FUSION PROTEIN AND USE THEREOF
20230270856 · 2023-08-31
Inventors
Cpc classification
A61K39/4611
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a fusion protein and the use thereof. The fusion protein includes, from N to C terminals, a first moiety, an Fc segment, a linking moiety comprising a moiety selected from a linker and a protein or polypeptide selected from IL2 or scFv, and a substrate moiety of transpeptidase A; the linker includes a sequence selected from the group consisting of (1) (GGGGS)n, wherein when the linking moiety includes the protein or polypeptide and the linker, n≥1; when the linking moiety only includes the linker, n≥3; and (2) (EAAAK)n, n≥1; the substrate moiety includes LPXTG. The fusion protein can be directly connected to cells to enable the cells to have a targeting property, is more simple than an existing method for preparing targeting cells by means of cell transfection, and can also reduce the risk possibly generated by an effector cell genome operation.
Claims
1. A fusion protein comprising, from N terminus to C terminus, a first moiety, an Fc segment, a linking moiety, and a substrate moiety of Sortase A, wherein: the linking moiety comprises a region selected from a linker, and a protein or polypeptide selected from IL2 or scFv, preferably SEQ ID No. 33 or 34; the linker comprises a sequence selected from the group consisting of: (1) (GGGGS)n, wherein n≥1 when the linking moiety comprises the protein or polypeptide, and the linker, and n≥3 when the linking moiety comprises only the linker; (2) (EAAAK).sub.n, n≥1; the substrate moiety comprises the sequence as shown in LPXTG.
2. The fusion protein according to claim 1, wherein the first moiety is selected from F(ab′), F(ab′).sub.2, Fab, Fv, scFv, a receptor, and a ligand.
3. The fusion protein according to claim 1, wherein the Fc segment is a wild-type Fc segment or a variant Fc segment, preferably, the Fc segment is selected from Fc segments of IgG, IgM, IgA, IgD and IgE, preferably from Fc segments of IgG1, IgG2, IgG3 and IgG4.
4. The fusion protein according to claim 1, comprising the structure of any one of: full-length antibody-GGGGS-GGGGS-GGGGS-LPETGG; full-length antibody-EAAAK-LPETGG; full-length antibody-IL2-LPETGG; full-length antibody-scFv-GGGGS-LPETGG; full-length antibody-scFv-EAAAK-LPETGG; scFv-Fc segment-GGGGS-GGGGS-GGGGS-LPETGG; or scFv-Fc segment-EAAAK-LPETGG.
5. A nucleic acid encoding the fusion protein according to claim 1.
6. The nucleic acid according to claim 5, comprising the encoding sequence of SEQ ID Nos. 4 and 12, SEQ ID Nos. 4 and 14, SEQ ID Nos. 4 and 16, SEQ ID Nos. 4 and 18, SEQ ID Nos. 4 and 20, SEQ ID No. 28, SEQ ID No. 30, or SEQ ID No. 32.
7. A vector comprising the nucleic acid according to claim 6.
8. A host cell comprising the vector according to claim 7.
9. A method of generating the fusion protein, comprising (1) mixing equimolarly the vector of claim 7 expressing a heavy chain and a vector expressing a light chain in the presence of the light chain; (2) introducing the vector mixture into a host cell and expressing which for a suitable time under conditions suitable for the expression of the fusion protein; and (3) recovering the medium supernatant and purifying the fusion protein.
10. A method for connecting the fusion protein according to claim 1 to the surface of a cell, comprising a step of contacting the cell with the fusion protein and Sortase A.
11. The method according to claim 10, wherein the cell is an effector cell, preferably an NK cell or a T cell, preferably a peripheral blood NK cell, a peripheral blood T cell and a cord blood NK cell, preferably an NK92-FcγRIII cell.
12. A cell prepared by the method according to claim 10.
13. A pharmaceutical composition comprising the cell according to claim 12 and a pharmaceutically acceptable carrier.
14. Use of the pharmaceutical composition according to claim 13 in the manufacture of a medicament used for a disease.
15. The use according to claim 14, wherein the disease is a disease caused by abnormal cell proliferation and/or function, preferably a tumor, an autoimmune disease and/or an infectious disease.
16. A method for preventing and/or treating a disease caused by abnormal cell proliferation and/or function, preferably a tumor, an autoimmune disease and/or an infectious disease, comprising the step of providing the composition of claim 13.
17. A vector comprising the nucleic acid according to claim 5.
18. A cell prepared by the method according to claim 11.
19. Use of the cell according to claim 12 in the manufacture of a medicament used for a disease.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0054] The present invention is based on the inventor's findings that the directly fusion and expression of the LPXTG region specifically recognized by Sortase A at the C-terminus of the intact antibody or Fc fusion protein using the protein modification method described in the existing patents and literatures (US20160122707A1; Jeong H J, et al., Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling. PLoS One. 2017 Dec. 4; 12(12):e0189068; Chen I, et al., A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci USA. 2011 Jul. 12; 108(28):11399-404), i.e., no linking sequence added between the intact antibody or Fc fusion protein and the LPXTG sequence, is unable to achieve the direct connection of the intact antibody or Fc fusion protein to the cell surface by Sortase A. In contrast, using the fusion proteins in a specific configuration of the present invention, the intact antibody or Fc fusion protein may be directly connected to the cell surface via Sortase A.
[0055] As used herein, Sortase A is a membrane bound enzyme that enables a protein containing an enzyme substrate recognition sequence covalently linked to a bacterial cell membrane. The specific substrate recognition motif of Sortase A is LPXTG, and the enzyme cleaves between residues threonine (T) and glycine (G) of this substrate sequence, and further reacts with a substrate containing an oligoglycine sequence at the N-terminus for ligation. The type and source of Sortase A herein may not be limited as long as the Sortase A retains its functional properties as described above. For example, Sortase A may be a natural Sortase A or may be a variant of Sortase A (see, for example, CN201610726374.0).
[0056] As used herein, “first moiety” refers to the binding region in the fusion protein. The binding region may be an antigen-binding region of an antibody, a receptor for a ligand, or a ligand for a receptor, provided it is capable of binding a target, for example, on a target cell. The first moiety and the Fc region herein may be homologous or heterologous. For example, the first moiety and the Fc region are naturally occurring antibodies. The first moiety may be an antibody fragment. The first moiety may bind cancer antigens, infectious disease antigens, autoimmune disease antigens. For example, the first moiety may bind HER2 protein.
[0057] “Antibodies” which encompass a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they display desired antigen-binding activities. Antibodies, also known as immunoglobulins (Igs for short), can be classified into five classes, IgG, IgM, IgA, IgE, and IgD, according to their physicochemical properties and biological functions. The Fab (fragment of antigen binding, Fab) segment of an antibody is an antigen-binding fragment, composed of a complete light chain and VH and CH1 domains of the heavy chain; the Fc segment is a crystallizable segment (fragment crystallizable, Fc), which region is composed of two or three constant domains of the heavy chains depending on the type of the antibody. For example, the Fc domain of IgG contains the heavy chain CH2 and CH3 domains.
[0058] The term “Fc segment” is used to define the C-terminal region of the immunoglobulin heavy chain, which contains at least a portion of the constant region. The term includes both a natural sequence Fc segment and a variant Fc segment. A “variant Fc segment” comprises an amino acid sequence that differs from a “natural” or “wild-type” sequence Fc segment due to at least one “amino acid modification”. The variant Fc region may have at least one amino acid substitution, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the natural sequence Fc region or in the Fc segment of the parental polypeptide, as compared to the natural sequence Fc region or to the Fc segment of the parental polypeptide. The variant Fc segment herein will preferably have at least about 80% homology, and most preferably have at least about 90% homology, and more preferably have at least about 95% homology with the natural sequence Fc region and/or with the Fc segment of the parental polypeptide. The Fc region can bind various Fc receptors (FcRs) and other immune molecules, a process that triggers different target cell killing effects, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), and is the key effector segment of therapeutic antibody to exert its utility in vivo. The effector function of an antibody is a function contributed by the Fc effector domain of IgG (e.g., the Fc region of immunoglobulins). This function can be achieved, for example, by binding of the Fc effector domain to Fc receptors on immune cells with phagocytic or cytolytic activity, or by binding of the Fc effector domain to components of the complement system. Typical effector functions are ADCC, ADCP, and CDC. The “effector function” refers to those biological activities attributable to the Fc region of an antibody, which vary by antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation.
[0059] As used herein, the “linking moiety” refers to the moiety connecting the Fc region to the Sortase A substrate LPXTG. This moiety may comprise only a linker. In this case, the linking moiety may be the linker itself. Alternatively, the linking moiety may comprise only a protein or polypeptide, such as IL2 or scFv. The linking moiety may also comprise both the protein or polypeptide and the linker. For example, the linking moiety comprises IL2 or scFv and the linker, from the N-terminus to the C-terminus. There is no special restriction on the type of the linker provided that Fc-containing fusion proteins can be connected to the cell surface. For example, the linker can be a flexible linking sequence, such as G(n)S(n), n≥1, preferably, the linking sequence is GGGGSGGGGSGGGGS. The linker may also be a rigid linker sequence, such as (EAAAK)n, n≥1, preferably, the linking sequence is EAAAK. In an embodiment where the linking moiety comprises a protein or polypeptide and a linker, the linker is (GGGGS)n, n≥1, and the linker may also be a rigid linker sequence (EAAAK)n, n≥1. In an embodiment where the linking moiety comprises only a linker, the linker is (GGGGS)n, n≥3. In one embodiment, the linker is (EAAAK).sub.n, n≥1. Herein, n≥3 refers to, for example, an integer where n is 3, 4, 5, 6, 7, 8, 9, 10, or 11. n≥1 refers to, for example, an integer where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
[0060] The term “moiety” is equivalent to “region” in the context used in a first moiety, a linking moiety, and a substrate moiety of Sortase A. The first moiety, the linking moiety and the substrate moiety may be referred to as the first region, the linking region and the substrate region.
[0061] The term “Fc receptor” or “FcR” is used to describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a natural sequence human FcR. In addition, FcRs can be FcRs (gamma receptor) that bind IgG antibodies and include receptors for the subclasses FcγRI, FcγRII and FcγRIII, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA (activating receptor) and FcγRIIB (“inhibitory receptor”), which share similar amino acid sequences and differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA comprises an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB comprises an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain. The “FcRs” encompasses other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn responsible for the transfer of maternal IgG to the fetus. Herein, the effector cells can express FcγRIII.
[0062] “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to cell-mediated responses in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing FcRs recognize antibodies bound on target cells and subsequently cause lysis of the target cells. The major cells (NK cells) used to mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9(1991)457-492.
[0063] The terms “antibody-dependent cellular phagocytosis” and “ADCP” refer to a process that antibody-coated cells are fully or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) bound to the Fc region of immunoglobulins.
[0064] As used herein, “Fc fusion protein” or “fusion protein” refers to the fusion of the Fc region of an antibody with a protein molecule having a specific biological function using genetic engineering techniques, which not only has the original activity of the functional protein, but also has certain properties of the antibody, such as ADCC, CDC and ADCP. It can thus be seen that if an antigen-binding molecules with an Fc segment (such as an antibody and antigen-binding region-Fc fusion protein) is directly connected to the surface of an effector cell, not only can the cell have targeting for recognition of specific antigens, but also, the physiological effects triggered by the Fc region can further enhance the killing activity of the effector cell. Herein, the Fc fusion protein can have structuress of, from the N-terminus to the C-terminus: [0065] full-length antibody-GGGGS-GGGGS-GGGGS-LPETGG; [0066] full-length antibody-EAAAK-LPETGG; [0067] full-length antibody-IL2-LPETGG; [0068] full-length antibody-scFv-GGGGS-LPETGG; [0069] full-length antibody-scFv-EAAAK-LPETGG; [0070] scFv-Fc segment-GGGGS-GGGGS-GGGGS-LPETGG; [0071] scFv-Fc segment-EAAAK-LPETGG.
[0072] The full-length antibody in these Fc fusion proteins can be IgG1, IgG2, IgG3 or IgG4 antibody.
[0073] “Antibody fragment” refers to a molecule of a non-intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabody; linear antibody; single chain antibody molecule (e.g., scFv); and multispecific antibody formed from an antibody fragment. Herein, the antibody fragment that binds a specific antigen may be used as the first moiety of the present invention.
EXAMPLES
[0074] The present invention is described in further detail below in combination with specific examples, which are given merely to illustrate the invention and not to limit the scope of the invention.
[0075] The experimental methods in the following examples, if not otherwise specified, are conventional methods.
[0076] The materials, reagents and the like used in the following examples, if not otherwise specified, are available from commercial sources.
Example 1: Expression and Purification of Sortase a Proteins
[0077] I. Construction of Recombinant Expression Plasmid pET-SrtA
[0078] 1. The sequence of Sortase A gene is shown in Sequence 1, and primer F: 5′-CGGCAGCCATATGGCTAAACCTCAAATTCCGA-3′ (the underlined is the restriction recognition sequence of restriction endonuclease NdeI, sequence 35) and primer R: 5′-GTGGTGCTCGAGTTATTTGACTTCTGTAGCTAC-3′ (the underlined is the restriction recognition sequence of restriction endonuclease XhoI, sequence 36) were designed and synthesized according to Sequence 1.
TABLE-US-00002 Sequence 1: (SEQ ID No. 1) ATGGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGCTATAT TGAAATTCCAGATGCTGATATTAAAGAACCAGTATATCCAGGACCAGCAA CAAGCGAACAATTAAATAGAGGTGTAAGCTTTGCAGAAGAAAATGAATCA CTAGATGATCAAAATATTTCAATTGCAGGACACACTTTCATTGACCGTCC GAACTATCAATTTACAAATCTTAAAGCAGCCAAAAAAGGTAGTATGGTGT ACTTTAAAGTTGGTAATGAAACACGTAAGTATAAAATGACAAGTATAAGA AACGTTAAGCCTACAGATGTAGGAGTTCTAGATGAACAAAAAGGTAAAGA TAAACAATTAACATTAATTACTTGTGATGATTACAATGAAAAGACAGGCG TTTGGGAAACCCGTAAAATCTTTGTAGCTACAGAAGTCAAATAA
[0079] 2. The DNA fragment shown in Sequence 1 above was synthesized and used as a template (reference: Chen I, et al., A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci USA. 2011 Jul. 12; 108(28):11399-404.), and PCR amplification was performed using primer F and primer R synthesized in step 1 (reaction procedure: 95° C. for 5 min; 95° C. for 30 s, 55° C. for 30 s, 72° C. for 2 min, 30 cycles), and then the PCR amplification product of about 457 bp was recovered using a PCR product recovery kit.
[0080] 3. The PCR amplification product recovered in step 2 was taken and digested with restriction endonuclease NdeI and restriction endonuclease XhoI, and the digested DNA fragment was recovered.
[0081] 4. The vector pET-28(a) was digested with restriction endonuclease NdeI and restriction endonuclease XhoI, and the digested vector backbone was recovered.
[0082] 5. The DNA fragment and the vector backbone were ligated to obtain the recombinant expression plasmid pET-SrtA.
[0083] The recombinant expression plasmid pET-SrtA was structurally described as follows: the small fragment between the restriction endonuclease NdeI and XhoI recognition sequences of vector pET-28(a) was replaced with the DNA molecule shown in Sequence 1 in the sequence listing. According to the sequencing results, the sequence SEQ ID No. 1 of Sortase A was correctly inserted into vector pET-28(a). The recombinant expression plasmid pET-SrtA expresses the protein shown in Sequence 2 in the sequence listing (hereinafter referred to as Sortase A protein).
TABLE-US-00003 Sequence 2: (SEQ ID No. 2) MGSSHHHHHHSSGLVPRGSHMAKPQIPKDKSKVAGYIEIPDADIKEPVYP GPATSEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKG SMVYFKVGNETRKYKMTSIRNVKPTDVGVLDEQKGKDKQLTLITCDDYNE KTGVWETRKIFVATEVK
[0084] II. Expression and Purification of Sortase a Protein
[0085] 1. The recombinant expression plasmid pET-SrtA was introduced into E. coli BL21(DE3) to obtain the recombinant strain, named BL21(DE3)-pET-SrtA.
[0086] 2. The single colony of BL21(DE3)-pET-SrtA was inoculated into LB medium containing 100 mg/mL kanamycin, cultured at 37° C., 200 rpm with shaking, to obtain culture broth 1 with OD600 of 0.6. IPTG was added into culture broth 1 to obtain culture broth 2 (the concentration of IPTG is 0.1 mM in culture broth 2); then cultured at 37° C., 200 rpm with shaking for 6 h to obtain culture broth 3.
[0087] 3. The culture broth 3 was taken for centrifugation at 5000 rpm for 10 min to collect the bacteria.
[0088] 4. The bacteria collected in step 3 were taken and resuspended with 10 mL TBS buffer (10 mmol/L, Tris, 0.9% NaCl), pH7.4 to obtain bacterial suspension; then the bacterial suspension was ultrasonicated. Ultrasonication parameters: ultrasonication frequency 30%; ultrasonication for 10 s, stop for 5 s, total ultrasonication time is 30 min.
[0089] 5. The system completed in step 4 was taken and centrifuged at 12000 rpm for 10 min to collect the supernatant.
[0090] 6. The supernatant collected in step 5 was mixed with Ni-NTA Resin and incubated for 10 min, then the supernatant was dicarded, and the Ni-NTA Resin was washed with TBS buffer containing 20 mM imidazole, pH 7.4 for 3 times.
[0091] 7. After step 6 was completed, elution was performed with TBS buffer containing 500 mM imidazole, pH 7.4 and the solution flowed through the column was collected, that is the Sortase A protein.
Example 2: Preparation and Binding Activity Identification of Fc Segment-Containing Fusion Proteins
[0092] I. Preparation of Fc Segment-Containing Fusion Proteins
[0093] 1. Construction of the Plasmid for Fc Segment-Containing Fusion Proteins
[0094] The structures of the Fc segment-containing fusion proteins (including recombinant antibodies and Fc fusion proteins, No. RP1-RP14) are shown in
[0095] 1.1. Construction of the Expression Plasmid for the Recombinant Antibody Ab-CH-LPETGG (No.: RP1):
[0096] The light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 3. In Sequence 3, nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-LPETGG (Sequence 4).
TABLE-US-00004 Sequence 3: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGA GCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAAC ACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCT GATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCG GCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGAC CTTTGGCCAGGGCACCAAACTCGAGATCAAACGTACGGTGGCGGCGCCAT CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGTACCGCT AGCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTGA (nucleotides 1-60 encode the signal peptide and the last three nucleotides TGA are the stop codon) Sequence 4: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
[0097] The heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 5. In Sequence 5, nucleotides 61-1428 encode the full-length heavy chain of the recombinant antibody Ab-CH-LPETGG (Sequence 6).
TABLE-US-00005 Sequence 5: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGC CGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAA GATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATG GGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCG TGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTAT CTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAG CCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC CCCGGGTAAACTGCCCGAGACCGGCGGCTGA
(nucleotides 1-60 encode the signal peptide and the last three nucleotides TGA are the stop codon)
TABLE-US-00006 Sequence 6: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LPETGG
[0098] 1.2. Construction of the Expression Plasmid for the Recombinant Antibody Ab-CH-GGGGS-LPETGG (No.: RP2):
[0099] The light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 3. In Sequence 3, nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-GGGGS-LPETGG (Sequence 4).
[0100] The heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 7. In Sequence 7, nucleotides 61-1443 encode the full-length heavy chain of the recombinant antibody Ab-CH-GGGGS-LPETGG (Sequence 8).
TABLE-US-00007 Sequence 7: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGC CGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAA GATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATG GGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCG TGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTAT CTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAG CCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC CCCGGGTAAAGGCGGCGGCGGCAGCCTGCCCGAGACCGGCGGCTGA
(nucleotides 1-60 encode the signal peptide and the last three nucleotides TGA are the stop codon)
TABLE-US-00008 Sequence 8: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSLPETGG
[0101] 1.3. Construction of the Expression Plasmid for the Recombinant Antibody Ab-CH-GGGGS(x2)-LPETGG (No.: RP3):
[0102] The light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 3. In Sequence 3, nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-GGGGS(x2)-LPETGG (Sequence 4).
[0103] The heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 9. In Sequence 9, nucleotides 61-1473 encode the full-length heavy chain of the recombinant antibody Ab-CH-GGGGS(x2)-LPETGG (Sequence 10).
TABLE-US-00009 Sequence 9: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGC CGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAA GATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATG GGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCG TGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTAT CTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAG CCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC CCCGGGTAAAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCTGCCCGAGA CCGGCGGCTGA
[0104] (nucleotides 1-60 encode the signal peptide and the last three nucleotides TGA are the stop codon)
TABLE-US-00010 Sequence 10: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSLPETGG
[0105] 1.4. Construction of the Expression Plasmid for the Recombinant Antibody Ab-CH-GGGGS(x3)-LPETGG (No.: RP4):
[0106] The light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 3. In Sequence 3, nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-GGGGS(x3)-LPETGG (Sequence 4).
[0107] The heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 11. In Sequence 11, nucleotides 61-1458 encode the full-length heavy chain of the recombinant antibody Ab-CH-GGGGS(x3)-LPETGG (Sequence 12).
TABLE-US-00011 Sequence 11: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGC CGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAA GATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATG GGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCG TGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTAT CTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAG CCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC CCCGGGTAAAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCG GCAGCCTGCCCGAGACCGGCGGCTGA
(nucleotides 1-60 encode the signal peptide and the last three nucleotides TGA are the stop codon)
TABLE-US-00012 Sequence 12: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGSLPETGG
[0108] 1.5. Construction of the Expression Plasmid for the Recombinant Antibody Ab-CH-EAAAK-LPETGG (No.: RP5):
[0109] The light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 3. In Sequence 3, nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-EAAAK-LPETGG (Sequence 4).
[0110] The heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 13. In Sequence 13, nucleotides 61-1443 encode the full-length heavy chain of the recombinant antibody Ab-CH-EAAAK-LPETGG (Sequence 14).
TABLE-US-00013 Sequence 13: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGC CGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAA GATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATG GGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCG TGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTAT CTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAG CCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC CCCGGGTAAAGAGGCCGCCGCCAAGCTGCCCGAGACCGGCGGCTGA Sequence 14: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK EAAAKLPETGG
[0111] 1.6. Construction of the Expression Plasmid for the Recombinant Antibody Ab-CH-IL2-LPETGG (No. RP6):
[0112] The light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 3. In Sequence 3, nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-IL2-LPETGG (Sequence 4).
[0113] The recombinant heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 15. In Sequence 15, nucleotides 61-1854 encode the full-length heavy chain of the recombinant antibody Ab-CH-IL2-LPETGG (Sequence 16).
TABLE-US-00014 Sequence 15: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGC CGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAA GATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATG GGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCG TGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTAT CTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAG CCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC CCCGGGTAAAGGCGGCGGCGGCAGCGGATCCGCCCCCACCTCCTCCTCCA CCAAGAAGACCCAGCTGCAGCTGGAGCACCTGCTGCTGGACCTGCAGATG ATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGACCAGGATGCT GACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCACCTGC AGTGCCTGGAGGAGGAGCTGAAGCCCCTGGAGGAGGTGCTGAACCTGGCC CAGTCCAAGAACTTCCACCTGAGGCCCAGGGACCTGATCTCCAACATCAA CGTGATCGTGCTGGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGT ACGCCGACGAGACCGCCACCATCGTGGAGTTCCTGAACAGGTGGATCACC TTCTGCCAGTCCATCATCTCCACCCTGACCCGTACGCTGCCCGAGACCGG CGGCTGA Sequence 16: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIV LELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTRTLPETGG
(The underlined part is the amino acid sequence of IL2)
[0114] 1.7. Construction of the Expression Plasmid for the Recombinant Antibody Ab1-CH-scFv.Ab2-GGGGS-LPETGG (No.: RP7):
[0115] The light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 3. In Sequence 3, nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab1-CH-scFv.Ab2-GGGGS-LPETGG (Sequence 4).
[0116] The recombinant heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 17. In Sequence 17, nucleotides 61-2178 encode the full-length heavy chain of the recombinant antibody Ab1-CH-scFv.Ab2-GGGGS-LPETGG (Sequence 18).
TABLE-US-00015 Sequence 17: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGC CGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAA GATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATG GGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCG TGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTAT CTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAG CCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC CCCGGGTAAAGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCA GCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCC AGCCAGGACGTGAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAA GGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGC CCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACCT GTACCACCCCGCCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAG CTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCT GAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGACAGCTGGATCCACTGGG TGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCTGGATCAGCCCC TACGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCAT CAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGA GGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCACTGGCCCGGC GGCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGG CGGCGGCAGCCTGCCCGAGACCGGCGGCTGA
(the coding sequence for scFv.Ab2 sequence is underlined)
TABLE-US-00016 Sequence 18: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPK LLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHP ATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA ASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISAD TSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGS LPETGG
(the scFv.Ab2 sequence is bolded and underlined)
[0117] 1.8. Construction of the Expression Plasmid for the Recombinant Antibody Ab1-CH-scFv.Ab2-EAAAK-LPETGG (No.: RP8):
[0118] The light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 3. In Sequence 3, nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab1-CH-scFv.Ab2-EAAAK-LPETGG (Sequence 4).
[0119] The recombinant heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 19. In Sequence 19, nucleotides 61-2178 encode the full-length heavy chain of the recombinant antibody Ab1-CH-scFv.Ab2-EAAAK-LPETGG (Sequence 20).
TABLE-US-00017 Sequence 19: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGC CGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAA GATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATG GGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCG TGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTAT CTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAG CCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCC CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC CCCGGGTAAAGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCA GCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCC AGCCAGGACGTGAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAA GGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGC CCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACCT GTACCACCCCGCCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAG CTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCT GAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGACAGCTGGATCCACTGGG TGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCTGGATCAGCCCC TACGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCAT CAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGA GGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCACTGGCCCGGC GGCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGAGGC CGCCGCCAAGCTGCCCGAGACCGGCGGCTGA
(the scFv.Ab2 sequence is underlined, and the linker sequence is GAGGCCGCCGCCAAG)
TABLE-US-00018 Sequence 20: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPK LLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHP ATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA ASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISAD TSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS LPETGG
(the scFv.Ab2 sequence is bold and underlined, and the linker sequence is EAAAK)
[0120] 1.9. Construction of the Expression Plasmid for the Fc Fusion Protein scFv-Fc-LPETGG (No. RP9):
[0121] The Fc fusion protein expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 21. In Sequence 21, nucleotides 61-1506 encode the full-length protein of scFv-Fc-LPETGG (Sequence 22).
TABLE-US-00019 Sequence 21: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGA GCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAAC ACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCT GATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCG GCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGAC CTTTGGCCAGGGCACCAAACTCGAGATCAAAGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGAAGTGCAGCTGGTGGAAAGCGGC GGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAG CGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGG GCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACC CGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAG CAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCG CGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGAT TATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCGAGCCCAA ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAACTGCCCGAGACC GGCGGCTGA Sequence 22: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFN IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKLPETGG
[0122] 1.10. Construction of the Expression Plasmid for the Fc Fusion Protein scFv-Fc-GGGGS-LPETGG (No.: RP10):
[0123] The Fc fusion protein expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 23. In Sequence 23, nucleotides 61-1521 encode the full-length protein of scFv-Fc-GGGGS-LPETGG (Sequence 24).
TABLE-US-00020 Sequence 23: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGA GCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAAC ACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCT GATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCG GCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGAC CTTTGGCCAGGGCACCAAACTCGAGATCAAAGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGAAGTGCAGCTGGTGGAAAGCGGC GGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAG CGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGG GCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACC CGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAG CAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCG CGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGAT TATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCGAGCCCAA ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGGCGGCGGCGGC AGCCTGCCCGAGACCGGCGGCTGA Sequence 24: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFN IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSLPETGG
[0124] 1.11. Construction of the Expression Plasmids for the Fc Fusion Protein scFv-Fc-(GGGGS)x2-LPETGG (No.: RP11):
[0125] The Fc fusion protein expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 25. In Sequence 25, nucleotides 61-1536 encode the full-length protein of scFv-Fc-(GGGGS)x2-LPETGG (Sequence 26).
TABLE-US-00021 Sequence 25: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGA GCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAAC ACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCT GATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCG GCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGAC CTTTGGCCAGGGCACCAAACTCGAGATCAAAGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGAAGTGCAGCTGGTGGAAAGCGGC GGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAG CGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGG GCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACC CGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAG CAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCG CGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGAT TATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCGAGCCCAA ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGGCGGCGGCGGC AGCGGCGGCGGCGGCAGCCTGCCCGAGACCGGCGGCTGA Sequence 26: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFN IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSLPETGG
[0126] 1.12. Construction of the Expression Plasmid for the Fc Fusion Protein scFv-Fc-(GGGGS)x3-LPETGG (No.: RP12):
[0127] The Fc fusion protein expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 27. In Sequence 27, nucleotides 61-1550 encode the full-length protein of scFv-Fc-(GGGGS)x3-LPETGG (Sequence 28).
TABLE-US-00022 Sequence 27: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGA GCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAAC ACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCT GATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCG GCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGAC CTTTGGCCAGGGCACCAAACTCGAGATCAAAGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGAAGTGCAGCTGGTGGAAAGCGGC GGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAG CGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGG GCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACC CGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAG CAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCG CGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGAT TATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCGAGCCCAA ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGGCGGCGGCGGC AGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCTGCCCGAGACCGGCGG CTGA Sequence 28: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFN IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSLPETGG
[0128] 1.13. Construction of the Expression Plasmid for the Fc Fusion Protein scFv-Fc-EAAAK-LPETGG (No.: RP13):
[0129] The Fc fusion protein expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 29. In Sequence 29, nucleotides 61-1521 encode the full-length protein of scFv-Fc-EAAAK-LPETGG (Sequence 30).
TABLE-US-00023 Sequence 29: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGA GCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAAC ACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCT GATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCG GCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGAC CTTTGGCCAGGGCACCAAACTCGAGATCAAAGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGAAGTGCAGCTGGTGGAAAGCGGC GGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAG CGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGG GCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACC CGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAG CAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCG CGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGAT TATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCGAGCCCAA ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGAGGCCGCCGCC AAGCTGCCCGAGACCGGCGGCTGA Sequence 30: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFN IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKEAAAKLPETGG
[0130] 1.14. Construction of the Expression Plasmid for the Recombinant Antibody Ab(IgG4)-EAAAK-LPETGG (No.: RP14):
[0131] The light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 3. In Sequence 3, nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab(IgG4)-EAAAK-LPETGG (Sequence 4).
[0132] The heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in vector pCDNA3.1(+) with the DNA molecule shown in Sequence 31. In Sequence 31, nucleotides 61-1800 encode the full-length heavy chain of the recombinant antibody Ab(IgG4)-EAAAK-LPETGG (Sequence 32).
TABLE-US-00024 Sequence 31: ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGG GTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGC CGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAA GATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATG GGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCG TGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTAT CTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAG CCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCA CCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTGCTCCCGCAGTACTTCTGAGAGCACAGCGGCCCTGGGCTG CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGG CACCAAGACCTACACGTGCAACGTGGATCACAAGCCCAGCAACACCAAGG TGGACAAACGCGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCA GCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACC CAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGG TGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCC TCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACA GGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACA AGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAA AGAGGCCGCCGCCAAGCTGCCCGAGACCGGCGGCTGA Sequence 32: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKEAA AKLPETGG
[0133] 2. Preparation of Fc Segment-Containing Fusion Proteins
[0134] 2.1 After equimolar mixing of the above heavy chain expression vectors and light chain expression vectors (if there is a light chain), CHO cells were cotransfected using ExpiFectamine™ CHO Transfection Kit (purchased from Therno Fisher, Inc., Cat. No.: A29129) (operated according to the instructions) to express recombinant antibodies; CHO cells were transfected with the above Fc fusion protein expression plasmids using ExpiFectamine™ CHO Transfection Kit (Cat. No.: A29129) (operated according to the instructions) to express Fc fusion proteins; CHO cells were then cultured at 37° C. and 5% CO.sub.2 using ExpiCHO™ Expression Medium for 8 days, and the supernatant was collected.
[0135] 2.2 The supernatant obtained in step 2.1 was taken and purified with Protein A. The purified product was collected.
[0136] 2.3 The purified product obtained in step 2.2 was taken and subjected to ultra-filtration concentration and exchange. The system was replaced with PBS buffer to obtain the solution of recombinant antibodies and Fc fusion proteins.
[0137] The protein concentration of the solution was detected by UV absorption method at A280 nm.
[0138] The polyacrylamide gel electrophoresis of the solution of Fc segment-containing fusion proteins (including recombinant antibodies and Fc fusion proteins) is shown in
[0139] II. ELISA Assay for the Binding Activity of Fc Segment-Containing Fusion Proteins 1. A microtiter plate was taken and the coating solution (100 μl/well) was added overnight at 4° C.
[0140] The coating solution consists of a coating antigen and the coating buffer, and the concentration of the coating antigen in the coating solution is 1 μg/mL. The coating antigen is HER2 protein (purchased from Sino Biological Inc., Cat. No.: 10004-H08H4). The coating buffer (pH 9.6): Na.sub.2CO.sub.3 1.59 g, NaHCO.sub.3 2.94 g, the balance being water.
[0141] 2, After completing step 1, the microtiter plate was taken and washed with PBST buffer for three times.
[0142] 3. After completing step 2, the microtiter plate was taken and added with PBST buffer containing 5 g/100 mL skimmed milk powder, and blocked at 37° C. for 1 h.
[0143] 4. The Fc segment-containing fusion protein solution prepared above was taken to prepare the stock solution with antibody concentration of 10 μg/mL by using PBST buffer containing 5 g/100 mL skimmed milk powder, followed by three-fold gradient dilution using PBST buffer containing 5 g/100 mL skimmed milk powder to obtain Fc segment-containing fusion protein solutions at different concentrations.
[0144] 5. The microtiter plate after completing step 3 was taken and added with Fc segment-containing fusion protein solutions (100 μL per well) at different concentrations obtained in step 4, and incubated at 37° C. for 1 h. 3 replicate wells were set for each concentration.
[0145] 6. After completing step 5, the microtiter plate was taken and washed with PBST buffer for three times (250 μL per well each time).
[0146] 7. After completing step 6, the microtiter plate was taken and added with HRP-labeled goat anti-human IgG secondary antibody dilution (HRP-labeled goat anti-human IgG secondary antibody diluted 1:40,000 in PBST buffer containing 5 g/100 mL skimmed milk powder), and incubated at 37° C. for 30 min.
[0147] 8. After completing step 7, the microtiter plate was taken and added with TMB chromogenic reagent (100 μL per well), and developed at room temperature for 10 min.
[0148] 9. After completing step 8, the microtiter plate was taken and the development was terminated by adding 2N H.sub.2SO.sub.4 solution (50 μL per well), and then the OD value was detected at a wavelength of 450 nm.
[0149] The results are shown in
Example 3: Connection of the Fc Segment-Containing Fusion Proteins to NK92-FcγRIII Cells Using Sortase a and Cell Activity Assay
[0150] I. Connection of the Fc Segment-Containing Fusion Proteins to NK92-FcγRIII Cells (Purchased from ATCC, Cat. No.: pta-8837) Using Sortase A
[0151] 1. 100 μL of NK92-FcγRIII cell suspension at a concentration of 1.0×10.sup.6/mL was taken and added with Sortase A to a final concentration of 20 ug/ml, and the Fc segment-containing fusion protein to a final concentration of 10 ug/ml, to obtain an incubation system. The incubation system was incubated at 25° C. for 90 min. The cells were collected after centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-SrtA-NK.
[0152] 2. 100 μL of NK92-FcγRIII cell suspension at a concentration of 1.0×10.sup.6/mL was taken and added with the Fc segment-containing fusion protein alone to a final concentration of 10 ug/ml to obtain an incubation system. The incubation system was incubated at 25° C. for 90 min. The cells were collected by centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-NK.
[0153] 3. The cells collected in steps 1 and 2 were taken and added with APC-labeled Donkey Anti-Human IgG antibody (APC AffiniPure F(ab′).sub.2 Fragment Donkey Anti-Human IgG, Fcγ fragment specific, purchased from Jackson ImmunoResearch Company, Cat. No.: 709-136-098) according to the instructions to obtain an incubation system. The incubation system was incubated at 25° C. for 30 min.
[0154] 4. After completing step 3, cells were collected by centrifugation, and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4.
[0155] 5. After completing step 4, cells without treatment were used as blank group cells, and the levels of cells labeled with Fc segment-containing fusion proteins with or without the addition of Sortase A were detected using flow cytometry.
[0156] The experimental results are shown in
[0157] II. Cells Connected with Fc Segment-Containing Fusion Proteins can Bind Specifically to the Corresponding Target Antigens
[0158] 1. 200 μL of NK92-FcγRIII cell suspension at a concentration of 1.0×10.sup.6/mL was taken and added with Sortase A to a final concentration of 20 ug/ml and Fc segment-containing fusion protein to a final concentration of 10 ug/ml, to obtain an incubation system. The incubation system was incubated at 25° C. for 90 min. The cells were collected by centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-SrtA-NK.
[0159] 2. 200 μL of NK92-FcγRIII cell suspension at a concentration of 1.0×10.sup.6/mL was taken and added with the Fc segment-containing fusion protein alone to a final concentration of 10 ug/ml to obtain an incubation system. The incubation system was incubated at 25° C. for 90 min. The cells were collected by centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-NK.
[0160] 3. 100 μL of the cell suspension after the completion of steps 1 and 2 at a concentration of 1.0×10.sup.6/mL was taken and added with biotin-labeled HER2 protein (purchased from ACRObiosysterms, Cat. No.: H822R) to obtain an incubation system. The incubation system was incubated at 25° C. for 60 min.
[0161] 4. After completing step 3, cells were collected by centrifugation, and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4.
[0162] 5. After completing step 4, the incubation system was obtained by adding FITC-labeled streptavidin (FITC Streptavidin, purchased from Biolegend Company, Cat. No.: 405201) according to the instructions. The incubation system was incubated at 25° C. for 30 min.
[0163] 6. After completing step 5, cells were collected by centrifugation, and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4.
[0164] 7. After completing step 6, cells without treatment were used as blank group cells, and the levels of binding of cells to target antigen HER2 were detected using flow cytometry.
[0165] The experimental results are shown in
[0166] III. NK92-FcγRIII Cells Successfully Connected with the Fc Segment-Containing Fusion Proteins can be Further Activated by Target Cells Expressing Specific Antigens.
[0167] 1. 400 μL of NK92-FcγRIII cell suspension at a concentration of 1.0×10.sup.6/mL was taken and added with Sortase A to a final concentration of 20 ug/ml and Fc segment-containing fusion proteins to a final concentration of 10 ug/ml, to obtain an incubation system. The incubation system was incubated at 25° C. for 90 min. The cells were collected by centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-SrtA-NK.
[0168] 2. 200 μL of NK92-FcγRIII cell suspension at a concentration of 1.0×10.sup.6/mL was taken and added with the Fc segment-containing fusion proteins alone to a final concentration of 10 ug/ml to obtain an incubation system. The incubation system was incubated for 90 min at 25° C. The cells were collected by centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-NK.
[0169] 3. A 24-well plate was taken and added with 200 μL of suspension of SKOV3 cells (which is an antigen HER2 protein high-expressing cell line) at a concentration of 4.0×10.sup.5/mL, and then with 200 μL of cell suspension after the completion of step 1 or 2 at a concentration of 2×10.sup.6/mL to obtain an incubation system. The incubation system was incubated at 37° C. for 60 min.
[0170] 4. The system after completing step 3 was taken, and cells were collected by centrifugation at 4° C. and washed thoroughly with pH 7.4, 0.01 mol/L PBS buffer in ice bath.
[0171] 5. After completing step 4, Alexa Fluor® 488-labeled anti-human CD107a antibody (Alexa Fluor® 488anti-human CD107a, purchased from Biolegend Company, Cat. No.: 328610) and APC-labeled anti-human CD56 antibody (APC anti-human CD56 (NCAM), purchased from Biolegend Company, Cat. No.: 318310) were added according to the instructions to obtain an incubation system. The incubation system was incubated at 4° C. for 30 min.
[0172] 6. After completing step 5, cells were collected by centrifugation at 4° C., and wash thoroughly with pH 7.4, 0.01 mol/L PBS buffer in an ice bath.
[0173] 7. After completing step 6, cells without treatment were used as blank group cells, and the level of cellular CD107a was detected using flow cytometry.
[0174] The experimental results are shown in
[0175] IV. NK92-FcγRIII Cells that have been Connected with the Fc Segment-Containing Fusion Proteins can Kill the Target Cells Expressing the Specific Antigens
[0176] 1. 400 μL of NK92-FcγRIII cell suspension at a concentration of 1.0×10.sup.6/mL was taken and added with Sortase A to a final concentration of 20 ug/ml and the Fc segment-containing fusion proteins to a final concentration of 10 ug/ml to obtain an incubation system. The incubation system was incubated at 25° C. for 90 min. The cells were collected by centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-SrtA-NK.
[0177] 2. 200 μL of NK92-FcγRIII cell suspension at a concentration of 1.0×10.sup.6/mL was taken and added with the Fc segment-containing fusion proteins alone to a final concentration of 10 ug/ml to obtain an incubation system. The incubation system was incubated at 25° C. for 90 min. The cells were collected by centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-NK.
[0178] 3. A 96-well plate was taken and added with 50 μL of suspension of SKOV3 cells (which is an antigen HER2 protein high-expressing cell line) at a concentration of 4.0×10.sup.5/mL, and then with 50 μL of cell suspension after completion of step 1 or 2 at a concentration of 2×10.sup.6/mL to obtain an incubation system. The incubation system was cultured at 37° C. for 4 hrs.
[0179] 4. A 96-well plate was taken and added with 50 μL of suspension of MCF-7 cells (which is an antigen HER2 protein negative cell line) at a concentration of 4.0×10.sup.5/mL, and then with 50 μL of cell suspension after completion of step 1 or 2 at a concentration of 2×10.sup.6/mL was added to obtain an incubation system. The incubation system was cultured at 37° C. for 4 hrs.
[0180] 5. The system after completing steps 3 and 4 was taken, added with 100 ul of working solution for detecting lactate dehydrogenase (LDH) activity (purchased from Dojindo Laboratories Company, Cat. No.: CK12), and developed in the dark for 15 min, followed by addition of 50 ul of termination solution and determination of OD490 nm absorbance.
[0181] The experimental results are shown in
[0182] V. Connection of the Fc Segment-Containing Fusion Proteins to Peripheral Blood NK Cells/Peripheral Blood T Cells/Cord Blood NK Cells Using Sortase A
[0183] 1. Peripheral blood NK cells were purchased from Beijing Junda Kean Technology Co., Ltd.
[0184] 2. Peripheral blood T cell preparation: mononuclear cells were firstly obtained from human peripheral blood by density gradient centrifugation, and then T cells therein were enriched using the T cell negative selection kit (purchased from Stemcell Company, Cat. No.: 710410) (operated according to the instructions).
[0185] 3. Cord blood NK cells were donated by Shandong Qilu Stem Cell Engineering Co. LTD.
[0186] 4. 100 μL of suspension of peripheral blood NK cells or peripheral blood T cells or cord blood NK cells at a concentration of 1.0×10.sup.6/mL was taken, and added with Sortase A to a final concentration of 20 ug/ml, and Fc segment-containing fusion proteins to a final concentration of 10 ug/ml to obtain an incubation system. The incubation system was incubated at 25° C. for 90 min. The cells were collected by centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-SrtA-cells.
[0187] 5. 100 μL of suspension of peripheral blood NK cells or peripheral blood T cells or cord blood NK cells at a concentration of 1.0×10.sup.6/mL was taken, and added with the Fc segment-containing fusion proteins alone to a final concentration of 10 ug/ml to obtain an incubation system. The incubation system was incubated at 25° C. for 90 min. The cells were collected by centrifugation and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4. The cells obtained above were named as: recombinant protein-cells.
[0188] 6. After completing steps 4 and 5, cells were collected by centrifugation, and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4.
[0189] 7. The cells collected in step 6 were taken and added with APC-labeled donkey anti-human IgG antibody (APCAffiniPure F(ab′).sub.2 Fragment Donkey Anti-Human IgG, Fcγ fragment specific, purchased from Jackson ImmunoResearch Company, Cat. No.: 709-136-098) according to the instructions to obtain an incubation system. The incubation system was incubated at 25° C. for 30 min.
[0190] 8. After completing step 7, cells were collected by centrifugation, and washed thoroughly with 0.01 mol/L PBS buffer, pH 7.4.
[0191] 9. After completing step 8, the cells without treatment were used as blank group cells, and the cell labeling level was detected using flow cytometry.
[0192] The experimental results are shown in
CONCLUSION
[0193] Our experimental results indicate that the fusion protein obtained through directly fusion and expression of the LPXTG region specifically recognized by Sortase A only at the C-terminus of the Fc segment-containing fusion protein (e.g., intact antibody or Fc fusion protein) (i.e., no linking sequence is added between the Fc segment in the intact antibody or Fc fusion protein and the LPXTG sequence) using the protein modification methods described in the existing patents and literatures (US20160122707A1; Jeong H J, et al., Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling. PLoS One. 2017 Dec. 4; 12(12):e0189068; Chen I, et al., A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci USA. 2011 Jul. 12; 108(28):11399-404), may not be directly connected to the cell surface by Sortase A. We have demonstrated that constructs that connecting the C-terminus of the Fc segment in an intact antibody or Fc fusion protein to the LPXTG region recognized by Sortase A via the specific linking sequences are capable of direct connection to the cell surface mediated by Sortase A. The constructs are RP4, RP5, RP6, RP7, RPB, RP12, RP13, and RP14 as described in the above examples.