ANTIBODY INHIBITING ACTIVATED RAS IN CELL BY INTERNALIZING INTO CYTOSOL OF CELL, AND USE THEREOF
20200339681 ยท 2020-10-29
Inventors
- Yong Sung Kim (Gyeonggi-do, KR)
- Dong Ki Choi (Daejeon, KR)
- Seung Min Shin (Seoul, KR)
- Seong-Wook Park (Gyeonggi-do, KR)
Cpc classification
C07K2317/60
CHEMISTRY; METALLURGY
C40B50/06
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
C40B30/04
CHEMISTRY; METALLURGY
C12N15/1037
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/82
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K14/70557
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
C07K14/705
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
C40B30/04
CHEMISTRY; METALLURGY
Abstract
A tumor-specific cytosol-internalized RAS-inhibiting antibody, in which modified heavy-chain variable region and a light-chain variable region are combined, according to the present disclosure facilitates development into a therapeutic drug due to a high production yield, and can effectively suppress mutant RAS by means of tumor-specific internalization into the cytosol, and thus effective anti-cancer activity can be expected as a stand-alone drug or in combination treatment with existing medicine.
Claims
1. A heavy-chain variable region specifically binding to Ras(RasGTP) activated by GTP bound thereto, the heavy-chain variable region comprising: CDR1 having an amino acid sequence represented by the following Formula 1; CDR2 having an amino acid sequence represented by the following Formula 2; and CDR3 having an amino acid sequence represented by the following Formula 3,
D-X.sub.11-SMS[Formula 1] wherein X.sub.11 is F or Y,
YISRTSHT-X.sub.21-X.sub.22-YADSVKG[Formula 2] wherein X.sub.21 is T, I or L, and X.sub.22 is Y, C, S, L or A,
G-F-X.sub.31-X.sub.32-X.sub.33-Y[Formula 3] wherein X.sub.31 is K, F, R or N, X.sub.32 is M or L, and X.sub.33 is D or N.
2. The heavy-chain variable region according to claim 1, wherein CDR1 having an amino acid sequence represented by the following Formula 1; CDR2 having an amino acid sequence represented by the following Formula 2; and CDR3 having an amino acid sequence represented by the following Formula 3,
D-X.sub.11-SMS[Formula 1] wherein X.sub.11 is F or Y,
YISRTSHT-X.sub.21-X.sub.22-YADSVKG[Formula 2] wherein X.sub.21-X.sub.22 is TY, IY, TC, TS, IS, LC, LL or IA,
G-F-X.sub.31-X.sub.32-X.sub.33-Y[Formula 3] wherein X.sub.31-X.sub.32-X.sub.33 is KMD, RMD, FMN, RLD or NLD.
3. The heavy-chain variable region according to claim 1, wherein the CRD1 sequence of the heavy-chain variable region is selected from the group consisting of amino acid sequences represented by SEQ ID NOS: 2 to 4, the CDR2 sequence of the heavy-chain variable region is selected from the group consisting of amino acid sequences represented by SEQ ID NO: 5 and SEQ ID NOS: 10 to 16, and the CDR3 sequence of the heavy-chain variable region is selected from the group consisting of amino acid sequences represented by SEQ ID NOS: 6 to 9 and SEQ ID NOS: 17 to 18.
4. The heavy-chain variable region according to claim 1, wherein the heavy-chain variable region is selected from the group consisting of: i) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 5 and CDR3 of SEQ ID NO: 7; ii) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5 and CDR3 of SEQ ID NO: 7; iii) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5 and CDR3 of SEQ ID NO: 8; iv) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5 and CDR3 of SEQ ID NO: 9; v) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5 and CDR3 of SEQ ID NO: 8; vi) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 10 and CDR3 of SEQ ID NO: 7; vii) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 11 and CDR3 of SEQ ID NO: 7; viii) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 12 and CDR3 of SEQ ID NO: 7; ix) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 13 and CDR3 of SEQ ID NO: 7; x) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 17; xi) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 15 and CDR3 of SEQ ID NO: 18; and xii) a heavy-chain variable region comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 16 and CDR3 of SEQ ID NO: 7.
5. The heavy-chain variable region according to claim 1, wherein the heavy-chain variable region is selected from the group consisting of amino acid sequences represented by SEQ ID NOS: 20 to 32.
6. An intact immunoglobulin antibody comprising the heavy-chain variable region according to claim 1.
7. The intact immunoglobulin antibody according to claim 6, wherein the antibody has cytoplasmic penetration ability.
8. The intact immunoglobulin antibody according to claim 6, wherein a light-chain variable region of the antibody is selected from the group consisting of amino acid sequences represented by SEQ ID NOS: 34 to 43.
9. The intact immunoglobulin antibody according to claim 6, wherein the light-chain variable region or the heavy-chain variable region of the antibody is fused with a peptide targeting EpCAM (epithelial cell adhesion molecule), integrin v3 or integrin v5.
10. The intact immunoglobulin antibody according to claim 9, wherein the light-chain variable region or the heavy-chain variable region is selected from the group consisting of amino acid sequences represented by SEQ ID NOS: 44 to 63.
11. The intact immunoglobulin antibody according to claim 6, wherein the antibody comprises a heavy-chain constant region or a light-chain constant region derived from human immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM.
12. The intact immunoglobulin antibody according to claim 11, wherein the heavy-chain constant region comprises at least one mutation of N434D of the CH3, L234A, L235A and P329G of the CH2, wherein the amino acid position is determined according to EU numbering.
13. The intact immunoglobulin antibody according to claim 12, wherein the mutation of the heavy-chain constant region is selected from the group consisting of amino acid sequences represented by SEQ ID NOS: 65 to 69.
14. The intact immunoglobulin antibody according to claim 6, wherein the antibody is selected from the group consisting of single-chain Fvs (scFV), single-chain antibodies, Fab fragments, F(ab) fragments, disulfide-binding Fvs (sdFV) and epitope-binding fragments of the antibodies.
15. The intact immunoglobulin antibody according to claim 6, wherein the antibody is a bispecific antibody (bispecific Ab).
16. The intact immunoglobulin antibody according to claim 6, wherein the antibody is fused with one or more selected from the group consisting of proteins, peptides, small-molecule drugs, toxins, enzymes, nucleic acids and nanoparticles.
17. A method for preparing an intact immunoglobulin-type antibody having improved affinity for intracellular RasGTP and tumor-tissue-specific cytoplasmic penetration ability, the method comprising: (1) preparing an endosomal escape heavy-chain expression vector cloned with nucleic acids, substituted with a humanized heavy-chain variable region (VH) having improved affinity for intracellular RasGTP from a heavy-chain variable region (VH) included in a heavy chain comprising a human heavy-chain variable region (VH) and a human heavy-chain constant region (CH1-hinge-CH2-CH3); (2) preparing a cytoplasmic penetration light-chain expression vector cloned with nucleic acids, substituted with a humanized light-chain variable region (VL) having cytoplasmic penetration ability and a humanized light-chain variable region (VL) having cytoplasmic penetration ability specific for tumor tissues from a light-chain variable region (VL) included in a light chain comprising a human light-chain variable region (VL) and a human light-chain constant region (CL); (3) co-transforming the prepared heavy- and light-chain expression vectors into animal cells for protein expression to express an intact immunoglobulin-type antibody including a heavy-chain variable region (VH) having improved affinity for intracellular RasGTP and a light-chain variable region (VL) having cytoplasmic penetration ability specific for tumor tissues; and (4) purifying and recovering the expressed intact immunoglobulin-type antibody.
18. A composition for preventing or treating cancer comprising the antibody according to claim 6.
19. The composition according to claim 18, wherein the cancer has a mutation associated with an activated intracellular Ras.
20. The composition according to claim 19, wherein the mutation associated with the activated intracellular Ras is cancer having a mutation in 12nd, 13rd or 61st amino acid of the Ras.
21. The composition according to claim 18, wherein the preventing or treating cancer is characterized in that the antibody according to claim 6 inhibits binding of activated Ras (RasGTP) to B-Raf, C-Raf or PI3K in the cytoplasm.
22. A composition for diagnosing tumors comprising the antibody according to claim 6.
23. A polynucleotide encoding the antibody according to claim 6.
24. A vector comprising the polynucleotide according to claim 23.
25. A method for constructing a heavy-chain variable region library specifically binding to RasGTP and having improved affinity therefor, the method comprising: (1) determining an amino acid site of three complementarity determining regions (CDRs) having high potential to bind to intracellular RasGTP involved in antigen binding of a RT11 heavy-chain variable region (VH) library template; (2) designing a degenerated codon primer capable of encoding an amino acid in need of inclusion in a library at the determined amino acid site; and (3) expressing the heavy-chain variable-region of designed library in a form of scFab or Fab using a yeast surface expression system.
26. A library of a heavy-chain variable region specifically binding to RasGTP and having improved affinity therefor, constructed by the method according to claim 25.
27. A method for screening a heavy-chain variable region specifically binding to RasGTP and having improved affinity therefor, the method comprising: (1) expressing the heavy-chain variable-region library capable of binding to RasGTP, prepared according to (3) in claim 25, using a yeast surface expression system; (2) constructing Avi-KRas.sup.G12D bound to GppNHp, a GTP analogue, in a stable form without deformation during biotinylation; (3) binding the heavy-chain variable-region library with the GppNHp-bound Avi-KRas.sup.G12D; and (4) measuring affinity of binding between the heavy-chain variable-region library and the GppNHp-bound Avi-KRas.sup.G12D.
Description
DESCRIPTION OF DRAWINGS
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BEST MODE
[0171] Hereinafter, the present disclosure will be described in more detail with reference to examples. However, it will be obvious to those skilled in the art that these examples are provided only for illustration of the present disclosure and should not be construed as limiting the scope of the present disclosure.
Example 1. Preparation of Avi-KRas.SUP.G12D .Protein Having GppNHp Bound Thereto
[0172] An Avi-KRas.sup.G12D antigen including an Avi tag (GLNDIFEAQKIEWHE) fused to an N-term thereof for use in library selection was constructed. The Avi-KRas.sup.G12D antigen was constructed in order to minimize structural denaturation which may cause problems with antigen biotinylation during library selection.
[0173] Specifically, DNA in which catalytic G domains (residues 1 to 169) of the KRas.sup.G12D protein, excluding the C-terminal hypervariable region are fused to an 8 his tag and an Avi-tag at the N-terminus using a GSG linker was constructed by PCR and was cloned using the restriction enzymes NcoI and HindIII in the pET23 vector, which is a vector for E. coli expression. Then, the constructed pET23-8Xhis-Avi-KRas.sup.G12D (1-169) vector was transformed through electroporation into the E. coli strain BL21(DE3)plysE along with a vector (pBirAcm) encoding BirA as a biotin ligase for in-vivo biotinylation, and then was selected in a selective medium containing 100 g/ml ampicillin and 10 g/ml chloramphenicol. After culturing the selected E. coli in the selective medium (LBCA) containing 5 mM MgCl.sub.2 at 37 C. until absorbance at 600 nm reached 0.6, 0.5 mM IPTG for protein expression and 50 M d-biotin for in-vivo biotinylation were added thereto, followed by further culturing at 30 C. for 4 hours. The stock solution for addition of 50 M d-biotin was prepared by adding 12 mg of d-biotin to 10 mL of 10 mM bicin buffer (pH 8.3). After culturing E. coli, the E. coli collected using a centrifuge were resuspended in buffer containing 20 mM Tris, 500 mM NaCl, 5 mM imidazole and 2 mM -ME, and E. coli (SONICS) was pulverized with ultrasound waves. Only the supernatant, from which the pulverized E. coli was removed, was collected using a centrifuge, and then purified using Ni-NTA resin for specifically purifying the protein fused with the His tag. The Ni-NTA resin was washed with 50 ml of washing buffer (20 mM Tris, pH 7.4, 300 mM NaCl, 20 mM imidazole, 2 mM MgCl.sub.2 and 2 mM -ME) and proteins were eluted with an elution buffer (20 mM Tris, pH 7.4, 300 mM NaCl, 250 mM imidazole, 2 mM MgCl.sub.2 and 2 mM -ME). The eluted proteins were buffered with a storage buffer (50 mM Tris-HCl, pH 8.0, 1 mM DTT, 2 mM MgCl.sub.2) using a dialysis method. The purified proteins were quantified using absorbance and the absorption coefficient measured at a wavelength of 280 nm. Purity of about 98% or more was identified through SDS-PAGE analysis.
[0174] The fusion of the purified Avi-KRas.sup.G12D protein with Avi-tag and biotin at high yield through the in-vivo biotinylation reaction was identified using a gel shift method. Specifically, the Avi-KRas.sup.G12D protein was diluted in an SDS-PAGE loading buffer (25 mM Tris-HCl, pH 6.8, 1% SDS, 10% glycerol, 175 mM -ME), allowed to react for 10 minutes and then allowed to react with 1 g of streptavidin at room temperature for 30 minutes, the protein separated through SDS-PAGE was transferred to the PVDF membrane and identified using the anti-His antibody fused with HRP.
[0175] The Avi-KRas.sup.G12D protein was reacted with GTP analog (GppNHp) or GDP and was then analyzed by SDS-PAGE. Specifically, in order to form a complex of the GTP analog (GppNHP) and Ras protein, the purified Ras protein was diluted in a substrate exchange buffer (40 mM Tris-HCl, pH 7.5, 200 mM (NH.sub.4).sub.2SO.sub.4, 10 M ZnCl.sub.2, 5 mM DTT) and an alkaline phosphatase fused with 2 units of beads per mg of Ras protein and GppNHp having a molar amount at least 10 times that of the Ras protein were added thereto, followed by allowing a reaction to proceed at room temperature for 1 hour. In order to form a complex of GDP and Ras protein, GDP in a molar amount at least 20 times that of the Ras protein and 20 mM EDTA were added and allowed to react at 30 C. for 30 minutes, and then 60 mM MgCl.sub.2 was added thereto, and the result was allowed to stand at 4 C. for 30 minutes to stop the reaction. The GppNHp or GDP-bound Ras protein obtained through the method was analyzed through SDS-PAGE after the buffer was exchanged with a storage buffer (50 mM Tris-HCl, pH 8.0, 1 mM DTT, 2 mM MgCl.sub.2) using a PD10 Sephadex G25 column. For long-term storage, the Ras protein bound to the substrate was stored at 80 C.
[0176] ELISA was performed to analyze the RT11 binding capacity of the Avi-KRas.sup.G12D protein prepared through the above method and the His-KRas.sup.G12D protein having no Avi-tag. Specifically, RT11, an anti-RasGTP iMab, was bound at a concentration of 5 g/ml in a 96-well EIA/RIA plate (COSTAR Corning) at room temperature for 1 hour, and was then washed with 0.1% TBST (12 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween20, 5 mM MgCl.sub.2) at room temperature for 10 minutes. After binding with 4% TBSB (12 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 4% BSA, 10 mM MgCl.sub.2) for 1 hour, the result was washed 3 times with 0.1% TBST for 10 minutes. Then, the KRas protein bound with GppNHp and the KRas protein bound with GDP were diluted in 4% TBSB and then bound at a concentration of 100 nM for 1 hour at room temperature, followed by washing three times with 0.1% TBST for 10 minutes. The result was bound to HRP-conjugated anti-his antibody (HRP-conjugated anti-his mAb) as a labeling antibody. The result was reacted with a TMB ELISA solution, and absorbance at 450 nm was quantified.
[0177] The above experiment showed that the KRas.sup.G12D protein fused with Avi-tag can be used to select the RT11 affinity-improvement library.
Example 2. Construction of Anti-RasGTP iMab RT11-Based High-Diversity Antibody Library and Selection of Heavy-Chain Variable Region (VH) with Enhanced RasGTP-Specific Affinity
[0178] The anti-RasGTP iMab RT11 in the conventional patent (Koran Patent No. 10-1602876) binds highly specifically to RasGTP and exhibits biological activity in various Ras mutant cell lines, but exhibits a level of affinity of about 12 nM for RasGTP, which is a lower affinity than the affinity of various antibodies in the IgG format. In addition, anti-RasGTP iMab RT11, which exhibits biological activity through inhibition of binding between RasGTP and effector molecules, can enhance biological activity through the improvement of affinity with RasGTP. Accordingly, the present inventors tried to increase the affinity of anti-RasGTP iMab RT11 for RasGTP in addition to modifying (improving) the light-chain variable region to impart tissue specificity thereto in order to increase the therapeutic efficiency of anti-RasGTP iMab.
[0179] The light-chain variable region used during selection of the RasGTP specific heavy-chain variable region with improved affinity based on RT11 is the hT4-3 light-chain variable region with improved endosomal escape ability, which is obtained by introducing CDR3 in which WYW (Trp-Tyr-Trp) is located at residues 92-94 of the hT4 light-chain variable region (VL) for cytoplasmic penetration used in the conventional patent (Korean Patent No. 10-1602876) and substituting, with phenylalanine (Phe), the residue 87 of the light-chain variable region framework region corresponding to the interface between the light-chain variable region (VL) and heavy-chain variable region (VH) in order to overcome the increased exposure of hydrophobic residues to solvents and thus decreased production yield due to the introduction of WYW (Korean Patent Application No. 10-2016-0065365).
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[0181] The binding structure between RasGTP and RT11 antibody was predicted using homology modeling and docking programs, and random mutations were introduced into CDRs and surrounding regions predicted to play an important role in antigen binding based on the prediction result.
[0182] Specifically, degenerated codons that may include a suitable amino acid sequence in the predicted binding structure were used for the residues of CDR1 (Nos. 31 to 33) and CDR2 (Nos. 52 to 56). The degenerate codons RNC, THC and KSG were sequentially used for CDR1 (Nos. 31 to 33) and the degenerate codons ASC, MRA, ASC, ASC, CRC and WMC were sequentially used for CDR2 (Nos. 52 to 56). ARG degenerate codons capable of encoding Arg and Lys present in the germline antibody sequence were used because the residue 94 of the CDR3-surrounding framework is a residue at a position that can affect the CDR structure. The residues (Nos. 95 to 97) of CDR3 were spiked oligomers capable of conserving the sequence of RT11 with a 50% probability. This is a technique that can maintain the wild-type amino acid at 50% during the PCR process by designing a primer such that, in each of 3 nucleotides encoding amino acids for each residue, wild-type nucleotides can be maintained at 79%, and the ratio of remaining nucleotides was adjusted to 7%. The CDR3 (No. 100a) residue is an important residue for VH/VL binding, and a WTK degenerate codon, which may include Phe, Ile and Leu present in the germline antibody sequence, including Met of the RT11 antibody was used therefor.
[0183] Specifically, hT4-3 VL with improved cytoplasmic penetration ability was used for the light-chain variable region in the affinity improvement library.
[0184] Specifically, the designed library-encoding DNA was amplified using a PCR technique and was then concentrated using an ethanol precipitation method. The yeast surface expression vector (C-aga2), which expresses aga2 protein at the c-terminus for homologous recombination, was treated with NheI and MluI restriction enzymes and then purified using agarose gel extraction and concentrated using ethanol precipitation. The 5 g vector treated with restriction enzymes for 12 g of each library-encoding DNA was transformed by electroporation into the yeast EBY100 for yeast surface expression (Baek D. S. and Kim Y. S., 2014), and the number of colonies grown in a selective medium, SD-CAA (20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids, 5.4 g/L Na 2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids) through serial dilution was measured to determine the library size.
Example 3. Selection of Heavy-Chain Variable Region (VH) with Improved Affinity for GppNHp-Bound KRas.SUP.G12D
[0185] The RT11-3-based affinity improvement library constructed in Example 2 was selected using the GppNHp-bound KRas.sup.G12D as an antigen.
[0186] Specifically, about 100 nM of the purified GppNHp-bound Avi-KRas.sup.G12D was reacted with yeast inducing expression of the single-chain Fab (scFab)-type heavy-chain variable region library on the cell surface using an SG-CAA medium (20 g/L galactose, 6.7 g/L yeast nitrogen base without amino acids, 5.4 g/L Na.sub.2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids) at room temperature for 1 hour. Then, the yeast expressing the library linked with GppNHp-bound Avi-KRas.sup.G12D was reacted with Streptavidin Microbead (Miltenyi Biotec) for 20 minutes at 4 C., and then a yeast expressing the heavy-chain variable region having high affinity for the GppNHp-bound Avi-KRas.sup.G12D was enriched using MACS (magnetic activated cell sorting). The yeast expressing the selected library was cultured in selective media SD-CAA (20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids, 5.4 g/L Na.sub.2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids) and SG-CAA to induce library expression, the GppNHP-bound Avi-KRas.sup.G12D and Avi-KRas.sup.G12D antigen bound with GDP undergoing no in-vivo biotinylation for primary FACS screening were competitively reacted at a ratio of 1:10 with the library-expressing yeast for 1 hour at room temperature, reacted with PE-conjugated Streptavidin-R-phycoerythrin (SA-PE, Invitrogen), and suspended through FACS (fluorescence activated cell sorting, FACS Caliber, BD biosciences). Then, secondary FACS screening was performed in the same manner as above at a ratio of 1:100 using 10 nM of the GppNHp-bound Avi-KRas.sup.G12D antigen and the Avi-KRas.sup.G12D antigen bound with GDP undergoing no in-vivo biotinylation, and tertiary FACS screening was performed in the same manner as above at a ratio of 1:100 using 1 nM of the GppNHp-bound Avi-KRas.sup.G12D antigen and the GDP-bound Avi-KRas.sup.G12D antigen undergoing no in-vivo biotinylation.
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[0189] Six unique clones (RT21, RT22, RT23, RT24, RT25, RT26) having high affinity and specificity to the GppNHp-bound Avi-KRas.sup.G12D were selected through the analysis of individual clone binding ability as described above.
[0190] The following Table 1 shows the heavy-chain variable region sequences of six individual clones that have high binding ability to the selected GppNHp-bound Avi-KRas.sup.G12D, and Table 2 shows the CDR sequences of the heavy-chain variable regions of Table 1.
TABLE-US-00006 TABLE1 SEQ VH ID name Sequence NO. RT11VH 102030405052a 19 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMSWVRQAPGKGLEWVSYISRTSHTTY 60708052a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFFMDYWGQGTLVTVSS RT21VH 102030405052a 20 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYSMSWVRQAPGKGLEWVSYISRTSHTTY 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFKMDYWGQGTLVTVSS RT22VH 102030405052a 21 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTTY 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFKMDYWGQGTLVTVSS RT23VH 102030405052a 22 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYSMSWVRQAPGKGLEWVSYISRTSHTTY 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFRMDYWGQGTLVTVSS RT24VH 102030405052a 23 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTTY 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGFFMNYWGQGTLVTVSS RT25VH 102030405052a 24 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTTY 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFRMDYWGQGTLVTVSS RT26VH 102030405052a 25 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTTY 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFFMNYWGQGTLVTVSS
TABLE-US-00007 TABLE2 KabatNo. SEQIDNO. 31 32 33 34 35 RT11VH- 1 S Y S M S CDR1 RT21VH- 2 D Y S M S CDR1 RT22VH- 3 D F S M S CDR1 RT23VH- 4 D Y S M S CDR1 RT24VH- 3 D F S M S CDR1 RT25VH- 3 D F S M S CDR1 RT26VH- 3 D F S M S CDR1 KabatNo. 50 51 52 52a 53 54 55 56 57 58 59 60 61 62 63 64 65 RT11VH- 5 Y I S R T S H T T Y Y A D S V K G CDR2 RT21VH- 5 Y I S R T S H T T Y Y A D S V K G CDR2 RT22VH- 5 Y I S R T S H T T Y Y A D S V K G CDR2 RT23VH- 5 Y I S R T S H T T Y Y A D S V K G CDR2 RT24VH- 5 Y I S R T S H T T Y Y A D S V K G CDR2 RT25VH- 5 Y I S R T S H T T Y Y A D S V K G CDR2 RT26VH- 5 Y I S R T S H T T Y Y A D S V K G CDR2 KabatNo. 95 96 97 98 99 100 100a 101 102 RT11VH- 6 G F F M D Y CDR3 RT21VH- 7 G F K M D Y CDR3 RT22VH- 7 G F K M D Y CDR3 RT23VH- 8 G F R M D Y CDR3 RT24VH- 9 G F F M N Y CDR3 RT25VH- 8 G F R M D Y CDR3 RT26VH- 9 G F F M N Y CDR3
Example 4. Analysis of Antigen-Binding Capacity of Anti-RasGTP iMab Antigen with Improved Affinity
[0191] In order to construct the cytoplasmic penetration antibody (cytotransmab) introduced with the mutation in the heavy-chain variable region, a heavy chain including a heavy-chain variable region having improved affinity for RasGTP and a heavy-chain constant region (CH1-CH2-CH3) based on RT11 were cloned into an animal expression vector, and RGD10 protopeptides having specificity for integrins (Integrin v3 and v5), which are overexpressed in neovascular cells and various tumors, were fused with the N-terminus of the cytoplasmic penetration humanized hT4-3 light-chain using two GGGGS linkers using a genetic engineering technique, and were cloned into an animal expression vector. The RGD10 protopeptide has affinity similar to that of the RGD4C protopeptide, but has only one disulfide bond between two cysteines, and it can be fused using a genetic engineering technique. The heavy-chain expression vector having improved affinity for RasGTP and the cytoplasmic penetration humanized light-chain expression vector (hT4-i3 LC) fused with the RGD10 protopeptide were simultaneously subjected to transient transfection in HEK293F protein-expressing cells to express an anti-RasGTP iMab mutation with improved affinity.
[0192] Specifically, in order to construct a heavy-chain expression vector for producing an intact immunoglobulin-type cytoplasmic penetration antibody, DNA encoding a heavy-chain including the heavy-chain constant region (CH1-CH2-CH3) and being introduced with the heavy-chain variable mutation having improved affinity based on RT11, which is fused with a DNA encoding a secretion signal peptide at the 5 end thereof, was cloned into the pcDNA3.4 vector with NotI/HindIII. Proteins were expressed and purified through transient transfection using the heavy-chain expression vector and the light chain of the previous cytoplasmic penetration antibody (Korean Patent Application No. 10-2016-0065365), and yields were compared. HEK293F cells suspended and grown in a serum-free FreeStyle 293 expression medium were transfected with a mixture of plasmids and polyethyleneimine (PEI) in a shake flask. During 200 mL transfection into the shake flask, the HEK293F cells were seeded in 100 ml of medium at a density of 2.010.sup.6 cells/ml and cultured at 120 rpm, 8% CO.sub.2, and 37 degrees. In order to produce the antibody, suitable heavy-chain and light-chain plasmids were diluted in a 10 ml FreeStyle 293 expression medium to a total of 250 g (2.5 g/ml) including 125 g of a heavy chain and 125 g of a light chain and filtered, then mixed with 10 ml of a medium, in which 750 g (7.5 g/ml) of PEI was diluted, and reacted at room temperature for 10 minutes. Then, 100 ml of the reacted mixed medium was added to previously seeded cells, and then the cells were cultured at 120 rpm and 8% CO.sub.2 for 4 hours, and the remaining 100 ml of FreeStyle 293 expression medium was added thereto, followed by culturing for 6 days. Proteins were purified from the cell culture supernatant collected in accordance with the standard protocol. Antibodies were applied to a Protein A Sepharose column and washed with PBS (pH 7.4). The antibodies were eluted at pH 3.0 using 0.1M glycine and 0.5M NaCl buffer, and then the sample was immediately neutralized with 1M Tris buffer. The eluted antibody fraction was concentrated in a fresh PBS (pH 6.5) buffer exchanged through a dialysis method. The purified protein was quantified using absorbance and absorption coefficient at a wavelength of 280 nm.
[0193]
[0194]
[0195] Specifically, RT11-i, an anti-RasGTP iMab, and six affinity-enhanced iMabs fused with RGD protopeptides were bound at a concentration of 5 g/ml in a 96-well EIA/RIA plate at room temperature for 1 hour. Then, the result was washed with 0.1% TBST (12 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween20, 5 mM MgCl.sub.2) three times for 10 minutes. Then, the result was bound to 4% TBSB (12 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 4% BSA, 10 mM MgCl.sub.2) for 1 hour and then washed 3 times with 0.1% TBST for 10 minutes. The GppNHp-bound KRas protein and the GDP-bound KRas protein were diluted in 4% TBSB, bound at various concentrations of 100 nM, 10 nM and 1 nM for 1 hour at room temperature, and washed 3 times with 0.1% TBST for 10 minutes. The result was bound to HRP-conjugated anti-his antibody (HRP-conjugated anti-his mAb) as a labeling antibody. The result was reacted with TMB ELISA solution, and the absorbance at 450 nm was quantified.
[0196] ELISA analysis showed that the selected 6 types of anti-RasGTP iMabs exhibited higher antigen-binding capacity than the conventional RT11-i3.
[0197]
[0198] Specifically, an SW480 cell line was diluted in 0.5 ml of 210.sup.4 cells/well in a 24-well plate, cultured for 12 hours at 37 C. under 5% CO.sub.2, and then treated with TMab4-i, RT11-i, and 6 types of 1 M anti-RasGTP iMab with improved affinity and then cultured at 37 C. for 12 hours. Then, the medium was removed, the residue was washed with PBS, and proteins attached to the cell surface were removed with a weakly acidic solution (200 mM glycine, 150 mM NaCl pH 2.5). After washing with PBS, 4% paraformaldehyde was added and cells were immobilized at 25 degrees for 10 minutes. After washing with PBS, the cells were cultured in a buffer containing PBS supplemented with 0.1% saponin, 0.1% sodium azide and 1% BSA at 25 degrees for 10 minutes and a hole was formed in the cell membrane. Then, the cells were washed with PBS again and reacted with a buffer containing 2% BSA in addition to PBS for 1 hour at 25 degrees in order to inhibit non-specific binding. Each antibody was stained with an antibody that specifically recognizes human Fc linked with Alexa-488 (green fluorescence). The nuclei were stained (blue fluorescence) using Hoechst33342, and KRas-labeled antibodies were stained (red fluorescence) and then observed with a confocal microscope. All of the anti-RasGTP iMabs except TMab4-i3 were found to have fluorescence overlapping that of the intracellular Ras.
[0199] Among them, the RT22-i3 iMab that exhibits the ability to bind to activated intracellular Ras and the highest binding capacity with Avi-KRas.sup.G12DGppNHp during ELISA experiments was subjected to SPR (surface plasmon resonance) analysis using a Biacore2000 instrument to more quantitatively analyze the binding ability thereof to GppNHp-bound KRas.sup.G12D.
[0200] Specifically, RT22-i3 was diluted to a concentration of 20 l/ml in 10 mM NaAc buffer (pH 4.0) and immobilized at approximately 1800 response units (RU) on a CM5 sensor chip (GE Healthcare). Subsequently, Tris buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM MgCl.sub.2, 0.01% Tween 20) was analyzed at a flow rate of 30 l/min, and GppNHp-bound GST-KRas.sup.G12D complete antibody was analyzed at a concentration of 50 nM to 3.125 nM. After binding and dissociation analysis, regeneration of the CM5 chip was performed by flowing a buffer (20 mM NaOH, 1M NaCl, pH10.0) at a flow rate of 30 l/min for 1 minute. Each sensorgram, obtained by binding for 3 minutes and dissociation for 3 minutes, was normalized with reference to a blank cell and subtracted, and the affinity was thus calculated.
[0201] The following Table 3 shows the results of affinity analysis of anti-RasGTP iMab RT11-i and RT22-i3 for complete-form GppNHp-bound GST-KRas.sup.G12D using SPR (BIACORE 2000).
TABLE-US-00008 TABLE 3 KRas.sup.G12DGppNHp k.sub.a (M.sup.1s.sup.1) k.sub.d (s.sup.1) K.sub.D (M) RT11-i 3.33 0.13 10.sup.4 6.84 0.32 10.sup.4 2.05 0.14 10.sup.8 RT22-i3 2.07 0.13 10.sup.4 2.98 0.12 10.sup.4 1.44 0.12 10.sup.9
Example 5. Library Construction and Selection for Additional Affinity Improvement Based on RT22 Heavy-Chain Variable Region (VH)
[0202] The anti-RasGTP iMab (RT22-i3) including a heavy-chain variable region (RT22 VH) with improved binding ability to RasGTP has high affinity of about 1.4 nM. However, it was selected through combination with the light-chain variable region (hT4-3 VL), which maintains binding to the cell membrane receptor, HSPG. Since HSPG is expressed in most tissues, a light-chain variable region in which germline antibody sequences were introduced into CDR1 and CDR2 was constructed, and the anti-RasGTP iMab constructed using the light-chain variable region was found to have a serious decrease in production yield during fusion of RGD protopeptides. Accordingly, the CDR of the heavy-chain variable region (VH) combined with the light-chain variable region having tissue specificity was modified to overcome the problems of reduced affinity and production yield.
[0203] A description of the light-chain variable region having reduced HSPG binding and tissue specificity is given in detail below.
[0204]
[0205] To improve the affinity, 3D structural models were analyzed using Galaxy modeling, and mutations were imparted to CDR2 (Nos. 55 to 58) and CDR3 (Nos. 95, 97, and 100a), considered to have a significant effect on antigen binding, based on the analysis result.
[0206] Specifically, a degeneration codon (VRC) was used for the residue 55 of CDR2 so that the residue amino acid sequence could be maintained at a probability of 16.67% and a hydrophilic or negatively charged amino acid could be located thereat, and a degeneration codon (MYT) was used for residue 57 of CDR3 so that the affinity with the antigen could be increased in consideration of the size and direction of the side chain while maintaining the conventional amino acid sequence with a 25% probability. A degeneration codon (VST) was used for residue 95 of CDR3 so that the binding ability to RasGTP could be maintained or improved in consideration of the size and direction of the side chain while maintaining the conventional amino acid sequence with a 16.67% probability, and a degeneration codon (WTK) was used for residue 100a so that a hydrophobic amino acid could be located thereat. The same spiked oligomer as in Example 2 was used for residues 56 and 58 of CDR2 and residue 97 of CDR3. This is a mutation method in which all amino acids can be applied while preserving the conventional wild-type RT22 VH sequence at 50% probability, and is a technique that can maintain the wild-type amino acid at 50% during the PCR process by designing a primer such that, in each of 3 nucleotides encoding amino acids, the wild-type nucleotide can be maintained at 79%, and the ratio of remaining nucleotides is adjusted to 7%.
[0207] Specifically, a yeast expressing the initial RT22-based library on the surface thereof was mated with a yeast secreting the cytoplasmic penetration light chain (hT4-33 VL) from which HSPG-binding ability was removed and the resulting product was expressed in the form of Fab on the yeast surface.
[0208] Specifically, in order to construct the yeast secreting the cytoplasmic penetration light chain (hT4-33 VL) to be conjugated with the heavy-chain variable region (VH) library, pYDS-K-hT4-33 VL, obtained by cloning the DNA encoding hT4-33 VL having cytoplasmic penetration ability and reduced HSPG binding ability into the light-chain variable region yeast secretion vector, pYDS-K using restriction enzymes NheI and BsiWI, was transformed by electroporation into the YVH10 strain, which is a mating--type yeast-mating strain, and was mated with a yeast selectively cultured in a selective medium SD-CAA+Trp (20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids, 5.4 g/L Na 2HPO.sub.4, 8.6 g/L NaH 2PO.sub.4, 5 g/L casamino acids, 0.4 mg/L tryptophan) (SIGMA).
[0209] Specifically, in the case of yeast mating, when absorbance at 600 nm of 1 indicates 110.sup.7 yeast individuals. Among cultured yeast, yeast expressing the heavy-chain variable region library based on RT22 and yeast containing hT4-33 VL were mixed in amounts of 1.510.sup.7 individuals and washed three times with YPD (20 g/L dextrose, 20 g/L peptone, 10 g/L yeast extract, 14.7 g/L sodium citrate, 4.29 g/L citric acid, pH 4.5) (SIGMA), and then resuspended with 100 l of YPD and dropped while preventing the same from spreading over the YPD plate, dried and cultured at 30 degrees for 6 hours. Then, the dried spreading yeast site was washed three times with YPD medium and cultured at 30 C. for 24 hours in a selective medium SD-CAA to a final yeast concentration of 110.sup.6 or less, and only the mated yeast was selected.
Example 6. Selection of Heavy-Chain Variable Region (VH) Having High Specific Binding Ability to GTP-Bound KRas G12D Based on RT22
[0210] GppNHp, a GTP analogue, was bound to the in-vivo biotinylated Avi-KRas.sup.G12D protein in the same manner as in Example 1, and was used for library selection. For the primary MACS selection, about 100 nM of the antigen was reacted with the Fab library expressed on the yeast surface at room temperature for 1 hour. Then, the yeast expressing the Fab library bound with the antigen was reacted with Streptavidin Microbead at 4 degrees for 20 minutes, and then the yeast expressing the heavy-chain variable region having high affinity to GppNHp-bound Avi-KRas.sup.G12D was enriched using magnetic activated cell sorting (MACS). The selected library-expressing yeast was cultured in selective media SD-CAA+URA (20 g/L D-glucose, 6.7 g/L yeast nitrogen base without amino acids, 5.4 g/L Na.sub.2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids, 0.2 mg/L uracil) and SG-CAA+URA (20 g/L Galactose, 6.7 g/L yeast nitrogen base without amino acids, 5.4 g/L Na.sub.2HPO.sub.4, 8.6 g/L NaH.sub.2PO.sub.4, 5 g/L casamino acids, 0.2 mg/L Uracil) to induce library expression and primary to tertiary FACS selection was performed.
[0211] As a result of analyzing 47 individual clones of the final primary MACS and tertiary FACS selected libraries in the same manner as in Example 3, seven heavy-chain variable regions having unique amino acid sequences having high affinity for the GppNHp-bound Avi-KRas.sup.G12D protein were selected.
[0212] The following Table 4 shows seven heavy-chain variable-region (VH) sequences having unique amino acid sequences of the individual clones selected from the affinity improvement library based on RT22 as a template.
[0213] The following Table 5 shows the sequences of CDRs 1, 2 and 3 of the selected heavy-chain variable regions (VH).
TABLE-US-00009 TABLE4 SEQ VH ID name Sequence NO. RT31VH 102030405052a 26 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTTY 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFKMDYWGQGTLVTVSS RT32VH 102030405052a 27 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTTC 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFKMDYWGQGTLVTVSS RT33VH 102030405052a 28 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTTS 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFKMDYWGQGTLVTVSS RT34VH 102030405052a 29 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTIS 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFKMDYWGQGTLVTVSS RT35VH 102030405052a 30 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTLC 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFRLDYWGQGTLVTVSS RT36VH 102030405052a 31 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTLL 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFNLDYWGQGTLVTVSS RT37VH 102030405052a 32 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTIA 60708082a90100a110 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFKMDYWGQGTLVTVSS
TABLE-US-00010 TABLE5 Name SEQIDNO. Sequence KabatNo. 31 32 33 34 35 RT22VH- 3 D F S M S CDR1 KabatNo. 50 51 52 52a 53 54 55 56 57 58 59 60 61 62 63 64 65 RT22VH- 5 Y I S R T S H T T Y Y A D S V K G CDR2 RT31VH- 10 Y I S R T S H T I Y Y A D S V K G CDR2 RT32VH- 11 Y I S R T S H T T C Y A D S V K G CDR2 RT33VH- 12 Y I S R T S H T T S Y A D S V K G CDR2 RT34VH- 13 Y I S R T S H T I S Y A D S V K G CDR2 RT35VH- 14 Y I S R T S H T L C Y A D S V K G CDR2 RT36VH- 15 Y I S R T S H T L L Y A D S V K G CDR2 RT37VH- 16 Y I S R T S H T I A Y A D S V K G CDR2 KabatNo. 95 96 97 98 99 100 100a 101 102 RT22VH- 7 G F K M D Y CDR3 RT31VH- 7 G F K M D Y CDR3 RT32VH- 7 G F K M D Y CDR3 RT33VH- 7 G F K M D Y CDR3 RT34VH- 7 G F K M D Y CDR3 RT35VH- 17 G F R L D Y CDR3 RT36VH- 18 G F N L D Y CDR3 RT37VH- 7 G F K M D Y CDR3
Example 7. Expression and Purification of RT22-Based Affinity-Improved Anti-RasGTP iMabs and Analysis of Binding Capacity Thereof to GppNHp-Bound KRas.SUP.G12D
[0214] The heavy-chain variable region selected from the RT22-based library was cloned into a heavy-chain animal expression vector in the same manner as in Example 4, subjected to transient co-transfection into the cytoplasm-penetrating humanized light chain expression vector and the HEK293F protein-expressing cell to express separate clones, and purified in the same manner as in Example 4. Among the seven heavy-chain variable regions with improved affinity, RT32 and RT35 clones were excluded from expression purification because they included the cysteine in the CDR2 sequence and thus could not be present as a monomer (alone), and had the potential to form a dimer or oligomer through an unnatural disulfide bond when purified with IgG.
[0215] The RT22-based affinity-enhanced heavy-chain variable region was expressed in combination with a light-chain variable region (hT4-i33 VL: SEQ ID NO: 38) fused with RGD protopeptide that has an inhibitory activity of HSPG binding ability and endosome escape ability through introduction of a WYW mutation in CDR3. The description of the light-chain variable region (hT4-i33 VL) having reduced binding affinity to HSPG, SEQ ID NO: 38 and tissue specificity will be described in detail below.
[0216] As a result of expression, anti-RasGTP iMabs having improved affinity similar to RT22-i33, used as a template, also showed a low production yield of 1 mg/L, as in Example 3.
[0217]
[0218] Specifically, a molecular weight of about 150 kDa was detected under non-reducing conditions, and the molecular weight of the heavy chain and the molecular weight of the light chain were found to be 50 kDa and 25 kDa, respectively, under reducing conditions. This indicated that the expressed and purified individual clones were present as monomers (alone) in the solution, and did not form dimmers or oligomers through unnatural disulfide bonds.
[0219]
[0220] Specifically, in the same manner as in Example 4, the anti-RasGTP iMab, RT11 and RT22-i33, used as a library template, were used as a control group, and five affinity-enhanced iMabs fused with RGD protopeptides were bound at a concentration of 5 g/ml in a 96-well EIA/RIA plate at room temperature for 1 hour and washed three times with 0.1% TBST (12 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween20, 5 mM MgCl.sub.2) for 10 minutes. Then, the result was bound in 4% TBSB (12 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 4% BSA, 10 mM MgCl.sub.2) for 1 hour and then washed 3 times with 0.1% TBST for 10 minutes. The GppNHp-bound KRas protein was diluted to various concentrations of 100 nM, 10 nM, and 1 nM, and the GDP-bound KRas protein was diluted in 4% TBSB to a concentration of 100 nM, bound for 1 hour at room temperature, and washed 3 times with 0.1% TBST for 10 minutes. The result was bound to an HRP-conjugated anti-his antibody (HRP-conjugated anti-his mAb) as a labeling antibody. The result was reacted with a TMB ELISA solution, and the absorbance at 450 nm was quantified.
[0221]
[0222] Specifically, in the same manner as in Example 4, the anti-RasGTP iMab, RT11 and RT22-i33, used as a library template, were used as a control group, and five affinity-enhanced iMabs fused with RGD protopeptides were bound at a concentration of 5 g/ml in a 96-well EIA/RIA plate at room temperature for 1 hour and washed three times with 0.1% TBST (12 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween20, 5 mM MgCl.sub.2) for 10 minutes. Then, the result was bound in 4% TBSB (12 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 4% BSA, 10 mM MgCl.sub.2) for 1 hour and then washed 3 times with 0.1% TBST for 10 minutes. The GppNHp-bound KRas protein was diluted to various concentrations of 100 nM, 10 nM and 1 nM, and the GDP-bound KRas protein was diluted to a concentration of 100 nM in 4% TBSB, bound for 1 hour at room temperature, and washed 3 times with 0.1% TBST for 10 minutes. The result was bound to an HRP-conjugated anti-his antibody (HRP-conjugated anti-his mAb) as a labeling antibody. The result was reacted with TMB ELISA solution, and the absorbance at 450 nm was quantified.
[0223] In addition, ELISA analysis was performed using a BIACORE2000 instrument to determine the quantitative affinity for GppNHp-bound KRas.sup.G12D, of the affinity-improved anti-RasGTP iMabs having higher antigen-binding ability than RT22-i33.
[0224] Specifically, in the same manner as in Example 4, RT22-i33 was used as a control group, and RT31-i33, RT34-i33 and RT36-i33 were diluted in 10 mM Na-acetate buffer (pH 4.0) and immobilized in an amount of approximately 1600 response units (RU) on a CM5 sensor chip (GE Healthcare). Subsequently, Tris buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl.sub.2, 0.01% Tween 20) was analyzed at a flow rate of 30 l/min, and GppNHp-bound KRas.sup.G12D was analyzed at a concentration of 100 nM to 6.25 nM. After binding and dissociation analysis, regeneration of the CM5 chip was performed by flowing a buffer (20 mM NaOH, 1M NaCl, pH 10.0) at a flow rate of 30 l/min for 1.5 minutes. Each sensorgram, obtained by binding for 3 minutes and dissociation for 3 minutes, was normalized with reference to a blank cell and subtracted and the affinity was thus calculated.
[0225] The following Table 6 shows the result of affinity analysis of anti-RasGTP iMab having improved affinity for GppNHp-bound KRas.sup.G12D using SPR (BIACORE 2000).
TABLE-US-00011 TABLE 6 KRas.sup.G12DGppNHp k.sub.a (M.sup.1s.sup.1) k.sub.d (s.sup.1) K.sub.D (M) RT22-i33 9.92 0.56 10.sup.4 8.44 0.51 10.sup.4 8.51 0.82 10.sup.9 RT31-i33 8.81 0.42 10.sup.4 1.52 0.13 10.sup.4 1.72 0.19 10.sup.9 RT34-i33 6.2 0.45 10.sup.4 2.28 0.13 10.sup.4 3.67 0.39 10.sup.9 RT36-i33 3.96 0.24 10.sup.4 1.26 0.05 10.sup.4 3.19 0.26 10.sup.9
[0226] The results showed that all of RT31-i33, RT34-i33, and RT36-i33, which are anti-RasGTP iMabs with improved affinity based on RT22 VH, have higher affinity than the conventional RT22-i33.
Example 8. Logic of Development of Light-Chain Variable Region (VL) with Improved Cytoplasmic Penetration Ability Specific for Tumor Tissue Cells and Endosomal Escape Ability
[0227] The results showed that the cytoplasm-penetrating anti-RasGTP iMab of the prior patents (Korean Patent Nos. 10-1602870 and 10-1602876) was constructed by substituting the heavy-chain variable region of the conventional cytoplasmic penetration antibody (cytotransmab), having cytoplasmic penetration ability, with the heavy-chain variable region (VH) having binding ability highly specific to RasGTP. The cytoplasmic penetration antibody (cytotransmab) is an antibody capable of penetrating into the cytoplasm based on the light-chain variable region (VL), and cell penetration is initiated through binding with heparan sulfate proteoglycan (HSPG) on the cell surface. However, HSPG is a cell-surface protein that is highly expressed in normal cells as well, and thus HSPG-binding ability thereof needs to be suppressed in order to modify the protein to be capable of penetrating specifically into the cytoplasm of tumor tissue cells.
[0228] Therefore, the present inventors found that CDR1 and CDR2, in hT4 VL of the light-chain variable region (VL) of the cytoplasmic penetration antibody (cytotransmab), which are expected to contribute to HSPG binding, were substituted with CDR sequences that have amino acid sequences having the same number of amino acids in the human germline sequence and not including a cationic patch sequence of CDR1, which is responsible for endocytosis. At this time, amino acids known to be important for the stability of the conventional light-chain variable region were preserved.
[0229] The light-chain variable region developed based on this logic is hT4-03 (Korean Patent Application No. 10-2016-0065365).
[0230] In order to further improve the endosome escape ability of this light-chain variable region, CDR3 in where WYW (Trp-Tyr-Trp) is located was introduced into residues 92-94 of the light-chain variable region (VL) (Koran Patent Application No. 10-2016-0065365), and residue 87 of the light-chain variable region framework region corresponding to the interface between the light-chain variable region (VL) and the heavy-chain variable region (VH) was substituted with phenylalanine (Phe) in order to overcome the increased exposure of hydrophobic residues to solvents and thus decreased production yield due to the introduction of WYW (Korean Patent Application No. 10-2016-0065365).
[0231] This was designated as hT4-33 light-chain variable region (VL).
[0232] The following Table 7 shows the sequence and name of the light-chain variable region (hT4-33 VL) having improved cytoplasmic penetration specific for tumor tissue cells and endosome escape ability constructed using an overlapping PCR technique.
TABLE-US-00012 TABLE7 SEQ VL ID name Sequence NO. hT4-3VL 1020abcdef304050 33 DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW 60708090100 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR hT4-03VL 1020abcdef304050 34 DLVMTQSPSSLSASVGDRVTITCKSSQSLLDSDDGNTYLAWYQQKPGKAPKLLIYW 60708090100 LSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYHMYTFGQGTKVEIKR hT4-33VL 1020acbdef304050 35 DLVMTQSPSSLSASVGDRVTITCKSSQSLLDSDDGNTYLAWYQQKPGKAPKLLIYW 60708090100 LSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR
Example 9. Selection of Protopeptides for Targeting Tumor-Tissue-Specific Cell Surface Proteins and Inducing Endocytosis
[0233] RT11, which is an anti-RasGTP iMab using hT4 VL, enters cells and binds to RasGTP in the cells to exhibit tumor growth inhibitory ability, but has the disadvantage of low tumor-tissue specificity due to the HSPG-binding ability thereof. In order to overcome this disadvantage, the light chain having reduced HSPG-binding capacity was obtained through Example 8 above and experiments were conducted using an iMab form introduced with a light-chain variable region fused at the N-terminal thereof with RGD10 protopeptides for targeting integrin receptors as in Examples above.
[0234] Additionally, various protopeptides to impart tumor-tissue specificity were selected in addition to the integrin-targeting protopeptides.
[0235] The EpCAM (epithelial cell adhesion molecule) receptor is a receptor that is mainly overexpressed in colorectal cancer cells, and is a target located on a suitable cell membrane for imparting tumor specificity to anti-RasGTP iMab. Accordingly, a light-chain variable region including an Ep133 protopeptide (EHLHCLGSLCWP) capable of targeting the EpCAM receptor (U.S. patent application Ser. No. 14/428,017) fused with the N-terminus thereof was constructed.
[0236] The following Table 8 shows the sequences and names of protopeptides for targeting tumor-tissue-specific cell surface proteins and inducing endocytosis.
TABLE-US-00013 TABLE8 Peptide Target name antigen Sequence RGD10 Integrinv3/ DGARYCRGDCFDG v5 EP133 EpCAM EHLHCLGSLCWP
Example 10. Construction and Purification of Anti-RasGTP iMab Introduced with Light-Chain Variable Region (VL) Fused with Target Protopeptide for Tumor-Tissue-Specific Cytoplasmic Penetration
[0237]
[0238] Specifically, for expression in animal cells of iMab fused with protopeptides, protopeptides were genetically fused to the N-terminus of hT4-33, the light-chain variable region (VL) using two G4S linkers. Subsequently, a DNA encoding a light chain including the protopeptide-fused hT4-light-chain variable region and a light-chain constant region (CL) was cloned with NotI/BsiWI into a pcDNA3.4 vector fused with a DNA encoding a secretory signal peptide at the 5 end thereof.
[0239] The heavy-chain variable region (RT22 VH) having specific binding ability to GTP-bound Ras and the cytoplasmic penetration humanized light chain expression vector were subjected to transient co-transfection into the HEK293F-protein-expressing cells to express individual clones in the same manner as in Example 4, and were purified in the same manner as in Example 4.
[0240] The following Table 9 shows the production yield of cytotransmab and anti-RasGTP iMab including a light-chain variable region (VL) fused with a tumor-tissue-specific protopeptide using a genetic-engineering technique.
TABLE-US-00014 TABLE 9 Production yield Heavy Light chain (mg/1 L of IgG1.sub.
[0241] It was found that the production yield of the anti-RasGTP iMab fused with integrin- or EpCAM-targeting protopeptide was significantly low, that is, 1 mg/l L.
Example 11. Determination of Specific Binding with Intracellular RasGTP of RT22-i33 MG, RT22-Ep33 MG
[0242]
[0243] Specifically, SW480 cells were prepared in the same manner as in Example 4, treated with RT22-33 1 M, RT22-i33 MG 0.5 M, RT22-ep33 MG 1 M, CT-33 1 M, CT-i33 MG 0.5 M, and CT-ep33 MG 1 M, and then cultured at 37 C. for 12 hours. Then, the cells were stained under the same conditions as in Example 4 and observed with a confocal microscope. RT22-i33 MG and RT22-ep33 MG, except for RT22-33, which does not undergo endocytosis, were found to have fluorescence overlapping that of intracellular Ras. In addition, CT-33, CT-i33 MG and CT-ep33 MG, not targeting intracellular Ras, were found to have no fluorescence overlapping that of intracellular Ras.
Example 12. Design of CDR1 and CDR2 Mutants of Light-Chain Variable Region (VL) to Improve Yield of Tumor-Tissue-Specific Anti-RasGTP iMab
[0244] Like the results of analysis of Example 10, it was found that the production yield did not decrease upon fusion of the protopeptide with the conventional light-chain variable region (hT4-3 VL) having HSPG-binding ability, but the production yield decreased upon fusion with the light-chain variable region (hT4-33 VL) having reduced HSPG-binding ability. In addition, CDR1 and CDR2 of the light-chain variable region (VL), which greatly affects HSPG-binding ability, were modified to improve production yield upon fusion with protopeptides recognizing specifically for tumor tissues (RGD10, Ep133).
[0245] Specifically, in order to maintain low binding capacity to HSPG, a mutation was formed in common by substituting the phenylalanine, residue 27c, which is considered to affect the formation of the loop structure of CDR1 of the light-chain variable region of hT4 VL, with leucine, preserved in the light-chain variable region having reduced HSPG-binding capacity, and mutations were formed in residues 27f to 30, which are considered to be located at the tip of the CDR1 loop structure due to the canonical structure and thus to be exposed to the side chain.
[0246] Specifically, in the case of hT4-34 VL, a point mutation was formed only in the residue 27c, which is considered to affect the formation of the loop structure of CDR1, among CDR1 and CDR2 of the light-chain variable region, using hT4 VL as a template.
[0247] Specifically, hT4-35 VL, hT4-36 VL and hT4-37 VL were mutated according to each strategy using hT4-33 VL having greatly reduced HSPG-binding ability as a template. The hT4-35 VL was designed by respectively mutating the aspartate at residues 27d and 27f of CDR1 to asparagine and arginine, preserved in hT4 VL. The hT4-36 VL was designed by respectively mutating aspartate and glycine at residues 27d and 29 of CDR1 to asparagine and arginine, and hT4-37 VL was designed by respectively mutating aspartate and asparagine at residues 27d and 30 of CDR1 to asparagine and lysine. The residue numbers followed the Kabat number.
[0248] Specifically, hT4-38 VL was designed by mutating arginine at residue 29 to glycine present in the sequence of hT4-33 VL and mutating asparagine at residue 31 to threonine present in the sequence of hT4-33 VL, while preserving arginine at residue 27f and lysine at residue 30, which had increased yield when maintaining the sequence of hT4 VL in order to minimize HSPG-binding capacity. Like hT4-38 VL, hT4-39 VL was designed by mutating threonine at residue 28 to aspartate and mutating arginine at residue 29 to glycine while preserving arginine at residue 27f and lysine at residue 30 in order to minimize HSPG-binding capacity using the sequence preserved in hT4-33 VL.
[0249] The following Table 10 shows the sequence of a light-chain variable region (VL) having a mutation for increasing production yield upon tumor-tissue-cell-specific cytoplasmic penetration and protopeptide fusion.
TABLE-US-00015 TABLE10 SEQ VL ID name Sequence NO. hT4-34VL 11020abcdef304050 36 DLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGKAPKLLIYW 60708090100 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR hT4-35VL 11020abcdef304050 37 DLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRDGNTYLAWYQQKPGKAPKLLIYW 60708090100 LSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR hT4-36VL 11020abcdef304050 38 DLVMTQSPSSLSASVGDRVTITCKSSQSLLNSDDRNTYLAWYQQKPGKAPKLLIYW 60708090100 LSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR hT4-37VL 11020abcdef304050 39 DLVMTQSPSSLSASVGDRVTITCKSSQSLLNSDDGKTYLAWYQQKPGKAPKLLIYW 60708090100 LSYRASGVPSRFSGSGSGTDFTLTISSLDPEDFATYFCQQYWYWMYTFGQGTKVEIKR hT4-38VL 11020abcdef304050 40 DLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRTGKTYLAWYQQKPGKAPKLLIYW 60708090100 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR hT4-39VL 11020abcdef304050 41 DLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRDGKNYLAWYQQKPGKAPKLLIYW 60708090100 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR
Example 13. Determination of HSPG-Binding Ability of Anti-RasGTP iMab Introduced with Mutants of CDR1 and CDR2 of Light-Chain Variable Region (VL) Having Improved Production Yield and Tumor-Tissue-Cell-Specific Cytoplasmic Penetration
[0250] The decrease degree in HSPG-binding ability of anti-RasGTP iMab introduced with mutants of CDR1 and CDR2 of a light-chain variable region (VL) for improving production yield and inhibiting HSPG-binding ability constructed in Example 12 above, compared to cytotransmab (TMab4) using a conventional hT4 light-chain, was observed using a confocal microscope and cell-based ELISA.
[0251]
[0252] Specifically, the HeLa cell line expressing HSPG was seeded at 510.sup.4 cells/well in a 24-well plate in 0.5 ml of a medium containing 10% FBS and cultured at 5% CO.sub.2 and 37 degrees for 12 hours. When the cells were stabilized, PBS, TMab4, RT22-33, RT22-34, RT22-35, RT22-36, RT22-37, RT22-38, RT22-39, CT-33, CT-38 and CT-39 were cultured at 1 M at 37 degrees for 6 hours. After washing with PBS and a weakly acidic solution in the same manner as in Example 4, the cells were immobilized, perforated and then blocked. Each antibody was stained with an antibody that specifically recognizes human Fc conjugated with Alexa488 (green fluorescence). Then, the nuclei were stained (blue fluorescence) using Hoechst33342 and observed under a confocal microscope.
[0253] The results showed that anti-RasGTP iMabs located in the cytoplasm through HSPG exhibited decreasing fluorescence intensity in the order of TMab4 (100%), RT22-34 (40%), RT22-36 (25%), RT22-38 (26%), RT22-39 (19%), RT22-37 (15.5%), RT22-(12.4%), and RT22-33 (6.8%).
[0254]
[0255] Specifically, the HeLa (HSPG.sup.+) cell line was cultured in a 96-well plate such that the cells completely filled the bottom of the well, and was then washed three times with a washing buffer (HBSS buffer, 50 mM HEPES). Then, TMab4, RT22-33, RT22-34, RT22-35, RT22-36, RT22-37, RT22-38, RT22-39, CT-33, CT-38 and CT-39 were diluted to concentrations of 100, 50, 25, 12.5, 6.25, 3.125 ng/ml in a blocking buffer (HBSS buffer, 50 mM HEPES, 1% BSA) and cultured for 2 hours at 4 C. After washing three times with a washing buffer, the cells were linked to an HRP-conjugated anti-human mAb as a labeling antibody. The cells were reacted with TMB ELISA solution, and the absorbance at 450 nm was quantified.
[0256] With the same behavior as in
[0257]
[0258] Specifically, 110.sup.5 HeLa (HSPG.sup.+) cells were prepared for each sample. The cells were cultured in PBSF (PBS buffer, 2% BSA) supplemented with 500 nM of TMab4, RT22-33, RT22-34, RT22-35, RT22-36, RT22-37, RT22-38, and RT22-39 for 1 hour at 4 C. Then, each antibody was reacted with an antibody specifically recognizing human Fc conjugated to Alexa488 (green fluorescence) for 30 minutes at 4 C. The resulting product was washed with PBS and analyzed using flow cytometry. The result showed that fluorescence intensity binding to the cells was measured in the order of TMab4, RT22-34, RT22-38, RT22-36, RT22-39, RT22-37, RT22-35 and RT22-33, in the same behavior as in the confocal microscopy results.
[0259] Anti-RasGTP iMab using the hT4-34 light chain having a remaining HSPG-binding capacity of 40%, which was found based on the result, was not used in subsequent biochemical identification experiments.
Example 14. Expression and Purification of Tumor-Tissue-Specific Anti-RasGTP iMab Introduced with Mutants of CDR1 and CDR2 of Light-Chain Variable Region (VL) Having Improved Production Yield and Tumor-Tissue-Cell-Specific Cytoplasmic Penetration
[0260] RGD or Ep133 protopeptides were fused to the light-chain variable regions (VL) introduced with mutants for providing tumor-tissue-cell-specific cytoplasmic penetration and improving production yield to conduct cloning upon fusion with protopeptides using a Mg linker using a genetic-engineering technique in the same manner as in Example 10.
[0261] The following Table 11 shows the sequence of the light-chain variable regions (VL) having improved production yield and tumor-tissue-cell-specific cytoplasmic penetration ability and being fused with an integrin-targeting protopeptide.
TABLE-US-00016 TABLE11 SEQ VL ID name Sequence NO. hT4-i34MGVL 1102030405060 44 DGARYCRGDCFDGMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAW 708090100110120 YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWM 130 YTFGQGTKVEIKR hT4-i35MGVL 1102030405060 45 DGARYCRGDCFDGMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRDGNTYLAW 708090100110120 YQQKPGKAPKLLIYWLSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWM 130 YTFGQGTKVEIKR hT4-i36MGVL 1102030405060 46 DGARYCRGDCFDGMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSDDRNTYLAW 708090100110120 YQQKPGKAPKLLIYWLSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWM 130 YTFGQGTKVEIKR hT4-i37MGVL 1102030405060 47 DGARYCRGDCFDGMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSDDGKTYLAW 708090100110120 YQQKPGKAPKLLIYWLSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWM 130 YTFGQGTKVEIKR hT4-i38MGVL 1102030405060 48 DGARYCRGDCFDGMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRTGKTYLAW 708090100110120 YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWM 130 YTFGQGTKVEIKR hT4-i39MGVL 1102030405060 49 DGARYCRGDCFDGMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRDGKNYLAW 708090100110120 YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWM 130 YTFGQGTKVEIKR
[0262] The following Table 12 shows the sequences of the light-chain variable regions (VL) having a mutation for improving production yield and inhibiting HSPG-binding ability, and fused with EpCAM-targeting protopeptide.
TABLE-US-00017 TABLE12 SEQ VL ID name Sequence NO. hT4-ep33MGVL 1102030405060 50 EHLHCLGSLCWPMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLDSDDGNTYLAWY 708090100110120 QQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMY 130 TFGQGTKVEIKR hT4-ep34MGVL 1102030405060 51 EHLHCLGSLCWPMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWY 708090100110120 QQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMY 130 TFGQGTKVEIKR hT4-ep35MGVL 1102030405060 52 EHLHCLGSLCWPMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRDGNTYLAWY 708090100110120 QQKPGKAPKLLIYWLSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMY 130 TFGQGTKVEIKR hT4-ep36MGVL 1102030405060 53 EHLHCLGSLCWPMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSDDRNTYLAWY 708090100110120 QQKPGKAPKLLIYWLSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMY 130 TFGQGTKVEIKR hT4-ep37MGVL 1102030405060 54 EHLHCLGSLCWPMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSDDGKTYLAWY 708090100110120 QQKPGKAPKLLIYWLSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMY 130 TFGQGTKVEIKR hT4-ep38MGVL 1102030405060 55 EHLHCLGSLCWPMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRTGKTYLAWY 708090100110120 QQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMY 130 TFGQGTKVEIKR hT4-ep39MGVL 1102030405060 56 EHLHCLGSLCWPMGSSSNDLVMTQSPSSLSASVGDRVTITCKSSQSLLNSRDGKNYLAWY 708090100110120 QQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMY 130 TFGQGTKVEIKR
[0263] A RT22 heavy-chain animal expression vector and a light-chain expression vector including a light-chain variable region having CDR1 and CDR2 mutations fused with integrin-targeting protopeptide for providing tumor-tissue specificity and improving production yield, were subjected to transient co-transfection into HEK293F-protein-expressing cells in the same manner as in Example 4, and then the process of purifying the antibody was conducted in the same manner as in Example 4.
[0264] In addition, the quantitative affinity for GppNHp-bound KRas.sup.G12D of the anti-RasGTP iMabs introduced with CDR1 and CDR2 mutations in the light-chain variable region (VL) for improving production yield and inhibiting HSPG-binding ability was analyzed using a BIACORE2000 instrument.
[0265] Specifically, RT22-i35 MG, RT22-i37 MG, RT22-i38 MG, RT22-i39 MG, RT22-ep37 MG, RT22-ep38 MG and RT22-ep39 MG were diluted to a concentration of 20 l/ml in 10 mM NaAc buffer (pH 4.0) and immobilized at approximately 1800 response units (RU) on a CM5 sensor chip (GE Healthcare). Subsequently, Tris buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM MgCl.sub.2, 0.01% Tween 20) was analyzed at a flow rate of 30 l/min, and complete GppNHp-bound GST-KRas.sup.G12D was analyzed at a concentration of 50 nM to 3.125 nM. After binding and dissociation analysis, regeneration of the CM5 chip was performed by flowing a buffer (20 mM NaOH, 1M NaCl, pH 10.0) at a flow rate of 30 l/min for 1 minute. Each sensorgram, obtained by binding for 3 minutes and dissociation for 3 minutes, was normalized with reference to a blank cell and subtracted, and the affinity was thus calculated.
[0266] The following Table 13 shows the production yield of the anti-RasGTP iMabs obtained by expressing an RT22 heavy chain in combination with hT4-i33 and hT4-ep33, and light chains (hT4-i34 MG to hT4-i39 MG and hT4-ep34 MG to hT4-ep39) introduced with mutations of CDR1 and CDR2 to improve production yield and inhibit HSPG-binding capacity, and of the cytotransmabs obtained by expressing a TMab4 heavy chain in combination with the light chains, and the result of analysis of the affinity of tumor-tissue-specific anti-RasGTP iMab based on SPR using a BIACORE2000 instrument.
TABLE-US-00018 TABLE 13 Heavy Light chain Production yield KRas.sup.G120. IgG1.sub.
[0267] It was found that the affinity for GppNHp-bound KRas.sup.G12D of tumor-tissue-specific anti-RasGTP iMabs introduced with mutants of light-chain variable region (VL) CDR1 and CDR2 to improve production yield was not changed.
[0268] The anti-RasGTP iMab using the hT4-36 light chain, which did not increase the production yield, found through the above results, was not used in subsequent biochemical identification experiments.
Example 15. Analysis of Binding Ability to GppNHp-Bound KRas.SUP.G12D .of Tumor-Tissue-Specific Anti-RasGTP iMab Introduced with Mutants of CDR1 and CDR2 of Light-Chain Variable Region (VL) Having Improved Production Yield and Tumor-Tissue-Cell-Specific Cytoplasmic Penetration
[0269]
[0270] Specifically, in order to compare the antigen-binding ability of the CDR1 and CDR2 mutant light chains, the heavy-chain variable region binding to GppNHp is fixed at RT22 VH, and analysis was conducted on RT11, RT22-i33 MG, RT22-i34 MG, RT22-i35 MG, RT22-i36 MG and RT22-i37 MG.
[0271] Specifically, ELISA was conducted in the same manner as Example 4, and tumor-tissue-specific anti-RasGTP iMabs introduced with mutants of CDR1 and CDR2 were fixed on a 96-well EIA/RIA plate, after which the GppNHp-bound KRas protein was bound at various concentrations of 100 nM, 10 nM and 1 nM and the GDP-bound KRas protein was bound at a concentration of 100 nM and was then bound to an HRP-conjugated anti-his antibody (HRP-conjugated anti-his mAb) as a labeling antibody. The result was reacted with TMB ELISA solution, and the absorbance at 450 nm was quantified.
[0272] The result of analysis of the antigen-binding ability by ELISA showed that, compared to the conventional RT22-i33, hT4-i34 VL, maintaining the cation patch of CDR1, had improved binding capacity to the GppNHp-bound KRas.sup.G12D antigen due to the light chain, while the heavy chain was the same as in the conventional case.
[0273]
[0274] Specifically, ELISA was conducted in the same manner as Example 4, and cytotransmabs and tumor-tissue-specific anti-RasGTP iMabs introduced with mutants of CDR1 and CDR2 were fixed on a 96-well EIA/RIA plate, after which the GppNHp-bound KRas protein was bound at various concentrations of 100 nM, 10 nM and 1 nM and the GDP-bound KRas protein was bound at a concentration of 100 nM and then was bound to an HRP-conjugated anti-his antibody (HRP-conjugated anti-his mAb) as a labeling antibody. The result was reacted with TMB ELISA solution, and the absorbance at 450 nm was quantified.
[0275] The result of analysis of the antigen-binding ability by ELISA showed that, compared to the conventional RT11-i, hT4-38 and hT4-39 light-chain variable regions improved binding capacity to the GppNHp-bound KRas.sup.G12D antigen.
[0276]
[0277] Specifically, SW480 cells were prepared in the same manner as in Example 4, and were treated with 1 M CT-i33 MG (=TMab4-i33 MG), CT-i38 MG, CT-i39 MG, RT22-i33 MG, RT22-i34 MG, RT22-i35 MG, RT22-i36 MG, RT22-i37 MG, RT22-i38 MG, RT22-i39 MG, CT-ep33 MG, CT-ep38 MG, CT-ep39 MG, RT22-ep38 MG and RT22-ep39 MG, and then cultured for 12 hours at 37 C. under 5% CO.sub.2. Then, the cells were stained under the same conditions as in Example 4 and observed with a confocal microscope. RT22-i33 MG, RT22-i34 MG, RT22-i35 MG, RT22-i36 MG, RT22-i37 MG, RT22-i38 MG, RT22-i39 MG, RT22-ep38 MG, and RT22-ep39 MG, excluding CT-i33 MG, CT-i38 MG, CT-i39 MG, CT-ep33 MG, CT-ep38 MG and CT-ep39 MG, were found to have fluorescence overlapping that of the intercellular Ras.
Example 16. Construction of Light-Chain Variable-Region (VL) Mutants with Modified Antibody Framework to Improve Production Yield
[0278] The light-chain variable region (VL) CDR1 mutants constructed in Example 12 increased yield when fused with the protopeptide for providing tumor-tissue specificity compared to the conventional hT4-33, but antibodies (RT22-ep38 MG, RT22-ep39 MG) fused with the Ep133 protopeptide, showing the best production yield, exhibited production yields that were approximately 2 times lower than antibodies fused with RGD10 protopeptides (RT22-i38 MG, RT22-i39 MG). Therefore, in order to further increase the stability and yield of the antibody fused with the Ep133 protopeptide, an additional mutant of the light-chain variable region (VL) antibody framework was constructed.
[0279] Specifically, most of sequences 2 and 3 of the antibody skeleton in the light chain used in the human complete IgG-type antibody include isoleucine and glutamine, present in human germline sequences, but the sequences 2 and 3 are preserved as leucine and valine in the light chain used in Anti-RasGTP iMab. Thus, a light-chain variable region (VL) antibody framework mutant was constructed by mutating these sequences 2 and 3 to isoleucine and glutamine, which are mainly present in human complete-IgG type antibodies, in order to increase the stability and yield.
[0280] The following Table 14 shows sequence information of a light-chain variable region (VL) having a modified antibody skeleton to improve the production yield based on hT4-38 and hT4-39 and a light-chain variable region (VL) fused with EpCAM-targeting protopeptide and having a modified antibody skeleton to improve the production yield.
TABLE-US-00019 TABLE14 SEQ VL ID name Sequence NO. hT4-58VL 11020abcdef304050 42 DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRTGKTYLAWYQQKPGKAPKLLIYW 60708090100 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR hT4-59VL 11020abcdef304050 43 DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRDGKNYLAWYQQKPGKAPKLLIYW 60708090100 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR hT4-ep58MGVL 1102030405060 57 EHLNCLGSLCWPMGSSSNDIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRTGKTYLAWY 708090100110120 QQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMY 130 TFGQGTKVEIKR hT4-ep59MGVL 1102030405060 58 EHLHCLGSLCWPMGSSSNDIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRDGKNYLAWY 708090100110120 QQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMY 130 TFGQGTKVEIKR
Example 17. Expression and Purification of Tissue-Cell-Specific Anti-RasGTP iMab Introduced with Light-Chain Variable-Region (VL) Mutants Having Modified Antibody Framework to Improve Production Yield
[0281] A RT22 heavy-chain animal expression vector and a light-chain expression vector including a light-chain variable region fused with EpCAM-targeting protopeptide for providing tumor-tissue specificity, and having a modified antibody framework to improve the production yield, were subjected to transient co-transfection into HEK293F-protein-expressing cells in the same manner as in Example 10, and then the process of purifying the antibody was conducted in the same manner as in Example 4.
[0282] Then, the quantitative affinity for GppNHp-bound KRas.sup.G12D of the tissue-cell-specific anti-RasGTP iMab introduced with the light-chain variable region (VL) having a modified antibody framework to improve production yield was analyzed using a BIACORE2000 instrument.
[0283] Specifically, RT22-ep58 MG and RT22-ep59 MG were diluted to a concentration of 20 l/ml in 10 mM NaAc buffer (pH 4.0) and immobilized at approximately 1800 response units (RU) on a CM5 sensor chip (GE Healthcare). Subsequently, Tris buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM MgCl.sub.2, 0.01% Tween 20) was analyzed at a flow rate of 30 l/min, and complete GppNHp-bound GST-KRas.sup.G12D was analyzed at a concentration of 50 nM to 3.125 nM. After binding and dissociation analysis, regeneration of the CM5 chip was performed by flowing a buffer (20 mM NaOH, 1M NaCl, pH 10.0) at a flow rate of 30 l/min for 1 minute. Each sensorgram, obtained by binding for 3 minutes and dissociation for 3 minutes, was normalized with reference to a blank cell and subtracted, and the affinity was thus calculated.
[0284] The following Table 15 shows the production yield of the anti-RasGTP iMab obtained by expressing an RT22 heavy chain in combination with light chains (hT4-ep58 MG and hT4-ep59 MG) fused with EpCAM-targeting protopeptide for providing tumor-tissue specificity and introduced with an antibody framework mutation to improve the production yield, and of the cytotransmabs obtained by expressing a TMab4 heavy chain in combination with the light chains, and the result of analysis of the affinity of tumor-tissue-specific anti-RasGTP iMab based on SPR using a BIACORE2000 instrument.
TABLE-US-00020 TABLE 15 Production yield Heavy Light chain (mg/1 L of KRas.sup.G120. IgG1.sub.
[0285] It was found that the affinity for GppNHp-bound KRas.sup.G12D of the anti-RasGTP iMab was not changed compared to RT22-ep38 MG and RT22-ep59 MG, tumor-tissue-specific anti-RasGTP iMabs introduced with mutants of light-chain variable region (VL) CDR1 and CDR2 to improve production yield.
[0286]
[0287] Specifically, ELISA was conducted in the same manner as in Example 4, and tumor-tissue-specific anti-RasGTP iMabs having light chains introduced with mutants of CDR1 and CDR2 were fixed on a 96-well EIA/RIA plate, after which the GppNHp-bound KRas protein was bound at various concentrations of 100 nM, 10 nM and 1 nM, and the GDP-bound KRas protein was bound at a concentration of 100 nM and was then bound to an HRP-conjugated anti-his antibody (HRP-conjugated anti-his mAb) as a labeling antibody. The result was reacted with TMB ELISA solution, and the absorbance at 450 nm was quantified.
[0288] The result of analysis on the antigen-binding ability by ELISA showed that, compared to the conventional RT11-i, hT4-58 and hT4-59 light-chain variable regions improved binding capacity to the GppNHp-bound KRas.sup.G12D antigen.
[0289]
[0290] Specifically, an SW480 cell line was diluted in 0.5 ml at 210.sup.4 cells/well in a 24-well plate, cultured for 12 hours at 37 C. under 5% CO.sub.2, and then treated with 1 M of CT-ep58 MG and anti-RasGTP iMabs, RT22-ep58 MG and RT22-ep59 MG with improved affinity and then cultured at 37 C. for 12 hours. Then, the medium was removed, the residue was washed with PBS, and proteins attached to the cell surface were removed with a weakly acidic solution (200 mM glycine, 150 mM NaCl pH 2.5). After washing with PBS, 4% paraformaldehyde was added, and cells were immobilized at 25 degrees for 10 minutes. After washing with PBS, the cells were cultured in a buffer containing PBS supplemented with 0.1% saponin, 0.1% sodium azide and 1% BSA at 25 C. for 10 minutes, and a hole was formed in the cell membrane. Then, the cells were washed with PBS again and reacted with a buffer containing 2% BSA in addition to PBS for 1 hour at 25 C. in order to inhibit non-specific binding. Each antibody was stained with an antibody that specifically recognizes human Fc linked with Alexa-488 (green fluorescence). The nuclei were stained (blue fluorescence) using Hoechst33342, and KRas-labeled antibodies were stained (red fluorescence) and then observed with a confocal microscope. All of the anti-RasGTP iMabs excluding CT-ep59 were found to have fluorescence overlapping that of the intracellular Ras.
Example 18. Determination of HSPG-Binding Ability of Anti-RasGTP iMab Introduced with Light-Chain Variable-Region (VL) Mutants Having Modified Antibody Framework to Improve Production Yield
[0291] The decrease level in HSPG-binding ability of cytotransmabs and the anti-RasGTP iMabs introduced with light-chain variable-region (VL) mutants having a modified antibody framework to improve production yield constructed in Example 17 above, compared to cytotransmab (TMab4) using the conventional hT4 light-chain, was observed using a confocal microscope and cell-based ELISA.
[0292]
[0293] Specifically, the HeLa cell line expressing HSPG was seeded at 510.sup.4 cells/well in a 24-well plate in 0.5 ml of a medium containing 10% FBS and cultured at 5% CO.sub.2 and 37 C. for 12 hours. When the cells were stabilized, PBS, TMab4, Avastin, RT22-58, RT22-59, CT-58 and CT-59 were cultured at 1 M at 37 C. for 6 hours. After washing with PBS and a weakly acidic solution in the same manner as in Example 4, the cells were immobilized, perforated and then blocked. Each antibody was stained with an antibody that specifically recognizes human Fc conjugated with Alexa488 (green fluorescence). Then, the nuclei were stained (blue fluorescence) using Hoechst33342 and observed under a confocal microscope.
[0294] The result showed that anti-RasGTP iMabs located in the cytoplasm through HSPG exhibited fluorescence intensity in the order of TMab4 (100%), RT22-58 (30%), CT-58 (27%), CT-59 and RT22-59 (20%).
[0295]
[0296] Specifically, the HeLa (HSPG.sup.+) cell line was cultured in a 96-well plate such that the cells completely filled the bottom of the well and then washed three times with a washing buffer (HBSS buffer, 50 mM HEPES). Then, PBS, TMab4, Avastin, RT22-58, RT22-59, CT-58 and CT-59 were diluted to concentrations of 100, 50, 25, 12.5, 6.25 and 3.125 ng/ml in a blocking buffer (HBSS buffer, 50 mM HEPES, 1% BSA) and the cells were cultured for 2 hours at 4 C. After washing three times with washing buffer, the cells were bound to an HRP-conjugated anti-human mAb as a labeling antibody. The cells were reacted with TMB ELISA solution, and the absorbance at 450 nm was quantified.
[0297] With the same behavior as in
[0298]
[0299] Specifically, 110.sup.5 HeLa (HSPG.sup.+) cells were prepared for each sample. The cells were cultured in PBSF (PBS buffer, 2% BSA) supplemented with 500 nM of TMab4, Avastin, RT22-58, RT22-59, CT-58 and CT-59 for 1 hour at 4 C. Then, each antibody was reacted with an antibody specifically recognizing human Fc conjugated to Alexa488 (green fluorescence) for 30 minutes at 4 C. The result was washed with PBS and analyzed with flow cytometry. The result showed that fluorescence intensity binding to the cells was measured in the order of TMab4, TMab4, RT22-58, CT-58, CT-59 and RT22-59, in the same behavior as in the confocal microscopy results.
[0300] The result showed that the light-chain variable region (VL) mutants having a modified antibody framework to improve production yield also have no HSPG-binding ability.
Example 19. Expression and Purification of Tumor-Tissue-Specific Anti-RasGTP iMab Introduced with RasGTP-Specific Heavy-Chain Variable Region (VH) Having Improved Affinity Based on RT22, and Light-Chain Variable Region (VL) Having Cytoplasmic Penetration Specific for Tumor Tissue Cells and Improved Production Yield
[0301] RT31, RT36 and RT37 heavy-chain animal expression vectors and light-chain expression vectors including a light-chain variable region fused with integrin or EpCAM-targeting protopeptides for providing tumor-tissue specificity and having inhibited HSPG-binding ability and improved production yield were subjected to transient co-transfection into HEK293F-protein-expressing cells in the same manner as in Example 4, and then the process of purifying the antibody was conducted in the same manner as in Example 4.
[0302] In addition, the quantitative affinity for GppNHp-bound KRas.sup.G12D of the anti-RasGTP iMabs introduced with the RT31, RT36 and RT37 heavy-chain animal expression vectors and the light-chain variable regions fused with integrin or EpCAM-targeting protopeptide for providing tumor-tissue specificity and having inhibited HSPG-binding ability and improved production yield was analyzed using a BIACORE2000 instrument.
[0303] Specifically, RT31-i37 MG, RT31-ep37 MG, RT36-i37 MG, RT36-i38 MG, RT36-i39 MG, RT36-ep37 MG, RT36-ep38 MG and RT36-ep39 MG were diluted to a concentration of 20 l/ml in 10 mM NaAc buffer (pH 4.0) and immobilized at approximately 1800 response units (RU) on a CM5 sensor chip (GE Healthcare). Subsequently, Tris buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM MgCl.sub.2, 0.01% Tween 20) was analyzed at a flow rate of 30 l/min, and complete GppNHp-bound GST-KRas.sup.G12D was analyzed at a concentration of 50 nM to 3.125 nM. After binding and dissociation analysis, regeneration of the CM5 chip was performed by flowing a buffer (20 mM NaOH, 1M NaCl, pH 10.0) at a flow rate of 30 l/min for 1 minute. Each sensorgram, obtained by binding for 3 minutes and dissociation for 3 minutes, was normalized with reference to a blank cell and subtracted, and the affinity was thus calculated.
[0304] The following Table 16 shows the production yield of the tumor-tissue-specific anti-RasGTP iMabs introduced with the RT31, RT36 and RT37 heavy-chain animal expression vectors and the light-chain variable regions fused with integrin or EpCAM-targeting protopeptide for providing tumor-tissue specificity, and having inhibited HSPG-binding ability and improved production yield, and the result of analysis of the affinity of tumor-tissue-specific anti-RasGTP iMabs based on SPR using a BIACORE2000 instrument.
TABLE-US-00021 TABLE 16 Production yield IgG1, Heavy (mg /1 L of KRas.sup.G12DGppNHp format chain Light chain transfected cells).sup.a K.sub.D (M).sup.b RT31-i37 MG RT31 hT4-i37 MG 1.4 0.20 3.1 0.12 10.sup.9 RT31-ep37 MG hT4-ep37 MG 4.7 2.12 ND RT36-i37 MG RT36 hT4-i37 MG 3.3 1.72 2.9 0.05 10.sup.9 RT36-i38 MG hT4-i38 MG 48.1 2.12 5.3 0.30 10.sup.9 RT36-i39 MG hT4-i39 MG 19.1 1.35 2.2 0.11 10.sup.8 RT36-ep37 MG hT4-ep37 MG 3.1 1.32 ND RT36-ep58 MG hT4-ep58 MG 24.4 1.99 1.2 0.11 10.sup.8 RT36-ep59 MG hT4-ep59 MG 35.8 19.86 1.13 0.07 10.sup.8 RT37-i37 MG RT37 hT4-i37 MG <1 ND .sup.aThe 2 plasmids that encode the HC and LC of each IgG antibody were co-transfected with the equivalent molar ratio into HEK293F cells in 1 L of culture media. After 6 d of culture, antibodies were purified from the cell culture supernatant using a protein-A affinity column. .sup.bND, not determined
[0305] The antibodies (RT31-i37 MG, RT31-ep37 MG, RT37-i37 MG) introduced with RT31 and RT37 heavy chains with improved affinity based on RT22 and light-chain variable region (VL) mutants for improving production yield and inhibiting HSPG-binding capacity have a problem of low production yield, and the antibodies (RT36-i38 MG, RT36-i39 MG, RT36-ep58 MG and RT36-ep59 MG) introduced with RT36 and light-chain variable-region (VL) mutants having improved production yield and tumor-tissue-cell-specific cytoplasmic penetration have a problem of low affinity for RasGTP, compared to RT22-i38 MG, RT22-i39 MG, RT22-ep58 MG and RT22-ep59 MG. Therefore, experiments were conducted on RT22-i38 MG, RT22-i39 MG, RT22-ep58 MG and RT22-ep59 MG, having high production yield and excellent affinity for RasGTP.
Example 20. Determination of Binding Ability to Integrin or EpCAM on Cell Surface of Anti-RasGTP iMab Including Light-Chain Variable Region (VL) Fused with Integrin or EpCAM-Targeting Protopeptide and Having Cytoplasmic Penetration Specific for Tumor Tissue Cells
[0306]
[0307] Specifically, 110.sup.5 of each of SW480, LoVo, K562 and K562 3 cell lines were prepared for each sample. The cells were cultured in a dilution of PE-conjugated anti-integrin 3 or PE-conjugated anti-integrin 5 at 1:100 at 4 C. for 1 hour. The cells were washed with PBS and analyzed by flow cytometry. The result showed that the 3 antibody bound to the K562 3-expressing cell line and the 5 antibody bound to SW480 and LoVo cells.
[0308]
[0309] Specifically, 110.sup.5 of each of SW480, LoVo, K562 and K562 3 cells were prepared for each sample. The cells were cultured for 1 hour at 4 C. in PBSF (PBS buffer, 2% BSA) supplemented with 500 nM of RT11-i, RT22-i38 MG, RT22-i39 MG and CT-i39 MG. Then, each antibody was reacted with an antibody specifically recognizing human Fc conjugated to Alexa488 (green fluorescence) for 30 minutes at 4 degrees. The cells were washed with PBS and analyzed by flow cytometry. The result showed that the fluorescence intensities of RT11-i, RT22-i38 MG, RT22-i39 MG and CT-i39 MG antibodies bound to SW480, LoVo and K562 3 cells expressing 3 or 5 were measured.
[0310]
[0311] Specifically, 110.sup.5 of each of SW480, LoVo, K562 and K562 3 cells were prepared for each sample. The anti-EpCAM antibody was diluted at 1:200 and cultured for 1 hour at 4 C. Then, the primary antibody was reacted with an antibody specifically recognizing murine Fc conjugated to which Alexa488 (green fluorescence) for 30 minutes at 4 C. The cells were washed with PBS and analyzed by flow cytometry. As a result, the fluorescence intensities of the 3 antibody bound to K562 3 cells and the 5 antibody bound to SW480 and LoVo cells were measured.
[0312]
[0313] Specifically, 110.sup.5 of each SW480, LoVo and HeLa cells were prepared for each sample. The cells were cultured for 1 hour at 4 C. in PBSF (PBS buffer, 2% BSA) supplemented with 500 nM of RT22-ep58 MG, RT22-ep59 MG, and CT-ep59 MG. Then, each antibody was reacted with an antibody specifically recognizing human Fc conjugated to Alexa488 (green fluorescence) for 30 minutes at 4 C. Then, the cells were washed with PBS and analyzed by flow cytometry. As a result, the fluorescence intensities of the RT22-ep58 MG, RT22-ep59 MG and CT-ep59 MG antibodies bound to SW480 and LoVo cells expressing EpCAM were measured.
Example 21. Determination of Growth Inhibition of Adherent Cells of Tumor-Tissue-Specific Anti-RasGTP iMab Including Combination of Light-Chain Variable Region (VL) Having Improved Production Yield and Tumor-Tissue-Cell-Specific Cytoplasmic Penetration Ability with Heavy-Chain Variable Region Having Improved Affinity for RasGTP
[0314]
[0315] Specifically, the cell line was diluted at a density of 110.sup.4 cells/well in 0.2 ml of a medium containing 10% FBS in a 96-well plate and cultured for 24 hours at 37 C. and at 5% CO.sub.2. Then, the resulting cells were treated with 0.5 M of TMab4-i, RT11-i, RT22-i38 MG and RT22-i39 MG for 48 hours, after which 10 l of a WST solution (Dojindo) was added thereto and the absorbance at 450 nm was quantified.
[0316] As can be shown from
[0317]
[0318] Specifically, the cell lines were diluted at a density of 110.sup.4 cells/well in 0.2 ml of a medium containing 10% FBS in a 96-well plate and cultured for 24 hours at 37 C. under 5% CO.sub.2. Then, the resulting cells were treated with 1 M of CT-ep59, RT11, RT22-ep58 MG and RT22-ep59 MG for 36 hours, after which 10 l of a WST solution (Dojindo) was added thereto and absorbance at 450 nm was quantified.
[0319] As can be shown from
[0320] As a result, when compared with the conventional anti-RasGTPs (RT11, RT11-i) in vitro, RT22-i38 MG, RT22-i39 MG, RT22-ep58 MG and RT22-ep59 MG have an improved cell growth inhibitory effect specific for Ras mutant cell lines. Thus, the subsequent experiment was conducted to determine whether or not the effect of inhibiting the tumor growth by the antibodies was also improved in an in-vivo animal model.
Example 22. Determination of Improved Tumor Growth Inhibitory Ability of Tumor-Tissue Integrin-Specific Anti-RasGTP iMab
[0321]
[0322]
[0323]
[0324] Specifically, in order to determine the improved tumor growth inhibition ability of RT22-i38 MG and RT22-i39 MG, tumor-tissue integrin-specific anti-RasGTP iMabs, having improved affinity and having no HSPG-binding ability, compared to the conventional tumor-tissue integrin-specific anti-RasGTP iMab, RT11-I in vivo, the KRas.sup.G12V mutant human colorectal cancer cell line SW480 was subcutaneously injected into BALB/c nude mice at a density of 510.sup.6 cells/mouse, and the KRas.sup.G13D mutant human colorectal cancer cell line LoVo was subcutaneously injected into BALB/c nude mice at a density of 210.sup.6 cells/mouse. After about 10 days, when the tumor volume reached about 50 to 80 mm.sup.3, the equivalent volume of a PBS vehicle control group, control group CT-i39 MG (TMab4 VH), and experimental groups RT11-i, RT22-i38 MG and RT22-i39 MG were injected intravenously at 20 mg/kg. A total of 8 intravenous injections were performed every 2 days, and the tumor volume was measured for 16 days using a caliper.
[0325] As can be shown from
[0326]
[0327] In addition, as can be seen from
Example 23. Determination of Tumor Growth Inhibitory Ability of Tumor Tissue EpCAM-Specific Anti-RasGTP iMab
[0328]
[0329]
[0330]
[0331] Specifically, in order to determine the in-vivo tumor growth inhibition of RT22-ep58 MG and RT22-ep59 MG, tumor-tissue EpCAM-specific anti-RasGTP iMabs, the KRas.sup.G12V mutant human colorectal cancer cell line SW480 was subcutaneously injected into BALB/c nude mice at a density of 510.sup.6 cells/mouse, and the KRas.sup.G13D mutant human colorectal cancer cell line LoVo was subcutaneously injected into BALB/c nude mice at a density of 210.sup.6 cells/mouse. After about 10 days, when the tumor volume reached about 50 to 80 mm.sup.3, the equivalent volume of a PBS vehicle control group, a control group, CT-ep59 MG, and experimental groups RT22-ep58 MG and RT22-ep59 MG were injected intravenously at 20 mg/kg. A total of 9 intravenous injections were performed every 2 days, and the tumor volume was measured for 18 days using a caliper.
[0332] As can be shown from
[0333]
[0334] In addition, as can be seen from
Example 24. Determination of Tumor Growth Inhibition Ability of Tumor-Tissue Integrin-Specific Anti-RasGTP iMab Depending on Dose, Administration Interval and Administration Route
[0335]
[0336]
[0337]
[0338] Specifically,
[0339] In order to determine the tumor growth inhibition effect, the KRas.sup.G13D mutant human colorectal cancer cell line LoVo was subcutaneously injected into BALB/c nude mice at a density of 210.sup.6 cells/mouse. After about 10 days, when the tumor volume reached about 50 to 80 mm.sup.3, the equivalent volume of a PBS vehicle control group, control group CT-i39 MG (TMab4 VH), and experimental group RT22-i39 MG were injected intravenously or intraperitoneally at 5, 10 and 20 mg/kg. A total of 9 intravenous injections were performed every 2 days, or a total of 6 intravenous injections were performed every week, and the tumor volume was measured for 18 days using a caliper.
[0340] As can be shown from
[0341]
[0342] In addition, as can be seen from
Example 25. Determination of Tumor Growth Inhibition Ability of Tumor Tissue EpCAM-Specific Anti-RasGTP iMab Depending on Dose, Administration Interval and Administration Route
[0343]
[0344]
[0345]
[0346] Specifically, in order to determine the in-vivo tumor growth inhibition of RT22-ep59 MG, tumor-tissue EpCAM-specific anti-RasGTP iMab, depending on dose, administration interval and administration route, the KRas.sup.G13D mutant human colorectal cancer cell line LoVo was subcutaneously injected into BALB/c nude mice at a density of 210.sup.6 cells/mouse. After about 10 days, when the tumor volume reached about 50 to 80 mm.sup.3, the equivalent volume of a PBS vehicle control group, control group CT-ep59 MG, and experimental group RT22-ep59 MG were injected intravenously or intraperitoneally at 5, 10, 20 mg/kg. A total of 9 intravenous injections were performed every 2 days, or a total of 6 intravenous injections were performed every week, and the tumor volume was measured for 18 days using a caliper.
[0347] As can be shown from
[0348]
[0349] In addition, as can be seen from
Example 26. Design and Expression/Purification of Heavy-Chain Modified Variant to Improve Intracytoplasmic Stability, In-Vivo Persistence and Tumor-Tissue-Targeting Ability of Anti-RasGTP iMab
[0350] Intracytoplasmic proteins are generally degraded by a ubiquitin-proteasome mechanism. Likewise, an intact immunoglobulin-type antibody in the cytoplasm is bound to TRIM21 E3 ligase and is degraded by the ubiquitin-proteasome mechanism, and the degradation of the antibody is inhibited when a mutation is introduced into the N434 amino acid in the region CH3 of the antibody that binds to TRIM21. Therefore, in order to improve the intracytoplasmic stability of the anti-RasGTP iMab, the intact immunoglobulin-type antibody, a variant in which a N434D mutation was introduced into the CH3 region was constructed. Specifically, in the same manner as in Example 4, the cytoplasmic penetration humanized light-chain expression vector (hT4-ep59 MG LC) and the heavy-chain expression vector (RT22 IgG1 N434D) of the anti-RasGTP iMab having improved intracytoplasmic stability were subjected to transient co-transfection into HEK293F-protein-expressing cells to express anti-RasGTP iMab mutations with improved intracytoplasmic stability.
[0351] In addition, the immunoglobulin-type antibody binds to the Fc receptor of immune cells in the body and is removed through an ADCC mechanism. Among the IgG subclass, IgG2 and IgG4 have weak binding force to the Fc gamma receptor and thus inhibition in this function. Therefore, in order to improve the in-vivo stability of the anti-RasGTP iMab, the intact immunoglobulin IgG1 antibody, a variant introduced with heavy-chain constant regions of IgG4 (CH1 to CH3) was constructed. At this time, the S228P mutation was further introduced into the Hinge site so that Fab-arm exchange, which is the characteristic of IgG4, does not occur in the body. Specifically, in the same manner as in Example 4, cytoplasm-penetrating humanized light-chain expression vector (hT4-ep59 MG LC) and the heavy-chain expression vector (RT22 IgG4 S228P) of the anti-RasGTP iMab having improved in-vivo persistence were subjected to transient co-transfection into HEK293F protein-expressing cells to express an anti-RasGTP iMab mutation with improved in-vivo persistence.
[0352] The following Table 17 shows the sequence information of the heavy-chain constant region (CH1-CH3) modified to improve the intracytoplasmic stability and in-vivo persistence of anti-RasGTP iMab.
TABLE-US-00022 TABLE17 Heavy Chain SEQ Constant ID Region Sequence NO. IgG1 1102030405060 64 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS 708090100110120 GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG 130140150160170180 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN 190200210220230240 STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE 250260270280290300 LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW 310320330 QQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG1N434D 1102030405060 65 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS 708090100110120 GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG 130140150160170180 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN 190200210220230240 STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE 250260270280290300 LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW 310320330 QQGNVFSCSVMHEALHDHYTQKSLSLSPGK IgG4S228P 1102030405060 66 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS 708090100110120 GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV 130140150160170180 FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY 190200210220230240 RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK 250260270280290300 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG 310320327 NVFSCSVMHEALHNHYTQKSLSLSLGK IgG4S228P, 1102030405060 67 N434D ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS 708090100110120 GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV 130140150160170180 FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY 190200210220230240 RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK 250260270280290300 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG 310320327 NVFSCSVMHEALHDHYTQKSLSLSLGK
[0353] In addition, in order to improve the tumor-tissue-targeting ability of anti-RasGTP iMab, a clone that binds to the ep133 peptide having EpCAM targeting ability to the N-terminus of the anti-RasGTP iMab heavy-chain variable region was constructed, expressed and purified. Specifically, in the same manner as in Example 4, the cytoplasm-penetrating humanized light-chain expression vector (hT4-ep59 MG LC) and the heavy-chain expression vectors (epRT22 GS, epRT22 MG, epRT22 (G4S)2) of the anti-RasGTP iMab having improved tumor-tissue-targeting ability were subjected to transient co-transfection into HEK293F protein-expressing cells to express anti-RasGTP iMab mutations having improved tumor-tissue-targeting ability.
[0354] The following Table 18 shows the heavy-chain variable-region sequences of anti-RasGTP iMab having improved tumor-tissue-targeting ability.
TABLE-US-00023 TABLE18 SEQ ID VH Sequence NO. epRT22GS 102030405060 61 EHLHCLGSLCWPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLE 708090100110120 WVSYISRTSHTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFKMDYWGQ 128 GTLVTVSS epRT22MG 102030405060 62 EHLHCLGSLCWPMGSSSNEVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPG 708090100110120 KGLEWVSYISRTSHTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFKMD 130 YWGQGTLVTVSS epRT22(G4S).sub.2 102030405060 63 EHLHCLGSLCWPGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVR 708090100110120 QAPGKGLEWVSYISRTSHTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARG 130 FKMDYWGQGTLVTVSS
[0355] The following Table 19 shows the production yield of anti-RasGTP iMab having improved intracytoplasmic stability, in-vivo persistence and tumor-tissue-targeting ability
TABLE-US-00024 TABLE 19 IgG1, Heavy chain Production yield format VH CH1-CH3 Light chain (mg/1 L of transfected cells).sup.a epRas03 MG RT22 IgG1 hT4-ep59 MG 69.8 6.6 (= R122-ep59 MG) epRas13 MG IgG1 N434D 62.1 2.9 epRas23 MG epRT22 GS IgG1 58.2 4.8 epRas33 MG IgG1 N434D ND epRas03 MG IgG4 RT22 IgG4 S228P 48.1 3.7 epRas13 MG IgG4 IgG4 S228P, N434D 48.2 1.2 epRas23 MG IgG4 epRT22 GS IgG4 ND epRas33 MG IgG4 IgG4 S228P, N434D 50.2 2.0
[0356] In Table 19 above, the conventional RT22-ep59 MG was newly named epRas03 MG, the RT22-i39 MG was named inRas03, and the heavy chain constant region variants were named based thereon.
Example 27. Determination of Reduced Intracytoplasmic Degradation and Non-Adherent Cell Growth Inhibition of Anti-RasGTP iMab Having Improved Intracytoplasmic Stability
[0357] An improved split green fluorescent protein complementation system (Korean Patent Application No. 10-2015-0143278) was used to determine the decrease in degradation of the anti-RasGTP iMab having improved intracytoplasmic stability constructed in Example 25. For this purpose, epRas03-GFP11-SBP2 MG and epRas13-GFP11-SBP2 MG, having a GFP11-SBP2 peptide fused to the heavy-chain C-terminus of epRas03 MG and epRas13 MG, were constructed.
[0358]
[0359] Specifically, an SW480 cell line expressing SA-GFP1-10 at a density of 110.sup.4 cells/well in a 96-well assay plate was diluted in 0.1 ml of medium containing 10% FBS and cultured for 24 hours at 37 C. and 5% CO.sub.2. Then, the cells were treated with 2 M of epRas03 MG, epRas03-GFP11-SBP2 MG and epRas13-GFP11-SBP2 MG for 6 hours, and then the medium was replaced with fresh medium. Then, the cells were cultured for 0, 2, 4, and 6 hours, and green fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 528 nm was quantified using a fluorescent plate reader.
[0360] As shown in
[0361]
[0362] Specifically, 0.2 ml of a mixture of a 1.2% agarose solution and a 2 RPMI+20% FBS medium in a ratio of 1:1 was spread on a 24-well plate. After the bottom agar was hardened, a 0.7% agarose solution and a medium of 2RPMI+20% FBS containing 110.sup.3 cells +1 M or 4 M antibody were mixed at a ratio of 1:1, and 0.2 ml of the mixture was spread thereon. After the top agar was hardened, 0.2 ml of medium was added thereto. Subsequently, the medium was replaced with a medium containing 0.5 M or 2 M of the antibody every 3 days. After to 3 weeks, the colonies were stained with a BCIP/NBT solution, and then the number of colonies having a size of 200 m or more was measured.
[0363] As shown in
[0364]
[0365] Specifically, the cell lines were diluted at a density of 110.sup.3 cells/well in 0.1 ml of 10% FBS medium in a low-attachment 96-well plate, and cultured along with a 0.5 M or 2 M concentration of antibody for 48 hours at 37 C. and 5% CO.sub.2. Subsequently, the cells were further treated with 0.5 and 2 M antibody concentrations and cultured for 48 hours. After culturing for a total of 96 hours, 50 l of CellTiterGlo (Promega) was added to quantify luminescence.
[0366] As shown in
[0367]
[0368] Specifically, the cell lines were diluted at a density of 110.sup.3 cells/well in 0.1 ml medium containing 10% FBS in a low-attachment 96-well plate, and cultured along with a 2 M antibody for 48 hours at 37 C., and 5% CO.sub.2. Subsequently, the cells were further treated with the 2 M antibody and cultured for 48 hours. After culture for a total of 144 hours, 50 l of CellTiterGlo (Promega) was added to quantify luminescence.
[0369] As shown in
[0370] The result showed that both epRas13 MG and inRas13 had cytoplasmic stability superior to the conventional epRas03 MG and inRas03, and thus exhibited an improved cell growth inhibitory effect specific for the Ras mutant cell line.
Example 28. Pharmacokinetic Evaluation of Anti-RasGTP iMab Having Improved In-Vivo Persistence
[0371]
[0372] Specifically, both epRas03 MG and epRas03 MG IgG4 were found to have a molecular weight of about 150 kDa under non-reducing conditions, and the molecular weight of the heavy chain and the molecular weight of the light chain were found to be 50 kDa and 25 kDa, respectively, under reducing conditions. This indicated that the expressed and purified individual clones were present as monomers (alone) in the solution, and did not form dimers or oligomers through non-natural disulfide bonds.
[0373]
[0374] Specifically, 20 mg/kg of experimental group epRas03 MG IgG4 and control group epRas03 MG were intravenously injected into BALB/c nude mice. After 0.5, 1, 4, 8, 12 and 24 hours and 2, 4, 7, 14, 21 and 28 days, mouse blood was collected. The collected blood was centrifuged at 12,000 rpm for 10 minutes at 4 C. and the supernatant plasma was stored at 80 C. Thereafter, ELISA was performed to analyze the concentrations of epRas03 MG IgG4 and epRas03 MG in the plasma. Specifically, the anti-human Fab antibody (Sigma) was bound at a concentration of 2.5 g/ml to a 96-well half-area plate (Corning) for 1 hour at room temperature and washed three times with 0.1% PBST (PBS, pH 7.4, 0.1% Tween20). The antibody was bound to 1% PBSB (PBS, pH 7.4, 1% BSA) for 1 hour, the collected blood and purified antibody (for the reference curve) was diluted in 1% PBSB, bound for 2 hours, and then washed three times with 0.1% PBST. Then, the HRP-conjugated anti-human IgG, Fc antibody (Sigma) was bound as a labeling antibody for 1 hour, followed by washing 3 times with 0.1% PBST. The result was reacted with TMB ELISA solution, the reaction was stopped with a sulfuric acid solution, and the absorbance at 450 nm was quantified.
[0375] As can be seen from
Example 29. Determination of Improvement of EpCAM-Targeting Ability of Tumor-Tissue EpCAM-Specific Anti-RasGTP iMab Having Improved Tumor-Tissue-Targeting Ability
[0376]
[0377] Specifically, 110.sup.5 LoVo cells were prepared for each sample. The cells were cultured for 1 hour at 4 C. in PBSF (PBS buffer, 2% BSA) supplemented with 100 nM of Ras03, epRas03 MG and epRas23 MG. Then, each antibody was reacted with an antibody specifically recognizing human Fc conjugated to Alexa488 (green fluorescence) for 30 minutes at 4 C. Then, the cells were washed with PBS and analyzed by flow cytometry. As a result, the fluorescence intensities of epRas03 MG and epRas23 MG antibodies bound to LoVo cells expressing EpCAM were measured, and epRas23 MG fused with more EpCAM-targeting peptides had higher fluorescence intensity.
[0378]
[0379] Specifically, ELISA was conducted in the same manner as in Example 4, and epRas03 MG and epRas23 MG, tumor-tissue-specific anti-RasGTP iMabs including EpCAM-targeting peptide fused with the N-terminal of the heavy-chain variable region in order to improve tumor-tissue-targeting ability were fixed on a 96-well EIA/RIA plate, after which the GppNHp-bound KRas protein was bound at various concentrations of 100 nM, 50 nM and 25 nM, and the GDP-bound KRas protein was bound at a concentration of 100 nM and was then bound to an HRP-conjugated anti-his antibody (HRP-conjugated anti-his mAb) as a labeling antibody. The result was reacted with TMB ELISA solution, and the absorbance at 450 nm was quantified.
[0380] The result showed that the fusion of EpCAM-targeting peptide with the N-terminal of the heavy-chain variable region caused no difference in the affinity for Ras.
[0381]
[0382] Specifically, LoVo, DLD-1 and HCT116 cell lines were each diluted at a density of 110.sup.3 cells/well in 0.1 ml of a 10% FBS medium in a low-attachment 96-well plate, and cultured along with a 0.5 or 2 M antibody for 48 hours at 37 C. under 5% CO.sub.2. Subsequently, the cells were further treated with the 0.5 or 2 M antibody and cultured for 48 hours. After culture for a total of 96 hours, 50 l of CellTiterGlo (Promega) was added thereto to quantify luminescence.
[0383] As can be seen from
[0384]
[0385] Specifically, 20 g of DyLight fluorescently labeled anti-RasGTP iMabs, Ras03, epRas03 MG, and epRas23 MG were injected into BALB/c nude mice, and fluorescence emitted from the entire bodies of the mice was observed at 0, 6, 12, 24, 48 and 72 hours with an IVIS Lumina XRMS Series III (Perkin Elmer). At this time, the mice were anesthetized using 1.5-2.5% isoflurane (Piramal Critical Care). Each image shows fluorescence values of the entire body quantified using Living Image software (Perkin Elmer).
[0386] As can be seen from
[0387]
[0388] Specifically, following the experiment, 72 hours after antibody injection, the mice were euthanized, the tumors, heart, lungs, liver, kidneys, pancreas and spleen were extracted and the fluorescence in each of these organs was quantified using Living Image software (Perkin Elmer).
[0389] As can be seen from
Example 30. Design and Expression/Purification of Heavy-Chain Modified Variant to Improve In-Vivo Persistence of Anti-RasGTP iMab
[0390] An immunoglobulin-type antibody binds to the Fc receptor of immune cells in the body and is removed through an ADCC mechanism. IgG1 has weak binding force to the Fc gamma receptor due to the introduction of L234A, L235A and P239G mutations and thus inhibition of this function. Therefore, in order to improve the in-vivo stability of anti-RasGTP iMab, the intact immunoglobulin IgG1 antibody, a mutant (IgG1 LALA-PG) in which L234A, L235A and P239G mutations (LALA-PG) were introduced into the heavy-chain constant regions (CH1 to CH3) of IgG1 was constructed. Specifically, in the same manner as in Example 4, cytoplasm-penetrating humanized light-chain expression vector (hT4-ep59 GSSG LC) and the heavy-chain expression vector (RT22 IgG1 LALA-PG or RT22 IgG1 LALA-PG, N434D) of the anti-RasGTP iMab having improved in-vivo persistence were subjected to transient co-transfection into HEK293F protein-expressing cells to express an anti-RasGTP iMab mutant with improved in-vivo persistence.
[0391] The following Table 20 shows the sequence information of the heavy-chain constant region (CH1-CH3) introduced with the LALA-PG mutant to improve the in-vivo persistence of anti-RasGTP iMab.
TABLE-US-00025 TABLE20 SEQ ID VHname Sequence NO. IgG1LAL-PG 1102030405060 68 ASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSS 708090100110120 GLYSLSSVVTVPSGSLGTQTYLCHVNHKFSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG 130140150160170180 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVRFNWYVDGVEVHNAKTKPREEQYN 190200210220230240 STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDE 250260270280290300 LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW 310320330 QQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG1LALA-PG, 1102030405060 69 N434D ASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSS 708090100110120 GLYSLSSVVTVPSGSLGTQTYLCHVNHKFSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG 130140150160170180 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVRFNWYVDGVEVHNAKTKPREEQYN 190200210220230240 STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDE 250260270280290300 LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW 310320330 QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Example 31. Pharmacokinetic Evaluation of LALA-PG Mutant to Improve In-Vivo Persistence of Tumor-Tissue-Specific Anti-RasGTP iMab
[0392]
[0393] Specifically, the size-exclusion chromatography was performed using an Agilent Technologies HPLC 1200 series. The column used herein was a Zenix SEC-300 column. The experiment was conducted at a flow rate of 1 ml/min. The mobile phase included 150 mM sodium phosphate (pH 7.0). The diluent was the mobile phase, and analysis was performed with 280 nm UV. It was identified that the antibody was purified as a monomer, not a multimer, from all iMabs.
[0394]
[0395] Specifically, epRas03, epRas13, epRas33 and epRas83 were intravenously injected at 20 mg/kg into BALB/c nude mice. After 0.5, 1, 4, 8, 12 and 24 hours and 2, 4, 7, 14, 21 and 28 days, mouse blood was collected. The collected blood was centrifuged at 12,000 rpm for 10 minutes at 4 C. and only the supernatant plasma was stored at 80 C. Then, ELISA was performed to analyze the concentrations of epRas03, epRas13, epRas33 and epRas83 in the plasma. Specifically, in the same manner as in Example 28, an anti-human Fab antibody (Sigma) was bound on a 96-well half-area plate (Corning) at a concentration of 2.5 g/ml for 1 hour at room temperature and then washed three times with 0.1% PBST (PBS, pH 7.4, 0.1% Tween20). After binding with 1% PBSB (PBS, pH 7.4, 1% BSA) for 1 hour, the collected blood and purified antibody (for the reference curve) was diluted in 1% PBSB, bound for 2 hours, and then washed three times with 0.1% PBST 3. Then, the antibody was bound to an HRP-conjugated anti-human IgG, Fc antibody (Sigma) as a labeling antibody for 1 hour and then washed 3 times with 0.1% PBST. The result was reacted with TMB ELISA solution, the reaction was stopped with a sulfuric acid solution, and the absorbance at 450 nm was quantified.
[0396] As can be seen from
Example 32. Evaluation of Binding Ability to GppNHp-Bound KRas.SUP.G12D .and Neonatal Fc Receptor (FcRn) of LALA-PG Mutant to Improve In-Vivo Persistence of Anti-RasGTP iMab
[0397] The binding ability was analyzed based on SPR (surface plasmon resonance) using a Biacore2000 instrument to identify that the LALA-PG mutant to improve in-vivo persistence of anti-RasGTP iMab maintains the binding ability to GppNHp-bound KRas.sup.G12D and has no influence on the binding ability to FcRn.
[0398] Specifically, in order to determine the binding ability to GppNHp-bound KRas.sup.G12D, epRas03, epRas13, epRas83, inRas03 MG, inRas13 MG and inRas83 MG were diluted to a concentration of 20 l/ml in 10 mM NaAc buffer (pH 4.0) and immobilized at approximately 1800 response units (RU) on a CM5 sensor chip (GE Healthcare). Subsequently, Tris buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM MgCl.sub.2, 0.01% Tween 20) was analyzed at a flow rate of 30 l/min, and complete GppNHp-bound GST-KRas.sup.G12D was analyzed at a concentration of 100 nM to 6.25 nM. After binding and dissociation analysis, regeneration of the CM5 chip was performed by flowing a buffer (20 mM NaOH, 1M NaCl, pH 10.0) at a flow rate of 30 l/min for 1 minute. Each sensorgram, obtained by binding for 3 minutes and dissociation for 3 minutes, was normalized with reference to a blank cell and subtracted, and the affinity was thus calculated.
[0399] Specifically, in order to determine the binding ability to FcRn, FcRn was diluted to a concentration of 20 l/ml in 10 mM NaAc buffer (pH 4.0) and immobilized at approximately 650 response units (RU) on a CM5 sensor chip (GE Healthcare). Subsequently, a phosphate buffer (12 mM phosphate, pH 6.0, 137 mM NaCl, 0.01% Tween 20) was analyzed at a flow rate of 30 l/min, epRas03 was analyzed at a concentration of 200 nM to 6.25 nM, and epRas13 and epRas83 were analyzed at a concentration of 2000 nM to 62.5 nM. After binding and dissociation analysis, regeneration of the CM5 chip was performed by flowing a phosphate buffer (12 mM phosphate, pH 7.4, 137 mM NaCl, 0.01% Tween 20) at a flow rate of 30 l/min for 3 minutes. Each sensorgram, obtained by binding for 3 minutes and dissociation for 6 minutes, was normalized with reference to a blank cell and subtracted, and the affinity was thus calculated.
[0400] The following Table 21 shows the result of analysis, based on SPR using a BIACORE2000 instrument, of the affinity for complete GppNHp-bound KRas.sup.G12D and FcRn of the LALA-PG mutant to improve the in-vivo persistence of anti-RasGTP iMabs.
TABLE-US-00026 TABLE 21 hFcRn IgG1, Heavy chain KRas.sup.G12D.Math.GppNHp K.sub.D (M).sup.a format VH CH1-CH3 Light chain K.sub.D (M).sup.a at pH 6.0 epRas03 RT22 IgG1 hT4-ep59 GSSG 8.59 0.32 10.sup.9 1.48 10.sup.7 epRas13 IgG1 N434D 8.97 0.66 10.sup.9 2.7 10.sup.6 epRas33 IgG1 LALA-PG ND ND epRas83 IgG1 LALA-PG, N434D 8.94 10.sup.9 3.57 10.sup.6 inRas03 MG IgG1 hT4-i59 MG 6.4 10.sup.9 ND inRas13 MG IgG1 N434D 7.53 10.sup.9 ND inRas33 MG IgG1 LALA-PG ND ND inRas83 MG IgG1 LALA-PG, N434D 7.07 10.sup.9 ND Herceptin ND 2.28 10.sup.7 .sup.aND, not determined
Example 33. Determination of Tumor Growth Inhibition Ability of LALA-PG Mutant to Improve In-Vivo Persistence of Tumor-Tissue Integrin-Specific Anti-RasGTP iMab
[0401]
[0402] Specifically, size-exclusion chromatography was performed using an Agilent Technologies HPLC 1200 series. The column used herein was a Zenix SEC-300 column. The experiment was conducted at a flow rate of 1 ml/min. The mobile phase included 150 mM sodium phosphate (pH 7.0). The diluent was the mobile phase, and analysis was performed with 280 nm UV. It was identified that the antibody was purified as a monomer in 95% or more of all iMabs.
[0403]
[0404] Specifically, the KRas.sup.A146T mutant human colorectal cancer cell line LS1034 was subcutaneously injected into BALB/c nude mice at a density of 110.sup.7 cells/mouse. After about 14 days, when the tumor volume reached about 120 mm.sup.3, the equivalent volume of a PBS vehicle control group, control groups (inCT03 MG, inCT13 MG, inCT33 MG, inCT83 MG), and experimental groups (inRas03 MG, inRas13 MG, inRas33 MG, inRas83 MG) were injected intravenously at 20 mg/kg. A total of 7 intravenous injections were performed every 3 to 4 days, that is, twice a week, and the tumor volume was measured for 24 days using a caliper.
[0405] As can be seen from
[0406]
[0407] Specifically, the KRas.sup.G12V mutant human colorectal cancer cell line SW403 was subcutaneously injected into BALB/c nude mice at a density of 110.sup.7 cells/mouse. After about 14 days, when the tumor volume reached about 120 mm.sup.3, the equivalent volume of a PBS vehicle control group, control groups (inCT03 MG, inCT13 MG, inCT33 MG, inCT83 MG), and experimental groups (inRas03 MG, inRas13 MG, inRas33 MG, inRas83 MG) were injected intravenously at 20 mg/kg. A total of 5 intravenous injections were performed every 3 to 4 days twice for a week, and the tumor volume was measured for 17 days using a caliper.
[0408] As can be seen from
[0409] As can be seen from
INDUSTRIAL AVAILABILITY
[0410] The method for improving the tumor inhibition efficiency of an antibody that specifically penetrates into the cytoplasm of tumor tissue cells in the form of an intact immunoglobulin to directly inhibit intracellular RasGTP, provided by the present disclosure, is accomplished by selecting heavy-chain variable regions with improved affinity for RasGTP and developing light-chain variable regions with improved cytoplasmic penetration ability specifically for tumor tissues, and is capable of targeting RasGTP, which is located in certain tumor cells and is always activated through mutation, and of effectively inhibiting the activity thereof.
[0411] In addition, the light-chain variable region, imparting improved tumor-tissue-specific cytoplasmic penetration ability to the antibody provided by the present disclosure or the antibody including the same, lowers the binding ability to HSPG expressed in most normal cells and thereby makes it possible to provide endocytosis and endosomal escape through receptors expressed specifically for tumor tissues by the peptide fused for targeting tumor tissues, and the heavy-chain variable region (VH) having improved affinity for RasGTP has improved Ras inhibition ability when the antibody reaches the cytoplasm. The antibody, which specifically penetrates into the cytoplasm of tumor tissue cells in the form of an intact immunoglobulin including a combination of the modified light-chain variable region and the heavy-chain variable region, and directly inhibits intracellular RasGTP, exhibits improved tumor growth inhibitory ability based thereon.
[0412] The cytoplasmic penetration Ras-inhibitory antibody including a combination of the improved light-chain variable region and the heavy-chain variable region according to the present disclosure can be easily developed into a therapeutic drug owing to high production yield, is capable of effectively inhibiting mutant Ras through tumor-tissue-specific cytoplasmic penetration, and is thus expected to exert effective anti-cancer activity through treatment using a single drug or a combination thereof with a conventional therapeutic drug.
SEQUENCE LISTING FREE TEXT
[0413] An electronic file is attached.