IL-8 overcomes cancer stem cells by blocking the EMT pathway.

Abstract

Compositions, methods, and uses of recombinant IL-8 antibodies, fragments thereof, or single-chain variable fragments (scFv) thereof with high affinity for IL-8 for targeting tumor-expressed or endogenous IL-8 are presented. Preferably, the recombinant IL-8 antibody or scFv fragment comprises a VFV comprising a first amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15, 31-32. H segment, and / or a second amino acid sequence selected from the group consisting of SEQ ID NOs: 16-30, 33-34 L The recombinant IL-8 antibody or scFv fragment can be formulated as a pharmaceutical composition for administration to tumor-bearing patients to reduce tumor metastasis, reduce immunosuppression in the tumor microenvironment, or reduce a Th2-mediated immune response.

Description

[Technical Field]

[0001] This application claims priority to the inventors' concurrently pending U.S. Provisional Patent Application No. 62 / 580,232, filed on November 1, 2017.

[0002] The technical aspect of the present invention relates to compositions, methods, and uses of recombinant scFv or antibodies against interleukin-8 (IL-8) for treating patients with tumors, reducing tumor metastasis, or reducing immunosuppression in the tumor microenvironment. [Background technology]

[0003] The background information includes information that may be useful for understanding the present invention. This does not constitute an endorsement that any information provided herein is prior art or relating to the claimed invention, nor does it constitute an endorsement that any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications herein are invoked by reference in such a manner as if each individual publication or patent application were specifically and individually indicated as being invoked by reference. If a definition or use of a term in an invoked reference conflicts with or contradicts a definition of that term provided herein, the definition provided herein shall apply, and the definition of that term in the reference shall not apply.

[0005] Interleukin-8 (IL-8), also known as a neutrophil chemotactic factor, is primarily produced by macrophages and epithelial cells and is known to play a crucial role in the immune system by recruiting immune cells (e.g., neutrophils) to the site of infection. In recent years, there has been growing interest in identifying the relationship between tumor-derived IL-8 and tumor development, particularly the mechanisms of tumor metastasis and immunosuppression in the tumor microenvironment. For example, several studies have discovered that tumor-derived IL-8 is highly expressed in metastatic tumor cells, inducing epithelial-mesenchymal transition in tumor cells and actually producing tumor-initiating cells (e.g., tumor stem cells). Other studies have shown that tumor-derived IL-8 can attract bone marrow-derived suppressor cells (MDSCs), and that MDSCs can provide an immunosuppressive microenvironment around tumors by interfering with T cell-mediated immune responses in the tumor microenvironment.

[0006] To mitigate the effects of IL-8 on tumorigenesis, efforts have been made to neutralize tumorigenic or endogenous IL-8 by providing human antibodies or humanized antibodies against IL-8, or antisense oligonucleotides or microRNAs against IL-8. For example, U.S. Patent Publication No. 2003 / 0068319, granted to Bar-Eli, discloses the inhibition of tumor angiogenesis and metastasis by fully humanized and isolated monoclonal or polyclonal IL-8. In another example, U.S. Patent No. 5,849,903, granted to Petrzkowski, teaches a 20-base pair antisense oligonucleotide against IL-8 that is effective in reducing the growth of melanoma or lung cancer. However, the effectiveness of antisense oligonucleotides or microRNAs may vary depending on the type of tumor and the method of delivery of such compositions. Furthermore, isolated human antibodies or humanized antibodies may not be effective in some tumor microenvirons where isolated human antibodies or humanized antibodies are cleaved by endogenous metalloproteinases in the tumor microenvironment.

[0007] Thus, despite the study of several approaches to inhibit IL-8 expression or activity in the tumor microenvironment, targeting IL-8 using recombinant IL-8 antibodies or single-stranded variable fragments (scFv) with amino acid sequences highly affinity to IL-8 has been largely unexplored. Therefore, there remains a need for improved compositions, methods, and uses of recombinant IL-8 antibodies or single-stranded variable fragments (scFv) to target tumor-expressed or endogenous IL-8 in the tumor microenvironment to enhance the efficacy of cancer treatment. [Overview of the project]

[0008] The subject of the present invention relates to various compositions, methods, and uses of recombinant IL-8 antibodies, single-strand variable fragments (scFv), or other parts of antibodies (including fusion products, particularly fusion products in TxM) that have high affinity for IL-8, target tumorigenic or endogenous IL-8 to neutralize the effect of IL-8 that promotes EMT (epithelial-mesenchymal transition) in tumor cells, and / or promote immunosuppression in the tumor microenvironment.

[0009] Therefore, one aspect of the subject includes a single-stranded variable fragment (scFv) peptide. The scFv peptide comprises a V-shaped amino acid sequence. H V comprising a segment and / or a second amino acid sequence L Includes segments. The first and second amino acid sequences are selected from SEQ ID NOs: 1-15, 31-32 or SEQ ID NOs: 16-30, 33-34, respectively.

[0010] In another aspect of the subject matter of the present invention, the inventors consider a pharmaceutical composition for treating patients with cancer. The pharmaceutical composition comprises a first amino acid sequence V H V comprising a segment and / or a second amino acid sequence LIt includes a single-chain variable fragment (scFv) that contains segments. The first and second amino acid sequences are each selected from SEQ ID NOs: 1-15, 31-32 or SEQ ID NOs: 16-30, 33-34. Preferably, the scFv peptide is present in a pharmaceutically acceptable carrier.

[0011] Yet another aspect of the subject matter of the present invention is directed to a recombinant nucleic acid. The recombinant nucleic acid has a V segment having a first amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15, 31-32 H A first nucleic acid segment encoding the segment, and / or a second nucleic acid segment having a second amino acid sequence selected from the group consisting of SEQ ID NOs: 16-30, 33-34 and encoding the V segment. L The first and second segments are optionally present within the same reading frame.

[0012] In yet another aspect of the subject matter of the present invention, the inventors consider a recombinant isolated antibody or a fragment thereof. The recombinant isolated antibody or a fragment thereof has a V domain and / or a V domain having first and second amino acid sequences respectively. H domain and / or V L domain. Preferably, the first amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15, 31-32, and the second amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 16-30, 33-34.

[0013] In yet another aspect of the subject matter of the present invention, the inventors also consider a method of reducing the IL-8 effect in a tissue. In this method, a V segment comprising the first amino acid sequence, and / or a V H segment, and / or V LA single-stranded variable fragment (scFv) containing a segment is provided. Preferably, the first amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15 and 31-32, and the second amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 16-30 and 33-34. The method is followed by a step of treating the tissue with the scFv peptide at a dose and schedule effective in reducing the IL-8 effect in the tissue.

[0014] In yet another aspect of the subject matter of the present invention, the inventors consider a method for treating a patient having a tumor. In this method, a V comprising a first amino acid sequence H Segment, and / or V L A pharmaceutical composition is provided comprising a single-stranded variable fragment (scFv) having segments. Preferably, the first amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15 and 31-32, and the second amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 16-30 and 33-34. The pharmaceutical composition is administered to the patient in a dose and schedule effective for treating the tumor.

[0015] In yet another aspect of the subject matter of the present invention, the inventors consider a method for reducing immunosuppression in patients with tumors. In this method, a V comprising a first amino acid sequence H Segment, and / or V L A pharmaceutical composition is provided comprising a single-stranded variable fragment (scFv) having segments. Preferably, the first amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15 and 31-32, and the second amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 16-30 and 33-34. The pharmaceutical composition is administered to the patient in a dose and schedule effective in reducing the presence of bone marrow-derived suppressor cells in the tumor microenvironment.

[0016] In yet another aspect of the subject matter of the present invention, the inventors consider a method for reducing the Th-2-mediated immune response in patients with tumors. In this method, a V comprising a first amino acid sequence H Segment, and / or V L A pharmaceutical composition is provided comprising a single-stranded variable fragment (scFv) having segments. Preferably, the first amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15 and 31-32, and the second amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 16-30 and 33-34. The pharmaceutical composition is administered to the patient in a dose and schedule effective in reducing the patient's Th-2-mediated immune response.

[0017] In yet another aspect of the subject matter of the present invention, the inventors consider a method for reducing epithelial-mesenchymal transition of tumor cells in patients. In this method, a V comprising a first amino acid sequence H Segment, and / or V L A pharmaceutical composition is provided comprising a single-stranded variable fragment (scFv) having segments. Preferably, the first amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15 and 31-32, and the second amino acid sequence is at least 95% identical to a third amino acid sequence selected from the group consisting of SEQ ID NOs: 16-30 and 33-34. The pharmaceutical composition is administered to the patient in a dose and schedule effective in reducing epithelial-mesenchymal transition in tumor cells.

[0018] In yet another aspect of the subject matter of the present invention, the inventors consider the use of the above-mentioned scFv peptide to reduce the Th-2-mediated immune response in patients with tumors. The inventors also consider the use of the above-mentioned scFv peptide to reduce epithelial-mesenchymal transition of tumor cells in patients. Furthermore, the inventors further consider the use of the above-mentioned scFv peptide to reduce tumor metastasis in patients with tumors.

[0019] Various objects, features, aspects, and advantages of the subject matter of the present invention will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings. [Brief explanation of the drawing]

[0020] [Figure 1A] This graph shows the binding kinetics between one of the scFv peptides (43-12) and IL-8 or its orthologue or paralog. [Figure 1B] This graph shows the binding kinetics between one of the scFv peptides (49-31) and IL-8 or its orthologue or paralog. [Figure 2A-D] The graphs show neutrophil migration during IL-8 treatment (2A, 2C) and during simultaneous treatment with IL-8 and scFv peptide (2B, 2D). [Figure 3] The graphs show neutrophil migration during IL-8 treatment (CTRL) and during simultaneous treatment with IL-8 and scFv peptide (43-2, 43-12). [Figure 4A-C] The graph (4A) shows the binding dynamics between one of the scFv peptides (43-12) and IL-8 under specific pH conditions, and the graphs also show the binding dynamics of selected affinity-mature mutant scFv peptides (43-12a and 43-12b) under specific pH conditions. [Modes for carrying out the invention]

[0021] The inventors have now discovered that by mitigating the effects of IL-8, various IL-8-mediated effects, including tumor development and IL-8-mediated tumor metastasis via the epithelial-mesenchymal transition pathway, can be significantly reduced or suppressed. Such mitigating effects can be achieved by capturing or otherwise binding IL-8 in the tumor microenvironment using recombinant IL-8 antibodies or fragments thereof (e.g., scFv fragments for IL-8 that have high affinity for IL-8).

[0022] To this end, the inventors have discovered that various recombinant antibodies or fragments thereof, such as scFv, which have a high affinity for IL-8, can be generated so that the recombinant antibody or fragment, or the scFv fragment, can capture oncogenic or endogenous IL-8 in the tumor microenvironment. Binding or capture of IL-8 from the tumor microenvironment is thought to reduce the IL-8 effect on tumor cells, which initiates tumor cell metastasis. Furthermore, binding or capture of IL-8 may reduce the IL-8 effect that increases immunosuppression in the tumor microenvironment through the accumulation of myeloid-derived suppressor cells (MDSCs). In addition, binding or capture of IL-8 may reduce the IL-8 effect that increases the Th2-mediated immune response in the tumor microenvironment, which may contribute to the recruitment of myeloid-derived suppressor cells in the tumor microenvironment. While we do not wish to be bound by any particular theory or hypothesis, it is considered that IL-8 bound to an antibody or fragment may no longer be able to exert its biological signaling function, possibly due to steric effects. Furthermore, if binding is mediated on a particle or other surface to capture IL-8, the IL-8 concentration required for signal transduction may be reduced so as to diminish or suppress the IL-8 effect. Therefore, the terms binding and capture are used synonymously herein.

[0023] In the context of this specification, the term “tumor” is used synonymously with one or more cancer cells, cancerous tissue, malignant tumor cells, or malignant tumor tissue located in or found in one or more anatomical locations within the human body.

[0024] In the usage of this specification, the term “to combine” means K D However, 10 -3 M or less, 10 -4 M, 10 -5 M, 10 -6 M, or 10 -7 The terms "recognize" and / or "detect" interactions between two molecules with high affinity (M or less) can be used synonymously.

[0025] In the context of this specification, the terms “provide” or “providing” mean, and include, any act of manufacturing, producing, arranging, making available, or making immediately available.

[0026] In one exemplary and particularly preferred embodiment of the subject matter of the present invention, the inventors provide a V bonded to IL-8. H Segments and V L We consider single-stranded variable fragment (scFv) peptides having segments. While it is considered that the scFv peptide sequence may be any suitable sequence that provides a desirable binding affinity to IL-8, we have found that scFv peptides can be generated using at least one or more of the peptide sequences SEQ ID NOs: 1 to 34 in Table 1.

[0027] As an alternative, and in addition thereto, the inventors also present V shown in Table 1 H Segment and / or V L It is also considered that recombinant isolated antibodies or fragments thereof may be generated using the amino sequences of segments. In the use of this specification, the term “antibody” refers to immunoglobulin molecules and molecules containing the immunologically active portion of an immunoglobulin molecule, i.e., an antigen-binding site that binds immunospecifically to an antigen. Therefore, “antibodies and fragments thereof” include whole immunoglobulin molecules (e.g., full size, whole IgG1, etc.) and fragments of whole antibody molecules. Thus, fragments include scFv, Fab fragments, Fab' fragments, F(ab')2, disulfide-linked Fvs (sdFvs), Fvs, and V H Segment and / or V LAny fragment comprising either of the segments may be, but is not limited thereto. If the antibody is an immunoglobulin, it is considered that the immunoglobulin may constitute different types of immunoglobulins by comprising heavy chains or constant domains of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) and any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Furthermore, "antibodies" may include, but are not limited to, human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, and polyclonal antibodies. Therefore, as shown in Table 1, V H and / or V L It should be understood that the domain can be grafted onto any existing (typically human or humanized) antibody or antibody fragment using methods well known in the art.

[0028] The inventors of the present invention have identified V as shown in Table 1. H Segment and / or V L The amino sequence of the segment is used to couple with a carrier protein, and V is placed on its surface so that IL-8 can be captured by the hybrid protein. H Segment and / or V L Further consideration will be given to the possibility of generating hybrid proteins with segments. H Segment and / or V L Any suitable form of the carrier protein that can stably retain the segment and preferably provide access to the tumor microenvironment is considered. A particularly preferred carrier protein is one or more V H Segment and / or V L Examples include albumin, refolded albumin, and other proteins that have affinity for the antibody moiety (e.g., protein A, protein G, protein Z) coupled to the segment.

[0029] Typically, V H Segment and / or V L The segment is conjugated with an anchor molecule, thereby V HSegment and / or V L The segment can be coupled to a carrier protein. For example, if the carrier protein is albumin, the anchor molecule can be any appropriately sized hydrophobic peptide or glycolipid that fits into Sudlow sites I and II of albumin, or any other hydrophobic region of albumin. For example, the recombinant immunoglobulin protein for IL-8 described above can be coupled to the carrier protein via its Fc domain. In other embodiments, the anchor molecule may include a hydrophobic peptide (of a length of at least 10, 15, 20, or 30 amino acids). In these embodiments, V H Segment and / or V L Various configurations of segments (e.g., as scFv morphologies) and hydrophobic peptides can be considered. For example, a monovalent scFv domain may be directly linked to a hydrophobic peptide, or a polyvalent scFv may be directly linked to a hydrophobic peptide. Alternatively, one scFv domain may be directly linked to multiple hydrophobic peptides, or multiple scFv domains may be directly linked to multiple hydrophobic peptides.

[0030] Alternatively, or in addition to the above, one or more V H Segment and / or V L The segment can be coupled with an intermediate molecule having an anchor portion that binds to a carrier protein. In a preferred embodiment, the inventors have configured the intermediate molecule such that multiple target recognition domains may be vested via a single binding site on the carrier protein. H Segment and / or V LWe consider providing multiple binding sites to the segment. Suitable intermediate molecules may include any protein, glycolipid, organic molecule, or inorganic molecule that does not impose any significant toxicity to naive tissue. For example, suitable intermediate molecules may include nanoparticles (e.g., quantum dots, gold nanoparticles, magnetic nanoparticles, nanotubes, polymer nanoparticles, dendrimers, etc.) or beads (e.g., polystyrene beads, latex beads, DynaBeads, etc.). Preferably, the nanoparticles and / or beads have dimensions of less than 1 μm, preferably less than 100 nm. The nanoparticles may be crosslinked to or partially coated with hydrophobic tails that provide anchors to a carrier protein (e.g., albumin). One or more V H Segment and / or V L The segments can also be crosslinked onto or partially coated onto nanoparticles (for example, via additional tail domains linked to the target recognition domain for crosslinking purposes).

[0031] In another example, a suitable intermediate molecule may include beads (e.g., polystyrene beads, latex beads, DynaBeads, etc.) conjugated with an antibody against a carrier protein. Therefore, if the carrier protein is albumin, the beads may be conjugated with an α-albumin antibody (e.g., crosslinked, coated, etc.) so that the beads can bind to the carrier protein with high affinity and specificity. One or more V H Segment and / or V L The segments can also be crosslinked onto the beads or partially coated onto them (e.g., via additional tail domains linked to the target recognition domain for crosslinking, thiol-mediated crosslinking, etc.).

[0032] In some embodiments, the scFv peptide may form a recombinant immunoglobulin protein complex comprising or mimicking an ALT-803 (IL-15 superagonist complex, see, e.g., Blood 2015 126:1957) or TxM (targeted ALT-803-based scaffold platform, see, e.g., URL, altorbioscience.com / our-science / il-15-protein-superagonist-and-scaffold-technology / ) structure. Preferably, we consider that if the immunoglobulin protein complex mimics a TxM structure, the scFv peptide may be directly (or indirectly via a linker) coupled to one or more interleukin-15 (IL-15) binding motifs and / or one or more ligands for IL-15 binding motifs (e.g., IL-15, IL-15N72D, etc.). Therefore, if the recombinant immunoglobulin protein complex mimics the TxM IgG1 structure, the recombinant immunoglobulin protein complex may also contain the 1-4 scFv peptide for IL-8.

[0033] Furthermore, recombinant immunoglobulin protein complexes that mimic a TxM structure having one or more scFv peptides coupled to an IL-15 binding motif or its ligand may also include binding domains to tumor-specific antigens or patient-specific and tumor-specific neoepitopes (e.g., scFv peptides for neoepitopes). For example, a recombinant immunoglobulin protein complex may include two scFv peptides for IL-8 coupled to two IL-15 binding motifs, and two scFv peptides for neoepitopes coupled to two IL-15 binding motif ligands. Preferably, the neoepitopes are patient-specific and tumor-specific, and are identified by omics analysis of sequence data, as disclosed in U.S. Patent Application Publication No. 2012 / 0059670A1 and U.S. Patent Application Publication No. 2012 / 0066001A1.

[0034] In some embodiments, scFv, recombinant antibody, or fragment thereof is V of SEQ ID NOs. 1-15, 31-32. H It can be generated using a single sequence that encodes the segment. In other embodiments, the scFv, recombinant antibody, or fragment thereof is V of SEQ ID NOs. 16-30, 33-34. L It can be generated using a single sequence that encodes the segment. In other embodiments, the scFv, recombinant antibody, or fragment thereof may be V of SEQ ID NOs. 1-15, 31-32. H A single array that codes the segment, and V for sequence numbers 16-30, 33-34. L It can be generated using a single array that encodes the segments. In these embodiments, V H The sequence that codes for the segment, and V L The arrays encoding the segments are preferably pairs of arrays (for example, in scFv 49-31, arrays 1 and 16; in scFv 49-22, arrays 2 and 17; in scFv 49-7, arrays 3 and 18, etc.). However, any pair of V H Segments and V L The segment is one V H Select a segment from sequence numbers 1-15 and 31-32, and one V L It is considered that the segment can be generated by selecting segments from sequence numbers 16-30 and 33-34.

[0035] [Table 1]

[0036] [Table 2]

[0037] [Table 3]

[0038] The inventors also provide a method in which scFv, recombinant antibody, or fragment thereof is at least 85% identical, preferably at least 90% identical, and more preferably at least 95% identical to any one of SEQ ID NOs: 1-15, 31-32, or any one of SEQ ID NOs: 16-30, 33-34. H Segment and / or V L It is also considered that the segments may be generated using amino acid sequences that encode them. In such embodiments, the binding affinity of the scFv peptide, recombinant antibody, or fragment thereof is preferably 60% or more, preferably 70% or more, and more preferably 80% or more, of the binding affinity of the scFv peptide, recombinant antibody, or fragment thereof generated using any one of SEQ ID NOs. 1-15, 31-32, or any one of SEQ ID NOs. Indeed, and as will be discussed in more detail below, the inventors subjected selected scFv(43-12) to affinity maturation via random mutagenesis in the CDR regions of VH and VL. Notably, and among several binders in particular, two scFv(43-12a and 43-12b) were isolated from the affinity maturation process with improved binding characteristics.

[0039] Most typically, V in scFv peptides, recombinant antibodies, or fragments thereof. H Segments and V L The segments are typically 5 to 40 amino acids, preferably 10 to 30 amino acids, more preferably 20 to 30 amino acids, and are conjugated via linkers or spacers. In some embodiments, the linker is V H N-terminus and V of the segment L It can be conjugated to the C-terminus of the segment. In other embodiments, the linker is V L N-terminus and V of the segment H It can be conjugated to the C-terminus of the segment. The inventors of the present invention have found that V H Segment and V L A glycine-rich sequence for the linker (e.g., (G4S)) is provided to offer structural flexibility between the segments. nFor example, it is considered preferable that n is 1 to 5 in the formula). It is also considered to increase the solubility of the scFv peptide, recombinant antibody or fragment thereof by including one or more serine or threonine residues in the linker. There are a number of known methods for making linkers and scFvs in the art, and all such known methods are considered suitable for use herein.

[0040] Furthermore, the scFv peptide may contain multiple V H segments and V L segments and can form divalent or multivalent scFvs. In some embodiments, the multiple V H segments and / or V L segments may have the same amino acid sequence (for example, a multivalent scFv having three V H segments and three V L segments, where all V H segments have 49-31 V H (SEQ ID NO: 1) and all V L segments have 49-31 V L (SEQ ID NO: 16). In other embodiments, at least two of the V H segments and / or V L segments may have different amino acid sequences (for example, a multivalent scFv having three V H segments and three V L segments, where two of the V H segments are 49-31 V H (SEQ ID NO: 1), one of the V H segments is 49-22 V H (SEQ ID NO: 2), two of the V L segments are 49-31 V L (SEQ ID NO: 16), and one of the V H segments is 49-22 V L (SEQ ID NO: 17).

[0041] Preferably, the binding affinity (Kd) of scFv, recombinant antibody, or fragment thereof to IL-8 (at least one of 72-mer and 77-mer IL-8) is at least 1 × 10⁻¹⁶ when measured at a temperature of 25°C to 37°C and in a pH range of 5.5 to 7.5. -7 Less than M, preferably 1 × 10 -8 Less than M, more preferably 1 × 10 -9 It is less than M. The inventors of this invention have found that V H Segment and / or V L We consider that even if scFv, recombinant antibody, or fragment thereof is generated using the same amino acid sequence of a segment, the binding affinity of the scFv, recombinant antibody, or fragment thereof to IL-8 may differ due to structural differences. For example, Table 2 provides different affinities (measured by KD values) of scFv and recombinant antibody (IgG1) of several clones. Clones 49-31 are V H Segment (SEQ ID NO: 1) and V L The scFv or recombinant antibody (IgG1) generated using the amino acid sequence encoding the segment (SEQ ID NO: 16) is, and clone 43-2 is V H Segment (SEQ ID NO: 8) and V L The scFv or recombinant antibody (IgG1) generated using the amino acid sequence encoding the segment (SEQ ID NO: 23) is V H Segment (SEQ ID NO: 10) and V L This is an scFv or recombinant antibody (IgG1) generated using the amino acid sequence encoding the segment (SEQ ID NO: 25). D The preliminary values ​​are measured by surface plasmon resonance measurements, and the highest and lowest values ​​are shown to estimate the affinity range for IL-8. All Kd values ​​in Table 2 are in units of 10. -9 It is M.

[0042] [Table 4]

[0043] Table 3 provides further exemplary data for molecules having the sequences shown in Table 1. Here, using the sequences shown, scFv and the corresponding humanized IgG1 are prepared, and the KD is determined by SPR using immobilized IL-8 as the analyte on the chip surface. All values ​​are expressed as nano-M values ​​at the temperatures shown.

[0044] [Table 5]

[0045] scFv peptide (or antibody or fragment thereof V) H Segments and V L It is also considered that the segment may be encoded by a single recombinant nucleic acid. In this embodiment, the recombinant nucleic acid is V H The first nucleic acid segment (sequence element) that codes the segment, and V L It comprises at least two nucleic acid segments, including a second nucleic acid segment that codes for the first segment. Preferably, the first nucleic acid segment is selected to code for at least one of the amino acid sequences of SEQ ID NOs: 1-15 and 31-32, and the second nucleic acid segment is selected to code for at least one of the amino acid sequences of SEQ ID NOs: 16-30 and 33-34. However, each V H and V L It is also considered that the first and second nucleic acid segments encoding the segment are at least 85% identical, preferably at least 90% identical, and more preferably at least 95% identical, to one of sequence numbers 1-15, 31-32, or one of sequence numbers 16-30, 33-34. Most preferably, the two nucleic acid segments are within the same reading frame so that the two nucleic acid segments can be translated into a single protein having two peptide segments.

[0046] Furthermore, the recombinant nucleic acid may include a third nucleic acid segment between the first and second nucleic acid segments that encodes a linker peptide (preferably a G-rich or otherwise flexible linker peptide), which is typically 5 to 40 amino acids, preferably 10 to 30 amino acids, and more preferably 20 to 30 amino acids. In this embodiment, it is particularly preferable that the three nucleic acid segments are within the same reading frame so that the three nucleic acid segments can be translated into a single protein having three peptide segments.

[0047] In some embodiments, the recombinant nucleic acid may comprise multiple sets of first and second nucleic acid segments, each set comprising one of each of the first and second nucleic acid segments. In these embodiments, the recombinant nucleic acid preferably comprises a fourth nucleic acid segment encoding a connector peptide located between each set of the first and second nucleic acid segments. Thus, one exemplary recombinant nucleic acid may comprise [first set]-fourth nucleic acid encoding a connector peptide-[second set]-fourth nucleic acid encoding a connector peptide-[third set], where each of the first, second, and third sets comprises [V H First nucleic acid encoding the segment - third nucleic acid encoding the linker - V L The first and second nucleic acids include a second nucleic acid that encodes the segment, and the positions of the first and second nucleic acids may be arranged alternately with respect to each other. The sequence of the connector peptide may vary by the number of sets in one peptide. Preferably, the connector peptide may consist of 5 to 50 amino acids, preferably 10 to 40 amino acids, and more preferably 20 to 30 amino acids. The inventors also include V H and V L We also consider that glycine-rich sequences (e.g., G4S) are preferable for providing flexibility to connector peptides between segment sets.

[0048] The inventors further consider that scFv peptides, antibodies, or fragments thereof may be formulated as pharmaceutical compositions so as to be administered to patients with tumors to reduce or inhibit the endogenous effects of IL-8. Accordingly, it is considered that scFv peptides, antibodies, or fragments thereof may be formulated in any pharmaceutically acceptable carrier (e.g., as a sterile injection composition) in an amount of at least 1 ml, preferably at least 5 ml, more preferably at least 20 ml per dose unit for a therapeutic formulation. However, alternative formulations are also considered suitable for use herein, and all known routes and modes of administration are considered herein. In the usage herein, the term “administer” refers to both direct and indirect administration of the compounds and compositions considered herein, where direct administration is typically carried out by a healthcare professional (e.g., a physician, nurse, etc.), while indirect administration typically involves the step of providing or making available the compounds and compositions for direct administration to a healthcare professional.

[0049] In some embodiments, the pharmaceutical formulation is administered by systemic injection, including subcutaneous, subdermal, or intravenous injection. In other embodiments where systemic injection may not be efficient (for example, in the case of brain tumors), administration of the formulation by intratumor injection is considered.

[0050] The above V H and V L One exemplary method and use of a pharmaceutical composition comprising a segment, an scFv peptide, an antibody, or a fragment thereof, is to reduce IL-8-mediated effects in a target tissue. In the uses herein, IL-8-mediated effects refer to any biological consequences directly or indirectly induced by the presence of IL-8 in or in the tissue microenvironment. Thus, IL-8 effects may be attributable to IL-8 released (or secreted) by tumor cells and / or non-tumor cells in the tissue (e.g., immune-competent cells such as lymphocytes) or outside the tissue (e.g., healthy tissue near a tumor) and present in the tumor microenvironment at any given time.

[0051] In the embodiments considered in particular, the inventors consider IL-8-mediated effects, including, but not limited to, initiating cell migration, initiating epithelial-mesenchymal transition of tumors, initiating tumor metastasis, enhancing immunosuppression in the tumor microenvironment, stimulating MDSC development, and initiating a Th2-biased immune response in the tumor microenvironment, especially in the tumor microenvironment of patients. Therefore, without wishing to be bound to any particular theory, the inventors consider that reducing the amount of IL-8 from the tumor microenvironment by capturing free IL-8 (IL-8 not bound to the IL-8 receptor) reduces the IL-8 effect, or even reverses the effect of IL-8 on tumor cells: reducing epithelial-mesenchymal transition of tumors to reduce or prevent tumor metastasis, reducing a Th2-biased immune response or re-equalizing Th1 and Th2-mediated immune responses, and reducing or preventing immunosuppression in the tumor microenvironment. In particular, when excessive or abnormal increases in IL-8 expression (or accumulation) are observed in certain types of cancer (e.g., pancreatic cancer, triple-negative breast cancer, glioblastoma, etc.) compared to corresponding healthy tissue, it is expected that IL-8 binding or capture may more effectively alter the prognosis of these types of cancer.

[0052] With regard to the dose and schedule of administration of the pharmaceutical composition to the patient, it is considered that the dose and / or schedule may vary depending on the type of peptide (e.g., scFv, antibody, antibody fragment, any two combinations thereof, all combinations, etc.), the type and prognosis of the disease (e.g., tumor type, size, location), and the patient's health status (e.g., age, sex, etc.). Although it may vary, the dose and schedule may be selected and adjusted so that the formulation does not cause any significant toxic effect on the host's normal cells, but is still sufficient to reduce the effect of IL-8 in the tumor microenvironment by at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50% within 3 hours, 6 hours, 12 hours, 24 hours, 72 hours, or 1 week.

[0053] In some embodiments, the effect of IL-8 and the reduction of the IL-8 effect can be measured by the amount of free IL-8 (either 72-mer or 77-mer) in the tissue. In these embodiments, the administration conditions are typically adjusted so that at least one amount or concentration of 72-mer or 77-mer free IL-8 decreases by at least 30%, more preferably at least 40%, and most preferably at least 50% within 3 hours, 6 hours, 12 hours, 24 hours, 72 hours, or 1 week after administration of the pharmaceutical composition. In other embodiments, the effect of IL-8 and the reduction of the IL-8 effect can be measured via in vitro or in vivo measurements of biological activity. For example, doses and treatment schedules for reducing tumor cell migration or metastasis can be determined by an in vitro cell migration assay with IL-8. In this example, the administration conditions are typically adjusted to reduce the number of cells migrating from their original position or the migration distance of migrating cells by at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50%, within 1, 3, 6, and 12 hours after treatment with the pharmaceutical composition (or scFv, antibody, or fragment thereof without a pharmaceutically acceptable carrier) or within 1 hour, 3 hours, 6 hours, and 12 hours. As another example, the dose and treatment schedule for reducing immunosuppression by bone marrow-derived suppressor cells may be determined by measuring the accumulation or presence of bone marrow-derived suppressor cells in the tumor. Therefore, in this example, the administration conditions are typically adjusted to reduce the number of bone marrow-derived suppressor cells (in the total tumor tissue, or per 1 cm of tumor tissue) 2 The amount per serving is adjusted to decrease by at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50% within 3 hours, 6 hours, 12 hours, 24 hours, 72 hours, or 1 week after administration of the pharmaceutical composition.

[0054] In other embodiments, the effect of IL-8 and the reduction of the IL-8 effect may be measured by measuring the amount or concentration of local cytokine molecules. For example, the dose and treatment schedule for reducing the Th-2-mediated immune response may be determined by measuring the amount or concentration of at least one of IL-4, IL-5, IL-6, IL-9, IL-19, and IL-13 in the tumor microenvironment. In this example, the administration conditions are typically adjusted so that the amount or concentration of at least one of IL-4, IL-5, IL-6, IL-9, IL-19, and IL-13 decreases by at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50% within 3 hours, 6 hours, 12 hours, 24 hours, 72 hours, or 1 week after administration of the pharmaceutical composition.

[0055] In other embodiments, the effect of IL-8 and the reduction of the IL-8 effect may be measured by measuring the in vitro or in vivo expression levels of markers in one or more cells. For example, doses and treatment schedules for reducing epithelial-mesenchymal transition in tumor cells may be determined by measuring the expression of E-cadherin epithelial markers and N-cadherin mesenchymal markers in tumor cells. Therefore, the administration conditions are typically adjusted so that the expression level of N-cadherin mesenchymal markers decreases by at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50% within 3 hours, 6 hours, 12 hours, 24 hours, 72 hours, or 1 week after administration of the pharmaceutical composition, or the expression level of E-cadherin epithelial markers (compared to treatment with IL-8 alone) increases by at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50% within 3 hours, 6 hours, 12 hours, 24 hours, 72 hours, or 1 week after administration of the pharmaceutical composition. It is also considered that the ratio of N-cadherin to E-cadherin expression in tumor cells may also serve as an indicator of the effect of IL-8 and the decrease in the effect of IL-8. In this example, the administration conditions are typically adjusted so that the ratio of E-cadherin:N-cadherin expression levels increases by at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50%, within 3, 6, 12, 24, 72 hours, or 1 week after administration of the pharmaceutical composition.

[0056] From a different perspective, a decrease in IL-8-mediated effects in vivo can also be observed through physiological phenomena. For example, a decrease in IL-8 concentration can be observed through a decrease or elimination of EMT (epithelial-mesenchymal transition) in tumor cells and a reduction in associated signaling pathways. Similarly, a decrease in IL-8 concentration in patients, particularly in the tumor microenvironment, reduces the stem cell properties of tumor cells, as readily observable by appropriate stem cell markers. Another physiological effect of IL-8 reduction is the typical decrease in MDSC development, and the Th2-biased immune response in T cells within tumors is also reduced (thus shifting the Th1 / Th2 balance towards a Th1-type response).

[0057] Furthermore, the inventors consider that the effect of scFv, antibodies, or fragments thereof on IL-8 to reduce epithelial-mesenchymal transition in tumor cells in certain types of cancer may be enhanced by the simultaneous administration of one or more cancer drug therapies. These cancer drug therapies include fulvestrant, aldoxorubicin, docetaxel, and tumor necrosis treatment agents (e.g., 131 Examples of cancer drug therapies include, but are not limited to, I-chTNT-3, avelumab (a human monoclonal IgG1 antibody that interferes with the interaction between PD-L1 and its receptor), Braquili-targeted vaccines (e.g., ETBX-051 (Ad5[E1-,E2b-]-Braquili)), Her2-targeted vaccines (e.g., ETBX-021), MUC-1-targeted vaccines (e.g., ETBX-061 (Ad5[E1-,E2b-]-MUC1)), and yeast vaccines (e.g., GI-4000 (GI-4014, GI-4015, GI-4016, GI-4020), GI-6207, GI-6301). Details of these cancer drug therapies are described in international publication PCT / US17 / 40297, which is incorporated herein by reference in its entirety.

[0058] In some embodiments, the inventors consider that the effect of scFv, antibodies, or fragments thereof on IL-8 may be enhanced by the co-administration of one or more checkpoint inhibitors. With respect to proteins that interfere with or downregulate checkpoint inhibition, it is considered that any suitable peptide ligand that binds to the checkpoint receptor may be considered. Most typically, the binding inhibits or at least reduces receptor-mediated signaling, particularly CTLA-4 (especially CD8). + In the case of cells), PD-1 (especially CD4 + In the case of cells, receptors such as the TIM1 receptor, 2B4, and CD160 are considered. For example, suitable peptide binders may include not only antibody fragments that specifically bind to receptors, particularly scFv, but also small molecule peptide ligands (e.g., isolated via RNA display or phage panning). It should be understood here that the expression of peptide molecules is preferably coordinated so that the neoepitope or polytope is expressed simultaneously with one or more peptide ligands. Therefore, it is typically considered that peptide ligands are generated from a single transcript (which may or may not include the sequence portion encoding the polytope) or from multiple transcripts, for example, using an internal ribosome entry site or a 2A sequence.

[0059] In other embodiments, the inventors consider that the effect of scFv, antibodies, or fragments thereof on IL-8 may be modulated by the simultaneous administration of one or more immunostimulatory cytokines. For example, immunostimulatory cytokines may be selected based on the desired immune response or the direction of CD4+ T cell / naive Th cell polarization. For example, in one embodiment where polarization of Treg cells from naive CD4+ T cells is desired, the immunostimulatory cytokines may be selected to include IL-2 and TGF-β. In another embodiment where polarization of Th17 cells from naive CD4+ T cells is desired, the immunostimulatory cytokines may be selected to include IL-6 and TGF-β. Similarly, immunostimulatory cytokines for Th1 cell polarization may include IL-12 and IFN-γ, and immunostimulatory cytokines for Th2 cell polarization may include IL-4. Furthermore, immunostimulatory cytokines for Tfh cell (follicular helper T cell) polarization may include IL-6 and IL-12, and immunostimulatory cytokines for CD4+ cytotoxic T cell polarization may include IL-2.

[0060] The inventors further consider that scFv, antibodies, or fragments of the same agent against IL-8 may be co-treated (or co-administered) to cancer patients with activated or modified immune cells that can provide better access to the scFv, antibodies, or fragments of the same agent against IL-8 by reducing the immunosuppressive effect of tumor cells or bone marrow-derived suppressor cells in the tumor microenvironment, thereby maximizing the effect of scFv, antibodies, or fragments of the same agent against IL-8. For example, the activated or modified immune cells may include naive NKT cells or genetically modified NKT cells that express chimeric proteins or T cell receptor complexes to induce an NKT cell immune response and / or alter the tumor microenvironment (e.g., by suppressing the activity of bone marrow-derived suppressor cells). Preferably, the chimeric proteins or T cell receptors of genetically modified NKT cells bind to tumor (neo)epitopes, tumor-associated antigens, or autolipids presented on tumor cells. As another example, modified immune cells may include NKT cells that have been genetically modified to express at least one of CD40L and Fas-L, preferably on their cell surface. Details of genetically modified NKT cells and / or naive NKT cells, activated NKT cells for reducing immunosuppression in the tumor microenvironment are described in International Publication PCT / US18 / 53506 (and its corresponding U.S. National Phase Publication), which is incorporated herein by reference in whole. Similarly, activated or modified immune cells may include naive T cells or genetically modified T cells that express chimeric proteins or T cell receptor complexes to induce a T cell immune response and / or alter the tumor microenvironment (e.g., by suppressing the activity of bone marrow-derived suppressor cells). Preferably, the chimeric proteins or T cell receptors of genetically modified NKT cells bind to tumor (neo)epitopes, tumor-associated antigens, or autolipids presented on tumor cells.

[0061] As yet another example, activated or modified immune cells may include genetically modified NK cells expressing killer-activated receptors (KARs) that target soluble NK cell receptor ligands (e.g., NKG2D, Nkp-30, Nkp-44, Nkp-46, etc.), which inhibit effective NK cell activity by acting as decoy ligands for the NK cell receptor. Details of genetically modified NK cells having KARs are described in U.S. Provisional Patent Application No. 62 / 569503, which is incorporated herein by reference in its entirety.

[0062] In some embodiments, one or more of the above-mentioned cancer drug therapies, immunostimulatory cytokines, checkpoint inhibitors, and / or naive or genetically modified NK cells or NKT cells may be formulated in the same pharmaceutical composition as scFv, antibodies, or fragments thereof against IL-8. In other embodiments, the cancer drug therapy may be incorporated into a separate pharmaceutical composition which may be provided together with the pharmaceutical composition of scFv, antibodies, or fragments thereof, or into a separate pharmaceutical composition which may be administered before the pharmaceutical composition of scFv, antibodies, or fragments thereof is administered to the patient.

[0063] Therefore, it should be understood that the IL-8 conjugating molecules presented herein may form a therapeutic regimen including the administration of the IL-8 conjugating molecules presented herein. For example, it is conceivable that the treatment of cancer patients (e.g., pancreatic cancer, triple-negative breast cancer, glioblastoma) may be carried out in a modified manner, such as administering a first drug (e.g., fulvestrant) to reverse the mesenchymal-to-epithelial transition (MET) from EMT and a second drug (e.g., aldoxorubicin) to specifically target the hypoxic tumor microenvironment. Such a therapeutic regimen is thought to reduce the immunosuppressive environment, increase cellular stress on tumor cells, and consequently lead to increased immunogenicity of tumor cells against the immune system. Furthermore, the treatment may also include tumor necrosis-targeting antibodies that "label" necrotic tumor cells, and thus increase susceptibility to attack by NK cells and / or cytotoxic T cells. The IL-8 conjugating molecules presented herein may be administered concomitantly to further suppress the IL-8-mediated immunosuppressive effects described above. Furthermore, in the event of a decline in the immunosuppressive state as described above, the patient may then receive immunotherapy using a recombinant vaccine that typically expresses one or more of the following: Braquili, tumor or cancer-associated antigens, and / or patient-specific and tumor-specific neogenic antigens. Thus, cancer treatment may include tumor necrosis-targeting agents, drugs that target the hypoxic tumor microenvironment, drugs that reverse the MET conversion of EMT, vaccine components (recombinant viruses, yeasts, and / or bacteria), and at least two (or at least three, or at least four) IL-8 binding molecules as presented herein. [Examples]

[0064] The inventors of the present invention, V H Segment code 10 (43-12 V) H ), and V L Segment code 25(43-12 V) LUsing the nucleic acid sequence of ), the scFv molecule (43-12) was constructed, and its binding affinity to IL-8 (72-mer and 77-mer) and cross-reactivity with other orthologues or paralogs of IL-8 were determined. As shown in Figure 1A, the 43-12 scFv molecule showed strong affinity (KD of 385 pM and 440 pM) to both the 72-mer and 77-mer of IL-8, and showed only very slight cross-reactivity with paralogs (hCXCL1, hCXCL2, hCXCL7) or orthologues (mCXCL1) when present.

[0065] The inventors of the present invention, V H Segment code 1 (49-31 V) H ), and V L Segment code 16(49-31 V) L Using the nucleic acid sequence of ), the scFv molecule (49-31) was constructed, and its binding affinity to IL-8 (72-mer and 77-mer) and cross-reactivity with other orthologues or paralogs of IL-8 were determined. As shown in Figure 1B, the 49-31 scFv molecule showed strong affinity (KD of 147 pM and 120 pM) to both the 72-mer and 77-mer of IL-8, and showed only very slight cross-reactivity with paralogs (hCXCL1, hCXCL2, hCXCL7) or orthologues (mCXCL1) when present.

[0066] Next, the inventors tested the scFv molecules thus prepared (43-2, 43-12, 49-31, etc., all also shown in Table 3) to determine their IL-8 neutralizing effect. In a set of exemplary experiments, primary human neutrophils were isolated from blood, and the ability of scFv molecules (here 43-2, 43-12, 49-31) to neutralize IL-8-mediated neutrophil chemotaxis was evaluated. In a 3 mM pore size Transwell plate, 1 nM recombinant human IL-8 was incubated with antibody dose settings as shown in the left panel, and 50,000 calcein-AM labeled neutrophils were placed in the upper chamber and cultured for 1.5 hours and 3 hours. Neutrophil migration was assessed by assaying total fluorescence in the lower chamber, subtracting the background from wells containing only blank medium. Figure 2, panels A-D, show graphs representing in vitro neutrophil migration after treatment with IL-8 alone (Figures 2A and 2C) and with IL-8 and one of the scFv molecules (43-2, 43-12, or 49-31) for 1.5 hours (Figure 2B) or 3 hours (Figure 2D). As shown in Figures 2B and 2D, all scFv molecules (43-2, 43-12, and 49-31) reduced neutrophil migration by 30-70% at concentrations below 1 μM at both 1.5 and 3 hours, suggesting that all scFv molecules (43-2, 43-12, and 49-31) are effective in mitigating the effects of IL-8 by capturing free IL-8 in serum (or culture medium).

[0067] Figure 3 shows graphs illustrating neutrophil migration after treatment with IL-8 alone (CTRL) and with IL-8 and one of the scFv molecules (43-2, 43-12) at a concentration of 1 nM. Both scFv molecules (43-2, 43-12) completely reversed the effect of IL-8, so that the level of neutrophil migration with scFv molecule (43-2, 43-12) treatment was the same as or nearly the same as that with the medium alone. On the other hand, IL-8 treatment alone increased neutrophil migration by almost three times compared to the medium alone.

[0068] In yet another set of experiments, the inventors performed SPR analysis on selected mutant morphologies using affinity maturation (here, random mutagenesis and mRNA presentation selection of the CDR region of the VH and VL chains) of a previously identified IL-8 binder (here, 43-12, sequence in Table 1) under two different pH conditions, pH 7.4 and pH 6.0. As can be seen from the results in Figures 4A-C (Figure 4A depicts the results for the parent scFv 43-12), both derivative mutant morphologies exhibited improved binding affinity compared to the parent scFv. Specifically, Figure 4B shows exemplary results for 43-12a (sequence shown in Table 1), and Figure 4C shows exemplary results for 43-12b (sequence shown in Table 1).

[0069] It will be apparent to those skilled in the art that many more modifications beyond those already described are possible without departing from the inventive concept of this specification. Therefore, the subject matter of the present invention is not limited, except to the appended claims. Furthermore, in the interpretation of both this specification and the claims, all terms should be interpreted in the broadest possible form in context. In particular, the terms “comprises” and “comprising” should be interpreted in a non-exclusive manner to refer to an element, component, or step, suggesting that the referenced element, component, or step may exist, be used, or be combined with other elements, components, or steps not explicitly referenced. In the description herein and throughout the subsequent claims, the meanings of “a,” “an,” and “the” include plural references unless the context explicitly states otherwise. As used in this description, the meaning of “in” includes “in” and “on” unless the context explicitly states otherwise. Where a claim in this specification refers to at least one selected from the group consisting of A, B, C, ..., and N, the text should be interpreted as requiring only one element from the group, and not A+N or B+N, etc.

Claims

1. A V comprising a first amino acid sequence Ｈ V comprising a segment and a second amino acid sequence Ｌ A single-chain variable fragment (scFv) peptide comprising a segment, which specifically binds to interleukin-8 (IL-8), The aforementioned V Ｈ Segment and the V Ｌ The segment is (i) Sequence ID 8 and Sequence ID 23, (ii) Sequence IDs 10 and 25, (iii) Sequence IDs 12 and 27, (iv) Sequence ID 7 and Sequence ID 22, (v) Sequence IDs 13 and 28, (vi) Sequence ID 3 and Sequence ID 18, (vii) Sequence IDs 11 and 26, (viiii) Sequence IDs 14 and 29, (ix) Sequence ID 6 and Sequence ID 21, (x) Sequence ID 2 and Sequence ID 17, (xi) Sequence ID 15 and Sequence ID 30, (xii) Sequence ID 1 and Sequence ID 16, (xiii) Sequence ID 4 and Sequence ID 19, (xiv) Sequence ID 5 and Sequence ID 20, (xv) Sequence ID 9 and Sequence ID 24, (xvi) Sequence IDs 31 and 33, and (xvii) Sequence IDs 32 and 34 A peptide in which scFv is formed by a pair selected from the group consisting of the following.

2. The aforementioned V Ｈ Segments and V Ｌ The peptide according to claim 1, wherein the segment is conjugated with a linker peptide.

3. The peptide according to claim 2, wherein the linker peptide is a glycine-rich peptide.

4. The peptide according to any one of claims 1 to 3, wherein the peptide is present in a pharmacologically acceptable carrier.

5. The peptide is V Ｈ Segments and V Ｌ The peptide according to any one of claims 1 to 4, further comprising at least two pairs of segments, wherein the at least two pairs are linked to form a polyvalent scFv.

6. A fusion protein comprising an IL-8 antibody or its IL-8 specific binding domain, where the antibody comprises a V Ｈ segment and a V Ｌ segment, and A pharmaceutical composition for treating a patient with cancer, wherein the antibody or fusion protein is contained in a pharmacologically acceptable carrier, The aforementioned V Ｈ Segment and the V Ｌ The segment is (i) Sequence ID 8 and Sequence ID 23, (ii) Sequence IDs 10 and 25, and (iii) Sequence ID 1 and Sequence ID 16 A pharmaceutical composition in which an antibody or fusion protein is formed in pairs selected from the group consisting of the following.

7. V having the first amino acid sequence Ｈ A first nucleic acid segment encoding a segment, and a V having a second amino acid sequence. Ｌ It comprises a second nucleic acid segment that codes for a segment, The aforementioned V Ｈ Segment and the V Ｌ The segment is (i) Sequence ID 8 and Sequence ID 23, (ii) Sequence IDs 10 and 25, (iii) Sequence IDs 12 and 27, (iv) Sequence ID 7 and Sequence ID 22, (v) Sequence IDs 13 and 28, (vi) Sequence ID 3 and Sequence ID 18, (vii) Sequence IDs 11 and 26, (viiii) Sequence IDs 14 and 29, (ix) Sequence ID 6 and Sequence ID 21, (x) Sequence ID 2 and Sequence ID 17, (xi) Sequence ID 15 and Sequence ID 30, (xii) Sequence ID 1 and Sequence ID 16, (xiii) Sequence ID 4 and Sequence ID 19, (xiv) Sequence ID 5 and Sequence ID 20, (xv) Sequence ID 9 and Sequence ID 24, (xvi) Sequence IDs 31 and 33, and (xvii) Sequence IDs 32 and 34 Recombinant nucleic acids, selected from the group consisting of the following, that encode an IL-8 specific scFv, an antibody, or an IL-8 binding fragment thereof, in pairs.

8. The recombinant nucleic acid according to claim 7, further comprising a third nucleic acid segment encoding a linker peptide.

9. The aforementioned V Ｈ Segments and V Ｌ The recombinant nucleic acid according to claim 8, wherein the segment is coupled with the linker peptide.

10. The recombinant nucleic acid according to claim 8 or 9, wherein the linker peptide is a glycine-rich peptide.

11. Recombinant isolated IL-8 specific antibody, its antigen-binding fragment, or a fusion protein comprising the same, The antibody has a first and a second amino acid sequence, V Ｈ Domain and / or V Ｌ It includes a domain, The aforementioned V Ｈ Domain and the aforementioned V Ｌ The domain is (i) Sequence ID 8 and Sequence ID 23, (ii) Sequence IDs 10 and 25, (iii) Sequence IDs 12 and 27, (iv) Sequence ID 7 and Sequence ID 22, (v) Sequence IDs 13 and 28, (vi) Sequence ID 3 and Sequence ID 18, (vii) Sequence IDs 11 and 26, (viiii) Sequence IDs 14 and 29, (ix) Sequence ID 6 and Sequence ID 21, (x) Sequence ID 2 and Sequence ID 17, (xi) Sequence ID 15 and Sequence ID 30, (xii) Sequence ID 1 and Sequence ID 16, (xiii) Sequence ID 4 and Sequence ID 19, (xiv) Sequence ID 5 and Sequence ID 20, (xv) Sequence ID 9 and Sequence ID 24, (xvi) Sequence IDs 31 and 33, and (xvii) Sequence IDs 32 and 34 Recombinant isolated IL-8 specific antibodies, their antigen-binding fragments, or fusion proteins comprising them, wherein an antibody or its antigen-binding fragment is formed in a pair selected from the group consisting of the following.

12. The antibody or its antigen-binding fragment is 10 －７ The antibody or antigen-binding fragment thereof according to claim 11, having a binding affinity to IL-8 of less than M.

13. The antibody or its antigen-binding fragment (a) the entire immunoglobulin molecule; (b) scFv; (c) Monoclonal antibody; (d) Human antibodies; (e) Humanized antibodies; (f) Chimeric antibody; (g) Fab fragment; (h) Fab′ fragment; (i) F(ab')2; (j) Fv; and (k) The antibody or antigen-binding fragment thereof according to claim 11 or 12, selected from the group consisting of disulfide-linked Fv.

14. (a) Human IgG1 constant domain; (b) Human IgG2 constant domain; (c) Human IgG3 constant domain; (d) Human IgG4 constant domain; and (e) Human IgA constant domain An antibody or antigen-binding fragment thereof according to any one of claims 11 to 13, further comprising a heavy chain immunoglobulin constant domain selected from the group consisting of the above.

15. The antibody or antigen-binding fragment according to any one of claims 11 to 14, wherein the antibody or antigen-binding fragment thereof is present in a pharmacologically acceptable carrier.

16. A pharmaceutical composition for reducing the IL-8 effect in tissue, comprising the antibody or fusion protein described in claim 6.

17. The pharmaceutical composition according to claim 16, further comprising at least one of fulvestrant, aldoxorubicin, docetaxel, and a tumor necrosis treatment agent (TNT).

18. A pharmaceutical composition for treating a patient having a tumor, comprising the antibody or fusion protein described in claim 6.

19. The pharmaceutical composition according to claim 18, further comprising at least one of fulvestrant, aldoxorubicin, docetaxel, and a tumor necrosis treatment agent (TNT).

20. The pharmaceutical composition according to claim 18 or 19, wherein the tumor is at least one of pancreatic cancer, triple-negative breast cancer, and glioblastoma.

21. A pharmaceutical composition according to any one of claims 18 to 20, for use in combination with at least one of fulvestrant, aldoxorubicin, docetaxel, tumor necrosis treatment agent (TNT), and brachyli.

22. A pharmaceutical composition according to any one of claims 18 to 21, for use in combination with an immunostimulatory cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-15 hyperagonist, IL-21, IPS1, and LMP1.

23. A pharmaceutical composition according to any one of claims 18 to 22, for use in combination with an immune checkpoint inhibitor comprising a peptide ligand that binds to an immune checkpoint receptor selected from the group consisting of CTLA-4, PD-1, TIM1, 2B4, and CD160.

24. A pharmaceutical composition for reducing immunosuppression in patients with tumors, comprising the antibody or fusion protein described in claim 6.

25. The pharmaceutical composition according to claim 24, further comprising fulvestrant, aldoxorubicin, docetaxel, and at least one tumor necrosis treatment agent (TNT).

26. The pharmaceutical composition according to claim 24 or 25, wherein the tumor is at least one of pancreatic cancer, triple-negative breast cancer, and glioblastoma.

27. A pharmaceutical composition according to any one of claims 24 to 26, for use in combination with at least one of fulvestrant, aldoxorubicin, docetaxel, tumor necrosis treatment agent (TNT), and brachyli.

28. A pharmaceutical composition according to any one of claims 24 to 27, for use in combination with an immunostimulatory cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-15 hyperagonist, IL-21, IPS1, and LMP1.

29. A pharmaceutical composition for reducing the Th2-mediated immune response in patients with tumors, comprising the antibody or fusion protein described in claim 6.

30. The pharmaceutical composition according to claim 29, further comprising fulvestrant, aldoxorubicin, docetaxel, and at least one of tumor necrosis agents (TNTs).

31. The pharmaceutical composition according to claim 29 or 30, wherein the tumor is at least one of pancreatic cancer, triple-negative breast cancer, and glioblastoma.

32. A pharmaceutical composition according to any one of claims 29 to 31, for use in combination with at least one of fulvestrant, aldoxorubicin, docetaxel, tumor necrosis treatment agent (TNT), and brachyli.

33. A pharmaceutical composition according to any one of claims 29 to 32, for use in combination with an immunostimulatory cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-15 hyperagonist, IL-21, IPS1, and LMP1.

34. A pharmaceutical composition for reducing epithelial-mesenchymal transition of tumor cells in a patient, comprising the antibody or fusion protein described in claim 6.

35. The pharmaceutical composition according to claim 34, further comprising fulvestrant, aldoxorubicin, docetaxel, tumor necrosis treatment agent (TNT), and at least one of blaquili.

36. The pharmaceutical composition according to claim 34 or 35, wherein the tumor is at least one of pancreatic cancer, triple-negative breast cancer, and glioblastoma.

37. A pharmaceutical composition according to any one of claims 34 to 36, for use in combination with at least one of fulvestrant, aldoxorubicin, docetaxel, tumor necrosis treatment agent (TNT), and brachyli.

38. A pharmaceutical composition according to any one of claims 34 to 37, for use in combination with an immunostimulatory cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-15 hyperagonist, IL-21, IPS1, and LMP1.

39. A pharmaceutical composition for reducing the metastasis of a tumor in a patient having a tumor, comprising the antibody or fusion protein described in claim 6.

40. V containing the amino acid sequence of Sequence ID No. 8 Ｈ V comprising the segment and the amino acid sequence of SEQ ID NO: 23 Ｌ A peptide, pharmaceutical composition, or antibody or antigen-binding fragment thereof according to any one of claims 1 to 39, comprising a segment.

41. V containing the amino acid sequence of SEQ ID NO: 10 Ｈ V comprising the segment and the amino acid sequence of SEQ ID NO: 25 Ｌ A peptide, pharmaceutical composition, or antibody or antigen-binding fragment thereof according to any one of claims 1 to 39, comprising a segment.

42. V containing the amino acid sequence of Sequence ID No. 31 Ｈ V comprising the segment and the amino acid sequence of SEQ ID NO: 33 Ｌ A peptide, pharmaceutical composition, or antibody or antigen-binding fragment thereof according to any one of claims 1 to 39, comprising a segment.

43. V containing the amino acid sequence of SEQ ID NO: 32 Ｈ V comprising the segment and the amino acid sequence of SEQ ID NO: 34 Ｌ A peptide, pharmaceutical composition, or antibody or antigen-binding fragment thereof according to any one of claims 1 to 39, comprising a segment.

Images