Composition for combined administration comprising VEGF-Grab and PD-1 or PD-L1 antagonist

Abstract

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

[0002] In order for cancer cells to grow and proliferate, new blood vessels that supply oxygen and nutrients are necessary, and it is known that vascular endothelial growth factor (VEGF) plays a central role in the formation of new blood vessels. VEGF is a dimer of about 46 KDa consisting of two subunits, and five types of VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PlGF) have been identified in mammals so far. The VEGF binds to three receptor tyrosine kinases (RTKs) called VEGF receptors (VEGFR)-1, VEGFR-2, and VEGFR-3, and these VEGF receptors induce cell migration, survival, proliferation, etc., transmit signals that can form three-dimensional blood vessels, which are not possible with other RTKs, and regulate vascular permeability.

[0003] Since it was revealed that the expression of VEGF molecules increases in tumor cells and that VEGF receptors increase in tumor-infiltrating vascular endothelial cells, while both VEGF and VEGF receptors are expressed at low levels in normal cells not involved in angiogenesis, VEGF has been used as a target for cancer therapy.

[0004] Accordingly, new anti-cancer therapies have been developed that block the generation of blood vessels that supply nutrients to cancer cells, rather than the cancer cells themselves, and it has been reported that anti-VEGF receptor antibodies, soluble receptor structures, antisense, RNA aptamers against VEGF, and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors can be used to disrupt VEGF signaling. In fact, it has been found that anti-VEGF neutralizing antibodies suppress the growth of various human tumor cell lines in nude mice (Warren et al. J. Clin.Invest. 95: 1789-1797(1995)). In addition, patent documents related to VEGF inhibitors disclose various VEGF inhibitors, such as quinazoline derivatives as VEGF inhibitors (U.S. Patent No. 9040548), VEGF receptor and HGF receptor signal transduction inhibitors for treating cell proliferation diseases mediated by angiogenesis or suppressing the growth of solid tumors (U.S. Patent No. 8470850), and substances that inhibit the binding between VEGF and its receptor and thus are used to treat diseases such as cancer (Korean Patent Publication No. 2003-0075947).

[0005] Meanwhile, our body's immune system regulates the overall function of T lymphocytes through the regulation of these costimulatory and inhibitory signals while recognizing antigens. This servo mechanism is called the immune checkpoint. Our body's immune system detects tumor-specific neo-antigens that are expressed due to changes such as mutations that occur in tumor cells, and eliminates tumor cells or viral infection sources through these.

[0006] However, some tumor cells attempt to avoid such immune attacks by suppressing immune function by modifying the tumor microenvironment or by evading the immune system through T cell immune tolerance or immuno-editing.

[0007] One such evasion strategy is to suppress the function of tumor-specific T lymphocytes through changes in immune checkpoint function. In other words, by activating such an inhibitory immune checkpoint in tumor cells, the attack of tumor-specific T lymphocytes can be avoided. In this regard, by suppressing the function of PD-1 or its ligand PD-L1 using a monoclonal antibody, the activity and effect of the suppressed tumor-specific T lymphocytes can be enhanced, resulting in an antitumor effect.

[0008] Under these technical background, the present inventors conducted research into cancer immunotherapy and discovered that the combined use of VEGF-Grab and a PD-1 or PD-L1 antagonist slows tumor growth and prevents cancer metastasis, thereby completing the present invention.

[0009] The information provided in this Background section is intended solely to provide a deeper understanding of the background of the invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the invention pertains. Summary of the Invention [Problem to be solved by the invention]

[0010] The object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises VEGF-Grab as an active ingredient and is to be administered in combination 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, which comprises administering in combination a VEGF-Grab or a pharmaceutical composition containing it as an active ingredient and a PD-1 or PD-L1 antagonist or a pharmaceutical composition containing the same.

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

[0013] Another object of the present invention is to provide a use of VEGF-Grab or a pharmaceutical composition containing it as an active ingredient for the manufacture of a medicament for preventing or treating cancer in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition containing it. [Means for solving the problem]

[0014] To achieve the above-mentioned object, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a fusion protein comprising VEGFR1 (vascular endothelial growth factor receptor 1) domain 2, VEGFR1 domain 3, and an antibody Fc region as an active ingredient, and is to be administered in combination with a PD-1 or PD-L1 antagonist.

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

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

[0017] The present invention also provides use of VEGF-Grab or a pharmaceutical composition containing it as an active ingredient for the manufacture of a medicament for preventing or treating cancer in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition containing it. [Brief description of the drawings]

[0018] [Figure 1]FIG. 1 shows an overview of combination immunotherapy with PB101 and PD-L1 immune checkpoint inhibitors in an MC38 colon cancer model. [Diagram 2] This figure confirms the tumor growth retardation effect in the group administered PB101 and αPD-L1 in combination. [Diagram 3] FIG. 1 shows an overview of combination immunotherapy with PB101 and PD-L1 immune checkpoint inhibitors in a Hepa-VEGF liver cancer model. [Figure 4] This figure confirms the tumor growth retardation effect in the group administered PB101 and αPD-L1 in combination. [Diagram 5] This figure confirms the survival benefit of the PB101 and αPD-L1 combination administration group. [Figure 6] FIG. 1 shows an outline of a cancer cell rechallenge experiment after combined immunotherapy with PB101 and PD-L1 immune checkpoint inhibitors in a Hepa-VEGF liver cancer model. [Figure 7] 1 shows the results of confirming that when cancer was inoculated into the combined administration group, the cancer did not grow. [Figure 8] 13 is a graph showing the results of confirming that when cancer was inoculated into the combined administration group, the cancer did not metastasize. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is one that is well known and commonly used in the art.

[0020] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient a fusion protein comprising VEGFR1 (vascular endothelial growth factor receptor 1) domain 2, VEGFR1 domain 3, and an Fc region of an antibody, and is to be administered in combination with a PD-1 or PD-L1 antagonist.

[0021] From another aspect, the present invention relates to a method for preventing or treating cancer, which comprises administering a fusion protein comprising VEGFR1 (vascular endothelial growth factor receptor 1) domain 2, VEGFR1 domain 3, and an Fc region of an antibody, or a pharmaceutical composition containing the same as an active ingredient, in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition containing the same.

[0022] From another aspect, the present invention relates to use of a fusion protein comprising VEGFR1 (vascular endothelial growth factor receptor 1) domain 2, VEGFR1 domain 3, and an antibody Fc region, 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.

[0023] From another aspect, the present invention relates to use of a fusion protein comprising VEGFR1 (vascular endothelial growth factor receptor 1) domain 2, VEGFR1 domain 3, and an antibody Fc region, or a pharmaceutical composition comprising the same as an active ingredient, for the manufacture of a medicament for preventing or treating cancer, to be used in combination with a PD-1 or PD-L1 antagonist or a pharmaceutical composition comprising the same.

[0024] The present invention will now be described in more detail.

[0025] VEGF-Grab 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.

[0026] "VEGFR1 (vascular endothelial growth factor receptor 1)" is a receptor for vascular endothelial growth factor (VEGF), and can activate the receptor tyrosine kinase to stimulate cell division, migration, differentiation, etc. VEGFR1 domain 2 and domain 3 are domains that recognize VEGF.

[0027] The VEGFR1 domain 2 and domain 3 are not limited to their origin or sequence, since there may be differences in the amino acid sequence of the protein exhibiting activity depending on the species, and may include the wild type or a mutant having activity. The VEGFR1 domain 2 of the present invention may include the amino acid sequence of SEQ ID NO: 1, and the VEGFR1 domain 3 may include the amino acid sequence of SEQ ID NO: 2.

[0028] Amino acid sequence of VEGFR1 domain 2 (SEQ ID NO:1): EFGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGH

[0029] Amino acid sequence of VEGFR1 domain 3 (SEQ ID NO:2): LYKTNYLTHRQTNTIIDVQISTPSPVTLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKA

[0030] The term "antibody Fc region" refers to the heavy and light chain variable regions of an immunoglobulin, and the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) excluding the heavy chain constant region 1 (CH1) and light chain constant region (CL1), and may include a hinge portion 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, the immunoglobulin Fc region has a relatively low molecular weight compared to the entire immunoglobulin molecule, which is advantageous in terms of the production, purification, and yield of the conjugate. In addition, by removing the Fab portion, which shows high heterogeneity due to the different amino acid sequences for each antibody, the homogeneity of the substance is significantly increased, and the possibility of inducing antigenicity in blood is also expected to be reduced.

[0031] The Fc region of the antibody may be an Fc region derived from IgA, IgD, IgE, IgG, IgM, or a combination or hybrid thereof, or specifically derived from IgG or IgM, which are most abundant in human blood, and more specifically, may be human-derived IgG1, but is not limited to these.

[0032] The fusion protein may refer to a protein comprising VEGFR1 domain 2 (R1D2)-VEGFR1 domain 3 (R1D3)-hinge-human antibody Fc region (CH2 and CH3), and may be used interchangeably with the name "VEGF-Grab."

[0033] The fusion protein binds to VEGF, a ligand of VEGFR1 (vascular endothelial growth factor receptor 1), and binds to and inhibits the activity of VEGF-A, VEGF-B, or PlGF. The fusion protein may comprise the amino acid sequence of SEQ ID NO:3.

[0034] Amino acid sequence of the VEGF-Grab fusion protein (SEQ ID NO:3): EFGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTP SPVTLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKNASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKALEDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0035] In one embodiment of the present invention, the VEGF-Grab fusion protein is designated "PB101."

[0036] The fusion protein may be linked from the N-terminus in the order of VEGFR1 domain 2, VEGFR1 domain 3 and the antibody fragment, or VEGFR1 domain 3, VEGFR1 domain 2 and the antibody fragment, and in the fusion protein, VEGFR1 domain 2, VEGFR1 domain 3 and the antibody Fc region may be linked directly to each other or via a linker.

[0037] The linker is not particularly limited as long as it expresses the activity of the fusion protein. Specifically, the linker may be an amino acid such as glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, arginine acid, etc., and more specifically, a plurality of amino acids such as valine, leucine, aspartic acid, glycine, alanine, proline, etc. may be used for linking. Even more specifically, taking into consideration the ease of genetic manipulation, 1 to 20 amino acids each of glycine, valine, leucine, aspartic acid, etc. may be linked for use.

[0038] Specifically, the linker may be a peptide linker and may comprise about 5-25 aa residues, or specifically about 5-30 aa residues, including, but not limited to, hydrophilic amino acids such as glycine and / or serine.

[0039] In order to provide structural flexibility, the linker may be, for example, a glycine linker (G, Gly)p (p is 1 to 10), a GS linker (G n S) m (n, m are each 1 to 10). Specifically, the linker may include GGGGS or (GGGGS)2, or may include 1 to 10 aa glycines in (G, Gly)p, where p is 1 to 10.

[0040] PD-1 or PD-L1 antagonists The PD-1 antagonist includes agents capable of inhibiting the binding of PD-L1 to PD-1. For example, the PD-1 antagonist may be an anti-PD-1 antibody.

[0041] The PD-L1 antagonist includes agents that can inhibit the binding of PD-L1 to PD-1. For example, the PD-L1 antagonist may be an anti-PD-L1 antibody.

[0042] By "antibody" is meant an anti-PD-1 or anti-PD-L1 antibody that specifically binds to PD-1 or PD-L1. The scope of the invention includes intact antibody forms that specifically bind to PD-1 or PD-L1 as well as antigen-binding fragments of the antibody molecules.

[0043] An intact antibody has a structure with two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by disulfide bonds.

[0044] The term "heavy chain" as used herein refers to both a full-length heavy chain and a fragment thereof that includes a variable region domain VH and three constant region domains CH1, CH2, and CH3 that contain an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and the term "light chain" as used herein refers to both a full-length light chain and a fragment thereof that includes a variable region domain VL and a constant region domain CL that contain an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

[0045] The antibodies as a whole include IgA, IgD, IgE, IgM and IgG subtypes, and in particular IgG includes IgG1, IgG2, IgG3 and IgG4. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2) subclasses. The light chain constant region has kappa (κ) and lambda (λ) types.

[0046] The antigen-binding fragment of an antibody or antibody fragment means a fragment that retains antigen-binding function, and includes Fab, F(ab'), F(ab')2, Fv, etc. Among antibody fragments, Fab has a structure that has a light chain and a heavy chain variable region, a light chain constant region, and the first constant region of the heavy chain (CH1), and has one antigen-binding site. Fab' differs from Fab in 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 bond formation between cysteine ​​residues in the hinge region of Fab'.

[0047] Fv corresponds to the minimum antibody fragment having only the heavy chain variable region and the light chain variable region. In two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, and can form a dimer-like structure like two-chain Fv. Such antibody fragments can be produced by using proteolytic enzymes (for example, Fab is obtained by restrictive cleavage of an intact antibody with papain, and F(ab')2 is obtained by cleavage with pepsin) or by using genetic recombination technology.

[0048] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region is a dimer of one heavy-chain and one light-chain variable domain.

[0049] The "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments usually contain a pair of Fab' fragments covalently linked by cysteines in the hinge region located at the C-terminus of the Fab' fragments.

[0050] "Single-chain Fv (scFv)" antibody fragments are constructs consisting of a single polypeptide chain comprising the VH and VL domains of an antibody, and optionally further comprising a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0051] In the present invention, the antibody includes, but is not limited to, a monoclonal antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a scFv, a Fab fragment, a F(ab')2 fragment, a disulfide-linked Fvs (sdFv) and an anti-heterotype (anti-Id) antibody, or an epitope-binding fragment of the antibody.

[0052] The heavy chain constant region may be selected from any of the gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) isotypes. For example, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be of the kappa or lambda type.

[0053] The monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigen site. In contrast to conventional (polyclonal) antibodies, which usually contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0054] "Epitope" refers to a determinant on a protein to which an antibody can specifically bind. Epitopes are usually composed of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0055] The "humanized" forms of non-human (e.g., murine) antibodies described above are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from a hypervariable region of a receptor have been replaced with residues from a hypervariable region of a non-human species (donor antibody), e.g., mouse, rat, rabbit or non-human primate, that has the desired specificity, affinity and capacity.

[0056] The "human antibody" refers to a molecule derived from human immunoglobulin, and all of the amino acid sequences constituting the antibody, including the complementarity determining regions and structural regions, are entirely composed of human immunoglobulin.

[0057] These include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0058] The "variable region" of an antibody as used in the present invention refers to the light and heavy chain portions of an antibody molecule that contain 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.

[0059] "Complementary determining region (CDR)" refers to the amino acid residues of an antibody variable domain which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.

[0060] "Framework regions (FR)" are those variable domain residues other than the CDR residues. Each variable domain normally has four FRs, identified as FR1, FR2, FR3 and FR4.

[0061] In one embodiment of the present invention, the anti-PD-1 antibody or anti-PD-L1 antibody may specifically be, but is not limited to, the murine PD-L1 antibody 10F.9G2.

[0062] The anti-PD-1 antibody may be, for example, AMP-224, GLS-010, PDR-001, Pidilimab, PF-06801591, Genolimzumab, CA-170, MEDI-0680, JS-001, LZM-009, AK-103, Nivolumab, Dostarlimab, Pembrolizumab, Sintilimab, Tislelizumab, Toripalimab, Camrelizumab, or Cemiplimab.

[0063] The anti-PD-L1 antibody may be, for example, CX-072, WBP-3155, KN035, A167, Cosibelimab, Atezolizumab, Avelumab, Durvalumab, Adebrelimab, or BMS-936559.

[0064] Nucleic acid molecules, expression vectors and host cells An isolated nucleic acid molecule encoding said fusion protein or said antagonist is provided.

[0065] The term "nucleic acid" refers to a term that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include not only natural nucleotides but also analogues in which the sugar or base moiety has been modified. The sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. The modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0066] An expression vector comprising the isolated nucleic acid molecule is provided. As used herein, the term "vector" includes a plasmid vector, a cosmid vector, a bacteriophage vector, an adenovirus vector, a retrovirus vector, a virus vector such as an adeno-associated virus vector, and the like, as a means for expressing a gene of interest in a host cell. Components of a vector typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, a transcription termination sequence. The nucleic acid encoding the antibody is operably linked to the promoter, the transcription termination sequence, and the like.

[0067] "Operably linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or detoxification of the other nucleic acid sequence.

[0068] When a prokaryotic cell is used as the host, it generally contains 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, T7 promoter, etc.), a ribosome binding site for initiating detoxification, and a transcription / detoxification termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) may be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0069] As used herein, the term "expression of a heterologous nucleic acid sequence" or "expression" of a protein of interest refers to the transcription of the inserted DNA sequence, translation of the mRNA transcript, and production of a fusion protein product or an antibody or antibody fragment.

[0070] The vectors contain as selection markers antibiotic resistance genes commonly used in the art, such as ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline resistance genes.

[0071] A host cell comprising the expression vector is provided.

[0072] As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, the terms "transformed" and "transduced" refer to the introduction of a nucleic acid (e.g., a vector) into a cell by a number of techniques known in the art.

[0073] Prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilus, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus), may be used.

[0074] However, of most interest to animal cells, examples of useful host cell lines may be, 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, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0075] The host cell may be cultured in various media. Any commercially available medium may be used as the culture medium without limitation. Any other necessary supplements known to those skilled in the art may be included at appropriate concentrations. The culture conditions, such as temperature, pH, etc., are already used with the host cell selected for expression, and will be clear to those skilled in the art.

[0076] The fusion protein or the antagonist may be recovered, for example, by removing impurities by centrifugation or ultrafiltration, and the resulting product may be purified, for example, by affinity chromatography, etc. Additional purification techniques, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., may also be used.

[0077] Pharmaceutical Compositions The pharmaceutical composition according to the invention may be administered by any route. The composition of the invention may be administered to an animal by any suitable means, either directly (e.g., locally, by injection, implantation, or topical administration to a tissue site) or systemically (e.g., parenterally or orally). When the composition of the invention is administered parenterally, such as intravenously, subcutaneously, intraocularly, intraperitoneally, intramuscularly, orally, rectally, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracistenal, intracapsular, intranasal, or aerosol administration, the pharmaceutical composition may, for example, comprise part of an aqueous or physiologically compatible body fluid suspension or solution. Thus, a physiologically acceptable carrier or vehicle may be added to the composition to deliver it to a patient. Thus, a carrier such as a body fluid for the formulation may typically include physiological saline.

[0078] Furthermore, 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, two or more doses spaced apart in time, or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of practice routinely performed by those skilled in the art.

[0079] The unit dose is 0.01 μg / kg to 100 mg / kg in humans, specifically 1 μg / kg to 10 mg / kg of body weight. The above content is the optimum amount, but may vary depending on the disease to be treated and the presence or absence of side effects, and the optimum dose can be determined using routine experiments. The fusion protein may be administered by periodic bolus injections, or by continuous intravenous, subcutaneous, or intraperitoneal administration from an external reservoir (e.g., an intravenous bag) or an internal source (e.g., a bioerodable implant). The PD-1 or PD-L1 antagonist may be administered intravenously or subcutaneously.

[0080] The fusion protein may be administered at a dose of 1 mg / kg to 15 mg / kg, specifically, 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.

[0081] The PD-1 or PD-L1 antagonist may be administered at a dose of 1 mg / kg to 20 mg / kg. Specifically, the PD-1 or PD-L1 antagonist may be administered at a dose of 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, or 20 mg / kg.

[0082] Use in combination The VEGF-Grab, PD-1 or PD-L1 antagonists preferably have complementary activities, so that they do not adversely affect each other.

[0083] Compositions according to the invention may be (1) co-formulated as a combined dosage form and administered or delivered simultaneously, or (2) administered or delivered simultaneously or sequentially as separate dosage forms.

[0084] In the case of the combined dosage form, the VEGF-Grab, PD-1 or PD-L1 antagonist may be present in the same composition. The dosage form may be, for example, but not limited to, a dry powder composition, a solution or a suspension.

[0085] The VEGF-Grab, PD-1 or PD-L1 antagonists may be administered simultaneously or sequentially. The VEGF-Grab, PD-1 or PD-L1 antagonists may be administered simultaneously or sequentially, usually separately from each other. When administered sequentially, they may be administered in two or more doses. When administered sequentially, either one of the VEGF-Grab, PD-1 or PD-L1 antagonists may be administered followed by the other, or the two doses of the VEGF-Grab, PD-1 or PD-L1 antagonists may be administered alternately with an interval between them.

[0086] The appropriate dosage of the drug may be the amount currently used in the industry, and the dosage may be reduced when used in combination.

[0087] Administration of the Composition The compositions of the present invention may be administered by any route appropriate to the disease to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intravenous, inhalation, intradermal, intraspinal, epidural, and injection techniques), transdermal, rectal, intranasal, topical (including buccal and sublingual), intravaginal, intraperitoneal, pulmonary, and intranasal administration. Topical administration may include utilizing transdermal administration, such as transdermal patches or iontophoretic devices. Drug formulations are described in the following references: See Remington's Pharmaceutical Sciences, 18th Ed., (1995) Mack Publishing Co., Easton, PA. Other examples of drug formulations are described in the following references: See Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, Vol 3, 2001, pp. 211-214, 2001 ... nd Ed., New York, NY].

[0088] The preferred route will vary depending, for example, on the condition of the recipient. For oral administration, it can be formulated as a pill, capsule, tablet, etc., along with a pharma- ceutically acceptable carrier, lubricant, or excipient. For parenteral administration, it can be formulated as a unit dose injectable along with a pharma- ceutically acceptable parenteral vehicle or diluent.

[0089] Prevention or treatment The compositions of the present invention are used for the prevention or treatment of cancer, particularly for the immunotherapy of cancer.

[0090] The cancers include all solid cancers and blood cancers, and specifically include liver cancer, colorectal cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, perianal cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal carcinoma, vulvar carcinoma, 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 ureter cancer, renal cell carcinoma, renal pelvis kidney cancer, central nervous system (CNS) cancer, and / or other cancers. The cancer may be, but is not limited to, a primary central nervous system tumor, a primary central nervous system lymphoma, a spinal cord tumor, a brain stem glioma, a pituitary adenoma, an intrahepatic biliary tract cancer, a capillary hemangioma, a pancreatic neuroendocrine tumor, or the like, and more specifically, may be a solid cancer among the above cancers.

[0091] The cancer may be, for example, but is not limited to, colon cancer or liver cancer.

[0092] The present invention provides applications in the prevention or treatment of cancer, specifically in cancer immunotherapy.

[0093] The present invention provides a method for preventing or treating cancer, specifically for immunotherapy of cancer. The individual may be an individual suffering from cancer. The individual may be a mammal, preferably a human.

[0094] "Treatment" refers to any indication of success in treating or ameliorating an injury, medical condition or condition, including any subjective or objective parameter, for example, relief, remission, making symptoms or damage more tolerable to the patient, decreasing the disease state or condition, slowing the rate of regression or decline, making a final regression point less debilitating, improving the physical or mental well-being of the patient. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including a physical exam, a neuropsychiatric exam, and / or a psychiatric evaluation.

[0095] An "effective amount" is generally an amount sufficient to reduce the severity, frequency of a symptom, eliminate a symptom, the underlying cause of a symptom, prevent the occurrence or underlying cause of a symptom, or ameliorate or correct damage resulting from or associated with a disease state. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to correct a disease state or symptom, particularly a condition or symptom associated with a disease state, or to prevent, hinder, delay, or reverse the progression of a disease state or other undesirable symptoms associated with a disease in any way. A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, provides the intended prophylactic effect, e.g., preventing or delaying the onset of a disease state, or reducing the likelihood of onset (or recurrence) of a disease state or associated symptoms.

[0096] product Kits containing VEGF-Grab, PD-1 or PD-L1 antagonists useful for treating the aforementioned cancers can be provided. Such kits contain a single container when co-formulated into a combined dosage form. Such kits contain containers each containing a VEGF-Grab, PD-1 or PD-L1 antagonist.

[0097] The kit may further include a label or package insert associated with or on the container. The term "package insert" refers to instructions typically included in commercial packaging for therapeutic products, including information regarding indications, applications, dosages, administration, contraindications, and / or warnings regarding the use of such therapeutic products. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container may be formed from a variety of materials, such as glass or plastic. The label or package insert indicates that the composition is used for the prevention or treatment of a selected disease, such as cancer, or for cancer immunotherapy. The kit may further include a second container containing a pharma- ceutically acceptable buffer, such as sterile water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. Other materials desirable by commerce and the user, such as other buffers, diluents, fillers, needles, and syringes, may further be included.

[0098] It may also include further instructions for simultaneous, sequential or separate administration of the VEGF-Grab, PD-1 or PD-L1 antagonists to a patient in need thereof.

[0099] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding the present invention. However, the examples according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following examples.

[0100] Example 1: Combination immunotherapy of PB101 and PD-L1 immune checkpoint inhibitors in an MC38 colon cancer model 1.1 Method 2x10 5 MC38 colon cancer cell line was injected subcutaneously. The tumor size was approximately 50 mm. 3 When the dose reached 100 mg / kg, the drug was administered via intraperitoneal injection (ip) (Figure 1). Tumor size was measured in all groups, and euthanasia was performed using CO2 gas at the end of observation.

[0101] 1.2 Results In an MC38 colon cancer model, compared to the control group, the group treated with αPD-L1 (10F.9G2, a murine PD-L1 antibody) alone showed a 29.5% tumor growth delay, and the group treated with PB101 alone showed a 54.7% tumor growth delay. In particular, the group treated with PB101 and αPD-L1 in combination showed a significant increase in tumor growth delay to 71.4% (Figure 2).

[0102] In an MC38 colon cancer model, combined treatment with PB101 and αPD-L1 increased the antitumor response effect compared to either treatment alone.

[0103] Example 2: Combination immunotherapy of PB101 and PD-L1 immune checkpoint inhibitors in a Hepa-VEGF liver cancer model 2.1 Method 5x10 6 Hepa-VEGF colon cancer cell line was injected subcutaneously. The tumor size was approximately 50 mm 3 When the dose reached 10 mg / kg, the drug was administered via intraperitoneal injection (ip) (Figure 3).

[0104] Tumor size was measured in all groups, and survival was followed up to 85 days to ensure durable efficacy.

[0105] 2.2 Results In the Hepa-VEGF liver cancer model, tumor growth delay was 48.2% in the αPD-L1 group and 60.3% in the PB101 alone group compared to the control group at day 26, and tumor growth delay was significantly increased to 84.1% in the PB101 and αPD-L1 combination group. Furthermore, unlike the other experimental groups, complete tumor regression was observed in 27.2% (3 / 11) of the PB101 and αPD-L1 combination group (Figure 4).

[0106] After an initial 2-week treatment, the drug was discontinued and monitored for 60 days, and a survival benefit was observed with both monotherapy and combination therapy (Figure 5).

[0107] Example 3: Cancer cell rechallenge experiment after combined immunotherapy with PB101 and PD-L1 immune checkpoint inhibitors in a Hepa-VEGF liver cancer model 3.1 Method After two experiments, four mice that were in CR or close to CR were selected from the PB101 + aPD-L1 combination treatment group and were then randomly assigned to receive 2x10 6 Hepa-VEGF colon cancer cell line was injected subcutaneously and 1x10 6 Hepa-VEGF colon cancer cell line was injected intravenously. Six mice in the combination treatment group were injected with cancer cells in the same manner and observed for up to 20 days before being euthanized (Figure 6).

[0108] 3.2 Results When cancer was inoculated into the combined treatment group, it was confirmed that the cancer did not grow, unlike in the control group (Figure 7), and this is presumably due to the formation of immune memory cells.

[0109] In addition, in the case of intravenously injected cancer cells, metastasis to the lungs was confirmed in the control group, but was hardly confirmed in the combined administration group (Figure 8). This is presumably due to the formation of immune memory cells. [Industrial Applicability]

[0110] The pharmaceutical composition according to the present invention can exert effects of retarding tumor growth and preventing cancer metastasis.

[0111] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0112] Sequence Listing Free Text Attach electronic files.

Claims

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

2. The pharmaceutical composition according to claim 1, characterized in that the VEGFR1 domain 2 comprises the amino acid sequence of SEQ ID NO:

1.

3. The pharmaceutical composition according to claim 1, characterized in that the VEGFR1 domain 3 comprises 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. 5. The pharmaceutical composition of claim 4, wherein the anti-PD-1 antibody is AMP-224, GLS-010, PDR-001, Pidilimab, PF-06801591, Genolimzumab, CA-170, MEDI-0680, JS-001, LZM-009, AK-103, Nivolumab, Dostarlimab, Pembrolizumab, Sintilimab, Tislelizumab, Toripalimab, Camrelizumab, or Cemiplimab.

6. 5. The pharmaceutical composition of claim 4, wherein the anti-PD-L1 antibody is CX-072, WBP-3155, KN035, A167, Cosibelimab, Atezolizumab, Avelumab, Durvalumab, Adebrelimab, or BMS-936559.

7. The pharmaceutical composition of claim 1, wherein the fusion protein and the PD-1 or PD-L1 antagonist are contained in a combined dosage form.

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

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