COMBINATION THERAPEUTIC COMPOSITIONS COMPRISING VEGF-Grab AND PD-1 OR PD-L1 ANTAGONISTS

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, which contains VEGF-Grab as an active ingredient and is intended to be administered in combination with a PD-1 or PD-L1 antagonist.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising VEGF-Grab as an active ingredient, for co-administration with a PD-1 or PD-L1 antagonist. Background Art

[0002] In order to proliferate and grow cancer cells, new blood vessels are needed to supply them with oxygen and nutrients, and vascular endothelial growth factor (VEGF) is known to play a key role in the formation of new blood vessels. VEGF is a dimer of about 46KDa composed of two subunits, and five types of VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF) have been identified in mammals. These VEGFs bind to three receptor tyrosine kinases (RTKs), namely VEGF receptor (VEGFR)-1, VEGFR-2 and VEGFR-3. These VEGF receptors trigger cell migration, survival and proliferation, and have the ability to transmit signals that form three-dimensional blood vessels that do not exist in other RTKs, or regulate vascular permeability.

[0003] VEGF has been used as a target for cancer therapy since it was discovered that VEGF expression is increased in tumor cells and VEGF receptors are increased in tumor-infiltrating vascular endothelial cells, but that VEGF and its receptors are expressed at low levels in normal cells not involved in angiogenesis.

[0004] Therefore, new anticancer therapies have been developed to prevent angiogenesis that nourishes cancer cells, rather than the cancer cells themselves, and it has been reported that anti-VEGF receptor antibodies, soluble receptor constructs, antisense, RNA aptamers against VEGF, and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors can be used to interfere with VEGF signaling. In fact, anti-VEGF neutralizing antibodies have been shown to inhibit the growth of various human tumor cell lines in nude mice (Warren et al. J. Clin. Invest. 95: 1789-1797 (1995)). In addition, patent literature related to VEGF inhibitors discloses a variety of VEGF inhibitors, such as quinazoline derivatives as VEGF inhibitors (U.S. Patent No. 9040548), inhibitors of VEGF receptor and HGF receptor signaling for treating angiogenesis-mediated cell proliferative diseases or inhibiting the growth of solid tumors (U.S. Patent No. 8470850), substances that inhibit angiogenesis by interfering with the binding of VEGF to its receptors, all of which are used to treat diseases such as cancer (Korean Patent Publication No. 2003-0075947).

[0005] On the other hand, the body's immune system regulates overall T lymphocyte function by simultaneously regulating these co-stimulatory and co-inhibitory signals and antigen recognition. This regulatory mechanism is called immune checkpoints. The body's immune system detects tumor-specific neoantigens expressed by changes such as mutations in tumor cells and eliminates tumor cells or viral infectious agents.

[0006] However, some tumor cells try to avoid this immune attack by changing the tumor microenvironment to suppress immune function or by immune escape through T cell immune tolerance or immune editing.

[0007] One such escape strategy is to inhibit the function of tumor-specific T lymphocytes by changing the function of immune checkpoints, that is, activating inhibitory immune checkpoints on tumor cells to avoid the attack of tumor-specific T lymphocytes. In this regard, monoclonal antibodies against PD-1 or its ligand PD-L1 can be used to inhibit its function, thereby enhancing the activity and effectiveness of suppressed tumor-specific T lymphocytes to achieve anti-tumor effects.

[0008] Based on this technical background, the inventors of the present invention discovered that the combination of VEGF-Grab and PD-1 or PD-L1 antagonists delayed tumor growth and prevented cancer metastasis when studying cancer immunotherapy, thereby completing the present invention.

[0009] The above information in this related background section is only intended to enhance relative understanding of the present invention and may not include prior art information that is already known to one of ordinary skill in the art. Summary of the invention

[0010] The object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising VEGF-Grab as an active ingredient, for co-administration with a PD-1 or PD-L1 antagonist.

[0011] Another object of the present invention is to provide a method for preventing or treating cancer, by administering VEGF-Grab or a pharmaceutical composition comprising the same as an active ingredient in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same.

[0012] Another object of the present invention is to provide the use of VEGF-Grab or a pharmaceutical composition comprising the same as an active ingredient for preventing or treating cancer, in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same.

[0013] Another object of the present invention is to provide the use of VEGF-Grab or a pharmaceutical composition comprising the same as an active ingredient in the preparation of a medicament for preventing or treating cancer, in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same.

[0014] To achieve the above objectives, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a fusion protein as an active ingredient, wherein the fusion protein comprises vascular endothelial growth factor receptor 1 (VEGFR1) domain 2, VEGFR1 domain 3 and the Fc region of an antibody, for co-administration with a PD-1 or PD-L1 antagonist.

[0015] The present invention also provides a method for preventing or treating cancer, comprising administering VEGF-Grab or a pharmaceutical composition comprising the same as an active ingredient in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same.

[0016] The present invention also provides the use of VEGF-Grab or a pharmaceutical composition comprising the same as an active ingredient for preventing or treating cancer, in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same.

[0017] The present invention also provides a use of a pharmaceutical composition comprising VEGF-Grab or a pharmaceutical composition comprising the same as an active ingredient in the preparation of a medicament for preventing or treating cancer, in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 An overview of the combination immunotherapy of PB101 with a PD-L1 immune checkpoint inhibitor in the MC38 colon cancer model is provided.

[0019] Figure 2 The tumor growth retardation effect in the PB101 and αPD-L1 combination group is illustrated.

[0020] Figure 3 An overview of the combination immunotherapy of PB101 with a PD-L1 immune checkpoint inhibitor in the Hepa-VEGF liver cancer model is provided.

[0021] Figure 4 The tumor growth retardation effect in the PB101 and αPD-L1 combination group is illustrated.

[0022] Figure 5 A survival benefit in the PB101 and αPD-L1 combination group is illustrated.

[0023] Figure 6 An overview of the cancer cell rechallenge experiment after combined immunotherapy of PB101 and PD-L1 immune checkpoint inhibitor in the Hepa-VEGF liver cancer model is shown.

[0024] Figure 7Results demonstrating that the cancer in the combination group did not grow upon rechallenge are shown.

[0025] Figure 8 Results demonstrating that the cancer in the combination group did not metastasize upon rechallenge are shown. DETAILED DESCRIPTION

[0026] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one skilled in the art. Generally, the terms used herein are well known and commonly used in the art.

[0027] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a fusion protein as an active ingredient, wherein the fusion protein comprises vascular endothelial growth factor receptor 1 (VEGFR1) domain 2, VEGFR1 domain 3 and the Fc region of an antibody, for co-administration with a PD-1 or PD-L1 antagonist.

[0028] In another aspect, the present invention relates to a method for prevention or treatment, administering a fusion protein comprising VEGFR1 (vascular endothelial growth factor receptor 1) domain 2, VEGFR1 domain 3 and the Fc region of an antibody or a pharmaceutical composition comprising the same, in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same.

[0029] On the other hand, the present invention relates to the use of a fusion protein comprising vascular endothelial growth factor receptor 1 (VEGFR1) domain 2, VEGFR1 domain 3 and the Fc region of an antibody or a pharmaceutical composition comprising the same as an active ingredient for preventing or treating cancer, in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same.

[0030] On the other hand, the present invention relates to the use of a fusion protein comprising vascular endothelial growth factor receptor 1 (VEGFR1) domain 2, VEGFR1 domain 3 and the Fc region of an antibody, or a pharmaceutical composition comprising the same as an active ingredient, in the preparation of a medicament for preventing or treating cancer, in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same.

[0031] Hereinafter, the present invention is described in more detail.

[0032] VEGF-Grab

[0033] The present invention includes a fusion protein comprising vascular endothelial growth factor receptor 1 (VEGFR1) domain 2, VEGFR1 domain 3 and an Fc region of an antibody.

[0034] "VEGFR1" (vascular endothelial growth factor receptor 1) is a receptor for vascular endothelial growth factor (VGEF), which can stimulate cell division, migration and differentiation by activating the receptor's tyrosine kinase. In addition, VEGFR1 domain 2 and domain 3 are domains that recognize VEGF.

[0035] VEGFR1 domains 2 and 3 are not limited to their origin or sequence, and may include wild-type or active variants thereof, since they may differ in the amino acid sequence of proteins exhibiting activity depending on species. VEGFR1 domain 2 of the present invention may include the amino acid sequence of SEQ ID NO: 1, and VEGFR1 domain 3 may include the amino acid sequence of SEQ ID NO: 2.

[0036] Amino acid sequence of VEGFR1 domain 2 (SEQ ID NO: 1):

[0037] EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIP DGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGH

[0038] Amino acid sequence of VEGFR1 domain 3 (SEQ ID NO: 2):

[0039] LYKTNYLTHRQTNTIIDVQISTPSPVTLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKA

[0040] "Antibody Fc region" refers to the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) (excluding heavy chain and light chain variable regions), heavy chain constant region 1 (CH1) and light chain constant region 1 (CL1) of an immunoglobulin, and may also include the hinge region in the heavy chain constant region. The antibody Fc region is a biodegradable polypeptide that is metabolized in vivo and is therefore safe to use as a drug carrier. In addition, compared with the total molecule of immunoglobulin, the immunoglobulin Fc region has a relatively small molecular weight, which has advantages in terms of preparation, purification and the yield of the conjugate, and because there are differences in the amino acid sequences of different antibodies, by eliminating the Fab portion that exhibits a high degree of heterogeneity, the homogeneity of the substance is greatly improved, reducing the possibility of inducing blood antigenicity.

[0041] The Fc region of the antibody may be, but is not limited to, an Fc region derived from IgA, IgD, IgE, IgG, IgM, or a combination thereof or a hybrid thereof, or more specifically, IgG or IgM, which are the most abundant in human blood, more specifically, humanized IgG1.

[0042] The fusion protein may refer to a protein including VEGFR1 domain 2 (R1D2)-VEGFR1 domain 3 (R1D3)-hinge-Fc region (CH2 and CH3) of a human antibody, and may be used interchangeably with the term "VEGF-Grab".

[0043] The fusion protein binds to VEGF, a ligand of VEGFR1 (vascular endothelial growth factor receptor 1), and inhibits activity by binding to VEGF-A, VEGF-B or PlGF. The fusion protein may include the amino acid sequence of SEQ ID NO:3.

[0044] Amino acid sequence of VEGF-Grab fusion protein (SEQ ID NO: 3):

[0045] EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTP SPVTLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKALEDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0046] In one embodiment of the present invention, the VEGF-Grab fusion protein is named "PB101".

[0047] The fusion protein can be connected from the N-terminus in the following order: VEGFR1 domain 2, VEGFR1 domain 3, antibody fragment, or in the following order: VEGFR1 domain 3, VEGFR1 domain 2, antibody fragment, wherein VEGFR1 domain 2, VEGFR1 domain 3 and the Fc region of the antibody in the fusion protein can be directly connected to each other or connected to each other via a linker.

[0048] The linker is not particularly limited as long as it can make the fusion protein show activity, but in particular, amino acids such as glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, or arginine can be used. More specifically, multiple amino acids such as valine, leucine, aspartic acid, glycine, alanine, proline, etc. can be used and connected in multiple arrays, and more specifically, considering the convenience of genetic manipulation, multiple amino acids such as glycine, valine, leucine, aspartic acid, etc. can be used and connected in one to twenty groups.

[0049] Particularly, the linker can be a peptide linker and can include about 5-25 amino acid (aa) residues, or more specifically, can include about 5-30 aa residues.For example, it can include but is not limited to hydrophilic amino acids, such as glycine and / or serine.

[0050] The linker may include, for example, a glycine linker (G, Gly) p (where p is 1 to 10), GS linker (G n S) m (wherein n and m are 1 to 10, respectively) to provide structural flexibility. Specifically, the linker may include GGGGS or (GGGGS)2, or may include (G, Gly) p 1-10 aa of glycine in which p is 1 to 10.

[0051] PD-1 or PD-L1 antagonists

[0052] PD-1 antagonists include agents that can inhibit the binding of PD-L1 to PD-1. For example, the PD-1 antagonist can be an anti-PD-1 antibody.

[0053] PD-L1 antagonists include agents that can inhibit the binding of PD-L1 to PD-1. For example, the PD-L1 antagonist can be an anti-PD-L1 antibody.

[0054] "Antibody" refers to an anti-PD-1 antibody or an anti-PD-L1 antibody that specifically binds to PD-1 or PD-L1. The scope of the present invention includes antigen-binding fragments of antibody molecules, as well as complete antibody forms that specifically bind to PD-1 or PD-L1.

[0055] An intact antibody has a structure of two full-length light chains and two full-length heavy chains, each light chain connected to a heavy chain by disulfide bonds.

[0056] As used herein, the term "heavy chain" refers to a full-length heavy chain and a fragment thereof, the full-length heavy chain including a variable region domain VH (which includes an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen) and three constant region domains CH1, CH2 and CH3. In addition, as used herein, the term "light chain" refers to a full-length light chain and a fragment thereof, the full-length light chain including a variable region domain VL (which includes an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen) and a constant region domain CL.

[0057] Whole antibodies include IgA, IgD, IgE, IgM and IgG subtypes, especially IgG including IgG1, IgG2, IgG3 and IgG4. Heavy chain constant regions include gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subtypes include gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1) and alpha2 (α2). Light chain constant regions include kappa (κ) and lambda (λ) types.

[0058] The antigen-binding fragment of an antibody or antibody fragment refers to a fragment with antigen-binding function, including Fab, F(ab'), F(ab')2 and Fv. Among antibody fragments, Fab has a structure of a light chain variable region, a heavy chain variable region, a light chain constant region and a heavy chain first constant region (CH1), and has one antigen-binding site. The difference between Fab' and Fab is that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is generated by disulfide bonding of cysteine ​​residues in the hinge region of Fab'.

[0059] Fv corresponds to the smallest antibody fragment with only heavy chain variable region and light chain variable region. Double-chain Fv has non-covalently linked heavy chain variable region and light chain variable region, while single-chain Fv (scFv) has covalently linked heavy chain variable region and light chain variable region, usually connected via a peptide linker, or directly connected at the C-terminus, which can form a dimer-like structure similar to double-chain Fv. These antibody fragments can be produced by using protease (for example, using papain to limit the cutting of complete form of antibody to produce Fab, using pepsin to cut to produce F (ab ') 2) or by genetic recombination technology.

[0060] The "Fv" fragment is an antibody fragment that contains the recognition and binding sites of an intact antibody. These regions are a dimer of one heavy chain variable domain and one light chain variable domain.

[0061] The "Fab" fragment includes the variable domain and the constant domain in the light chain, and the variable domain and the first constant domain (CH1) in the heavy chain. F(ab')2 antibody fragments typically include a pair of Fab' fragments covalently linked by cysteine ​​located in the hinge region present at the C-terminus of the Fab' fragments.

[0062] The structure of a "single-chain Fv (scFv)" antibody fragment is composed of a single polypeptide chain containing an antibody VH domain and a VL domain. A polypeptide linker may also be included between the VH domain and the VL domain to allow the scFv to form a structure required for binding to an antigen.

[0063] In the present invention, antibodies include but are not limited to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFv, Fab fragments, F(ab')2 fragments, disulfide-bonded Fv (sdFv) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the above antibodies.

[0064] The heavy chain constant region can be selected from any of the following isotypes: gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). For example, the constant region is gamma1 (IgG1), gamma2 (IgG2), gamma3 (IgG3) or gamma4 (IgG4). The light chain constant region can be κ type or λ type.

[0065] A monoclonal antibody is an antibody obtained from a population of substantially homogeneous antibodies, in other words, identical except for possible naturally occurring mutations (which may be present in trace amounts in individual antibodies of the population). Monoclonal antibodies are highly specific because they are directed against a single antigenic site. Unlike conventional (polyclonal) antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0066] "Epitope" refers to a protein determinant to which an antibody can specifically bind. An epitope usually consists of a group of chemically active surface molecules, such as amino acids or sugar side chains, and usually has specific charge characteristics as well as specific three-dimensional structural features. The difference between amorphous epitopes and steric epitopes is that the binding of the former will disappear in the presence of denaturing solvents, while the binding of the latter will not disappear.

[0067] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity.

[0068] "Human antibody" refers to a molecule derived from human immunoglobulin, wherein the entire amino acid sequence constituting the antibody (including complementary crystal regions and structural regions) consists of human immunoglobulin.

[0069] "Chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while one or more of the remaining chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and include antibody fragments that exhibit the desired biological activity.

[0070] As used herein, the "variable region" of an antibody refers to the light chain and heavy chain portions of an antibody molecule, which include the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0071] "Complementarity determining region (CDR)" refers to the amino acid residues in the variable domain of an antibody that are required for antigen binding. Each variable domain usually has three CDR regions, which are designated CDR1, CDR2, and CDR3.

[0072] "Framework regions (FR)" are the variable domain residues other than the CDR residues. Each variable domain usually has four FRs, which are designated FR1, FR2, FR3, and FR4.

[0073] In one embodiment of the present invention, the anti-PD-1 antibody or anti-PD-L1 antibody may be but is not limited to 10F.9G2, specifically a mouse PD-L1 antibody.

[0074] The anti-PD-1 antibody can be, for example, AMP-224, GLS-010, PDR-001, Pidilizumab, PF-06801591, Genolizumab, CA-170, MEDI-0680, JS-001, LZM-009, AK-103, Nivolumab, Dostarlimab, Pembrolizumab, Sintilimab, Tislelizumab, Toripalimab, Camrelizumab, or Cemiplimab.

[0075] The anti-PD-L1 antibody can be, for example, CX-072, WBP-3155, KN035, A167, Cosibelimab, Atezolizumab, Avelumab, Durvalumab, Adebrelimab, or BMS-936559.

[0076] Nucleic acid molecules, expression vectors and host cells

[0077] An isolated nucleic acid molecule encoding a fusion protein or an antagonist is provided.

[0078] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides (the basic building blocks of nucleic acids) include not only naturally occurring nucleotides, but also analogs with modified sugar or base sites. The nucleic acid sequences encoding the heavy and light chain variable regions of the present invention can be modified. Modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0079] An expression vector comprising an isolated nucleic acid molecule is provided. As used herein, the term "vector" refers to a tool for expressing a target gene in a host cell, including viral vectors such as plasmid vectors, cosmid vectors, phage vectors, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, etc. The components of the vector generally include, but are not limited to, one or more of the following: a signal sequence, a replication site, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the antibody is operably linked to the promoter and the transcription termination sequence.

[0080] "Operably linked" means a functional relationship between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulatory binding sites) and another nucleic acid sequence, whereby the control sequence regulates the transcription and / or decoding of the other nucleic acid sequence.

[0081] In the case of prokaryotes as hosts, it generally includes a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site for initiation of decoding, and a transcription / decoding termination sequence. In addition, for example, when eukaryotic cells are used as hosts, promoters derived from mammalian cell genomes (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and they usually have a polyadenylation sequence as a transcription termination sequence.

[0082] As used herein, the term "expression of a heterologous nucleic acid sequence" or "expression" of a target protein refers to the transcription of an inserted DNA sequence, the translation of an mRNA transcript, and the production of a fusion protein product or an antibody or antibody fragment.

[0083] The vector includes an antibiotic resistance gene conventionally used in the art as a selective marker, for example, resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0084] Host cells comprising expression vectors are provided.

[0085] As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector may be introduced. As used herein, the terms "transformation" and "transfection" refer to the introduction of a nucleic acid (e.g., a vector) into a cell by any of a number of techniques known in the art.

[0086] Prokaryotic host cells can be used, such as strains of Bacillus, such as Escherichia coli, Bacillus subtilus, and Bacillus thuringiensis; Streptomyces; Pseudomonas (e.g., Pseudomonas putida); Proteus mirabilis; or Staphylococcus (e.g., Staphylococcus carnosus).

[0087] However, of greatest interest are animal cells, and examples of useful host cell lines may include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, and HT1080.

[0088] Host cells can be cultured in a variety of culture media. Any commercially available culture media can be used as culture media without restriction. All other necessary supplements known to those skilled in the art may also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc. have been used to select host cells for expression and are apparent to those skilled in the art.

[0089] The recovery of the fusion protein or antagonist can be achieved by removing impurities (e.g., by centrifugation or ultrafiltration) and purifying the resulting product (e.g., by affinity chromatography). Additional other purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc., can be used.

[0090] Pharmaceutical composition

[0091] Pharmaceutical composition according to the present invention can be administered by any route.Composition of the present invention can be provided to animal by any suitable means, whether directly (for example, by injection, implantation or topical administration to tissue site, or by topical administration) or whole body (for example, parenteral or oral).When composition of the present invention is administered parenterally, such as intravenously, subcutaneously, ocularly, intraperitoneally, intramuscularly, orally, rectum, orbitally, intracerebrally, intracranial, intraspinal, intraventricularly, intrathecally, intracisternally, or intracapsularly, intranasally, or via atomization, pharmaceutical composition can include a part of a body fluid suspension or solution that is for example aqueous or physiologically applicable.Therefore, carrier or support body are physiologically acceptable, can be added to composition and delivered to patient.Therefore, the carrier (such as body fluid) of preparation can generally include normal saline.

[0092] In addition, the frequency of administration depends on the pharmacokinetic parameters of the VEGF-Grab, PD-1 or PD-L1 antagonist in the formulation used. Typically, the clinician will administer the pharmaceutical composition until a dose is reached that achieves the desired effect. Thus, the pharmaceutical composition may be administered as a single dose, as two or more spaced doses, or as a continuous infusion via an implant device or catheter. Those skilled in the art can routinely further refine the appropriate dosage, and this is within the scope of their routine work.

[0093] In addition, in humans, the unit dose is 0.01 μg / kg to 100 mg / kg, more specifically 1 μg / kg to 10 mg / kg. Although the above amounts are optimal, they may vary depending on the disease to be treated and the presence or absence of side effects, and the optimal dose can be determined using routine experiments. Administration of the fusion protein can be by regular bolus injection or by continuous intravenous, subcutaneous or intraperitoneal administration from an external reservoir (e.g., an intravenous infusion bag) or an internal reservoir (e.g., a bioerodible implant). PD-1 or PD-L1 antagonists can be administered intravenously or subcutaneously.

[0094] The dosage of fusion protein can be administered at 1 mg / kg to 15 mg / kg. Specifically, it can be administered at 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg or 15 mg / kg.

[0095] The dosage of PD-1 or PD-L1 antagonists can be 1 mg / kg to 20 mg / kg. Specifically, the dosage of the PD-1 or PD-L1 antagonists can be 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg.

[0096] combination

[0097] Preferably, the VEGF-Grab, PD-1 or PD-L1 antagonists have complementary activities such that they do not adversely affect each other.

[0098] The compositions according to the present invention can be (1) co-formulated into a combined formulation and administered or delivered simultaneously; or (2) administered or delivered simultaneously or sequentially as separate formulations.

[0099] For combined preparations, VEGF-Grab, PD-1 or PD-L1 antagonists may be present in the same composition. The preparation may be, for example, but not limited to, a dry powder composition, a solution or a suspension.

[0100] VEGF-Grab, PD-1 or PD-L1 antagonists may be administered simultaneously or sequentially. VEGF-Grab, PD-1 or PD-L1 antagonists are generally administered separately from one another and may be administered simultaneously or sequentially. If administered sequentially, they may be administered in two or more doses. When administered sequentially, one of the VEGF-Grab, PD-1 or PD-L1 antagonists may be administered first and then the other, or the VEGF-Grab, PD-1 or PD-L1 antagonists may be administered alternately with an interval between administrations of the VEGF-Grab, PD-1 or PD-L1 antagonists.

[0101] Suitable dosages of the above-mentioned agents may be those currently used in the art, and may be reduced due to combined use.

[0102] Administration of the composition

[0103] The compositions of the present invention may be administered by any route appropriate to the condition to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, inhalation, intradermal, intrathecal, intrathecal and infusion techniques), transdermal, rectal, intranasal, topical (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal administration. Topical administration may include the use of transdermal administration, such as a transdermal patch or iontophoresis device. The formulation of the drug is discussed in the following literature [Reference: Remington's Pharmaceutical Sciences, 18 th Ed., (1995) Mack Publishing Co., Easton, PA]. Other examples of drug formulation are described in the following literature [Reference: Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, Vol 3, 2 nd Ed.,NewYork,NY].

[0104] The preferred route may vary, for example, depending on the condition of the recipient. If administered orally, it can be formulated into pills, capsules, tablets, etc., and added with a pharmaceutically acceptable carrier, excipient or excipient. For parenteral administration, it can be formulated into a unit dose injection and added with a pharmaceutically acceptable parenteral carrier or diluent.

[0105] Prevention or treatment

[0106] The composition of the present invention is used for preventing or treating cancer, more specifically for cancer immunotherapy.

[0107] Cancer includes solid cancers and blood cancers, more specifically including but not limited to liver cancer, colorectal cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, head and neck cancer, skin or eye melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, hilar rectal cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or urinary tract cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, primary central nervous system lymphoma, spinal cord tumors, brain stem gliomas, pituitary adenomas, intrahepatic biliary tract cancer (intrahepatic biliary tract cancer), ovarian cancer, rectal cancer, gastric cancer, hilar rectal cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or urinary tract cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, primary central nervous system lymphomas, spinal cord tumors, brain stem gliomas, pituitary adenomas, intrahepatic biliary tract cancer (intrahepatic biliary tract cancer), cancer), medulloblastoma and pancreatic neuroendocrine tumors, and more specifically solid tumors among the above cancers.

[0108] The cancer may be, for example but not limited to, colorectal cancer or liver cancer.

[0109] The present invention provides a method for preventing or treating cancer, and more particularly, for cancer immunotherapy.

[0110] The present invention provides a method for preventing or treating cancer, and more specifically, provides a method for cancer immunotherapy. The subject may be an individual suffering from cancer. In addition, the individual may be a mammal, preferably a human.

[0111] "Treatment" means any indication of success by any subjective or objective parameter, such as alleviation; remission; producing a symptom or injury more acceptable to the patient, and a reduction in pathology or condition; slowing the rate of regression or decline; producing a final, debilitating point of regression; or curing or ameliorating an injury, pathology or condition, including improving the patient's physical or mental health. Treatment or amelioration of symptoms can be based on objective or subjective parameters, including physical examination, neuropsychiatric examination, and / or psychiatric evaluation.

[0112] An "effective amount" is generally an amount sufficient to reduce the severity or frequency of symptoms, eliminate symptoms or their potential causes, prevent the occurrence of symptoms or their potential causes, or improve or correct damage caused by or associated with a disease state. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactic effective amount. A "therapeutically effective amount" refers to an amount sufficient to correct a disease state or symptom (particularly a condition or symptom associated with a disease state), or an amount sufficient to prevent, prevent, delay or reverse the progression of a disease state or any other adverse symptom associated with a disease state in any way. A "prophylactic effective amount" refers to an amount that has an expected preventive effect when a pharmaceutical composition is administered to a subject, for example, preventing or delaying the onset of a disease state, or reducing the likelihood of an onset (or recurrence) of a disease state or associated symptoms.

[0113] product

[0114] A kit comprising VEGF-Grab and a PD-1 or PD-L1 antagonist may be provided for treating any of the above cancers. Such a kit may comprise a single container when co-formulated as a co-preparation. Such a kit comprises a container comprising VEGF-Grab or a PD-1 or PD-L1 antagonist.

[0115] The kit may be associated with a container, or may further include a lid or package insert thereon. The term "package insert" may refer to instructions typically included in the commercial packaging of a therapeutic product, and includes information about the indications, uses, dosage, administration, contraindications and / or warnings of such therapeutic products. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container may be made of various materials, such as glass or plastic. The label or package insert indicates that the composition is used to prevent or treat a selected disease, such as cancer, or for cancer immunotherapy. The composition may further include a second container, which includes a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution or dextrose solution. Other materials that are desirable from a commercial and user perspective may further be included, such as other buffers, diluents, fillers, needles and syringes.

[0116] The method may further include instructions for administering VEGF-Grab or a PD-1 or PD-L1 antagonist simultaneously, sequentially or separately to a patient in need thereof.

[0117] Hereinafter, the present invention will be described in detail with reference to examples to facilitate understanding. However, the examples according to the present invention can be modified in many other ways, and the scope of the present invention should not be interpreted as being limited to the following examples.

[0118] Example 1: Combination immunotherapy of PB101 and PD-L1 immune checkpoint inhibitor in MC38 colon cancer model

[0119] 1.1 Methods

[0120] Eight-week-old C57BL / 6 immune-protected mice were injected subcutaneously on the right flank with 2x 10 5 MC38 colon cancer cell line. When the tumor size reaches about 50mm 3 When the drug is administered via ip (intraperitoneal injection) Figure 1 ).

[0121] Tumor sizes were measured for all groups, and euthanasia was performed using CO2 gas at the end of observation.

[0122] 1.2 Results

[0123] In the MC38 colon cancer model, it was observed that compared with the control group, the αPD-L1 (10F.9G2, a mouse PD-L1 antibody) group showed a tumor growth inhibition rate of 29.5%, the PB101 group showed a tumor growth inhibition rate of 54.7%, and in particular, the PB101 and αPD-L1 combination group showed a significant increase in tumor growth inhibition rate to 71.4% ( Figure 2 ).

[0124] Combination treatment of PB101 and αPD-L1 enhanced the anti-tumor response in the MC38 colon cancer model compared with the single treatment groups.

[0125] Example 2: Combination immunotherapy of PB101 and PD-L1 immune checkpoint inhibitor in Hepa-VEGF liver cancer model

[0126] 2.1 Methods

[0127] Eight-week-old C57BL / 6 immune-protected mice were injected subcutaneously on the right flank with 5x 10 6 Hepa-VEGF liver cancer cell line. When the tumor size reaches about 50mm 3 When the drug is administered via ip (intraperitoneal injection) Figure 3 ).

[0128] Tumor size was measured in all groups, and survival was followed up to 85 days to ensure sustained effect.

[0129] 2.2 Results

[0130] In the Hepa-VEGF liver cancer model, it was observed that compared with the control group, the αPD-L1 group showed a tumor growth inhibition rate of 48.2%, the PB101 group showed a tumor growth inhibition rate of 60.3%, and in particular, the PB101 and αPD-L1 combination group showed a significant increase in tumor growth inhibition rate to 84.1%. In addition, it was observed that unlike the other experimental groups, 27.2% (3 / 11) of the tumors in the PB101 and αPD-L1 combination group completely regressed ( Figure 4 ).

[0131] After the first two weeks of drug administration, survival benefits were observed for both monotherapy and co-administration after drug discontinuation and monitoring for 60 days ( Figure 5 ).

[0132] Example 3: Cancer cell re-stimulation experiment after combined immunotherapy of PB101 and PD-L1 immune checkpoint inhibitor in Hepa-VEGF liver cancer model

[0133] 3.1 Methods

[0134] After two experiments, four CR and near-CR mice were selected from the PB101+aPD-L1 combination group and injected subcutaneously with 2x10 6 Hepa-VEGF hepatoma cell line, and 1x 10 6 Six mice in the combination group were injected with cancer cells in the same way, followed for 20 days, and euthanized ( Figure 6 ).

[0135] 3.2 Results

[0136] When the cancer was re-stimulated in the combination group, unlike in the control group, the cancer did not grow ( Figure 7 ), which is speculated to be due to the formation of immune memory.

[0137] In addition, for the intravenously injected cancer cells, metastasis to the lung was identified in the control group but was rarely seen in the combination group ( Figure 8 ). This may be due to the induction of long-lasting antitumor immune memory against recurrence and metastasis.

[0138] Industrial Applicability

[0139] The pharmaceutical composition according to the present invention can be used to delay tumor growth and prevent cancer metastasis.

[0140] Although certain aspects of the present invention have been described in detail above, it is obvious to those skilled in the art that these specific descriptions are only preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the essential scope of the present invention will be defined by the appended claims and their equivalents.

[0141] An electronic file accompanying the free text of the sequence listing.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising a fusion protein as an active ingredient, wherein the fusion protein comprises VEGFR1 (vascular endothelial growth factor receptor 1) domain 2, VEGFR1 domain 3 and the Fc region of an antibody, for co-administration with a PD-1 antagonist or a PD-L1 antagonist.

2. The pharmaceutical composition according to claim 1, wherein The VEGFR1 domain 2 includes the amino acid sequence of SEQ ID NO:

1.

3. The pharmaceutical composition according to claim 1, wherein The VEGFR1 domain 3 includes the amino acid sequence of SEQ ID NO:

2.

4. The pharmaceutical composition according to claim 1, wherein The PD-1 or PD-L1 antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody.

5. The pharmaceutical composition according to claim 4, wherein The anti-PD-1 antibody is AMP-224, GLS-010, PDR-001, pidilizumab, PF-06801591, genoluzumab, CA-170, MEDI-0680, JS-001, LZM-009, AK-103, nivolumab, dotalizumab, pembrolizumab, sintilimab, tislelizumab, toripalizumab, carrelizumab or cemiprilimab.

6. The pharmaceutical composition according to claim 4, wherein The anti-PD-L1 antibody is CX-072, WBP-3155, KN035, A167, coxilimab, atezolizumab, avelumab, durvalumab, adebelimumab or BMS-936559.

7. The pharmaceutical composition according to claim 1, wherein The fusion protein and the PD-1 or PD-L1 antagonist are included in a co-formulation.

8. The pharmaceutical composition according to claim 1, wherein The fusion protein and the PD-1 or PD-L1 antagonist are administered simultaneously or sequentially.

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