice from group 5 had survived and achieved CR or near-CR.

[0136] The survival rate and complete remission (CR) rate of mice in group 6 were both lower than those in group 2. By the end of the experiment, only one mouse in group 6 had survived and achieved CR. However, the overall tumor growth in group 6 mice was slower, and their survival was longer. This is significant for patients with low drug response, not only in terms of extended survival but also in the opportunity to explore new therapies during that extended survival period.

[0137] Thus, it can be seen that extending the administration interval between CTLA-4 and the recombinant fusion protein, and reducing the competition between them for effector cell binding or space, allows CTLA-4 to better create a tumor microenvironment favorable to immune cells, especially effector cells, and subsequent drugs in the early stages of treatment, thereby enhancing the anti-tumor effects of subsequent drugs and immune cells. In other words, CTLA-4 and the recombinant fusion protein only exhibit a synergistic effect when administered at an appropriate time.

[0138] The sequence information of this application is summarized as follows.

[0139] Although this application has been described in conjunction with one or more embodiments, it should be understood that this application is not limited to these embodiments. The description in this application is intended to cover all variations and equivalents, all of which are included within the spirit and scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

Claims

1. A pharmaceutical composition comprising: i) a CTLA-4 antibody or antigen binding portion thereof, and ii) a recombinant fusion protein comprising a PD-L1 antibody or antigen binding portion thereof, and a VEGF binding peptide, wherein the CTLA-4 antibody or antigen binding portion thereof comprises a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3, wherein the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequences set forth in GFTFSSYT (SEQ ID NO: 1), ISYDGNNK (SEQ ID NO: 2), ARTGWLGPFDY (SEQ ID NO: 3), QSVGSSY (SEQ ID NO: 4), GAF, and QQYGSSPWT (SEQ ID NO: 5), respectively, wherein the heavy chain constant region comprises FcR binding capacity, wherein the PD-L1 antibody or antigen binding portion thereof comprises a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3, wherein the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 comprise the amino acid sequences set forth in GFTFSDSW (SEQ ID NO: 8), ISPYGGST (SEQ ID NO: 9), ARRHWPGGFDY (SEQ ID NO: 10), QDVSTA (SEQ ID NO: 11), SAS, and QQYLYHPAT (SEQ ID NO: 12), or GYTFTSNW (SEQ ID NO: 21), IHPNSGSS (SEQ ID NO: 22), ARSYYGSSPYYFDY (SEQ ID NO: 23), QDIINY (SEQ ID NO: 24), YTS, and QQGDTLPWT (SEQ ID NO: 25), respectively, wherein the heavy chain constant region comprises FcR binding capacity, wherein the VEGF binding peptide comprises the second extracellular Ig-like domain of vascular endothelial growth factor receptor 1 (VEGFR1D2), wherein the VEGF binding peptide is linked to the N-terminus of the heavy chain variable region or the light chain variable region of the PD-L1 antibody or antigen binding portion thereof.

2. The pharmaceutical composition of claim 1, wherein the VEGFR1D2 is linked to the N-terminus of the heavy chain variable region of the PD-L1 antibody or antigen binding portion thereof.

3. The pharmaceutical composition of claim 2, wherein the VEGFR1D2 comprises an amino acid sequence set forth in SEQ ID NO:

17.

4. The pharmaceutical composition of claim 1, wherein the heavy chain variable region and the light chain variable region of the CTLA-4 antibody or antigen-binding portion thereof comprise the amino acid sequences set forth in SEQ ID NOs: 6 and 7, respectively.

5. The pharmaceutical composition of claim 1, wherein the heavy chain variable region and the light chain variable region of the PD-L1 antibody or antigen-binding portion thereof comprise the amino acid sequences set forth in SEQ ID NOs: 13 and 14, or SEQ ID NOs: 26 and 27, respectively.

6. The pharmaceutical composition of claim 1, wherein the heavy chain constant region in the CTLA-4 antibody or antigen-binding portion thereof comprises an amino acid sequence set forth in SEQ ID NO: 15, and the heavy chain constant region in the PD-L1 antibody or antigen-binding portion thereof comprises an amino acid sequence set forth in SEQ ID NO:

15.

7. The pharmaceutical composition of claim 1, wherein the VEGFR1D2 is linked via a linker to the N-terminus of the heavy chain variable region or the light chain variable region of the PD-L1 antibody or antigen-binding portion thereof.

8. The pharmaceutical composition of claim 7, wherein the linker comprises an amino acid sequence set forth in SEQ ID NO:

18.

9. The pharmaceutical composition of claim 1, wherein the recombinant fusion protein comprises i) a VEGFR1D2-linker-PD-L1 antibody heavy chain variable region-heavy chain constant region chain, and ii) a PD-L1 antibody light chain variable region-light chain constant region chain, wherein i) and ii) comprise the amino acid sequences set forth in SEQ ID NOs: 19 and 20, or SEQ ID NOs: 28 and 29, respectively.

10. The pharmaceutical composition of claim 1, further comprising instructions for use for indicating the use of the pharmaceutical composition, wherein the instructions for use indicate that the CTLA-4 antibody or antigen-binding portion thereof is administered first, and the recombinant fusion protein is administered after a number of days equivalent to about 1.5-2.5 half-lives of the CTLA-4 antibody or antigen-binding portion thereof.

11. Use of the pharmaceutical composition of any one of claims 1-10 in the manufacture of a medicament for treating a solid tumor.

12. The use of claim 11, wherein the solid tumor is lung cancer, breast cancer, melanoma, hepatocellular carcinoma, renal cancer, sarcoma, ovarian cancer, thymic carcinoma, or colorectal cancer.

13. The use of claim 11, wherein the solid tumor is colorectal cancer.

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