Recombinant fusion protein targeting PD-L1 and VEGF as well as preparation and application of recombinant fusion protein

Abstract

The invention provides a recombinant fusion protein which comprises 1) a PD-L1 antibody or an antigen binding fragment thereof and 2) a VEGF binding peptide, and the N end of a heavy chain or a light chain of the PD-L1 antibody or the antigen binding fragment thereof is connected with the VEGF binding peptide through a linker. Also provided are nucleic acid molecules encoding the recombinant fusion protein, expression vectors comprising the nucleic acid molecules, methods of preparing the recombinant fusion protein, and methods of using the recombinant fusion protein to treat diseases associated with PD-L1 and / or VEGF pathways.

Description

Recombinant fusion protein targeting PD-L1 and VEGF and its preparation and use

[0001] This application claims priority to Chinese patent application CN202210639281.X filed on May 31, 2022. Field of the Invention

[0002] The present application relates to a recombinant fusion protein targeting PD-L1, VEGF and / or FcR, and its preparation and use, in particular its use in tumor treatment. Background Art

[0003] Cancer cells have developed several mechanisms to evade host immune surveillance. For example, many tumor cells or cancer cells express high levels of PD-L1 and PD-L2 on their surfaces, both of which can bind to PD-1 on the surface of T cells, thereby inducing T cell apoptosis.

[0004] Furthermore, the growth of cancer cells depends on an adequate nutrient supply. Cancer cells themselves can secrete factors that promote blood vessel growth, such as vascular endothelial growth factor (VEGF). Inhibiting the activity of VEGF or its receptors will terminate the blood supply to solid tumors, thereby inhibiting their growth.

[0005] PD-L1 and PD-1

[0006] PD-1 / PD-L1 is one of the most studied inhibitory immune checkpoints.

[0007] PD-L1, also known as programmed death ligand 1 or CD274, is a transmembrane protein that plays an important role in suppressing the immune system in certain events such as allogeneic tissue transplantation, autoimmune diseases and cancer. + Th1 helper cells and CD8 + T cells produce interferon-γ (IFN-γ), which activates macrophages to kill tumors and induces PD-L1 expression in macrophages and tumor cells. + PD-1 on T cells binds to PD-L1 expressed by cells in the TME, triggering CD8 + T cell dysfunction (Topalian, S. et al., (2016) Nat Rev Cancer 16(5):275–287). PD-1 can also bind to PD-L2 and downregulate the immune system.

[0008] An analysis of 196 tumor samples from patients with renal cell carcinoma showed that high expression of PD-L1 in tumors was associated with increased tumor aggressiveness and a 4.5-fold increased risk of death (Thompson RH, et al., (2004) PNAS. 101(49):17174–17179). Approximately 1 / 5 to 1 / 4 of patients with non-small cell lung cancer, melanoma, or renal cell carcinoma have an objective response to a PD-1 antibody, BMS-936558 (Suzanne L. Topalian, et al., (2012) N Engl J Med 366:2443-2454).

[0009] VEGF and VEGFR

[0010] VEGF is a pleiotropic growth factor that plays a crucial role in controlling tissue and wound repair processes. It is divided into VEGF-A (also known as VEGF165), VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PIGF. During tissue healing, VEGF, particularly VEGF-A, drives angiogenesis and downregulates immunity (Canic M, et al., (2009) J Surg Res. 153:347-358; Leung DW, et al., (1989) Science. 246:1306–1309; Voron T, et al., (2014) Front Oncol. 4:70). Both of these properties of VEGF are crucial for carcinogenesis: the generation of tumor blood vessels provides blood and nutrients to cancerous tissues, while suppressing anti-cancer immunity promotes cancer cell growth (Hanahan D, Weinberg RA. (2011) Cell. 144:646–674). Specifically, VEGF is believed to exert its immunosuppressive effects through three main mechanisms: inhibiting dendritic cell (DC) maturation, reducing T cell tumor infiltration, and increasing suppressive cells in the tumor microenvironment. Abnormal VEGF expression can also promote the generation of structurally abnormal, leaky new blood vessels, leading to, for example, diabetic retinopathy and wet age-related macular degeneration. Bevacizumab (Avastin), an FDA-approved VEGF monoclonal antibody, treats cancer (colon cancer, lung cancer), non-squamous non-small cell lung cancer, renal cell carcinoma, glioblastoma multiforme, ovarian cancer, and cervical cancer by inhibiting the biological activity of VEGF. Another protein drug targeting VEGF, Aflibercept, is approved in the United States and Europe for the treatment of wet macular degeneration (trade name Eylea) and metastatic colorectal cancer (Zaltrap).

[0011] There are three subtypes of VEGF receptors (VEGFRs): VEGFR-1, VEGFR-2, and VEGFR-3, all of which have an extracellular portion composed of seven immunoglobulin-like domains, a transmembrane region, and an intracellular portion. VEGFR-2 appears to mediate nearly all known cellular responses to VEGF, while VEGFR-1 blocks VEGF from binding to VEGFR-2, modulating the VEGFR-2 signaling pathway. VEGF-A, the most dangerous type to human health, binds to both VEGFR-1 and VEGFR-2. VEGFR antagonists are most commonly used to treat cancer or in cancer therapy research. Lenvatinib (Lenvima), a multikinase inhibitor of VEGFR-1, VEGFR-2, and VEGFR-3 kinases, was approved in 2015 for the treatment of differentiated thyroid cancer and in 2016 for the treatment of advanced renal cell carcinoma in combination with everolimus.

[0012] Fc and FcR

[0013] The crystallizable region (Fc region) is the tail region of an antibody and is the domain that determines the antibody's effector functions (ie, how the antibody establishes a relationship with specific cell receptors or other defense proteins).

[0014] Fc receptors (FcRs) are proteins expressed on the surface of certain cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. These cells contribute to the protective functions of the immune system.

[0015] The Fc region can interact with Fc receptors and some proteins of the complement system to activate the immune system and the complement system.

[0016] Therapeutic bispecific or multispecific fusion proteins / antibodies

[0017] Antibodies targeting a single antigen may have limited efficacy. For example, the approved PD-L1 antibody, Avelumab The overall remission rate was only 33%.

[0018] In recent years, bispecific or trispecific fusion proteins have been developed, and these fusion proteins have shown considerable efficacy in preclinical and clinical trials.

[0019] Although conceptually, attaching additional binding groups to traditional antibodies is not very complex, such modifications can significantly alter the structure of the antibody, and the antibody and the additional binding groups may interact to affect binding affinity and / or drug efficacy (Wang S et al., (2021) EMBO Mol Med. 13(9): e14291). To optimize in vivo efficacy and drug properties, careful design and modification are required in terms of the selection of primary and secondary groups (sequences), the balance of target binding affinity, the selection of binding sites (N- or C-terminus of the heavy or light chain), structural stability, linker length, and sequence (Shim H. (2020) Biomolecules. 10(3): 360).

[0020] The citation of any document in this application does not constitute an admission that these documents are prior art to this application.

[0021] Summary of the Invention

[0022] The present application provides a new recombinant fusion protein comprising a PD-L1 antibody and a VEGF binding peptide. Compared to PD-L1 antibodies and VEGF binding proteins (including VEGF antibodies), the recombinant fusion protein of the present application can bind to PD-L1 with comparable activity. + , VEGF + , and / or PD-L1 + VEGF + cells, for PD-L1 + VEGF + The cells induce considerable antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP), and / or block PD-1-PD-L1 interaction and VEGF-VEGFR interaction with considerable activity. In addition, the recombinant fusion protein of the present application shows better anti-tumor effect than the combination of VEGF binding protein and PD-L1 antibody in in vivo experiments.

[0023] Specifically, the present application discloses a recombinant fusion protein comprising 1) a PD-L1 antibody or an antigen-binding fragment thereof that specifically binds to PD-L1 and 2) a VEGF-binding peptide that specifically binds to VEGF, wherein the VEGF-binding peptide is linked to the PD-L1 antibody or antigen-binding fragment thereof. The PD-L1 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region may comprise HV-CDR1, HV-CDR2, and HV-CDR3, wherein the HV-CDR1, HV-CDR2, and HV-CDR3 may comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable region may comprise LV-CDR1, LV-CDR2, and LV-CDR3, wherein the LV-CDR1 and LV-CDR3 may comprise the amino acid sequences set forth in SEQ ID NOs: 4 and 5, respectively, and the LV-CDR2 may comprise the amino acid sequence set forth in "YTS." The PD-L1 antibody or antigen-binding fragment thereof may further comprise a heavy chain constant region, which may be linked to the C-terminus of the heavy chain variable region. The heavy chain constant region may have FcR binding ability. In certain embodiments, the PD-L1 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region, a heavy chain constant region, and a light chain variable region, the heavy chain variable region may comprise HV-CDR1, HV-CDR2, and HV-CDR3, HV-CDR1, HV-CDR2, and HV-CDR3 may comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, the light chain variable region may comprise LV-CDR1, LV-CDR2, and LV-CDR3, LV-CDR1 and LV-CDR3 may comprise the amino acid sequences shown in SEQ ID NOs: 4 and 5, respectively, LV-CDR2 may comprise the amino acid sequence shown as "YTS", the heavy chain constant region may have FcR binding ability and be linked to the C-terminus of the heavy chain variable region. The VEGF-binding peptide can bind 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 fragment.

[0024] The VEGF-binding peptide can be the extracellular Ig-like domain of vascular endothelial growth factor receptor 1 (VEGFR1). The VEGF-binding peptide can be the second extracellular Ig-like domain of VEGFR1 (VEGFR1D2). In some embodiments, the VEGF-binding peptide can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11.

[0025] The heavy chain variable region of the PD-L1 antibody or antigen-binding fragment thereof may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7. In one embodiment, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 7. The light chain variable region of the PD-L1 antibody or antigen-binding fragment thereof may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In one embodiment, the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 8. In one embodiment, the heavy chain variable region and the light chain variable region of the PD-L1 antibody or antigen-binding fragment thereof may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 7 and 8, respectively. In some embodiments, the heavy chain variable region and the light chain variable region of the PD-L1 antibody or antigen-binding fragment thereof may comprise the amino acid sequences shown in SEQ ID NOs: 7 and 8, respectively.

[0026] The heavy chain constant region can be a naturally occurring or engineered human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a functional fragment thereof. The heavy chain constant region with FcR binding can be a naturally occurring or engineered human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a functional fragment thereof. In some embodiments, the heavy chain constant region with FcR binding is a human IgG1 heavy chain constant region, or a functional fragment thereof. In some embodiments, the heavy chain constant region with FcR binding can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9.

[0027] The PD-L1 antibody or antigen-binding fragment thereof may further comprise a light chain constant region, such as a human kappa light chain constant region, or a functional fragment thereof, connected to the C-terminus of the light chain variable region. In some embodiments, the light chain constant region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10.

[0028] The heavy chain of the PD-L1 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region and a heavy chain constant region. The heavy chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15. In one embodiment, the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 15. The light chain of the PD-L1 antibody or antigen-binding fragment thereof may comprise a light chain variable region and a light chain constant region. The light chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In one embodiment, the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 14. In one embodiment, the heavy chain and light chain of the PD-L1 antibody or antigen-binding fragment thereof may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 15 and 14, respectively. In some embodiments, the heavy chain and light chain of the PD-L1 antibody or antigen-binding fragment thereof may comprise the amino acid sequence shown in SEQ ID NOs: 15 and 14, respectively.

[0029] At least one paratope of the PD-L1 antibody or antigen-binding fragment thereof can be linked to a VEGF-binding peptide at the N-terminus of the heavy chain variable region or light chain variable region that constitutes the paratope. In some embodiments, each paratope of the PD-L1 antibody or antigen-binding fragment thereof is linked to a VEGF-binding peptide at the N-terminus of the heavy chain variable region or light chain variable region that constitutes the paratope. In some embodiments, each paratope of the PD-L1 antibody or antigen-binding fragment thereof is linked to a VEGF-binding peptide at the N-terminus of the heavy chain variable region that constitutes the paratope. In some embodiments, each paratope of the PD-L1 antibody or antigen-binding fragment thereof is linked to a VEGF-binding peptide at the N-terminus of the light chain variable region that constitutes the paratope.

[0030] The PD-L1 antibody or antigen-binding fragment thereof of the present application can be linked to a VEGF-binding peptide via a linker. The linker can be a peptide of 5-30, 10-30, 10-20, or 15 amino acids in length. The linker can be, for example, a GS linker, such as -(Gly-Gly-Gly-Gly-Ser)3- (SEQ ID NO: 12).

[0031] The recombinant fusion protein of the present application may comprise a VEGF-binding peptide-linker-PD-L1 antibody heavy chain, and a PD-L1 antibody light chain, wherein the VEGF-binding peptide-linker-PD-L1 antibody heavy chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13, and the PD-L1 antibody light chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the recombinant fusion protein of the present application may comprise a VEGF-binding peptide-linker-PD-L1 antibody heavy chain and a PD-L1 antibody light chain, wherein the VEGF-binding peptide-linker-PD-L1 antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 13, and the PD-L1 antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 14. The amino acid sequences of SEQ ID NOs: 13 and 14 may be encoded by the nucleotide sequences set forth in SEQ ID NOs: 23 and 24, respectively.

[0032] In one embodiment, the recombinant fusion protein of the present application comprises:

[0033] i) a first polypeptide chain comprising, from N-terminus to C-terminus, a VEGF-binding peptide, a linker, a heavy chain variable region that specifically binds to PD-L1, and a heavy chain constant region;

[0034] ii) a second polypeptide chain, comprising, from N-terminus to C-terminus, a light chain variable region that specifically binds to PD-L1 and a light chain constant region;

[0035] i) a third polypeptide chain comprising, from N-terminus to C-terminus, a VEGF-binding peptide, a linker, a heavy chain variable region that specifically binds to PD-L1, and a heavy chain constant region; and

[0036] ii) a fourth polypeptide chain, comprising, from N-terminus to C-terminus, a light chain variable region that specifically binds to PD-L1 and a light chain constant region,

[0037] The heavy chain variable region that specifically binds to PD-L1 in the first polypeptide chain and the light chain variable region that specifically binds to PD-L1 in the second polypeptide chain can combine to form an antigen binding domain that specifically binds to PD-L1, the heavy chain variable region that specifically binds to PD-L1 in the third polypeptide chain and the light chain variable region that specifically binds to PD-L1 in the fourth polypeptide chain can combine to form an antigen binding domain that specifically binds to PD-L1, and the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain can be bound together by, for example, a knob-and-hole, covalent bond, or disulfide bond.

[0038] The recombinant fusion protein of the present application may comprise a PD-L1 antibody heavy chain and a VEGF-binding peptide-linker-PD-L1 antibody light chain, wherein the PD-L1 antibody heavy chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, and the VEGF-binding peptide-linker-PD-L1 antibody light chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the recombinant fusion protein of the present application may comprise a PD-L1 antibody heavy chain and a VEGF-binding peptide-linker-PD-L1 antibody light chain, wherein the PD-L1 antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 15, and the VEGF-binding peptide-linker-PD-L1 antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 16. The amino acid sequences set forth in SEQ ID NOs: 15 and 16 may be encoded by the nucleotide sequences set forth in SEQ ID NOs: 25 and 26, respectively.

[0039] In one embodiment, the recombinant fusion protein of the present application comprises:

[0040] i) a first polypeptide chain, comprising, from N-terminus to C-terminus, a heavy chain variable region that specifically binds to PD-L1 and a heavy chain constant region;

[0041] ii) a second polypeptide chain comprising, from N-terminus to C-terminus, a VEGF-binding peptide, a linker, a light chain variable region that specifically binds to PD-L1, and a light chain constant region;

[0042] i) a third polypeptide chain, comprising, from N-terminus to C-terminus, a heavy chain variable region that specifically binds to PD-L1, and a heavy chain constant region; and

[0043] ii) a fourth polypeptide chain comprising, from N-terminus to C-terminus, a VEGF-binding peptide, a linker, a light chain variable region that specifically binds to PD-L1, and a light chain constant region,

[0044] The heavy chain variable region that specifically binds to PD-L1 in the first polypeptide chain and the light chain variable region that specifically binds to PD-L1 in the second polypeptide chain can combine to form an antigen binding domain that specifically binds to PD-L1, the heavy chain variable region that specifically binds to PD-L1 in the third polypeptide chain and the light chain variable region that specifically binds to PD-L1 in the fourth polypeptide chain can combine to form an antigen binding domain that specifically binds to PD-L1, and the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain can be bound together by, for example, a knob-and-hole, covalent bond, or disulfide bond.

[0045] The present application also protects the PD-L1 antibody or antigen-binding portion thereof of the present application, which may comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region may comprise HV-CDR1, HV-CDR2, and HV-CDR3, HV-CDR1, HV-CDR2, and HV-CDR3 may comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable region may comprise LV-CDR1, LV-CDR2, and LV-CDR3, LV-CDR1 and LV-CDR3 may comprise the amino acid sequences set forth in SEQ ID NOs: 4 and 5, respectively, and LV-CDR2 may comprise the amino acid sequence set forth in "YTS". The heavy chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7. The light chain variable region may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8. The PD-L1 antibody, or antigen-binding portion thereof, may further comprise a heavy chain constant region and / or a light chain constant region. The heavy chain constant region may be a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a functional fragment thereof, linked to the C-terminus of the heavy chain variable region. In some embodiments, the heavy chain constant region may be a heavy chain constant region having FcR binding ability, or a functional fragment thereof. In some embodiments, the heavy chain constant region may be a human IgG1 heavy chain constant region, or a functional fragment thereof. In some embodiments, the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 9. The light chain constant region may be a human kappa light chain constant region, or a functional fragment thereof, linked to the C-terminus of the light chain variable region. In some embodiments, the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 10. The PD-L1 antibody or its antigen-binding portion of the present application has comparable PD-L1 binding activity / affinity and comparable PD-1-PD-L1 blocking activity compared to prior art PD-L1 antibodies such as atelizumab. The PD-L1 antibody or its antigen-binding portion of the present application can also induce PD-L1 + ADCC and ADCP of cells.

[0046] The present application also provides nucleic acid molecules encoding the recombinant fusion proteins, PD-L1 antibodies, or antigen-binding portions thereof of the present application, as well as expression vectors comprising the nucleic acid molecules, and host cells comprising the expression vectors or in which the nucleic acid molecules are integrated into the genome. Also provided is a method for preparing the recombinant fusion proteins, PD-L1 antibodies, or antigen-binding portions thereof using the host cells of the present application, comprising (i) expressing the recombinant fusion protein, PD-L1 antibody, or antigen-binding portion thereof in the host cells, and (ii) isolating the recombinant fusion protein, PD-L1 antibody, or antigen-binding portion thereof from the host cells or cell cultures thereof.

[0047] The present application also provides a pharmaceutical composition, which may include the recombinant fusion protein of the present application, a PD-L1 antibody or its antigen-binding portion, a nucleic acid molecule, an expression vector or host cell, and at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition includes at least one pharmaceutically acceptable adjuvant.

[0048] The pharmaceutical composition of the present application can be used to treat diseases related to VEGF and / or PD-L1 overexpression / pathway, or to prepare drugs for treating diseases related to VEGF and / or PD-L1 overexpression / pathway.

[0049] In one aspect, the present application provides a method for treating or alleviating a disease associated with the VEGF and / or PD-L1 pathway / overexpression in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present application to the subject.

[0050] The pharmaceutical composition of the present application can be administered via intraperitoneal, subcutaneous and intravenous routes. In one embodiment, the pharmaceutical composition of the present application can be administered via intraperitoneal injection, subcutaneous injection and intravenous injection.

[0051] The disease associated with the VEGF and / or PD-L1 pathway / overexpression can be cancer, including solid cancer or blood cancer. The cancer can be acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, renal cell carcinoma, melanoma, glioblastoma multiforme, cervical cancer, or angiosarcoma.

[0052] The disease associated with VEGF and / or PD-L1 pathway / overexpression may be age-related macular degeneration (AMD), diabetic retinopathy, or liver fibrosis.

[0053] Based on the following detailed description and examples, other features and advantages of the present disclosure will be very clear, and the detailed description and examples should not be interpreted as being restrictive. All documents, Genbank registration numbers, patents and published patent applications cited in the specification are incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The following detailed description is given by way of example but is not intended to limit the present invention to the specific embodiments described, and can be better understood in conjunction with the accompanying drawings.

[0055] Figure 1 is a schematic diagram of the structure of the recombinant fusion proteins IMM2518 and IMM2519 of the present application. The circular structure at the top of the structure represents VEGFR1D2, which is connected to the N-terminus of the heavy chain (left figure, IMM2518) or light chain (right figure, IMM2519) of the PD-L1 antibody IMM2515H via a linker. VEGFR1D2 comprises the amino acid sequence set forth in SEQ ID NO: 11. The linker has the amino acid sequence set forth in SEQ ID NO: 12. The heavy chain of IMM2515H comprises the amino acid sequence set forth in SEQ ID NO: 15, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0056] FIG2 shows the binding activity of IMM2518 and IMM2519 to VEGF.

[0057] Figure 3 shows the relationship between IMM2518 and IMM2519 and PD-L1 + The binding activity of CHO was measured, IMM2515H was used as a positive control, and hIgG1-Fc was used as a negative control.

[0058] Figure 4 shows the relationship between IMM2518 and PD-L1 + The binding activity of CHO was analyzed. IMM2510, IMM2515H, and atelizumab were used as positive controls, and hIgG1-Fc was used as a negative control.

[0059] Figure 5 shows the effect of IMM2518 on PD-1 and PD-L1 + Blocking activity of CHO cell binding, IMM2510, IMM2515H, and atelizumab were used as positive controls, and hIgG1-Fc was used as a negative control.

[0060] Figure 6 shows the effects of IMM2518 on VEGF and VEGFR + For blocking activity of Jurkat binding, IMM2510, VEGFR1-Fc, and bevacizumab were used as positive controls, and hIgG was used as a negative control.

[0061] FIG7 shows the binding activity of IMM2518 to human PD-L1, rat PD-L1, mouse PD-L1, monkey PD-L1, and human PD-L2.

[0062] Figures 8A-8C show that IMM2518 triggers the expression of PD-L1 + ADCC activity of Raji cells (A), RKO cells (B), and HCC827 cells (C). IMM2515H, IMM2510, and atezolizumab were used as positive controls, and hIgG1-Fc was used as a negative control.

[0063] FIG9 shows the activity of IMM2518 in inducing ADCP on RKO cells, IMM2510, IMM2515H, and atezolizumab were used as positive controls, and hIgG1-Fc was used as a negative control.

[0064] FIG10 shows the changes in tumor size after administration of IMM2518 to tumor-bearing mice. IMM2510, IMM2515H, VEGFR1D2-Fc, and the combination of IMM2515H+VEGFR1D2-Fc were used as controls.

[0065] FIG11 shows the changes in tumor size in tumor-bearing mice after administration of IMM2518 via intraperitoneal, subcutaneous, and intravenous injections. DETAILED DESCRIPTION

[0066] In principle, there are three main approaches to targeting two or more pharmacological mechanisms of tumor growth. Most commonly, a combination of two or more different drugs can be given to the patient. While this option maximizes flexibility in possible drug combinations and dosages, it presents challenges: a) poor patient compliance due to the large number of drugs and their varying dosing schedules; b) potential incompatibilities due to drug-drug interactions; and c) an increased risk of drug side effects. These issues can reduce the effectiveness of treatment and hinder the achievement of treatment goals, particularly in the management of chronic diseases such as cancer.

[0067] The second approach is to use a fixed-dose combination of multiple drugs in a single dosage form. This approach reduces the medication burden and improves patient compliance. The main disadvantage of fixed-dose combinations is that the choice of possible dose ratios between the active ingredients is limited, making it more difficult to appropriately adjust the dose for individual patients to maximize efficacy and minimize adverse effects. Furthermore, the metabolic kinetics of the different drugs in the combination may lead to complex temporal shifts in drug effects in each target patient, thereby compromising overall efficacy.

[0068] A third approach involves the use of multifunctional drugs that combine two or more pharmacological mechanisms in a single molecule. The design and identification of these multifunctional molecules is more complex and requires extensive research to confirm the optimal ratio of target activity within the molecule, while the combined pharmacokinetics may result in matched pharmacokinetic activity at the molecular target level. Multifunctional molecules can also be engineered into fixed-dose combinations with other drugs, thereby combining three or even four pharmacological mechanisms in a single pill to produce further increases in efficacy.

[0069] After extensive experiments, the current inventors have invented a new recombinant multifunctional fusion protein that can attack tumors through three mechanisms of action: one is to relieve the PD-1-mediated inhibitory signal on immune cells such as T cells to check or inhibit; the second is to inhibit the formation of new blood vessels in the tumor microenvironment and relieve immunosuppression, thereby limiting the growth of target cells; and the third is to activate immune cells such as NK cells and macrophages to target cells such as PD-L1. + Killing of tumor cells.

[0070] The recombinant fusion protein of the present application comprises a PD-L1 antibody or an antigen-binding fragment thereof, wherein at least one paratope of the PD-L1 antibody or antigen-binding fragment thereof is connected to a VEGF-binding peptide via a linker at the N-terminus of the heavy chain or light chain constituting the paratope. The recombinant fusion protein can simultaneously bind to VEGF, PD-L1, and FcR, i) blocking the interaction between PD-L1 on the surface of cancer cells / tumor cells and PD-1 on the surface of immune cells such as T cells, thereby relieving the PD-1-mediated inhibitory signal on T cells, ii) binding to FcR on the surface of immune cells such as NK cells and macrophages to stimulate NK cells or macrophages to target cells such as PD-L1. + Killing of tumor cells; and iii) binding to, for example, VEGF in the immune microenvironment to reduce tumor angiogenesis and reduce blood and nutrient supply to tumor cells.

[0071] In one embodiment, at least one paratope of the PD-L1 antibody or its antigen-binding fragment is linked to a VEGF-binding peptide via a linker at the N-terminus of the heavy chain or light chain constituting the paratope. In another embodiment, each paratope of the PD-L1 antibody or its antigen-binding fragment is linked to a VEGF-binding peptide via a linker at the N-terminus of the heavy chain or light chain constituting the paratope. In one embodiment, each paratope of the PD-L1 antibody or its antigen-binding fragment is linked to a VEGF-binding peptide via a linker at the N-terminus of the heavy chain constituting the paratope. In one embodiment, each paratope of the PD-L1 antibody or its antigen-binding fragment is linked to a VEGF-binding peptide via a linker at the N-terminus of the light chain constituting the paratope. The recombinant fusion protein of the present application is small in size (150-180 kDa) and has a long half-life of 5-10 days.

[0072] The three main components included in the recombinant fusion protein of the present application are a VEGF binding peptide, a linker, and a PD-L1 antibody or an antigen-binding fragment thereof. Those skilled in the art will appreciate that there are many design options for the above three components. Preferably, human sequences are used in the treatment of human cancers because the strong immunogenicity of non-human animal proteins or peptides may cause allergic reactions and other adverse reactions. However, based on different application purposes, other animal proteins or peptides can also be used in the present application and can be humanized.

[0073] Any extracellular Ig-like domain of any VEGFR (VEGFR1, VEGFR2, and VEGFR3) that can bind to VEGF, particularly VEGF-A, can be used to construct the recombinant fusion protein of the present application. In one embodiment, the VEGFR in the recombinant fusion protein is VEGFR1, particularly the second extracellular Ig-like domain (VEGFR1D2) of VEGFR1.

[0074] VEGFR1D2 may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11, wherein VEGFR1D2 can bind to VEGF expressed by target cells, such as cancer cells / tumor cells, or in the vicinity of target cells, thereby limiting the growth of the target cells. In one embodiment, VEGFR1D2 may comprise the amino acid sequence set forth in SEQ ID NO: 11.

[0075] The linker primarily serves as a spacer between the VEGF binding peptide 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, preferably 5-30, 10-30, 10-20, or 15 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. One or more of these amino acids can be glycosylated or deglycosylated, as known to those skilled in the art. In one embodiment, 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 of amino acids that are mostly sterically hindered by empty bonds, such as glycine and alanine. Exemplary linkers are polyglycine (particularly Gly, poly(Gly-Ala)), and polyalanine. An exemplary suitable linker shown in the following examples is a GS linker, such as -(Gly-Gly-Gly-Gly-Ser)3- (SEQ ID NO: 12).

[0076] The linker can also be a non-peptide linker. For example, an alkyl linker such as -NH-, -(CH2)sC(O)-, where s = 2-20, can be used. These alkyl linkers can also be connected via any non-sterically hindered group such as a lower alkyl group (e.g., C 1-4 lower acyl), halogen (such as Cl, Br), CN, NH2, phenyl, etc.

[0077] In some embodiments, the PD-L1 antibody can be an isolated monoclonal antibody comprising two heavy chains and two light chains, each heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15, and each light chain comprising the amino acid sequence set forth in SEQ ID NO: 14. The Fab portion (or paratope) of the PD-L1 antibody can bind to PD-L1 on the surface of target cells, such as cancer cells / tumor cells, blocking the interaction between PD-L1 and PD-1 on the surface of immune cells, such as T cells, thereby relieving the immune cell from the inhibitory signal mediated by PD-1. The Fc portion of the PD-L1 antibody can bind to FcRs on the surface of immune cells, such as NK cells and macrophages, to stimulate targeted cell killing by NK cells or macrophages. In some embodiments, the heavy chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, wherein the PD-L1 antibody is capable of binding to PD-L1 and blocking the PD-1-PD-L1 interaction, and is capable of binding to FcR on the surface of NK cells or macrophages to activate NK cells and / or macrophages to kill cancer cells. In some embodiments, the light chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14, wherein the PD-L1 antibody is capable of binding to PD-L1 and blocking the PD-1-PD-L1 interaction.

[0078] The term "antibody" as used herein includes whole antibodies such as IgG, IgA, IgD, IgE, and IgM, and any antigen-binding fragment (or antigen-binding portion) or single chain thereof. Whole antibodies are glycoproteins comprising at least two heavy chains and two light chains, the heavy chains and light chains being connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region. The heavy chain constant region contains three domains, C H1 、C H2 and C H3 Each light chain comprises a light chain variable region (V L ) and the light chain constant region. The light chain constant region comprises a domain C L . V Hand V L The V region can be further divided into highly variable regions, namely CDR regions, with relatively conserved framework regions (FR) distributed between the CDR regions. H and V L It is composed of three CDRs and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that react with antigens. The constant region of an antibody can mediate the binding of immune proteins to host tissues or factors, including various immune system cells (such as effector cells) and the first component of the complement system (C1q). The "functional fragment" of the heavy chain constant region refers to the part of the heavy chain constant region that has the characteristics of binding to various immune system cells (such as effector cells) and the first component of the complement system (C1q), such as the Fc region, hinge-Fc region, etc.

[0079] An "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-L1 protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in an "antigen-binding fragment" of an antibody include (i) a Fab fragment, which consists of a V L 、V H 、C L and C H1 (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) V H and C H1 (iv) an Fd fragment consisting of an antibody single-arm V L and V H Fv fragment composed of V H (vi) an isolated complementarity determining region (CDR); and (vii) a fragment comprising a heavy chain variable region, a light chain variable region, and an Fc region or a hinge-Fc region. In addition, although the two domains V L and V H Encoded by different genes, they can be recombinantly linked via a synthetic linker that makes the two into a single protein chain, where V L and V H The antigen-binding fragments are obtained by conventional techniques known to those skilled in the art, and the fragments can be screened for function in the same manner as intact antibodies.

[0080] The heavy chain variable region CDRs and light chain variable region CDRs of the antibodies or antigen-binding fragments thereof of the present application are identified by the IMGT numbering system. As is well known in the art, heavy chain variable region and light chain variable region CDRs can be identified by, for example, the Chothia, Kabat, AbM or Contact numbering systems / methods.

[0081] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively lyse target cells, such as cancer cells, that are bound to cell membrane surface antigens and antibodies, such as PD-L1 antibodies, or to VEGF-binding peptides.

[0082] The term "antibody-dependent cell-mediated phagocytosis" or "ADCP" refers to the binding of an antibody to an antigen epitope on a target cell via its Fab segment and the binding of its Fc segment to the FcR of a macrophage or neutrophil, thereby promoting the phagocytosis of the target cell by the macrophage or neutrophil.

[0083] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows and horses, are preferred.

[0084] The term "EC 50 ", also known as half-maximal effect concentration, refers to the concentration of molecules (such as antibodies, fusion proteins, etc.) that cause 50% of the maximum effect.

[0085] The term "IC 50 "" refers to the half-inhibitory concentration, which is the concentration of an inhibitor (such as an antibody, fusion protein, etc.) required to inhibit a specified biological process by half.

[0086] The term "therapeutically effective amount" refers to an amount of a molecule (e.g., an antibody, a fusion protein) sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). The therapeutically effective amount is related to the disease being treated, and those skilled in the art can readily determine the actual effective amount.

[0087] As used herein, "sequence identity" refers to the percentage of nucleotides / amino acids in a sequence that are identical to the nucleotides / amino acid residues in a reference sequence after sequence alignment, with spaces introduced, if necessary, to achieve the maximum percentage of sequence identity between the two sequences. A person skilled in the art can perform pairwise sequence alignment or multiple sequence alignment to determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences by various methods, such as using computer software such as ClustalOmega, T-coffee, Kalign, and MAFFT.

[0088] At the same time, the present application provides polynucleotides encoding recombinant fusion proteins, PD-L1 antibodies or antigen-binding portions thereof, and expression vectors expressing the recombinant fusion proteins. Examples of vectors include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transformable artificial chromosomes (TACs), mammalian artificial chromosomes (MACs), and artificial episomal chromosomes (HAECs).

[0089] The application provides host cells comprising the above-mentioned expression vectors. Host cells can be transformed or transfected with the expression vectors. Suitable host cells include Escherichia coli (E. coli), yeast and other eukaryotic organisms. Preferably, E. coli, yeast or mammalian cell lines (e.g., COS or CHO) are used.

[0090] On the other hand, the present application provides a pharmaceutical composition comprising the recombinant fusion protein, PD-L1 antibody or its antigen-binding portion, nucleic acid molecule, expression vector or host cell of the present application formulated together with a pharmaceutically acceptable carrier. The pharmaceutical composition may contain an excipient and / or an adjuvant. The composition may optionally contain one or more other pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present application may also be administered in combination therapy with, for example, another immunostimulant, anticancer drug, antiviral agent or vaccine.

[0091] The pharmaceutical composition may contain any number of excipients. Useful excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, stabilizers, colorants, flavorings, 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.

[0092] The main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection, physiological saline or artificial cerebrospinal fluid, which can be supplemented with other materials common in injection. For example, the vehicle or carrier can be a neutral buffered saline solution or a saline solution mixed with serum albumin. Other exemplary pharmaceutical compositions include Tris buffer or acetate buffer, which can also include sorbitol or its suitable substitute. In one embodiment of the application, the composition can be prepared for storage in the form of lyophilized or aqueous solution by mixing the selected components with the required purity with any formulation (Remington's Pharmaceutical Sciences, as above). In addition, the therapeutic composition can be formulated as a lyophilized agent using a suitable excipient such as sucrose.

[0093] The pharmaceutical composition can be used for intravenous, intramuscular, subcutaneous, non-intestinal, spinal or epidermal administration (for example, by injection or push injection).Depending on the difference of route of administration, active molecule can be wrapped in material to protect it from the effect of acid and other natural conditions that may inactivate it.Term used herein " non-parenteral administration " refers to the mode of administration except the intestinal tract and topical administration that are usually carried out by injection, including but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, dura mater and intrasternal injection and infusion.Or, the antibody of the present application can be administered by non-injection route, for example local, epidermal or mucosal mode of administration, for example, intranasal, oral, vaginal, rectal, sublingual, or topical.In some embodiments, the pharmaceutical composition of the present application is used through peritoneal, subcutaneous or intravenous route.

[0094] 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 drug concentrations.

[0095] The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form varies depending on the subject to be treated and the specific route of administration, and is generally that amount of the composition that produces a therapeutic effect. Generally speaking, in terms of percentage, this amount is about 0.01% to about 99% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0096] The dosage regimen can be adjusted to achieve the optimal desired response (e.g., therapeutic response). For example, multiple divided doses are administered over time, or the dose is proportionally reduced or increased according to the severity of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage units for ease of administration and for uniformity of dosage. The dosage unit type used herein refers to a physically separate unit suitable for a single administration to a subject to be treated; each unit contains the amount of active compound calculated in advance to produce the desired therapeutic effect together with the drug carrier. Alternatively, the fusion protein can be administered in a sustained release dosage form, in which case the frequency of administration is reduced.

[0097] For administration of the fusion protein, the dosage range is about 0.0001-100 mg / kg recipient body weight. An exemplary treatment schedule is twice a week.

[0098] A "therapeutically effective amount" of the present fusion protein preferably causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevents damage or disability caused by the disease. For example, for the treatment of tumor-bearing subjects, a "therapeutically effective amount" means that, relative to untreated subjects, preferably, tumor growth is inhibited by at least about 40%, more preferably by at least about 60%, more preferably by at least about 80%, and more preferably by at least about 99%. A therapeutically effective amount of the present fusion protein can reduce tumor volume or alleviate symptoms in a subject (typically a human, or another mammal).

[0099] The pharmaceutical composition can be a controlled 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. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0100] The pharmaceutical compositions 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) osmotic devices (U.S. Patents 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

[0101] In certain embodiments, the fusion proteins of the present invention can be formulated to ensure suitable in vivo distribution. For example, to ensure that the therapeutic fusion proteins of the present invention cross the blood-brain barrier, the fusion proteins are formulated in liposomes and can additionally include targeting groups to enhance selective delivery to specific cells or organs. See, for example, U.S. Patents 4,522,811, 5,374,548, 5,416,016, and 5,399,331.

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

[0103] The compositions of the present disclosure can be used alone or in combination with other therapeutic agents to enhance their therapeutic efficacy or reduce potential side effects.

[0104] Another object of the present application is to provide a method for preparing the above-mentioned recombinant fusion protein, PD-L1 antibody or its antigen-binding portion. In one embodiment, the preparation method comprises the following steps: (1) providing a nucleic acid molecule encoding a recombinant fusion protein, PD-L1 antibody or its antigen-binding portion; (2) constructing an expression vector comprising the nucleic acid molecule of (1); (3) transfecting or transforming suitable host cells with the expression vector in (2) and culturing these host cells to express the recombinant fusion protein, PD-L1 antibody or its antigen-binding portion; and (4) purifying the recombinant fusion protein, PD-L1 antibody or its antigen-binding portion. The preparation can be performed by a person of ordinary skill in the art using well-known techniques.

[0105] Another object of the present application is to provide a method for treating cancer using the pharmaceutical composition of the present application, comprising administering an effective amount of the above-mentioned pharmaceutical composition to a patient or subject in need. In one embodiment, the pharmaceutical composition is used to treat cancers with VEGF and / or PD-L1 pathways / overexpression, including but not limited to acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, renal cell carcinoma, melanoma, glioblastoma multiforme, cervical cancer, and angiosarcoma.

[0106] The present application also provides methods of using the pharmaceutical compositions of the present application to treat other diseases related to the VEGF and / or PD-L1 pathway / overexpression, including age-related macular degeneration (AMD), diabetic retinopathy, and liver fibrosis.

[0107] The present application will be further described with reference to the following non-limiting examples.

[0108] Example

[0109] The structures of the exemplary fusion proteins IMM2518 and IMM2519, as well as the control proteins of the present application are briefly described below.

[0110] IMM2515H is an IgG antibody that specifically binds to PD-L1 and comprises two heavy chains and two light chains. The amino acid sequences of the heavy chain variable region, heavy chain constant region, light chain variable region, and light chain constant region are set forth in SEQ ID NOs: 7, 9, 8, and 10, respectively. The amino acid sequences of the heavy and light chains are set forth in SEQ ID NOs: 15 and 14, respectively.

[0111] IMM2518 is an exemplary recombinant fusion protein of the present application, comprising two VEGFR1D2s (SEQ ID NO: 11) connected to the N-termini of two heavy chains of IMM2515H via a linker (SEQ ID NO: 12), as shown in Figure 1. That is, IMM2518 comprises two long chains as shown in SEQ ID NO: 13, and two short chains as shown in SEQ ID NO: 14.

[0112] IMM2519 is an exemplary recombinant fusion protein of the present application, comprising two VEGFR1D2s (SEQ ID NO: 11) connected to the N-termini of two light chains of IMM2515H via a linker (SEQ ID NO: 12), as shown in Figure 1. That is, IMM2519 comprises two long chains as shown in SEQ ID NO: 15, and two short chains as shown in SEQ ID NO: 16.

[0113] IMM2510 is a recombinant fusion protein with a similar structure to IMM2518, except that it contains two long chains as shown in SEQ ID NO: 17 and two short chains as shown in SEQ ID NO: 18.

[0114] VEGFR1D2-Fc is a fusion protein of VEGFR1D2 and Fc, comprising the amino acid sequence shown in SEQ ID NO:19.

[0115] Example 1. Construction of expression vector for recombinant fusion protein

[0116] 1.1 IMM2518

[0117] The full-length coding sequence of IMM2518 was artificially designed. Specifically, for the long chain, 57 nucleotides encoding the mouse IgG1 heavy chain signal peptide (SEQ ID NO: 21) were added to the 5' end of the VEGF binding peptide-linker-PD-L1 antibody heavy chain coding sequence (SEQ ID NO: 23), and the Kozak sequence (GCCGCCACC) was added to the 5' end of the signal peptide sequence. Finally, HindIII and NheI restriction enzyme sites were added to the 5' and 3' ends of the resulting sequence, respectively. For the short chain, the same signal peptide sequence and Kozak sequence were added to the PD-L1 light chain coding sequence (SEQ ID NO: 24), and HindIII and XbaI restriction enzyme sites were added to the 5' and 3' ends of the resulting sequence, respectively. The two resulting sequences were artificially synthesized and subcloned into pMac-H and pMac-L vectors, respectively.

[0118] 1.2 IMM2519

[0119] The full-length coding sequence of IMM2519 was artificially designed. Specifically, for the long chain, 57 nucleotides encoding the mouse IgG1 heavy chain signal peptide (SEQ ID NO: 21) were added to the 5' end of the PD-L1 antibody heavy chain coding sequence (SEQ ID NO: 25), and the Kozak sequence (GCCGCCACC) was added to the 5' end of the signal peptide sequence. Finally, HindIII and NheI restriction enzyme sites were added to the 5' and 3' ends of the resulting sequence, respectively. For the short chain, the same signal peptide sequence and Kozak sequence were added to the VEGF binding peptide-linker-PD-L1 light chain coding sequence (SEQ ID NO: 26), and HindIII and XbaI restriction enzyme sites were added to the 5' and 3' ends of the resulting sequence, respectively. The two resulting sequences were artificially synthesized and subcloned into pMac-H and pMac-L vectors, respectively.

[0120] Example 2. Preparation and quality analysis of recombinant fusion protein

[0121] The constructed expression vector was used to transiently express the protein in CHO-S cells. The general process is as follows: 1) The day before transient transfection, CHO-S cells were cultured at a rate of 1×10 6 1) Prepare the DNA at a density of 1 μg / ml for each heavy / light (long / short) chain expression vector in a 1:1 ratio and add it to 1 / 20 the volume of OPTI-MEM medium (Gibco). 2) Prepare the DNA at a density of 1 μg / ml for each heavy / light (long / short) chain expression vector in a 1:1 ratio and add it to 1 / 20 the volume of OPTI-MEM medium (Gibco). 3) Prepare PEI (MW 40,000 polyethyleneimine hydrochloride, Polysciences) at a 1 mg / ml ratio of 4:1 for each PEI:DNA and add it to 1 / 20 the volume of OPTI-MEM medium (Gibco). 4) Slowly add the PEI dilution to the DNA dilution, mix well, and incubate at room temperature for 20 minutes. 5) Add the PEI / DNA mixture to the cell suspension and incubate the cells in a 37°C, 5% CO2, 110 rpm incubator with shaking. 6) Add a transfection enhancer (1 mM sodium butyrate, 0.25% v / v DMSO) and simultaneously lower the culture temperature to 33 degrees; 7) When the cell viability dropped below 50%, centrifuge at 3000 rpm for 5 minutes, collect the supernatant, and perform affinity purification using Protein A filler.

[0122] The SEC-HPLC spectrum showed that IMM2518 had high purity, low aggregate content and low degradation amount, as shown in Table 1.

[0123] Table 1. SEC-HPLC results of the recombinant fusion protein of this application

[0124] Example 3. Recombinant fusion protein binds to VEGF-165

[0125] For VEGF binding, recombinant human VEGF-165 (Cat#11066-HNAH, Sino Biologicals) was prepared in carbonate-bicarbonate coating buffer (Cat#C3041, Sigma-Aldrich) and transferred to an ELISA plate at 50 ng / well. The plate was placed in a 4°C refrigerator overnight. Blocking was performed with 150 μL / well of 3% skim milk for 2 hours at room temperature. 100 μL of serially diluted recombinant fusion protein was then added, and the plate was incubated at 37°C for 1 hour. The plate was then washed five times with PBS-T containing 0.05% Tween 20. HRP-rabbit anti-human IgG Fc (Cat#:309-036-008, Jackson ImmunoResearchLab) was then added to the plate, and the plate was incubated at 37°C for 1 hour. After washing the plate five times with PBS-T, 100 μL of TMB color development solution was added to each well, and 500 μL of 1N H 2 SO 4 was added to stop color development before reading in a microplate reader.

[0126] As shown in FIG2 , the two recombinant fusion proteins of the present application have similar VAGF-165 binding activities.

[0127] Example 4. Recombinant fusion protein binds to PD-L1

[0128] For PD-L1 binding analysis, 100 μl of 1 × 10 6 100 μl of serially diluted recombinant fusion protein and control proteins IMM2515H and hIgG1-Fc (SEQ ID NO: 20) were incubated at 4°C for 40 minutes. After washing with cold PBS, the cells were incubated with FITC-conjugated anti-human IgG-Fc antibody (Cat # F9512, Sigma) for 40 minutes. The cells were then washed twice and subjected to FACS analysis.

[0129] The results are shown in Figure 3. The PD-L1 + The cell binding activity was 2.6-fold and 4.2-fold lower than that of the monospecific PD-L1 antibody IMM2515H, respectively.

[0130] At the same time, the binding of the recombinant fusion protein and the control proteins IMM2515H, IMM2510, atelizumab, and hIgG1-Fc to CHO-PD-L1 was also analyzed using the same procedures as above.

[0131] As shown in Figure 4, PD-L1 of IMM2518+ The cell binding activity is similar to that of IMM2510.

[0132] Example 5. Binding affinity of recombinant fusion protein to VEGF and PD-L1

[0133] The Kinetics Assay mode was selected on a label-free biomolecular analyzer (Access Medical Systems, Gator bio). Q buffer (10 mM, pH 7.4, PBS + 0.02% Tween + 0.2% BSA) was used as the assay buffer. A human Fc probe was used as the assay probe. IMM2518 was initially diluted at a concentration of 10 μg / ml and then serially diluted 2-fold to five concentrations. 10 μg / ml VEGF165 (Cat#11066-HNAH, Sino Biological) and 10 μg / ml PD-L1-his (Cat#90251-C08H, Sino Biological) were used as antigens. The probe was regenerated using regeneration buffer (10 mM Gly + 150 mM NaCl, pH 2.0) at pH 2.0. The instrument temperature was set at 25°C.

[0134] Table 2. Affinity of recombinant fusion proteins to VEGF and PD-L1 detected by BLI technology

[0135] The test results showed that after binding to VEGF165, IMM2518 dissociated very slowly, and no specific dissociation constant was obtained. The affinity between IMM2518 and PD-L1 was 0.441nM.

[0136] Example 6. Blocking activity of recombinant fusion protein on PD-1-PD-L1

[0137] For the analysis of PD-1-PD-L1 blocking activity, 50 μl of 5 × 10 5 / ml CHO-PD-L1 (Cat# YMAK-C006, ImmuneOnco) was incubated with 50 μl of serially diluted recombinant fusion protein and control proteins IMM2515H, IMM2510, atelizumab, and hIgG1-Fc at 4°C for 45 minutes. 50 μl of PD1-mFc (prepared in-house at ImmuneOnco, SEQ ID NO: 22) was then added and incubated at 4°C for 45 minutes. After washing with cold PBS, the cells were incubated with PE-conjugated anti-mouse IgG-Fc antibody (Biolegend, Cat# 405307). Afterwards, the cells were washed twice and subjected to FACS analysis.

[0138] As shown in Figure 5, IMM2518 has a negative effect on PD-1 protein and PD-L1 + The cell binding blocking ability is 2.2-fold and 3-fold lower than that of IMM2515H and atilizumab, respectively, and is similar to that of IMM2510.

[0139] Example 7. Blocking of VEGF activity by recombinant fusion protein

[0140] The inhibition of VEGF activity was evaluated using Jurkat-CVR cells constructed by ImmuneOnco. CVR (chimeric VEGFR1D2 receptor) consists of the second extracellular domain of VEGFR1, the hinge region of CD8α, the transmembrane and intracellular regions of CD28, and the signal transduction region of CD3z, and contains the amino acid sequence shown in SEQ ID NO:6. 50 μl of 5×10 5 100 μl of Jurkat-CVR were incubated with 50 μl of serially diluted recombinant fusion protein and control proteins VEGFR1D2-Fc (SEQ ID NO: 19), IMM2510, bevacizumab, and hIgG1-Fc at 37°C for 45 minutes. 50 μl of VEGF-Fc (prepared in-house at ImmuneOnco, SEQ ID NO: 27) was then added and incubated overnight at 37°C. After washing with cold PBS, the cells were incubated with PE-conjugated anti-human CD69 antibody (Biolegend, Cat# 310906). The cells were then washed twice and analyzed by FACS.

[0141] As shown in Figure 6 , the blocking ability of MM2518 on VEGF activity was slightly lower than that of VEGFR1D2-Fc and bevacizumab, and similar to that of IMM2510.

[0142] Example 8. Binding specificity of recombinant fusion protein to PD-L1

[0143] Human PD-L1 (Cat#10084-H08H, Sino Biological), mouse PD-L1 (Cat#50010-M08H, Sino Biological), monkey PD-L1 (Cat#90251-C08H, Sino Biological), rat PD-L1 (Cat#80450-R08H, Sino Biological), and human PD-L2 (Cat#10292-H08H, Sino Biological) were coated in carbonate-bicarbonate coating buffer (Cat#C3041, Sigma-Aldrich) at 50 ng / well and placed in a 4°C refrigerator overnight. Block with 150 μL / well 3% skim milk at room temperature for 2 hours. 100 μL of serially diluted recombinant fusion protein was then added, the plate was incubated at 37°C for 1 hour, and washed five times with PBS-T containing 0.05% Tween 20. HRP-rabbit anti-human IgG Fc (Cat#: 309-036-008, Jackson ImmunoResearchLab) was added to the plate and incubated at room temperature for 1 hour. After washing the plate five times with PBS-T, 100 μL of TMB color development solution was added to each well, and 500 μL of 1N H2SO4 was added to stop color development before reading on a microplate reader.

[0144] The results are shown in Figure 7. IMM2518 binds to human PD-L1 and cross-binds to monkey PD-L1, but has no cross-binding to rat PD-L1, mouse PD-L1, or human PD-L2.

[0145] Example 9. Recombinant fusion protein induces antibody-dependent cell-mediated cytotoxicity (ADCC)

[0146] Raji-PD-L1 cells labeled with CFSE fluorescent dye (Cat#21888-25mg, Sigma), 6×10 5 / ml, 50μl, and 100μl 6×10 5NK92MI cells (Cat#CRL-2408, ATCC) stably expressing FcγRIIIa(158V) were mixed at a 1:2 ratio with 50 μl of serially diluted IMM2518 or IMM2515H per ml of 5% CO2 at 37°C for 4 hours. Propidium iodide (PI) (Cat#P4170, Sigma) was then added to the cell culture medium to a final concentration of 5 μg / ml, and the cell culture medium was analyzed by FACS for PI signals from CFSE-labeled target cells. The percentage of cell lysis due to ADCC was calculated based on the following formula: % Lysis (or %ADCC) = (%PI-positive cells treated with IMM2518 or positive control - %PI-positive cells treated with negative control protein) / (100 - %PI-positive cells treated with negative control protein) * 100.

[0147] The results are shown in Figure 8A. The ADCC activity of IMM2518 against Raji-PD-L1 cells was nearly 6-fold lower than that of IMM2515H.

[0148] RKO cells (Cat#TCHu116, Cell Bank of the Chinese Academy of Sciences) or HCC827 cells (Cat#TCHu153, Cell Bank of the Chinese Academy of Sciences) were trypsinized and 5×10 5 / ml, 50μl, and 50μl 5×10 5 NK92MI cells (Cat#CRL-2408, ATCC) stably expressing FcγRIIIa(158V) were mixed at a 1:1 ratio with 50 μl of serially diluted IMM2518, IMM2515H, IMM2510, atezolizumab, or hIgG1-Fc at 37°C overnight under 5% CO2. 20 μl of CCK-8 (Biyuntian, Cat#C0039) was then added to the cell culture medium, and incubation continued in the incubator for 3 hours. The absorbance was measured at a wavelength of 450 nm. The percentage of cell lysis due to ADCC was calculated based on the following formula: % lysis (or % ADCC) = (OD450 value of effector cells and target cells in the negative control group - OD450 value of effector cells and target cells treated with IMM2518 or the positive control) / (OD450 value of effector cells and target cells in the negative control group - OD450 value of effector cells in the negative control) * 100%.

[0149] As shown in FIG8B and FIG8C , the ADCC activity of IMM2518 against RKO cells and HCC827 cells was 6-fold and 33-fold weaker than that of IMM2515H, respectively.

[0150] Example 10. Recombinant fusion protein induces antibody-dependent cell-mediated phagocytosis (ADCP)

[0151] 2.5×10 5 / ml THP-1 (Cat#TCHu 57, Cell Bank of the Chinese Academy of Sciences) was seeded in 96-well flat-bottom plates in 100 μl complete medium containing PMA (Sigma, Cat#379346-1MG) at a working concentration of 200 ng / ml and cultured for 48 hours. RKO cells (Cat#TCHu116, Cell Bank of the Chinese Academy of Sciences) were trypsinized and labeled with CFSE (Cat#21888-25mg, Sigma) as target cells. CFSE-labeled RKO cells, 5.0×10 5 100 μl of the solution was mixed with THP-1 effector cells seeded into a 96-well plate at a 1:2 effector-target ratio. The mixed cells were incubated with 50 μl of serially diluted IMM2518, IMM2515H, IMM2510, atezolizumab, or hIgG1-Fc at 37°C for 2 hours under 5% CO2. The supernatant was discarded and the cells were washed five times with 250 μl / well PBS buffer to remove target cells not adsorbed to the plate bottom. After washing, the THP-1 cells were repeatedly suspended by pipetting and the CFSE signal of THP-1 macrophages was analyzed by FACS.

[0152] As shown in FIG9 , IMM2518, IMM2515H, and IMM2510 had similar phagocytic activities against RKO tumor cells.

[0153] Example 11. Recombinant fusion protein has good anti-tumor effect

[0154] The in vivo anti-tumor efficacy of the recombinant fusion protein of this application was evaluated using C57BL / 6 hPD-1 humanized mice (Gempharmtech). Mouse colon cancer cell line MC38-hPD-L1 (WuXi AppTec) was cultured as a monolayer in vitro and passaged twice weekly using trypsin-EDTA for routine digestion. When cell saturation reached 80%-90% and the required number of cells was reached, cells were harvested and counted.

[0155] Contains 3×10 5 100 μL PBS of MC38-hPD-L1 cells was inoculated subcutaneously in the right forelimb axilla of mice. The average tumor volume was 60-90 mm. 336 animals within the range were randomly divided into 6 groups of 6 mice each, and this day was set as day 0. From this day on, mice were intraperitoneally injected with DPBS, IMM2510 (6.0 mg / kg), IMM2518 (6.0 mg / kg), IMM2515H (5.0 mg / kg), VEGFR1D2-Fc (2.5 mg / kg), and a combination of IMM2515H (5.0 mg / kg) and VEGFR1D2-Fc (2.5 mg / kg) twice a week for 4 weeks. Tumor volume and animal body weight were measured twice a week.

[0156] Tumor volume (V) was calculated as (length × width 2 ) / 2. The tumor growth inhibition rate (TGI) was calculated using the following formula: tumor growth inhibition rate = (1-tumor volume change in the treatment group / tumor volume change in the control group) × 100%.

[0157] The results are expressed as mean ± SEM. Comparisons between two groups were performed by Dunnett's multiple comparison, with P < 0.05 considered significant.

[0158] As shown in Figure 10, compared to DPBS, mice treated with a single targeted drug or a combination of two targeted drugs showed slower tumor growth, while mice treated with IMM2510 and IMM2518 showed significantly slower tumor growth. IMM2518 demonstrated a higher anti-tumor efficacy than IMM2510, with a TGI of 74.07% for IMM2510 and 97.47% for IMM2518.

[0159] Example 12. Antitumor effects of recombinant fusion proteins under different administration methods

[0160] According to the experimental steps of Example 11, the in vivo anti-tumor effect of the recombinant fusion protein at a lower dose and administered in different ways was tested.

[0161] Contains 3×10 5 100 μL PBS of MC38-hPD-L1 cells was inoculated into the right forelimb axilla of C57BL / 6hPD-1 humanized mice. 3 Animals within the range of 100 mg / kg were randomly divided into 4 groups of 6 mice each, and this day was set as day 0. From this day on, mice in each group received DPBS intraperitoneal (IP) injection, IMM2518 (4.0 mg / kg) intraperitoneal (IP) injection, IMM2518 (4.0 mg / kg) subcutaneous (SC) injection, and IMM2518 (4.0 mg / kg) tail vein (IV) injection twice a week for 4 weeks. Tumor volume and animal body weight were measured twice a week.

[0162] The tumor sizes of mice in each group during the experiment are shown in Figure 11. It can be seen that compared with DPBS, administration of a lower dose of IMM2518 still significantly inhibited tumor growth, and the effects of intraperitoneal injection, subcutaneous injection, and intravenous injection were comparable.

[0163] The sequence information of this application is summarized below.

[0164] Although the present application has been described in conjunction with one or more embodiments, it should be understood that the present application is not limited to these embodiments. The description in this application is intended to cover all variants and equivalents, all of which are included in the subject matter and scope of the appended claims. All documents cited in this article are incorporated herein by reference in their entirety.

Claims

1. A recombinant fusion protein comprising a PD-L1 antibody or an antigen-binding fragment thereof and a VEGF-binding peptide, The PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, a heavy chain constant region, and a light chain variable region, wherein the heavy chain variable region comprises HV-CDR1, HV-CDR2, and HV-CDR3, and the light chain variable region comprises LV-CDR1, LV-CDR2, and LV-CDR3, wherein HV-CDR1, HV-CDR2, HV-CDR3, LV-CDR1, and LV-CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, and 5, respectively, and LV-CDR2 comprises the amino acid sequence shown in "YTS", wherein the heavy chain constant region is connected to the C-terminus of the heavy chain variable region and has FcR binding ability, The VEGF-binding peptide comprises the second extracellular Ig-like domain (VEGFR1D2) of vascular endothelial growth factor receptor 1 (VEGFR1), 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 fragment thereof. 2 . The recombinant fusion protein according to claim 1 , wherein the VEGF-binding peptide comprises the amino acid sequence shown in SEQ ID NO:

11.

3. The recombinant fusion protein according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8. 4 . The recombinant fusion protein according to claim 1 , wherein the VEGF-binding peptide is linked to the N-terminus of the heavy chain variable region of the PD-L1 antibody or antigen-binding fragment thereof. The recombinant fusion protein according to claim 1 , wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

9. The recombinant fusion protein according to claim 1 , wherein the VEGF-binding peptide is linked to the PD-L1 antibody or antigen-binding fragment thereof via a linker. The recombinant fusion protein according to claim 6 , wherein the linker is -(Gly-Gly-Gly-Gly-Ser) 3 -(SEQ ID NO: 12).

8. The recombinant fusion protein according to claim 1, further comprising a light chain constant region, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO: 10 and is connected to the C-terminus of the light chain variable region.

9. The recombinant fusion protein according to claim 1, comprising: i) a VEGF-binding peptide-linker-PD-L1 antibody heavy chain variable region-heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 13, and a PD-L1 antibody light chain variable region-light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 14; or ii) a PD-L1 antibody heavy chain variable region-heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 15, and a VEGF-binding peptide-linker-PD-L1 antibody light chain variable region-light chain constant region comprising the amino acid sequence shown in SEQ ID NO:

16.

10. A nucleic acid molecule encoding the recombinant fusion protein according to any one of claims 1 to 9. An expression vector comprising the nucleic acid molecule according to claim 10 . 12 . A host cell comprising the expression vector according to claim 11 , or having the nucleic acid molecule according to claim 10 integrated into its genome.

13. A pharmaceutical composition comprising the recombinant fusion protein of any one of claims 1 to 9, the nucleic acid molecule of claim 10, the expression vector of claim 11, or the host cell of claim 12, and at least one pharmaceutically acceptable carrier.

14. Use of the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating a disease associated with the PD-L1 and / or VEGF pathway, wherein the disease is cancer, age-related macular degeneration (AMD), diabetic retinopathy, or liver fibrosis.

15. The method of claim 14, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, renal cell carcinoma, melanoma, glioblastoma multiforme, cervical cancer, and angiosarcoma.

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