Composition for overcoming resistance to EGFR-targeting agent
11548915 · 2023-01-10
Assignee
Inventors
Cpc classification
C07K16/2863
CHEMISTRY; METALLURGY
C07K2319/33
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
International classification
C07K16/28
CHEMISTRY; METALLURGY
Abstract
The present invention relates to methods of overcoming the resistance to an EGFR (Epidermal Growth Factor Receptor)-targeting antibody through a peptide that binds specifically to neuropilin-1 (NRP1). Moreover, the present invention relates to a fusion antibody in which a peptide that binds specifically to NRP1 is fused to an EGFR-targeting antibody, and to a composition of overcoming the resistance to an EGFR-targeting antibody alone by co-administration of the EGFR-targeting antibody and an NRP1-binding peptide-fused Fc. In addition, the fusion antibody according to the present invention, in which the NRP1-specific binding peptide is fused to an EGFR-targeting antibody, overcomes the resistance to the EGFR-targeting antibody alone in pancreatic cancer. Furthermore, the fusion antibody, in which the NRP1-specific binding peptide is fused to the EGFR-targeting antibody, also overcomes resistance to the EGFR-targeting antibody alone even in lung cancer. Thus, the NRP1-specific binding-fused EGFR-targeting antibody according to the present invention may be highly effective in the treatment of various tumors resistant to EGFR-targeting antibody alone.
Claims
1. A composition for treatment of cancer consisting comprising one or more neuropilin-1 (NRP1)-specific binding peptide(s) selected from the group consisting of SEQ ID NOS: 1 to 3.
2. The composition of claim 1, wherein the NRP1-specific binding peptide is fused to an EGFR-targeting antibody or fragment thereof.
3. The composition of claim 2, wherein the EGFR-targeting antibody or fragment thereof is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab.
4. The composition of claim 2, or wherein the cancer treated is pancreatic cancer or lung cancer.
5. The composition of claim 2, wherein the NRP1-specific binding peptide is fused to the C-terminus of the EGFR-targeting antibody or fragment thereof.
6. The composition of claim 5, wherein the NRP1-specific binding peptide fused to the C-terminus of an the EGFR-targeting antibody or fragment thereof further comprises a linker.
7. The composition of claim 6, wherein the linker comprises a sequence of (GGGGS)n (SEQ ID NO: 16), where n is an integer ranging from 1 to 20.
8. The composition of claim 2, wherein the antibody fragment is an Fc, Fab, scFv, V.sub.H or V.sub.L, of an EGFR antibody.
9. The composition of claim 8, wherein the NRP1-specific binding peptide is fused to the C-terminus of an antibody Fc fragment.
10. An anticancer drug comprising the composition of claim 8.
11. An anticancer adjuvant comprising the composition of claim 8.
12. A composition for co-administration with a cancer treatment, comprising a neuropilin-1 (NRP1)-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 3, wherein the NRP1-binding peptide is fused to an Fc antibody fragment or an EGFR-targeting antibody.
13. The composition of claim 12, wherein the EGFR-targeting antibody is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab.
14. The composition of claim 13, wherein the cancer is pancreatic cancer or lung cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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BEST MODE FOR CARRYING OUT THE INVENTION
(68) Unless defined otherwise, all the technical and scientific terms used herein have the same meaning as those generally understood by one of ordinary skill in the art to which the invention pertains. Generally, the nomenclature used herein and the experiment methods, which will be described below, are those well known and commonly employed in the art.
(69) In one aspect, the present invention is directed to a composition for treating cancer, which comprises a neuropilin-1 (NRP1)-specific binding peptide-fused EGFR targeting antibody that overcomes the resistance to an EGFR-targeting antibody alone.
(70) The present invention is also directed to a method for treating cancer, which comprises administering to a patient with a NRP1-binding peptide fused EGFR-targeting antibody that overcomes the resistance an EGFR-targeting antibody, particularly to a method for treating cancer exhibiting resistance to an EGFR-targeting antibody alone.
(71) The peptide may comprise one or more sequences that bind specifically to neuropilin-1, and for example, are selected from the group consisting of the following SEQ ID NOS: 1 to 3:
(72) TABLE-US-00001 (TPP11, SEQ ID NO: 1) HTPGNSKPTRTPRR, (TPP1, SEQ ID NO: 2) HTPGNSNQFVLTSTRPPR, and (TPP8, SEQ ID NO: 3) HTPGIATRTPR.
(73) The NRP1-binding peptide fused EGFR-targeting antibody can or the combination of NRP1-binding peptide-fused Fc and EGFR-targeting antibody can overcome the resistance to the EGFR-targeting antibody alone.
(74) by: i) reducing the expression level of active integrin β1 on the cell surface, thereby inhibiting the phosphorylation of Src and Akt; or ii) regulating the expression amount of NRP1 and integrin β3 on the cell surface.
(75) In one example of the present invention, it was shown that as the peptide did bind specifically to neuropilin-1 in pancreatic cancer, it reduced the expression of active integrin β1 on the cell surface, which results from the endocytosis of NRP1/active integrin β1. In addition, it was shown that the peptide inhibited the integrin β1-induced phosphorylation of FAK, Src and Akt. Accordingly, it was confirmed that the peptide according to the present invention, which binds specifically to neuropilin-1, can overcome the resistance of cancer to an EGFR-targeting agent (e.g., cetuximab or panitumumab) and increase the sensitivity of cancer to the EGFR-targeting agent.
(76) In another example of the present invention, it was confirmed that the peptide according to the present invention, which binds specifically to neuropilin-1, can overcome the resistance of lung cancer to an EGFR-targeting agent (e.g., cetuximab or panitumumab) and increase the sensitivity of lung cancer to the EGFR-targeting agent by regulating the expression amount of NRP1 and integrin β3 on the cell surface.
(77) The EGFR-targeting agent may be a gene expression inhibitor or an activity inhibitor, and the type thereof is not limited. For example, it may be an EGFR expression inhibitor or an agent that inhibits expression of active integrin β1 and expression of FAK, Src and Akt. The expression inhibitor may be an antisense nucleotide, a small hairpin RNA (shRNA), a small interfering RNA (siRNA) or a ribozyme, which binds complementarily to the mRNA of a protein gene, and the activity inhibitor may be an EGFR activity inhibitor or an agent that inhibits the activity of integrin β1, FAK, Src and Akt, and may also be a compound, a peptide, a peptide mimic, a substrate analogue, an aptamer, an antibody, or an antagonist.
(78) In one embodiment, the EGFR-targeting agent may be, for example, a compound that specifically inhibits EGFR activity, or an antibody or its fragment that binds specifically to EGFR, and the peptide may bind to the C-terminus of an antibody or its fragment.
(79) Specifically, the EGFR-targeting agent may be one or more antibodies selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab, or may be one or more selected from the group consisting of gefitinib, erlotinib, and lapatinib, but is not limited thereto.
(80) The antibody fragment means the heavy-chain or light-chain domain of the antibody, or a fragment thereof. For example, the antibody fragment may be Fc, Fab, a heavy-chain constant region fragment (CH1, CH2, or CH3) of an antibody, a heavy-chain variable region fragment (VH), a light-chain constant region fragment (CL), a light-chain variable region fragment (VL), a single-chain variable fragment (scFv), or a fragment thereof. Preferably, the antibody fragment may be a heavy-chain constant region crystalizable fragment (Fc) composed of hinge-CH2-CH3 of the antibody.
(81) Fab has a structure including variable regions of a light chain and a heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain with one antigen-binding site. Fab′ differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminal of the heavy chain CH1 domain. The F(ab′).sub.2 antibody is produced when the cysteine residue of the hinge region of the Fab′ forms a disulfide bond.
(82) An “Fv” fragment is an antibody fragment that contains complete antigen recognition and binding sites. Such region includes a heavy chain variable domain and a light chain variable domain, for example, dimers substantially tightly covalently associated with scFv.
(83) “Single chain Fv” or “scFv” antibody fragment comprises VH and VL domains of the antibody. Such domains are within a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH domain and the VL domain such that the scFv can form the desired structure for antigen binding.
(84) A single chain Fv (scFv) is connected to a heavy chain variable region and a light chain variable region via a peptide linker by a covalent bond or directly at the C-terminal. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, when the whole antibody is cleaved with papain, Fab can be obtained, and when whole antibody is cut with pepsin, F(ab′)2 fragment can be obtained), and it can also be produced through gene recombinant technology.
(85) The heavy chain constant region can be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε). Sub-classes have gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2) types. A constant region of the light chain has kappa (κ) and lambda (λ) types.
(86) The term “heavy chain” as used herein refers to a full-length heavy chain and fragments thereof including a variable region domain VH including an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2, and CH3. The term “light chain” as used herein refers to a full-length heavy chain and fragments thereof including a variable region domain VL including an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen and a constant region domain CL.
(87) Moreover, the antibody fragment may be a monomer, a dimer, or a multimer. A peptide binding to the neuropilin-1 may bind to a heavy chain constant region (Fc) fragment of an antibody specific for EGFR, preferably to the C-terminus of of Fc.
(88) According to an embodiment of the present invention, there is provided a strategy that targets both NRP1 and EGFR, by linking TPP11 (which is a peptide that binds specifically to NRP1) to the C-terminus of the heavy chain of each of cetuximab (Ctx) and panitumumab (Pnm), which are anti-EGFR antibodies that bind specifically to EGFR, suggesting that this strategy makes it possible to overcome resistance to the EGFR-targeting antibody.
(89) The “linking” means integrating two molecules having different or same functions or structures, and may be used interchangeably with “fusing”. It may be linking or fusing by all physical, chemical or biological methods by which the peptide can be linked.
(90) In some cases, the peptide may further comprise a linker and may be linked to the EGFR-targeting agent. In one embodiment, the linker may be a peptide linker.
(91) For example, a peptide linker comprising a sequence of (GGGGS)n SEQ ID NO: 16, (where n is an integer ranging from 1 to 20) may be linked to the EGFR-targeting antibody.
(92) The composition of the present invention may be formulated using a pharmaceutically acceptable carrier and/or an excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention pertains so as to be provided in a unit dosage form or enclosed into a multi-dose container. Here, the formulations of the composition may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of extracts, grains, suppositories, powders, granules, tablets or capsules, and may additionally include dispersing or stabilizing agents. Meanwhile, the composition includes an antibody or an antigen-binding fragment, and thus may be formulated into immuno liposome. Liposome including an antibody may be prepared according to a method well known in the pertinent art. The immuno liposome is a lipid composition including phosphatidylcholine, cholesterol and polyethyleneglycol-derived phosphatidylethanolamine, and may be prepared by reverse phase evaporation method. For example, a Fab′ fragment of antibody may be conjugated to liposome through disulphide exchange reaction. Liposome may further include chemical therapeutic agents such as Doxorubicin.
(93) The pharmaceutical composition of the present invention may be a pharmaceutical composition, and may be administered orally or parenterally. The parenteral administration is carried out by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and the like. Because a protein or peptide is digested when administered orally, a composition for oral administration should be formulated to coat an active drug agent or to be protected against degradation in stomach. Also, the composition may be administered by any device which can transport active substances to target cells.
(94) In one embodiment, the present invention is directed to a method for treating cancer, which comprises: administering a composition comprising a peptide that binds specifically to neuropilin-1 to a subject in need of the treatment of cancer, thereby adjusting resistance or sensitivity to an EGFR (Epidermal Growth Factor Receptor)-targeting agent.
(95) The appropriate dosage of the composition according to the present invention may vary depending on factors such as the formulation method, the administration method, patient's age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and reaction sensitivity. Preferably, a proper dosage of the composition is within the range of 0.001 and 100 mg/kg based on an adult. The term “pharmaceutically effective dose” as used herein refers to an amount sufficient to prevent or treat cancer or angiogenesis-related diseases.
(96) The composition and the treatment method according to the present invention are applied to cancer. The cancer is cancer that can be treated by an EGRF-targeting anticancer drug, and examples of the cancer include, but are not limited to, ACTH-producing tumor, acute lymphocytic or lymphoblastic leukemia, acute or chronic lymphoblastic leukemia, acute non-lymphoblastic leukemia, bladder cancer, brain tumor, breast cancer, carcinoma of the cervix, chronic myelogenous leukemia, kidney cancer, T-zone lymphoma, endometriosis, esophageal cancer, gallbladder cancer, Ewing's sarcoma, head and neck cancer, tongue cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin Lymphoma, osteosarcoma, ovarian cancer, ductal carcinoma in situ, prostate cancer, pancreatic cancer, colorectal cancer, penis cancer, retinoblastoma, skin cancer, gastric cancer, thyroid cancer, uterine cancer, testicular cancer, Wilms' tumor, and trophoblastoma. Most preferably, the cancer may be pancreatic cancer or lung cancer.
(97) In one embodiment, the cancer may be cancer in which NRP1 is expressed. In one example of the present invention, it was shown that EGFR and NRP1 were expressed in tumor cells, and in non-NRP1-expressing cell lines, the peptide that binds specifically to NRP1 did not reduce resistance to the EGFR-targeting antibody. Accordingly, it was confirmed that expression of NRP1 should be premised so that the peptide that binds specifically to NRP1 can reduce resistance to the EGFR-targeting antibody.
(98) In still another aspect, the present invention is directed to an anticancer drug or an anticancer adjuvant comprising the composition. The composition can exhibit direct anticancer effects through a composition comprising the peptide according to the present invention (e.g., a peptide itself or Fc fragment), which binds specifically to neuropilin-1, or it may be used as an anticancer adjuvant that reduces resistance to other anticancer drugs (e.g., EGFR-targeting antibody Ctx or Pnm) and increases sensitivity to the anticancer drugs.
(99) In yet another aspect, the present invention is directed to a composition for co-administration for cancer treatment, which comprises: a Fc-fused peptide that binds specifically to neuropilin-1 and an EGFR-targeting antibody, the combination of which can overcome the resistance to the EGFR-targeting antibody alone. The present invention is also directed to a method for treating cancer, which comprises co-administering a composition comprising a NRP1-binding peptide-fused Fc and an EGFR-targeting antibody, the combination of which can overcome the resistance to the EGFR-targeting antibody alone.
(100) When the Fc-fused peptide that binds specifically to neuropilin-1 is co-administered with the EGFR-targeting antibody, the NRP1-binding peptide fused Fc may act as a sensitizer to reduce resistance to the EGFR-targeting antibody and to increase sensitivity to the EGFR-targeting antibody, thereby improving the cancer treatment effect of the EGFR-targeting antibody alone.
(101) The composition for co-administration includes the NRP1-binding peptide fused Fc, and the components related thereto are the same as the components included in the above-described composition and treatment method. Thus, the description of each constitution applies equally to the method of treating cancer by co-administration.
(102) As used herein, the term “co-administration” means that the peptide specifically binding to neuropilin-1 and the EGFR-targeting agent may be administered simultaneously, sequentially, or in reverse order, and the NRP1-binding peptide fused Fc and the EGFR-targeting antibody may be administered in a combination of appropriate effective amounts of the active ingredients within the range determined by those skilled in the art. For example, the NRP1-binding peptide fused Fc and the EGFR-targeting antibody may be respectively stored in separate containers, and then administered simultaneously, sequentially, or in reverse order.
EXAMPLES
(103) Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.
Example 1: Analysis of Characteristics of Pancreatic Cancer Cell Lines Having or not Having Intrinsic Resistance to Ctx
(104) The characteristics of pancreatic cancer cell lines used in the present invention, which have or do not have intrinsic resistance to Ctx, were analyzed. This may be an important marker that predicts whether pancreatic cancer has intrinsic resistance to Ctx.
(105)
(106) Specifically, Miapaca-2, AsPC-1, BxPC-3, PANC-1, Capan-2 and SW1990 cell lines were prepared at a density of 2×10.sup.5 per sample. The cells were washed with PBS, and incubated with an antibody (R&D System) recognizing NRP1 and an FITC-conjugated antibody (e-Bioscience) recognizing each of EGFR and integrin β1 at 4° C. for 1 hour. Additionally, the NRP1 antibody bound to the cells were stained with the FITC-conjugated antibody, and then washed with PBS, followed by analysis with a flow cytometer (FACS Calibur) (BD Bioscience).
(107)
(108) Specifically, each of Miapaca-2, AsPC-1, BxPC-3, PANC-1, Capan-2 or SW1990 cell lines was added to each well of 6-well plates at a density of 6×10.sup.5 cells per well and cultured in 1 ml of 10% FBS-containing medium for 24 hours under 5% CO.sub.2 at 37° C. After the culture, lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% SDS, 1 mM EDTA, inhibitor cocktail (Sigma)) was added to obtain cell lysate. The cell lysate was quantified using a BCA protein assay kit. After performing SDS-PAGE, the gel was transferred to a PVDF membrane, and incubated with antibodies (SantaCruz), which recognize NRP1, EGFR, integrin β1 and β-actin, respectively, at 25° C. for 2 hours, and then incubated with HRP-conjugated secondary antibodies (SantaCruz) at 25° C. for hour, followed by detection. Analysis was performed using ImageQuant LAS4000 mini (GE Healthcare).
(109) As a result, as shown in
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(111) As shown in
(112) As a result, Ctx.sup.R pancreatic cancer cell lines maintain high phosphorylation levels of FAK, Src and Akt, even when they were treated with Ctx, unlike Ctx.sup.S pancreatic cancer cell lines. However, phosphorylation levels of EGFR (Y1173) and ERK1/2 showed no difference between the Ctx.sup.S pancreatic cancer cell lines and the Ctx.sup.R pancreatic cancer cell lines.
Example 2: Examination of the Effects of Inhibition of Integrin β1 Expression and Inhibition of Phosphorylation of Src and Akt on the Resistance of Pancreatic Cancer to Ctx
(113) It was found that the expression level of integrin β1 and the phosphorylation levels of Src and Akt were higher in the Ctx.sup.R pancreatic cancer cell lines than in the Ctx.sup.S cell lines. In fact, whether the resistance of the Ctx.sup.R pancreatic cancer cell lines to Ctx is associated with overexpressed integrin β1, Src and Akt was examined.
(114)
(115) Specifically, 3×10.sup.5 BxPC-3, PANC-1 or SW1990 cells were added to and cultured in each well of 6-well plates, and then subjected to transient transfection with siRNA. For transient transfection, 100 nM of each of a control siRNA having no target and an siRNA targeting the inhibition of integrin β1 expression was incubated with 500 μl of Opti-MEM media (Gibco) and 3.5 μl of RNAiMax (Invitrogen, USA) in a tube at room temperature for 15 minutes, and then added to each well. In addition, 500 μl of antibiotic-free DMEM medium was added to each well which was then incubated for 6 hours at 37° C. under 5% CO.sub.2, followed by replacement with 1 ml of 10% FBS-containing DMEM medium. After 24 hours of incubation, 7×10.sup.3 cells were added to each well of 96-well plates and incubated for 12 hours. Then, Ctx was diluted in 10% FBS-containing medium at a concentration of 2 μM, and the cells were incubated with the dilution for 48 hours. Next, for a cell proliferation assay, 20 μl of MTT reagent (Sigma) was added to each well, followed by incubation at 37° C. for 2 hours. The formed formazan was dissolved in DMSO, and the absorbance at 570 nm was measured using a microplate reader (Molecular Devices).
(116)
(117)
(118) Specifically, each of BxPC-3 and PANC-1 cell lines was added to 96-well plates at a density of 7×10.sup.3 cells per well and cultured in 10% FBS-containing medium for 12 hours. Then, each of 50 μM LY294002, 5 μM SU6656 and 2.5 μM sorafenib was diluted together with 2 μM Ctx, and the cells were incubated with the dilution for 72 hours. Then, cell viability was analyzed by MTT assay.
(119) As a result, it could be seen that, regardless of expression of NRP1 in pancreatic cancer, inhibiting the expression of integrin β1 or inhibiting the phosphorylation of Src and Akt could overcome resistance to Ctx. However, when phosphorylation of Raf was inhibited, resistance to Ctx could not be overcome. This suggests that, independently of the signaling pathway of KRas-BRaf, the signaling pathways of integrin β1, Src and Akt may be major markers of resistance to Ctx.
(120) Table 1 summarizes the results of analyzing the characteristics of the pancreatic cancer cell lines used in the present invention. The cell surface expression levels shown were classified by the FACS results shown in
(121) TABLE-US-00002 TABLE 1 Cell Mutational state Cell surface expression levels Responsiveness lines EGFR KRAS BRAF NRP1 EGFR Integrin β1 to Cetuximab Miapaca-2 WT G12C WT − + + Sensitive AsPC-1 WT G12D WT + ++ + Sensitive BxPC-3 WT WT WT + +++ ++ Resistant PANC-1 WT G12D WT ++ ++ ++ Resistant Capan-2 WT G12V WT + ++ +++ Resistant SW1990 WT G12D WT − ++ ++ Resistant
Example 3: Expression and Purification of Ctx-TPP11
(122) In order to examine whether Ctx-TPP11 can inhibit the proliferation of Ctx.sup.R pancreatic cancer cells, Ctx-TPP11 was expressed and purified.
(123) Specifically, for a vector for producing a fusion protein composed of a TPP11 peptide and an antibody heavy-chain constant region (Fc), the DNA of the TPP11-fused portion in the antibody heavy-chain constant region (amino acid (AA) sequence of SEQ ID NO: 4, and DNA sequence of SEQ ID NO: 5) obtained by treatment with BsrGI and HindIII restriction enzymes was cloned into a vector encoding a wild-type Ctx heavy chain (AA sequence of SEQ ID NO. 6; DNA sequence of SEQ ID NO: 7). As a light chain-encoding DNA (AA sequence of SEQ ID NO: 8, and DNA sequence of SEQ ID NO: 9), a wild-type Ctx light chain expression vector was used.
(124) TABLE-US-00003 TABLE 2 Constitution Sequences SEQ ID NO: Heavy chain MGWSCIILFLVATATGVHSQVQLKQSGPGLVQ SEQ ID NO: 4 amino acid PSQSLSITCTVSGFSLTNYGVHWVRQSPGKGL sequence of Ctx- EWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQ TPP11 VFFKMNSLQSNDTAIYYCARALTYYDYEFAYW GQGTLVTVSAASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSGG GGSHTPGNSKPTRTPRR Heavy chain DNA CAGGTGCAGCTGAAACAGAGCGGCCCGGGCCT SEQ ID NO: 5 sequence of Ctx- GGTGCAGCCGAGCCAGAGCCTGAGCATTACCT TPP11 GCACCGTGAGCGGCTTTAGCCTGACCAACTAT GGCGTGCATTGGGTGCGCCAGAGCCCGGGCAA AGGCCTGGAATGGCTGGGCGTGATTTGGAGCG GCGGCAACACCGATTATAACACCCCGTTTACC AGCCGCCTGAGCATTAACAAAGATAACAGCAA AAGCCAGGTGTTTTTTAAAATGAACAGCCTGC AGAGCAACGATACCGCGATTTATTATTGCGCG CGCGCGCTGACCTATTATGATTATGAATTTGC GTATTGGGGCCAGGGCACCCTGGTGACCGTGA GCGCGGCCTCCACCAAGGGCCCATCGGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGGTGACGGTGTCGTGG AACTCAGGCGCCCTGACCAGCGGCGTGCACAC CTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAA CGTGAATCACAAGCCCAGCAACACCAAGGTGG ACAAGAAAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGA ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGGTGGTGGA CGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCAAGGTCTCCAACAAAGCCCTCCC AGCCCCCATCGAGAAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTACACC CTGCCCCCATCCCGGGATGAGCTGACCAAGAA CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACT ACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT AAAGGTGGAGGAGGATCTGGAGGAGGAGGAAG TGGAGGTGGAGGATCACATACTCCTGGAAATA GCAAACCAACACGCACACCAAGGCGT Heavy chain MGWSCIILFLVATATGVHSQVQLKQSGPGLVQ SEQ ID NO: 6 amino acid PSQSLSITCTVSGFSLTNYGVHWVRQSPGKGL sequence of Ctx EWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQ VFFKMNSLQSNDTAIYYCARALTYYDYEFAYW GQGTLVTVSAASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK Heavy chain DNA CAGGTGCAGCTGAAACAGAGCGGCCCGGGCCT SEQ ID NO: 7 sequence of Ctx GGTGCAGCCGAGCCAGAGCCTGAGCATTACCT GCACCGTGAGCGGCTTTAGCCTGACCAACTAT GGCGTGCATTGGGTGCGCCAGAGCCCGGGCAA AGGCCTGGAATGGCTGGGCGTGATTTGGAGCG GCGGCAACACCGATTATAACACCCCGTTTACC AGCCGCCTGAGCATTAACAAAGATAACAGCAA AAGCCAGGTGTTTTTTAAAATGAACAGCCTGC AGAGCAACGATACCGCGATTTATTATTGCGCG CGCGCGCTGACCTATTATGATTATGAATTTGC GTATTGGGGCCAGGGCACCCTGGTGACCGTGA GCGCGGCCTCCACCAAGGGCCCATCGGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGGTGACGGTGTCGTGG AACTCAGGCGCCCTGACCAGCGGCGTGCACAC CTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAA CGTGAATCACAAGCCCAGCAACACCAAGGTGG ACAAGAAAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGA ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGGTGGTGGA CGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCAAGGTCTCCAACAAAGCCCTCCC AGCCCCCATCGAGAAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTACACC CTGCCCCCATCCCGGGATGAGCTGACCAAGAA CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACT ACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT AAA Light chain MGWSCIILFLVATATGVHSDILLTQSPVILSV SEQ ID NO: 8 amino acid SPGERVSFSCRASQSIGTNIHWYQQRTNGSPR sequence of Ctx LLIKYASESISGIPSRFSGSGSGTDFTLSINS VESEDIADYYCQQNNNWPTTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC Light chain DNA GATATTCTGCTGACCCAGAGCCCGGTGATTCT SEQ ID NO: 9 sequence of Ctx GAGCGTGAGCCCGGGCGAACGCGTGAGCTTTA GCTGCCGCGCGAGCCAGAGCATTGGCACCAAC ATTCATTGGTATCAGCAGCGCACCAACGGCAG CCCGCGCCTGCTGATTAAATATGCGAGCGAAA GCATTAGCGGCATTCCGAGCCGCTTTAGCGGC AGCGGCAGCGGCACCGATTTTACCCTGAGCAT TAACAGCGTGGAAAGCGAAGATATTGCGGATT ATTATTGCCAGCAGAACAACAACTGGCCGACC ACCTTTGGCGCGGGCACCAAACTGGAACTGAA ACGTACGGTGGCTGCACCATCTGTCTTCATCT TCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTT CTATCCCAGAGAGGCCAAAGTACAGTGGAAGG TGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTC GCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTGA
(125) The light-chain and heavy-chain expression vectors were subjected to transient transfection, thereby expressing and purifying protein. In a shaking flask, HEK293-F cells (Invitrogen) suspension-growing in serum-free FreeStyle 293 expression medium (Invitrogen) were transfected with a mixture of a plasmid and polyethylenimine (PEI) (Polyscience). For 200 mL transfection in a shaking flask (Corning), HEK293-F cells were seeded into 100 ml of medium at a density of 2×10.sup.6 cells/ml and cultured at 130 rpm under 8% CO.sub.2. Heavy-chain and light-chain plasmids suitable for producing each monoclonal antibody were diluted in 10 ml of FreeStyle 293 expression medium (Invitrogen) to 125 μg heavy chain and 125 μg light chain (a total of 250 μg (2.5 μg/ml)), and the dilution was mixed with 10 ml of medium containing 750 μg (7.5 μg/ml) of PEI and was incubated at room temperature for 10 minutes. Next, the incubated mixed medium was added to 100 ml of the above-seeded cells and incubated for 4 hours at 150 rpm under 8% CO.sub.2, and then the remaining 100 ml of FreeStyle 293 expression medium was added thereto, followed by incubation for 7 days. With reference to a standard protocol, protein was purified from the collected cell culture supernatant. Antibody was applied to a Protein A Sepharose column (GE healthcare), followed by washing with PBS (pH 7.4). The antibody was eluted with 0.1 M glycine buffer at pH 3.0, and then the sample was immediately neutralized with 1M Tris buffer. The eluted antibody fraction was concentrated by dialysis while replacing buffer with PBS (pH 7.4). The purified protein was quantified using the absorbance at 280 nm and absorption coefficient.
Example 4: Evaluation of the Simultaneous Binding Affinity of Ctx-TPP11 for Both NRP1 and EGFR
(126) The binding affinity of Ctx-TPP11 (expressed and purified in Example 2) for EGFR and NRP1-b1b2 was analyzed comparatively with that of wild-type antibody Ctx.
(127)
(128) Specifically, EGFR (0.5 μg/well) was coated on a 96-well plate (SPL, Korea) for 1 hour, and then incubated with each of Fc-TPP11, Ctx and Ctx-TPP11 (10 nM) at 25° C. for 1 hour. After washing with TBST (TBS with 0.1% Tween-20), each well was incubated with serially diluted biotinylated NRP1-b1b2 (1 μM-1 pM) at 25° C. for 1 hour, and then bound with AP-conjugated goat anti-biotin antibody at 25° C. for 1 hour. Next, the bound biotinylated protein was detected using a p-nitrophenyl phosphate substrate (Sigma-Aldrich), and the absorbance at 405 nm was measured using a microplate reader.
(129) Table 3 below shows the results of SPR (Surface plasmon resonance) performed using the Biacore2000 instrument (GE healthcare) in order to more quantitatively analyze the binding affinity of Ctx-TPP11 for NRP1-b1b2 and EGFR in comparison with Fc-TPP11 and Ctx.
(130) Specifically, each of Fc-TPP11, Ctx and Ctx-TPP11 protein was immobilized on a CM5 sensor chip (GE healthcare, USA) at a level of about 1,000 response units (RUs). For analysis, HBS-EP buffer [10 mM Hepes, 3 mM ethylenediaminetetraacetic acid, 0.005% surfactant P20 (pH 7.6), GE Healthcare] was used at a flow rate of 30 μl/min, and treatment with NRP1-b1b2 protein was performed. After analysis of association and dissociation, regeneration of the CM5 chip was performed by running buffer (20 mM NaOH, 1M NaCl, pH10.0) at a flow rate of 30 μl/min for 2 minutes. Each sensorgram obtained for 3 min of association and 6 min of dissociation was normalized relative to a blank cell and subtracted, thereby calculating the affinity.
(131) TABLE-US-00004 TABLE 3 Antibodies k.sub.on (M.sup.−1s.sup.−1) k.sub.off (s.sup.−1) K.sub.D (nM) EGFR Cetuximab (3.77 ± 1.34) × 10.sup.5 (5.36 ± 0.90) × 10.sup.−4 (1.64 ± 0.96) × 10.sup.−9 Ctx-TPP11 (3.50 ± 0.80) × 10.sup.5 (5.30 ± 0.64) × 10.sup.−4 (1.57 ± 0.44) × 10.sup.−9 NRP1-b1b2 Fc-TPP11 (5.38 ± 1.12) × 10.sup.4 (8.65 ± 0.61) × 10.sup.−4 (1.62 ± 0.23) × 10.sup.−8 Ctx-TPP11 (6.69 ± 2.00) × 10.sup.4 (9.85 ± 0.82) × 10.sup.−4 (1.55 ± 0.38) × 10.sup.−8
(132) As shown in Table 3 above, the affinity for EGFR was the same between TPP11-fused Ctx-TPP11 and wild-type antibody Ctx, and the affinity for NRP1-b1b2 was also the same between Ctx-TPP11 and Fc-TPP11. For analysis, at least five sensorgrams were used, and the results obtained in triplicate were statistically processed. ± indicates the results of independent experiments and standard deviation values.
Example 5: Evaluation of Cell Growth Inhibitory Ability of Ctx-TPP11 in Ctx.SUP.R .Pancreatic Cancer Cell Lines
(133) In order to examine whether Ctx-TPP11 that targets NRP1 and EGFR can inhibit the proliferation of Ctx.sup.R pancreatic cancer cells that express NRP1, a cell growth assay was performed on various cell lines.
(134)
(135) Specifically, pancreatic cancer cell lines were prepared in the same manner as described in Example 2. When the cells were stabilized, the cells were treated with various concentrations (0, 1, 2, 4 μM) of each of Fc-TPP11, Ctx and Ctx-TPP11 twice at 48-hour intervals and incubated for a total of 96 hours. In addition, in order to examine whether the inhibition of cell proliferation would result from targeting both NRP1 and EGFR, the cells were co-treated with the same concentration of Fc-TPP11 and Ctx and then incubated. In the same manner as described in Example 2, 20 μl of MTT reagent (Sigma) was added to each well which was then incubated at 37° C. for 2 hours, and the formed formazan was dissolved in DMSO. The absorbance at 570 nm was measured using a microplate reader (Molecular Devices). In Ctx.sup.S (Miapaca-2, AsPC-1) pancreatic cancer cell lines, Ctx and Ctx-TPP11 showed the same cell growth inhibitory ability, and in three Ctx.sup.R (BxPC-3, PANC-1, Capan-2) pancreatic cancer cell lines, Ctx-TPP11 and a combination of Fc-TPP11 and Ctx showed cell growth inhibition, unlike Ctx. However, as confirmed in Example, in SW1990 which is a Ctx.sup.R pancreatic cancer cell line expressing no NRP1, Ctx-TPP11 showed no effect. This suggests that Ctx-TPP11 shows an effect specific for its target NRP1.
(136) In addition, in order to examine whether the inhibition of Ctx.sup.R pancreatic cancer cell proliferation by Ctx-TPP11 is attributable to induction of apoptosis, an apoptosis assay was performed.
(137)
(138) Specifically, 2×10.sup.5 Miapaca-2, AsPC-1, BxPC-3 or PANC-1 cells were cultured in each well of 12-well plates. When the cells were stabilized, the cells were treated with 4 μM of each of Fc-TPP11, Ctx and Ctx-TPP11 and incubated for a total of 48 hours. Next, the cells were washed with cold PBS. 1×10.sup.6 cells were prepared per sample, and 5 μl of annexin V-FITC and 5 μl of propidium iodide were added thereto, followed by incubation at 25° C. for 15 minutes. Next, 400 μl of 1× binding buffer was added to each sample, followed by analysis with the flow cytometer FACS Calibur (BD Bioscience). The above-described experiment was performed in accordance with the manufacturer's protocol.
(139) After analysis, a dot plot for each sample is shown in
(140) In the Ctx.sup.S (Miapaca-2, AsPC-1) pancreatic cancer cell lines, Ctx and Ctx-TPP11 induced apoptosis in the same manner, and in the Ctx.sup.R (BxPC-3, PANC-1) pancreatic cancer cell lines, Ctx-TPP11 and a combination of Fc-TPP11 and Ctx induced apoptosis, unlike Ctx. This suggests that the inhibition of proliferation of Ctx.sup.R pancreatic cancer cells by Ctx-TPP11 is attributable to induction of apoptosis.
(141)
(142) Specifically, cells were prepared in the same manner as described in Example 2 and were subjected to transient transfection with siRNA. For transient transfection, 100 nM of each of a control siRNA having no target and an siRNA targeting the inhibition of integrin β1 expression was incubated with 500 μl of Opti-MEM media (Gibco) and 3.5 μl of RNAiMax (Invitrogen, USA) in a tube at room temperature for 15 minutes, and then added to each well. In addition, 500 μl of antibiotic-free DMEM medium was added to each well which was then incubated for 6 hours at 37° C. under 5% CO.sub.2, followed by replacement with 1 ml of 10% FBS-containing DMEM medium. After 24 hours of incubation, 7×10.sup.3 cells were added to each well of 96-well plates and incubated for 12 hours. Then, each of Fc-TPP11, Ctx and Ctx-TPP11 was diluted in 10% FBS-containing medium at a concentration of 2 μM, and the cells were incubated with the dilution for 48 hours. Next, for a cell proliferation assay, 20 μl of MTT reagent (Sigma) was added to each well, and the absorbance at 570 nm was measured using a microplate reader (Molecular Devices). When expression of NRP1 was inhibited, the cell growth inhibitory ability of Ctx-TPP11 disappeared, but resistance to Ctx still appeared. In addition, inhibition of cMet expression could not overcome resistance to Ctx. However, as shown in
(143)
Example 6: Evaluation of Ctx.SUP.R .Inhibitory Signal of Ctx-TPP11
(144) In order to confirm whether resistance to Ctx.sup.R is overcome by Ctx-TPP11 because Ctx-TPP11 inhibits the expression of the resistance marker integrin β1 identified in Example 1 and the phosphorylation of FAK, Src and Akt which are signaling factors downstream of integrin β1, the signal inhibitory effects of Ctx-TPP11 with control siRNA and integrin β1 siRNA were examined.
(145)
(146) Specifically, cells were prepared in the same manner as described in Example 2 and were subjected to transient transfection with siRNA. For transient transfection, 100 nM of each of a control siRNA having no target and an siRNA targeting the inhibition of integrin β1 expression was incubated with 500 μl of Opti-MEM media (Gibco) and 3.5 μl of RNAiMax (Invitrogen, USA) in a tube at room temperature for 15 minutes, and then added to each well. In addition, 500 μl of antibiotic-free DMEM medium was added to each well which was then incubated for 6 hours at 37° C. under 5% CO.sub.2, followed by replacement with 1 ml of 10% FBS-containing DMEM medium. After 12 hours of incubation, each of Fc-TPP11, Ctx and Ctx-TPP11 was diluted in 1 ml of 10% FBS-containing medium at a concentration of 2 μM, and the cells were incubated with the dilution for 24 hours at 37° C. under 5% CO.sub.2. After incubation, the cells were washed with cold PBS, and lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% SDS, 1 mM EDTA, Inhibitor cocktail(sigma)) was added thereto to obtain cell lysate. Next, Western blot analysis was performed in the same manner as described in Example 1.
(147) As a result, the Ctx.sup.R SW1990 cells expressing no NRP1 had the same Ctx resistance mechanism as BxPC-3 and PANC-1, but the effects of Fc-TPP11 and Ctx-TPP11 did not appear in the cells. In addition, it was shown that the cell growth inhibitory effect of Ctx-TPP11 was because of inhibition of the expression of the Ctx resistance marker integrin β1 identified in Example 1 and inhibition of the phosphorylation of FAK, Src and Akt which are signaling markers downstream of integrin β1. It was shown that when the expression of integrin β1 was inhibited, the phosphorylation of FAK, Src and Akt, which are resistance markers, was inhibited. This suggests that integrin β1 is a molecule upstream of the phosphorylation pathways of FAK, Src and Akt. However, Ctx-TPP11 did not reduce the whole cell expression of integrin β1 itself.
Example 7: Evaluation of Cell Proliferation Inhibitory Effect of Ctx-TPP11 in Ctx.SUP.R .Colorectal Cancer Cell Lines
(148) The mechanism of resistance of most colorectal cancers to Ctx includes KRas and BRaf mutations, unlike pancreatic cancer. In order to confirm whether Ctx-TPP11 also overcomes resistance in other resistance mechanisms, the cell proliferation inhibitory abilities of Fc-TPP11, Ctx and Ctx-TPP11 were evaluated.
(149)
(150) Specifically, cells were prepared in the same manner as described in Example 2. Then, each of Fc-TPP11, Ctx and Ctx-TPP11 was diluted in 1 ml of 10% FBS-containing medium at a concentration of 0.2 μM or 1 μM, and the cells were incubated with the dilution for 72 hours under 5% CO.sub.2 at 37° C., after which an MTT assay was performed. As a result, treatment with Ctx-TPP11 and a combination of Ctx and Fc-TPP11 did not overcome the resistance of the colorectal cancer cell lines having the resistance mechanism caused by KRas and BRaf mutations.
Example 8: Evaluation of Mechanism of the Down-Regulation of Active Integrin β1 by NRP1 Targeting
(151) Ctx-TPP11 inhibited the phosphorylation of the Ctx resistance markers (FAK, Src and Akt) by NRP1 targeting, but did not reduce the whole expression level of overexpressed integrin β1. Integrin β1 is classified into active integrin pi in an extended form which can send a signal, and inactive integrin β1 in a bent form which cannot send a signal. Thus, in order to examine whether Ctx-TPP11 down-regulates signaling from integrin β1 by actually reducing the expression of active integrin β1 which can send a signal, the endocytosis of active integrin β1 was analyzed after treatment with Ctx-TPP11.
(152)
(153) Specifically, a coverslip was added to a 24-well plate, and each of BxPC-3 or PANC-1 cell lines in 0.5 ml of 10% FBS-containing medium was added to each well of the plate at a density of 2.5×10.sup.4 cells per well and cultured for 12 hours under 5% CO.sub.2 at 37° C. When the cells were stabilized, the cells were serum-deprived with serum-free medium for 4 hours in order to eliminate the effect of serum. Next, the cells were treated with 2 μM of each of Fc-TPP11, Ctx, Ctx-TPP11 (diluted in 0.5 ml of serum-free medium) for 1 hour for 37° C., and then washed three times with cold PBS. Next, the cells were fixed with 4% paraformaldehyde at 25° C. for 10 minutes. Next, the cells were washed with PBS and incubated with a PBS buffer containing 0.1% saponin, 0.1% sodium azide and 1% BSA at 25° C. for 10 minutes, thereby forming holes in the cell membrane. Next, the cells were washed with PBS, and then incubated with a PBS buffer containing 2% BSA at 25° C. for 1 hour in order to inhibit non-specific binding. Next, the cells were incubated with primary antibodies, which recognize NRP1, active integrin β1 (antibody clone name: HUTS-21, BD Bioscience) and inactive integrin β1 (antibody clone name: mAb13, BD Bioscience), respectively, at 25° C. for 1 hour and 30 minutes. The cells were incubated with TRITC (red fluorescence) or FITC (green fluorescence)-conjugated secondary antibodies, which recognize the primary antibodies, respectively, at 25° C. for 1 hour, and the nucleus was stained (blue fluorescence) with Hoechst 33342, followed by confocal microscopic observation. NRP1 overlapped only with active integrin β1, indicating that NRP1 did bind specifically to active integrin β1.
(154)
(155) Specifically, cells were prepared in the same manner as described in Example 1. When the cells were stabilized, the cells were serum-deprived with serum-free medium for 4 hours in order to eliminate the effect of serum. Then, the cells were treated with 2 μM of each of Fc-TPP11, Ctx and Ctx-TPP11 (diluted in 1 ml of serum-free medium) at varying time points (0, 5, 15, 30 and 60 min) at 37° C., and then washed with cold PBS. BxPC-3 and PANC-1 cell lines were prepared at a density of 2×10.sup.5 cells per sample. The cells were incubated with primary antibodies, which recognize NRP1, active integrin β1 and inactive integrin β1, respectively, at 4° C. for 1 hour. Then, the cells were incubated with FITC-conjugated secondary antibodies, which recognize the primary antibodies, respectively, at 4° C. for 30 minutes, after which the cells were washed with cold PBS, and then analyzed by the flow cytometer FACS Calibur (BD Bioscience). After analysis, a histogram graph for each sample was obtained. Based on the mean fluorescence intensity of the histogram, the amounts of receptors remaining on the cell surface after the endocytosis of NRP1, EGFR, active integrin β1 and inactive integrin β1 after treatment with Fc-TPP11, Ctx and Ctx-TPP11 were quantitatively measured, and the results are shown in
(156) As a result, Fc-TPP11 and Ctx-TPP11, which bind to NRP1, increased the endocytosis of NRP1, and also increased the endocytosis of active integrin β1 bound to NRP1. In addition, Ctx and Ctx-TPP11 increased the endocytosis of EGFR. This suggests that Ctx-TPP11 binds to NRP1 and selectively reduces the cell surface expression levels of NRP1 and active integrin β1.
Example 9: Evaluation of the Inhibitory Activity of Ctx-TPP11 Against Cell Adhesion of Active Integrin β1
(157) Active integrin β1 expressed on the cell surface plays an important role in the binding of the cells to extracellular matrix (ECM). In particular, cells have the highest binding affinity for FN among extracellular matrix proteins. Thus, in order to examine whether Ctx-TPP11 reduces the adhesion of cells to FN by reducing the expression of active integrin β1, analysis was performed.
(158)
(159) Specifically, FN (Sigma), diluted in 0.5 ml of PBS at a concentration of 10 μg/ml, was coated on a 12-well uncoated plate at 37° C. for 30 minutes. BxPC-3 and PANC-1 were pretreated with 100 nM of each of Fc-TPP11, Ctx and Ctx-TPP11 at 37° C. for 30 minutes, and 3×10.sup.5 pretreated BxPC-3 cells and 1×10.sup.5 pretreated PANC-1 cells were added to the FN-coated plate. The BxPC-3 cells were incubated for 1 hour at 37° C., and the PANC-1 cells were incubated for 6 hours at 37° C., followed by washing of the cells with PBS. After washing, the cells were fixed with 4% paraformaldehyde at 25° C. for 10 minutes, and washed with PBS, and cells attached to FN were stained with 0.5% (w/v) crystal violet (diluted in 20% ethanol) at 25° C. for 15 minutes. The stained attached cells were analyzed with an optical microscope. Each well was imaged 10 times, and the number of the attached cells was counted.
Example 10: Evaluation of In Vivo Tumor Growth Inhibitory Activity of Ctx-TPP11
(160) In Example 5, the in vitro cell growth inhibitory activity of Ctx-TPP11 against Ctx.sup.R pancreatic cancer cell lines was confirmed. Whether the same effect of Ctx-TPP11 also appears in vivo was examined.
(161)
(162) Specifically, BxPC-3 cells (5×10.sup.6 cells/mouse), PANC-1 (1×10.sup.7 cells/mouse) or AsPC-1 (5×10.sup.6 cells/mouse) cells in a 1:1 mixture of 150 μl of PBS and 150 μl of Matrigel (BD Biosciences) were transplanted subcutaneously into 3-week-old female BALB/c nude mice (NARA Biotech, Korea). Mice with tumors having similar sizes (mean volume: 100-120 mm.sup.3) were randomly grouped, and each antibody (Ctx, Ctx-TPP11, and Fc-TPP11 and Ctx co-adminstered) was injected intravenously into the tail vein of each mouse. The tumor size was measured at least once, and the tumor volume (V) was calculated using the following equation: V=volume×width.sup.2/2.
(163) As shown in
(164)
(165) Specifically, 5 hours after the last administration of the antibody in
(166)
(167) Specifically, 5 hours after the last administration of the antibody in
Example 11: Analysis of Cell Surface Expression Levels of EGFR, NRP1 and Integrin β1 in Lung Cancer Cell Lines
(168) As shown in Example 1, in pancreatic cancer, the cell surface and whole cell expression levels of integrin β1 were higher in Ctx.sup.R pancreatic cancer cell lines than in Ctx.sup.S pancreatic cancer cell lines. In order to examine whether the mechanism of resistance of lung cancer to Ctx is also correlated with the expression level of integrin β1, the cell surface expression levels of EGFR, NRP1 and integrin β1 in lung cancer cell lines were analyzed.
(169)
(170) Specifically, lung cancer cell lines were prepared in the same manner as described in Example 1. The cells were washed with PBS, and then incubated with an antibody (R&D System), which recognizes NRP1, and FITC-conjugated antibodies(e-Bioscience) which recognize EGFR and integrin β1, respectively, at 4° C. for 1 hour. Additionally, the cells having the NRP1 antibody bound thereto were stained with FITC-conjugated antibody, washed with PBS, and then analyzed with the flow cytometer FACS Calibur (BD Bioscience).
(171)
(172) As a result, unlike the case of pancreatic cancer, resistance to Ctx and the cell surface expression level of integrin β1 showed no particular correlation in the lung cancer cell lines.
Example 12: Examination of the Effects of Inhibition of Expression of Various Cell Surface Receptors and Inhibition of Phosphorylation of Akt and Src on Ctx.SUP.R .in Lung Cancer
(173) As described in Example 11, unlike the case of pancreatic cancer, in lung cancer, the expression level of integrin β1 showed no particular correlation with resistance to Ctx. Thus, in order to examine which receptors on the cell surface are associated with resistance to Ctx, the effects of siRNA against receptors (with which NRP1 acts as co-receptor) among various cell surface receptors were examined.
(174)
(175) Specifically, 2×10.sup.5 A549 or HCC44 cells were cultured in each well of 6-well plates, and then subjected to transient transfection with siRNA. For transient transfection, 100 nM of each of a control siRNA having no target and an siRNA targeting each of NRP1, integrin β1, integrin β3, cMet, VEGFR1 and TGFβ2 was incubated with 500 μl of Opti-MEM media (Gibco) and 3.5 μl of RNAiMax (Invitrogen, USA) in a tube at room temperature for 15 minutes, and then added to each well. In addition, 500 μl of antibiotic-free DMEM medium was added to each well which was then incubated for 6 hours at 37° C. under 5% CO.sub.2, followed by replacement with 2 ml of DMEM medium containing 10% FBS and 1% antibiotic. After 24 hours of incubation, 5×10.sup.3 cells were added to each well of 96-well plates and incubated for 12 hours. Then, Ctx was diluted in 10% FBS-containing medium at a concentration of 2 μM, and the cells were incubated with the dilution for 48 hours, after which cell viability was analyzed by a WST-1 assay.
(176)
(177) As a result, it could be seen that inhibition of NRP1 expression and integrin β3 expression in the two lung cancer cell lines tested overcame resistance to Ctx.
(178) Furthermore, in order to examine which downstream signaling factors in addition to expression of NRP1 and integrin β3 are associated with resistance to Ctx, the effects of inhibitors of PI3K-Akt, Src and Raf were evaluated.
(179)
(180) Specifically, each of A549 and HCC44 cell lines was added to a 96-well plate at a density of 5×10.sup.3 cells per well and cultured in 10% FBS-containing medium for 12 hours. Then, 50, 20 and 10 μM of LY294002, 5, 2 and 1 μM of SU6656, and 5, 2 and 1 μM of sorafenib were diluted together with 2 μM of Ctx, and the cells were incubated with each of the dilutions for 48 hours, after which cell viability was analyzed by WST-1 assay.
(181) As a result, it could be seen that inhibition of NRP1 expression and integrin β3 in the lung cancer cell lines (HCC44 and A549) or inhibition of the phosphorylation of Akt and Src could overcome resistance to Ctx. However, inhibition of expression of integrin β1, cMet, VEGFR1 and TGFβ2 or inhibition of phosphorylation of Raf could not overcome resistance to Ctx.
(182) Table 4 below summarizes the results of analyzing the characteristics of the lung cancer cell lines used in the present invention.
(183) TABLE-US-00005 TABLE 4 Mutational state Responsiveness Cell lines EGFR RAS BRAF to Cetuximab Calu-3 WT WT WT Sensitive H1975 L858R, T790M WT WT Sensitive H522 WT WT WT Resistant H1299 WT NRAS Q61K WT Resistant A549 WT KRAS G12S WT Resistant Calu-1 WT KRAS G12C WT Resistant H358 WT KRAS G12C WT Resistant H441 WT KRAS G12V WT Resistant H2009 WT KRAS G12A WT Resistant HCC44 WT KRAS G12C WT Resistant HCC2108 WT KRAS Q61H WT Resistant SK-LU-1 WT KRAS G12D WT Resistant H460 WT KRAS Q61H WT Resistant
(184) As can be seen in
Example 13: Evaluation of Cell Proliferation Inhibitory Activity of Ctx-TPP11 in Ctx.SUP.R .Lung Cancer Cell Lines
(185) In order to examine whether Ctx-TPP11 overcomes resistance to Ctx even lung cancer resistant to Ctx, like the results obtained for pancreatic cancer, the cell proliferation inhibitory activities of Ctx and Ctx-TPP11 in Ctx.sup.S lung cancer cell lines and Ctx.sup.R lung cancer cell lines were evaluated.
(186)
(187)
(188) Specifically, lung cancer cells were added to 96-well plates at a density of 5×10.sup.3 cells per well and cultured in 10% FBS-containing medium for 12 hours. Then, the cells were incubated with various concentrations (0, 1, 2, and 4 μM) of Ctx or Ctx-TPP11 for 48 hours, after which 10 μl of Cyto-X reagent (LPS Solution) was added to each well, followed by incubation at 37° C. for 1 to 2 hours. The absorbance at 450 nm was measured using a microplate reader (Molecular Devices). In the Ctx.sup.S (Calu-3, H1975) lung cancer cell lines, Ctx and Ctx-TPP11 showed the same inhibitory activity against cell growth, and in eight Ctx.sup.R (H1299, A549, Calu-1, H358, H441, H2009, HCC44, SK-LU-1) lung cancer cell lines, only Ctx-TPP11 showed cell growth inhibitory activity, unlike Ctx. However, in three Ctx.sup.R lung cancer cell lines (HCC2108, H460, and H522) expressing no NRP1, Ctx-TPP11 showed no activity. This suggests that Ctx-TPP11 is specifically effect against its target NRP1.
(189)
(190) Specifically, cells were prepared in the same manner as described in
(191) As a result, it was shown that when expression of NRP1 was inhibited by NRP1 siRNA, the cell growth inhibitory activity of Ctx-TPP11 disappeared. This suggests that Ctx-TPP11 exhibits a specific effect against its target NRP1, like the results shown in
Example 14: Examination of Correlation Between NRP1, Integrin β3 and KRAS in Ctx.SUP.R .Lung Cancer Cell Lines
(192) Examples 11 and 12 above indicated that, in the lung cancer cell lines, resistance to Ctx was closely correlated with NRP1, integrin β3 and KRAS mutation, and in these Ctx.sup.R lung cancer cell lines, Ctx-TPP11 showed a specific effect against NRP1. Thus, in order to examine why Ctx-TPP11 can overcome resistance to Ctx in lung cancer cell lines, an immunoprecipitation assay was performed to confirm how NRP1 interacts with integrin β3 and KRAS.
(193)
(194) Specifically, each of lung cancer cell lines (HCC44 and A549; cell lines expressing NRP1 and integrin β3) was added to each well of a 100-mm.sup.3 plate at a density of 2×10.sup.6 cells and cultured in 10% FBS-containing medium for 12 hours. To obtain cell lysate, the cells were incubated with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% SDC, 0.1% SDS, 100× Protease inhibitor) at 4° C. for 30 minutes, and the cell debris was removed by precipitation. Next, the cell lysate was quantified using a BCA protein assay kit (Pierce), and then 0.5 mg of the cell lysate was incubated with 5 μg of anti-NRP1 antibody (Abacm) at 4° C. for 12 hours. Thereafter, Protein A/G agarose was added thereto, followed by incubation at 4° C. for 2 hours, after which the antibody was precipitated. Next, using anti-NRP1 antibody, anti-EGFR antibody, anti-integrin β3 antibody, anti-integrin β1 antibody, and anti-KRAS antibody, Western blot analysis was performed. As a result, it was shown that when NRP1 in HCC44 and A549 was immunoprecipitated, EGFR, integrin β3 and KRAS were observed together. This suggests that, in the Ctx.sup.R lung cancer cell lines (HCC44, and A549), NRP1 interacted with EGFR, integrin β3 and KRAS. However, the interaction of integrin β1 with NRP1 was observed in HCC44, but not observed in A549.
(195)
(196) Specifically, the A549 cell line was added to a 100 mm.sup.3 plate at a density of 1×10.sup.6 cells per well and cultured in 10% FBS-containing medium for 12 hours, after which it was treated with each of control siRNA and integrin β3 siRNA in the same manner as described in Example 12. Next, in the same manner as the method of
Example 15: Evaluation of Mechanism that Reduces Cell Surface Expression Level of Integrin β3 by NRP1 Targeting
(197) As described in Example 8 above, it was confirmed that, in pancreatic cancer, Ctx-TPP11 reduced the cell surface expression level of active integrin β1 by NRP1 targeting, thereby down-regulating signaling from integrin β1. In lung cancer, it was confirmed that NRP1 interacted with integrin β3. Thus, whether the cell surface expression level of integrin β3 can be reduced by NRP1 targeting was examined.
(198)
(199) Specifically, cells were prepared in the same manner as described in Example 8. When the cells were stabilized, the cells were serum-deprived with serum-free medium for 4 hours in order to eliminate the effect of serum. Then, 2 μM of each of Fc-TPP11, Ctx and Ctx-TPP11 was diluted in 1 ml of serum-free medium, and the cells were treated with the dilution for 15 minutes at 37° C., and then washed with cold PBS. HCC44 and A549 cell lines were prepared at a density of 1×10.sup.5 cells per sample. The cells were incubated with primary antibodies, which recognize NRP1 and integrin β3, respectively, at 4° C. for 1 hour. Then, the cells were incubated with FITC-conjugated secondary antibodies, which recognize the primary antibodies, respectively, at 4° C. for 30 minutes. Then, the cells were washed with PBS and analyzed using the flow cytometer FACS Calibur (BD Bioscience). After analysis, a histogram for each sample was obtained, and based on the mean fluorescence intensity of the histogram, the amounts of receptors on the cell surface after the endocytosis of NRP1 and integrin β3 by treatment with Fc-TPP11, Ctx or Ctx-TPP11 were measured. The results are quantitatively shown in
(200) As a result, it was shown that Fc-TPP11 and Ctx-TPP11, which did bind to NRP1, increased the endocytosis of NRP1 and also increased the endocytosis of integrin β3 bound to NRP1. This suggests that Ctx-TPP11 binds NRP1 and selectively reduces the cell surface expression levels of NRP1 and integrin β3.
Example 16: Expression and Purification of Pnm-TPP11
(201) Examples 1 to 13 described cell lines resistant to Ctx among EGFR-targeting antibodies. Additionally, in order to examine whether Pnm-TPP11, obtained by fusing TPP11 to Panitumumab (Pnm) among EGFR-targeting antibodies, can inhibit the proliferation of Pnm.sup.R lung cancer cells, Pnm-TPP11 was expressed and purified.
(202)
(203) Specifically, using a reverse primer for the C-terminus of the heavy chain of Pnm (amino acid (AA) sequence of SEQ ID NO: 12; and DNA sequence of SEQ ID NO: 13) and a (G.sub.4S).sub.3 linker and a forward primer for TPP11, polymerase chain reaction was performed, and a DNA fragment composed sequentially of a signal peptide, a Pnm heavy-chain, a (G.sub.4S).sub.3 linker, TPP11 and termination codon were obtained. Next, using 1% agarose gel and electrophoresis, DNA was recovered, and using NotI and BamHI restriction enzymes, a cohesive end was generated. Thereafter, cloning into a pcDNA3.4 vector by T4 ligase was performed, thereby constructing a vector capable of expressing a Pnm-TPP11 heavy chain (AA sequence of SEQ ID NO: 10, and DNA sequence of SEQ ID NO: 11) in animal cells. As a light chain-encoding DNA (AA sequence of SEQ ID NO: 14, and DNA sequence of SEQ ID NO: 15), a vector expressing a wild-type Pnm light chain was used.
(204) TABLE-US-00006 TABLE 5 Constitution Sequences SEQ ID NO: Heavy chain amino QVQLQESGPGLVKPSETLSLTCTVSGGSVSSG SEQ ID NO: 10 acid sequence of DYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPS Pnm-TPP11 LKSRLTISIDTSKTQFSLKLSSVTAADTAIYY CVRDRVTGAFDIWGQGTMVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCK VSNKGLPAPIEKTISKTKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGGG GSGGGGSGGGGSHTPGNSKPTRTPRR Heavy chain DNA CAGGTGCAGCTGCAGGAGTCCGGCCCCGGCCT SEQ ID NO: 11 sequence of Pnm- GGTGAAGCCCTCCGAGACCCTGTCCCTGACCT TPP11 GCACCGTGTCCGGCGGCTCCGTGTCCTCCGGC GACTACTACTGGACCTGGATTCGGCAGTCCCC CGGCAAGGGCCTGGAGTGGATCGGCCACATCT ACTACTCCGGCAACACCAACTACAACCCCTCC CTGAAGTCCCGGCTGACCATCTCCATCGACAC CTCCAAGACCCAGTTCTCCCTGAAGCTGTCCT CCGTGACCGCCGCCGACACCGCCATCTACTAC TGCGTGCGGGACCGGGTGACCGGCGCCTTCGA CATCTGGGGCCAGGGCACCATGGTGACCGTGT CCTCCGCCTCCACCAAGGGCCCATCGGTCTTC CCCCTGGCGCCCTGCTCCAGGAGCACCTCCGA GAGCACAGCGGCCCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGGTGACGGTGTCGTGG AACTCAGGCGCTCTGACCAGCGGCGTGCACAC CTTCCCAGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAACTTCGGCACCCAGACCTACACCTGCAA CGTAGATCACAAGCCCAGCAACACCAAGGTGG ACAAGACAGTTGAGCGCAAATGTTGTGTCGAG TGCCCACCGTGCCCAGCACCACCTGTGGCAGG ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAG GTCACGTGCGTGGTGGTGGACGTGAGCCACGA AGACCCCGAGGTCCAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAG CCACGGGAGGAGCAGTTCAACAGCACGTTCCG TGTGGTCAGCGTCCTCACCGTTGTGCACCAGG ACTGGCTGAACGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGGCCTCCCAGCCCCCATCGA GAAAACCATCTCCAAAACCAAAGGGCAGCCCC GAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGAGGAGATGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACACCTCCCAT GCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAG CAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGA GCCTCTCCCTGTCCCCGGGTAAAGGTGGAGGA GGATCTGGAGGAGGAGGAAGTGGAGGTGGAGG ATCACATACTCCTGGAAATAGCAAACCAACAC GCACACCAAGGCGT Heavy chain amino QVQLQESGPGLVKPSETLSLTCTVSGGSVSSG SEQ ID NO: 12 acid sequence of DYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPS Pnm LKSRLTISIDTSKTQFSLKLSSVTAADTAIYY CVRDRVTGAFDIWGQGTMVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCK VSNKGLPAPIEKTISKTKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain DNA CAGGTGCAGCTGCAGGAGTCCGGCCCCGGCCT SEQ ID NO: 13 sequence of Pnm GGTGAAGCCCTCCGAGACCCTGTCCCTGACCT GCACCGTGTCCGGCGGCTCCGTGTCCTCCGGC GACTACTACTGGACCTGGATTCGGCAGTCCCC CGGCAAGGGCCTGGAGTGGATCGGCCACATCT ACTACTCCGGCAACACCAACTACAACCCCTCC CTGAAGTCCCGGCTGACCATCTCCATCGACAC CTCCAAGACCCAGTTCTCCCTGAAGCTGTCCT CCGTGACCGCCGCCGACACCGCCATCTACTAC TGCGTGCGGGACCGGGTGACCGGCGCCTTCGA CATCTGGGGCCAGGGCACCATGGTGACCGTGT CCTCCGCCTCCACCAAGGGCCCATCGGTCTTC CCCCTGGCGCCCTGCTCCAGGAGCACCTCCGA GAGCACAGCGGCCCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGGTGACGGTGTCGTGG AACTCAGGCGCTCTGACCAGCGGCGTGCACAC CTTCCCAGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAACTTCGGCACCCAGACCTACACCTGCAA CGTAGATCACAAGCCCAGCAACACCAAGGTGG ACAAGACAGTTGAGCGCAAATGTTGTGTCGAG TGCCCACCGTGCCCAGCACCACCTGTGGCAGG ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAG GTCACGTGCGTGGTGGTGGACGTGAGCCACGA AGACCCCGAGGTCCAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAG CCACGGGAGGAGCAGTTCAACAGCACGTTCCG TGTGGTCAGCGTCCTCACCGTTGTGCACCAGG ACTGGCTGAACGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGGCCTCCCAGCCCCCATCGA GAAAACCATCTCCAAAACCAAAGGGCAGCCCC GAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGAGGAGATGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACACCTCCCAT GCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAG CAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGA GCCTCTCCCTGTCCCCGGGTAAA Light chain amino DIQMTQSPSSLSASVGDRVTITCQASQDISNY SEQ ID NO: 14 acid sequence of LNWYQQKPGKAPKLLIYDASNLETGVPSRFSG Pnm SGSGTDFTFTISSLQPEDIATYFCQHFDHLPL AFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC Light chain DNA GACATCCAGATGACCCAGTCTCCATCCTCCCT SEQ ID NO: 15 sequence of Pnm GTCTGCATCTGTAGGAGACAGAGTCACCATCA CTTGCCAGGCGAGTCAGGACATCAGCAACTAT TTAAATTGGTATCAGCAGAAACCAGGGAAAGC CCCTAAACTCCTGATCTACGATGCATCCAATT TGGAAACAGGGGTCCCATCAAGGTTCAGTGGA AGTGGATCTGGGACAGATTTTACTTTCACCAT CAGCAGCCTGCAGCCTGAAGATATTGCAACAT ATTTCTGCCAACACTTTGATCATCTCCCGCTC GCTTTCGGCGGAGGGACCAAGGTGGAGATCAA ACGTACGGTGGCTGCACCATCTGTCTTCATCT TCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTT CTATCCCAGAGAGGCCAAAGTACAGTGGAAGG TGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTC GCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GT
(205) The light-chain and heavy-chain expression vectors were subjected to transient transfection, thereby expressing and purifying protein. In a shaking flask, HEK293-F cells (Invitrogen) suspension-growing in serum-free FreeStyle 293 expression medium (Invitrogen) were transfected with a mixture of a plasmid and polyethylenimine (PEI) (Polyscience). For 200 mL transfection in a shaking flask (Corning), HEK293-F cells were seeded into 100 ml of medium at a density of 2×10.sup.6 cells/ml and cultured at 130 rpm under 8% CO.sub.2. Heavy-chain and light-chain plasmids suitable for producing each monoclonal antibody were diluted in 10 ml of FreeStyle 293 expression medium (Invitrogen) to 125 μg heavy chain and 125 μg light chain (a total of 250 μg (2.5 μg/ml)), and the dilution was mixed with 10 ml of medium containing 750 μg (7.5 μg/ml) of PEI and was incubated at room temperature for 10 minutes. Next, the incubated mixed medium was added to 100 ml of the above-seeded cells and incubated for 4 hours at 150 rpm under 8% CO.sub.2, and then the remaining 100 ml of FreeStyle 293 expression medium was added thereto, followed by incubation for 7 days. With reference to a standard protocol, protein was purified from the collected cell culture supernatant. Antibody was applied to a Protein A Sepharose column (GE healthcare), followed by washing with PBS (pH 7.4). The antibody was eluted with 0.1 M glycine buffer at pH 3.0, and then the sample was immediately neutralized with 1M Tris buffer. The eluted antibody fraction was concentrated by dialysis while replacing buffer with PBS (pH 7.4). The purified protein was quantified using the absorbance at 280 nm and absorption coefficient.
Example 17: Evaluation of Cell Growth Inhibitory Activity of Pnm-TPP11 in Pnm.SUP.R .Lung Cancer Cell Lines
(206) In order to examine whether Pnm-TPP11 can also inhibit the proliferation of NRP1-expressing Pnm.sup.R lung cancer cell lines, like Ctx-TPP11, a cell growth assay was performed using various lung cancer cell lines.
(207)
(208) Specifically, lung cancer cell lines were prepared in the same manner as described in Example 11. When the cells were stabilized, the cells were incubated with various concentrations (0, 1, 2, and 4 μM) of each of Pnm and Pnm-TPP11 for 48 hours. Then, 10 μl of Cyto-X reagent (LPS Solution) was added to each well which was then incubated at 37° C. for 1 to 2 hours, and the absorbance at 450 nm was measured using a microplate reader (Molecular Devices). In the Pnm.sup.S (Calu-3, A549, Calu-1, HCC44) lung cancer cell line, Pnm and Pnm-TPP11 showed the same inhibitory activity against cell growth, and in three Pnm.sup.R (H441, SK-LU-1, H1299) lung cancer cell lines, only Pnm-TPP11 showed cell growth inhibitory activity, unlike Pnm. However, in the non-NRP1-expressing Pnm.sup.R lung cancer cell line H460, Pnm-TPP11 was showed no effect. This suggests that Pnm-TPP11 shows an effect specific against its target NRP1.
INDUSTRIAL APPLICABILITY
(209) According to the present invention, when the peptide that binds specifically to neuropilin-1 (NRP1) is fused to an EGFR-targeting antibody or the NRP1-binding peptide fused Fc combined with an EGFR-targeting antibody, it acts on NRP1 in tumor cells to promote the endocytosis of NRP1/active integrin β1, thereby reducing the cell surface expression level of active integrin β1 and reducing the integrin β1-induced phosphorylation of FAK, Src and Akt. Accordingly, through the composition or combination formulation according to the present invention, the intrinsic resistance of cancer to an EGFR-targeting antibody can be overcome. Thus, the present invention may be used for the development of effective anticancer drugs or anticancer adjuvants.
(210) Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
(211) A 32 Kb sequence text file named “268824-459902 SEQUENCE-LISTING”, saved on Nov. 27, 2021, is herein incorporated by reference.