CN111423515A

CN111423515A - CD20/CD47 bispecific antibody and application

Info

Publication number: CN111423515A
Application number: CN202010208405.XA
Authority: CN
Inventors: 王雷; 尹建河; 董丽莎; 张崇骞; 赵永浩; 汤春; 张晓霞; J·彭; D·张
Original assignee: Beierda Pharmacy Suzhou Co ltd
Current assignee: Beierda Pharmacy Suzhou Co ltd; Beta Pharma Inc
Priority date: 2020-03-23
Filing date: 2020-03-23
Publication date: 2020-07-17

Abstract

The invention discloses a CD20/CD47 bispecific antibody and application, the bispecific antibody can simultaneously target CD20 and CD47, and comprises an anti-CD 20 antibody and a first functional region (SIRPaD1) of an extracellular domain of a human SIRPa membrane, wherein the N ends of heavy chains of the SIRPaD1 and CD20 antibodies are connected through a connecting peptide, and the Fc part adopts partial point mutation to enhance ADCC/CDC. The bispecific antibody is directed against the CD20 part, and shows stronger ADCC/CDC effect on tumor cells than the ADCC/CDC activated by the CD20 monoclonal antibody; the bispecific antibody can be directed against the CD47 partThe antibody can effectively block the binding of CD47 positive tumor cells and SIRPa, activate the phagocytic Activity (ADCP) of macrophages on the tumor cells, and is stronger than the ADCP activated by recombinant proteins SIRPaD1-Fc and CD47 monoclonal antibodies. The bispecific antibody was shown to be directed against CD20⁺/CD47⁺The double positive tumor cell has more specific target combination and killing effect, especially CD47⁺The single positive cell (such as red blood cell RBC) shows no binding or weak binding, human Peripheral Blood Mononuclear Cell (PBMC) shows no binding or weak binding, no side effect of ADCC/CDC and other immune killing on normal cells such as RBC and PBMC, and no side effect of agglutination on red blood cell.

Description

CD20/CD47 bispecific antibody and application

Technical Field

The invention relates to the field of biomedicine, in particular to the technical field of antibody engineering and the field of immunity, and more particularly relates to a CD20/CD47 bispecific antibody and application thereof.

Background

CD20 is expressed on the surface of B cells at various stages of developmental differentiation except plasma cells (immunoglobulin-secreting B cells), and plays an important regulatory role in B cell proliferation and differentiation by acting directly on B cells through the regulation of transmembrane calcium ion flux. The CD20 antigen is a B cell differentiation antigen, located only on pre-B cells and mature B cells, and is expressed in more than 95% of B cell lymphomas, but not in hematopoietic stem cells, plasma cells and other normal tissues. Aiming at the relative specificity of the CD20 antigen expressed in B cell lymphoma, a plurality of CD20 antibody medicaments are approved to be on the market, Rituxan developed under Roche always occupies the front of the antibody medicament market in recent years, and has good treatment effect on the lymphoma. Nevertheless, there is still a proportion of patients with drug relapse, where the mechanism is not yet clear.

CD47, also known as Integrin Associated Protein (IAP), is widely expressed on the cell surface and interacts with Signal regulatory Protein α (Signal regulatory Protein α α), Thrombospondin (Thrombospondin-1, TSP1) and Integrin (Integrins) to mediate a series of responses such as apoptosis, proliferation and immunity, CD47 was first identified as a tumor antigen of human ovarian cancer in the 80 th 19 th century, and CD47 was found to be expressed in various types of human tumors including acute myelocytic leukemia (AM L), chronic myelocytic leukemia (CM L), acute lymphocytic leukemia (A α), non-hodgkin lymphoma (NH L), Multiple Myeloma (MM), Bladder Cancer (BC) and other solid tumors, and various studies have shown that, at the cellular level, the effect of a single-function inhibitor of SIRP 6-47-related Protein (IAP) in vivo, inhibition of growth of CD 19-19 th lung cancer cells by a single-receptor agonist antibody, a single-ligand polypeptide ligand peptide (CTP) and a single-5-ligand peptide, and a single-peptide antibody against SIRP 9, which is effective in vivo in mice treated with a single-mouse anti-macrophage receptor agonist antibody, such as a single-11-5, a single-agonist antibody.

The CD47/CD20 is highly expressed in lymphoma cells, and a single-drug CD47 inhibitor or a CD20 inhibitor has clinical treatment value, but has certain side effects, particularly the binding of a CD47 antibody and erythrocytes causes erythrocyte agglutination. While the CD47 ligand SIRPa can obviously reduce the erythrocyte combination, non-specific combination still exists, and if the lymphoma treatment is carried out by targeting CD20 and CD47 at the same time, the specific killing of cancer cells can be improved, and the side effect can be reduced by improving the affinity. A bispecific antibody targeting CD47 and CD20 selective antibodies and lipids dual anti-expression lymphomas cells, CD20-selective inhibition of CD47-SIRPa "don't eat" signaling with a bispecific antibody-derivative activity of daratumab, and both indicate that the development of CD20-CD47 dual-target multifunctional protein has practical feasibility.

At present, no therapeutic recombinant protein which aims at CD47 and CD20 dual targets is on the market. The inhibition of CD47 target can stimulate the immunity of macrophage, and the new therapeutic mechanism is found after B cell and T cell treat tumor, and has wide application foreground. The CD47/CD20 has high expression in B, T lymphoma cells, so that the combined therapy aiming at two targets has potential clinical application value.

The invention patents of patent application numbers 201710151979.6 and WO2018/166507 disclose a novel recombinant bifunctional fusion protein, the recombinant bifunctional fusion protein (CD20mAb-SD1) provided by the invention is formed by directly connecting an anti-human CD20 monoclonal antibody and a first functional region (SIRPaD1) at the outer end of a human SIRPa membrane in series through the C end of Fc (without a linker), and the activation of ADCC/CDC/ADCP killing activity of immune cells on tumor cells is realized by simultaneously targeting CD20/CD 47. And another recombinant bifunctional fusion protein (SD1-CD20mAb), wherein the C end of the first functional region (SIRPaD1) at the outer end of the human SIRPa membrane is connected with the N end of the heavy chain of the CD20 antibody through GGSGGGGS.

In the patent of application numbers 201710151979.6 and WO2018/166507, SIRPaD1 carries out mutation of N80A, and SIRPaD1 and a CD20 antibody in CD20mAb-SD1 are directly connected in series through the C end of Fc, so that the ADCC effect on tumor cells is not obviously enhanced compared with that of CD20 monoclonal antibody. The CDC effect on tumor cells is reduced compared with that of a CD20 monoclonal antibody, and a certain steric hindrance exists on the simultaneous targeting of CD20/CD47 and a certain block exists on the maximum activation of immune functions. In addition, the N80A mutation has no obvious change effect on the blockade of SIRPa and CD47, and the patent CN201811187563.0 carries out experimental demonstration.

Disclosure of Invention

The invention aims to provide a CD20/CD47 bispecific antibody and a preparation method and application thereof, wherein the bispecific antibody is constructed by human SIRPa and CD20 antibodies in different structural modes, the bispecific antibody (9020-1RP) in one preferred structure has remarkably enhanced ADCC/CDC activity and ADCP function, Fc is subjected to combined mutation in the structure, and the bispecific antibody (9020-T1RP) in another Fc mutant form is obtained, and the 9020-T1RP has different degrees of enhancement in ADCC or CDC. The preferred structure in the present invention refers to the N-terminus of the heavy chains of human SIRPaD1 and CD20 antibodies, which are constructed as symmetric bispecific antibodies by linking peptides.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention discloses a CD20/CD47 bispecific antibody, which comprises a CD20 antibody of anti-CD 20 and a first structural domain SIRPaD1 of a human SIRPa extracellular segment, wherein the SIRPaD1 is connected to the N end of a heavy chain of a CD20 antibody through a connecting peptide.

Preferably, the CD20 antibody is of the IgG1 subtype.

Preferably, the CD20 antibody heavy chain Fc comprises IgG1 wild-type Fc-WT (SEQ ID No.9) (SEQ ID No.10) or IgG1 combined mutant ADCC-enhancing Fc-MT (SEQ ID No.11) (SEQ ID No.12), corresponding bispecific antibodies 9020-1RP, and 9020-T1RP, respectively; more preferably ADCC-enhanced, i.e., 9020-T1 RP.

Preferably, the human SIRPa is wild type V2(CAA 71403.130-504 aa GeneBank) and the human SIRPa extracellular segment is the first structural domain SIRPaD1(SEQ ID NO.1) of the human SIRPa extracellular segment.

Preferably, the linker peptide has the sequence (GGGGS) × 3.

Preferably, the structure of the CD20/CD47 bispecific antibody is a symmetric homodimer.

The invention also provides an amino acid encoding the CD20/CD47 bispecific antibody.

The invention also provides a polynucleotide encoding the amino acid.

The invention also provides an expression vector, which comprises the polynucleotide.

The invention also provides a host cell comprising the expression vector.

The invention also provides application of the CD20/CD47 bispecific antibody in preparation of a medicine for treating tumor diseases, namely CD20/CD47 overexpression.

Preferably, the neoplastic disorder includes at least one of a hematological neoplastic disorder including at least one of acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, mixed leukemia, non-hodgkin's lymphoma.

The invention has the following beneficial effects:

the invention discloses a group of bispecific antibodies of CD20/CD47, wherein a first functional region SIRPaD1 at the outer end of a CD47 preferably human SIRPa membrane is connected with the N end of a CD20 antibody heavy chain through a connecting peptide (linker) to form the bispecific antibody with a symmetrical homodimer structure. The heavy chain Fc of the CD20 antibody is optionally IgG1 wild type or mutant, preferably IgG1 combined with mutant ADCC enhancement to obtain the bispecific antibody 9020-T1 RP.

The invention evaluates the targeting specificity of the CD20/CD47 bispecific antibody at protein level and cell level, and proves that 9020-T1RP can simultaneously target CD20 and CD47 and can target a plurality of CD20 antibodies⁺/CD47⁺The tumor cell model of (1) is effective in activating antibody-mediated cell-dependent cytotoxicity (ADCC) and antibody complement-dependent complement killing (CDC) and activating phagocytic Activity (ADCP) of macrophages on tumor cells by blocking the binding of CD47 and its ligand SIRPa. The 9020-T1RP has stronger ADCC/CDC effect compared with the CD20 monoclonal antibody or other bispecific antibody, and has enhanced or equivalent ADCP effect compared with the CD47 fusion protein inhibitor or the CD47 monoclonal antibody or other bispecific antibody. Specifically, in the double-antibody structure, the combination of 9020-T1RP and CD47 is reduced by 10-50 times, and the side effect caused by CD47 off-target is obviously reduced.

The invention simultaneously carries out safety evaluation, verifies the off-target side effect, and proves that 9020-T1RP aims at CD20⁺CD47⁺Has higher specificity to single positive cell, especially CD47⁺Single positive cells (e.g., red blood cells RBC) show no or weak binding and Peripheral Blood Mononuclear Cells (PBMC) in humansIt has no binding or weak binding, no side effect of ADCC/CDC/ADCP on normal cells such as RBC and PBMC, and no coagulation on erythrocytes. In addition, CD20⁺/CD47⁺The tumor cells and Red Blood Cells (RBC) are mixed in different ratios, the targeting specificity of the double antibodies is detected, and the research finds that 9020-T1RP shows more specific binding to CD20 than the CD47 monoclonal antibody, SIRPa fusion protein, CD20 monoclonal antibody or other structural forms of double antibodies, no matter in the binding strength or the binding ratio⁺/CD47⁺A tumor cell.

Drawings

FIG. 1 is the structure diagram of 9020-T1RP and other forms of double antibodies 9020-1RP/9020-2RP/9020-6 RP.

FIG. 2 is an SDS-PAGE picture of the expression purification of bispecific antibodies according to the examples of the present invention.

FIG. 3 shows the result of analyzing the purity of 9020-T1RP by SEC-HP L C of the present invention.

FIG. 4 shows the results of the experiment for binding bispecific antibody CD20/CD47 in Experimental example 1 of the present invention, FIG. 4A shows the results of the experiment for measuring the amount of binding agent between double antibody and CD47 (E L ISA), and FIG. 4B shows the results of the experiment for measuring the amount of binding agent between double antibody and CD20 (FACS).

FIG. 5 shows the results of detecting the expression levels of CD20/CD47 on the cell surfaces of various tumor cell lines in Experimental example 2 of the present invention, FIG. 5A shows the results of detecting the expression levels of CD20/CD47 in SHP-77, CHO-K1-Cyno CD47, Ramous, H L60, Raji, Daudi, CCRF-CEM, CHO-K1-humanCD47, DU145, A431 and RBC, and FIG. 5B shows the results of detecting the expression levels of CD20/CD47 in CHO-K1, PC-3, MDA-MB-231, U87, RBC and JuRKAT.

FIG. 6 shows the results of bispecific antibody target binding agent effect test (FACS) in Experimental example 3 of the present invention, in which FIG. 6A shows the binding activity to Ramous cancer cell line expressing human CD20/CD47, FIG. 6B shows the binding activity to Raji cancer cell line expressing human CD20/CD47, and FIG. 6C shows the binding activity to Daudi cancer cell line expressing human CD20/CD 47.

FIG. 7 shows the results of the bispecific antibody target binding specificity experiment-I of Experimental example 4 of the present invention, FIG. 7A shows the binding of the antibody on CHO-K1/hCD47 and CHO-K1/hCD20-CD47 cells, and FIG. 7B shows the binding of the antibody on CHO-K1/hCD20 and CHO-K1/hCD20-CD47 cells.

Fig. 8 is the results of the bispecific antibody target binding specificity experiment-II of experimental example 5 of the present invention, fig. 8A is the binding of the antibody on Raji and RBC, respectively, fig. 8B is the binding of the antibody on Raji in Raji mixed RBC, and fig. 8C is the binding of the antibody on RBC in Raji mixed RBC.

FIG. 9 shows the results of the effect of the amount of binding agent of bispecific antibody and Red Blood Cells (RBC) in Experimental example 6 of the present invention, and FIG. 9B shows the effect of the amount of binding agent of bispecific antibody and red blood cells in further examination of FIG. 9A.

FIG. 10 is a graph showing the results of experiments on the binding of the bispecific antibody 9020-T1RP Fc terminal to the receptor in Experimental example 7 of the present invention, FIG. 10A is the results of experiments on the binding of the antibody to CD16a, and FIG. 10B is the results of experiments on the binding of the antibody to CD 32B.

FIG. 11 is a graph showing the effect of bispecific antibody of the present invention on erythrocyte agglutination.

FIG. 12 shows the ADCC activity of the bispecific antibody against tumor cells in Experimental example 9 of the present invention, FIG. 12A shows the ADCC activity of the bispecific antibody against tumor cells Daudi, FIG. 12B shows the ADCC activity of the bispecific antibody against tumor cells Ramous, FIG. 12C shows the ADCC activity of the bispecific antibody against tumor cells Raji, and FIGS. 12D and 12E show the ADCC activity of the bispecific antibody against tumor cells Ramous and Daudi as further experimental demonstration results.

FIG. 13 shows ADCC activities of the bispecific antibody on erythrocytes and PBMCs in Experimental example 10 of the present invention, FIG. 13A shows ADCC activities of the bispecific antibody on erythrocytes, FIG. 13B shows ADCC activities of the bispecific antibody on PBMCs, and FIG. 13C shows further demonstration results of ADCC activities of the bispecific antibody on erythrocytes.

FIG. 14 shows CDC activity of bispecific antibody against tumor cells, FIG. 14A shows CDC activity of bispecific antibody against Daudi, FIG. 14B shows CDC activity of bispecific antibody against Ramous, and FIG. 14C shows CDC activity of bispecific antibody against Raji, in Experimental example 11.

FIG. 15 shows CDC activities of bispecific antibodies against erythrocytes and PBMCs in Experimental example 12 of the present invention, FIG. 15A shows CDC activities of bispecific antibodies against erythrocytes, and FIG. 15B shows CDC activities of bispecific antibodies against PBMCs.

FIG. 16 is activation of phagocytic activity of macrophage by bispecific Antibody (ADCP) of Experimental example 13 of the present invention, FIG. 16A is activation of phagocytic activity of macrophage by bispecific antibody in Daudi cell, and FIG. 16B is activation of phagocytic activity of macrophage by bispecific antibody in Raji.

Detailed Description

The following examples are further illustrative of the present invention as to the technical content of the present invention, but the essence of the present invention is not limited to the following examples, and one of ordinary skill in the art can and should understand that any simple changes or substitutions based on the essence of the present invention should fall within the protection scope of the present invention.

The invention constructs bispecific antibodies based on CD20 antibody and SIRPa extracellular domain first (SIRPaD1), wherein SIRPaD1 can be connected with CD20 antibody by different ways, such as that SIRPaD1 can be connected to the N-terminal of CD20 antibody heavy chain, or connected to the N-terminal of CD20 antibody light chain, or connected to the C-terminal of CD20 antibody light chain through connecting peptide, corresponding to bispecific antibodies 9020-1RP, 9020-2RP and 9020-6 RP.

The research of the invention evaluates the binding capacity of bispecific antibodies 9020-1RP, 9020-2RP and 9020-6RP and CD20/CD47 on the level of protein and cells, and selects a stable cell line and a tumor cell line which are expressed by single factors of CD20 and CD47, a stable cell line and a tumor cell line which are co-expressed by CD20/CD47 and normal human cells which are expressed by single factors of CD47 on the level of cells.

The present invention continues to evaluate the differences in the ADCC/CDC function of bispecific antibodies 9020-1RP, 9020-2RP and 9020-6RP to tumor cells, PBMC/RBC as compared to CD20 antibody and other forms of bispecific antibodies, and the differences in ADCP macrophage immune activation as compared to CD47 monoclonal antibody, SIRPaD1 fusion protein and other forms of bispecific antibodies.

Research proves that the binding between 9020-1RP and CD47 is reduced by 10-50 times in the aspects of protein and cell level, the binding capability with CD20 can be continuously maintained, and the binding capability to CD20 is improved⁺CD47⁺The targeting specificity of the tumor cells reduces the off-targeting property of the combination with normal cells; and the ADCC aspect is obviously better than that of the CD20 monoclonal antibody or other bispecific antibody, and the ADCP aspect is better than that of the CD47 monoclonal antibody or fusion protein or other bispecific antibody.

The invention carries out continuous research on the basis of 9020-1RP, in particular to combined mutation on SIRPaD1, referring to a patent 201811187563.0 (application number), wherein SIRPaD1 is replaced by CD002 which corresponds to a bispecific antibody 9020-1 RP-C1; in addition, the research on the linker replaces the original linker with an antibody heavy chain hinge region ASTKG, the corresponding bispecific antibody is 9020-1RP-C2, and the research finds that the combination of 9020-1RP-C1/C2 and CD47 is remarkably enhanced, and the effect of reducing the side effect on CD20 is achieved⁺/CD47⁺The specificity of the tumor cells, 9020-1RP-C2 also generates certain blood coagulation; it was shown that the binding affinity of CD47 and the selection of linker had a significant impact on the safety of bispecific antibodies.

The present invention has continued to perform Fc function studies on 9020-1RP, and the human CD16 protein has higher affinity to antibody Fc than CD16-158F at position 158 due to the presence of valine (V) and phenylalanine (F), i.e., CD16-158V and CD 16-158F. CD16-158V, clinical data statistics show that patients with CD16-158V/V homozygotes have better therapeutic effect on Rituximab and Transtuzumab than patients with CD16-158F/F homozygotes or V/F heterozygotes.

The invention also provides an amino acid sequence and a nucleotide sequence for encoding the bispecific antibody, an expression vector for expressing the bispecific antibody and a preparation method.

The invention optimizes the structure of the bispecific antibody, evaluates the targeting specificity of CD47/CD20 under different structures (figure 1), and shows that 9020-T1RP can be used for detecting the bispecific antibodyEffectively improve CD20⁺/CD47⁺The specificity of tumor cells, the reduction of safety risk, the remarkable enhancement of ADCC/CDC function, and the certain improvement or maintenance of considerable level of ADCP.

A further optimization scheme of the invention is to select SIRPaD1 and compare the difference of the wild type SIRPaD1 and the mutant CD002 on the specificity of the bispecific antibody in tumor targeting.

The amino acid sequence of the wild type SIRPaD1 in the embodiment of the invention is as follows (SEQ ID NO. 1):

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

the corresponding nucleic acid sequence is as follows (SEQ ID NO. 2):

GAAGAGGAGCTGCAGGTCATCCAGCCCGACAAGTCTGTGTCCGTGGCAGCAGGAGAAAGCGCTATCCTGCATTGCACCGTGACCAGCCTGATTCCCGTGGGACCAATCCAGTGGTTCAGAGGAGCCGGACCAGCCAGAGAGCTGATCTACAACCAGAAGGAGGGCCACTTCCCCAGAGTGACAACAGTGTCCGAGAGCACCAAGCGGGAGAACATGGACTTCAGCATCAGCATCAGCAACATCACACCAGCCGACGCCGGCACATACTATTGCGTGAAGTTCCGGAAGGGCAGCCCAGATACCGAGTTCAAGAGCGGAGCCGGAACAGAGCTGAGCGTGAGAGCCAAGCCTAGC

the CD002 amino acid sequence in the examples of the present invention is as follows (SEQ ID NO. 3):

EEELQVIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARELIYNQRQGPFPRVTTVSETTKRENMDFSISISAITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS

the corresponding nucleic acid sequence is as follows (SEQ ID NO. 4):

GAAGAGGAGCTGCAGGTCATCCAGCCCGACAAGTCTGTGTCCGTGGCAGCAGGAGAAAGCGCTATCCTGCATTGCACCGTGACCAGCCTGTTTCCCGTGGGACCAATCCAGTGGTTCAGAGGAGCCGGACCAGCCAGAGAGCTGATCTACAACCAGAGGCAGGGCCCTTTCCCTAGAGTGACAACCGTGTCCGAGACCACCAAGAGGGAGAACATGGACTTCAGCATCAGCATCAGCGCCATCACACCAGCCGACGCCGGCACATACTATTGCGTGAAGTTCCGGAAGGGCAGCCCAGATACCGAGTTCAAGAGCGGAGCCGGAACAGAGCTGAGCGTGAGAGCCAAGCCTAGC

the further optimization scheme of the invention is to research the linker of the bispecific antibody, compare whether different linkers have influence on the structure of the bispecific antibody, and evaluate the tumor specificity and safety brought by the influence.

The linker sequence is (GGGGS) 3 or ASTKG/P.

The invention further optimizes the CD20 antibody in the bispecific antibody, and the preferred embodiment is rituximab (Ritxuximab) in candidate antibodies of rituximab (Ritxuximab), atolizumab (Obinuzumab), and Ofatumumab (Ofatumumab).

In a preferred embodiment of the invention, the variable region of the CD20 antibody heavy chain (Rituximab VH) has the following sequence (SEQ ID NO. 5):

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA

the corresponding nucleic acid sequence is as follows (SEQ ID NO. 6):

CAGGTGCAGCTGCAGCAGCCAGGAGCAGAACTGGTGAAGCCAGGCGCCAGCGTGAAGATGTCTTGCAAAGCCAGCGGCTACACCTTCACCAGCTACAACATGCATTGGGTGAAGCAGACCCCAGGAAGAGGCCTGGAGTGGATCGGTGCCATCTACCCCGGCAACGGCGACACCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACAGCCGATAAGAGCAGCAGCACCGCCTACATGCAGCTGTCTAGCCTGACCAGCGAGGATAGCGCCGTGTACTATTGCGCCAGGAGCACCTACTACGGCGGCGATTGGTACTTCAACGTCTGGGGAGCCGGAACAACAGTGACAGTGTCCGCA

in a preferred embodiment of the invention, the variable region of the light chain of the CD20 antibody (Rituximab V L) has the following sequence (SEQ ID NO. 7):

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK

the corresponding nucleic acid sequence is as follows (SEQ ID NO. 8):

CAGATCGTGCTGTCTCAGAGCCCAGCCATTCTGAGCGCTTCTCCAGGCGAGAAGGTCACCATGACTTGCAGAGCCAGCAGCAGCGTGTCCTACATCCATTGGTTCCAGCAGAAGCCAGGAAGCAGCCCTAAGCCTTGGATCTACGCCACCAGCAACCTGGCTAGCGGAGTGCCAGTGAGATTCAGCGGAAGCGGAAGCGGAACCAGCTACAGCCTGACCATCAGCAGAGTGGAGGCCGAAGACGCCGCTACATACTACTGCCAGCAGTGGACCAGCAACCCTCCTACCTTTGGCGGAGGCACCAAGCTGGAGATCAAG

the Fc (IgG1-WT) amino acid sequence of the invention is as follows (SEQ ID NO. 9):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

the corresponding nucleic acid sequence is as follows (SEQ ID NO. 10):

GCCAGCACCAAGGGACCTAGCGTGTTTCCTCTGGCCCCTTCTAGCAAGAGCACAAGCGGAGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTTCCCGAGCCCGTGACCGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGACTGTATAGCCTGAGCAGCGTGGTGACCGTGCCTTCTTCTTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGCCGAGCCCAAGTCTTGCGACAAGACCCACACTTGCCCCCCTTGTCCAGCTCCAGAACTCCTGGGAGGACCTAGCGTGTTCCTGTTCCCTCCCAAGCCTAAGGACACCCTGATGATCAGCCGGACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCCCACGAAGACCCCGAGGTCAAGTTCAATTGGTACGTGGACGGAGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCAGCCCCTATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAACCTCAGGTGTACACCCTGCCCCCTTCTAGAGACGAGCTGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAATCTAACGGTCAGCCAGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCCGCTGGCAGCAGGGCAACGTGTTCTCTTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCAGGAAAG

the scheme of the invention will be illustrated with reference to the following examples.

Example construction of bispecific antibody (double antibody) expression vector and preparation of protein expression

1. Construction of expression vectors

Human SIRP α possesses two pairs of wild type V1 or V2, the amino acid sequence 27-504 constitutes mature V1 (NP-542970.1 NCBI), V2 and V1 have 13 amino acid difference, the amino acid 30-504 constitutes mature V2(CAA71403.1GeneBank), the invention selects the amino acid sequence 31-148(SEQ ID NO:1) of wild type 2, V2, SIRP α D1.

The SIRP α D1 is connected with the N end of the heavy chain or the N end of the light chain of the CD20 antibody or the C end of the light chain through L inker, gene expression vectors 9020-1RP, 9020-2RP and 9020-6RP are constructed, the SIRPaD1 mutant CD002 is constructed to be a bispecific antibody corresponding to 9020-1RP-1C1, 9020-1RP is corresponding to 9020-1RP-1C2 after L inker is replaced by 9020-1RP, and the Fc mutation of the 9020-1RP is corresponding to 9020-T1 RP.

The bispecific antibody consists of 1 heavy chain and 1 light chain. 9020-1RP, 9020-T1RP, 9020-1RP-1C1 and 9020-1RP-1C2 are composed of 1 double heavy chain and a CD20 antibody light chain, and 9020-2RP and 9020-6RP are composed of 1 double light chain and a CD20 antibody heavy chain. See fig. 1.

And adding an optimized signal peptide into the N end of the amino acid of the bispecific antibody or the recombinant protein for secretion expression, performing codon optimization on the amino acid of the bispecific antibody or the recombinant protein, synthesizing the whole gene, adding a Kozak sequence GCCGCCACC into the 5' end of the nucleotide, adding EcoRI/HindIII enzyme cutting sites of pcDNA3.4 into the two ends of the nucleotide, and connecting the synthesized gene to the pcDNA3.4 after enzyme cutting.

And carrying out target gene sequencing on the constructed expression vector.

The amino acid and nucleotide composition of each fusion protein is as follows:

the 9020-1RP heavy chain has 1752 nucleotides in total (SEQ ID NO:15) and encodes 584 amino acids (SEQ ID NO: 14); 9020-2RP light chain has a total of 1038 nucleotides (SEQ ID NO:17) encoding 346 amino acids (SEQ ID NO: 16); 9020-6RP light chain has a total of 1038 nucleotides (SEQ ID NO:19) encoding 346 amino acids (SEQ ID NO: 18); the 9020-1RP-1C1 heavy chain has 1752 nucleotides (SEQ ID NO:21) in total and encodes 584 amino acids (SEQ ID NO: 20); the 9020-1RP-1C2 heavy chain has 1722 nucleotides in total (SEQ ID NO:23) and encodes 574 amino acids (SEQ ID NO: 22); the 9020-T1RP heavy chain has a total of 1752 nucleotides (SEQ ID NO:25) encoding 584 amino acids (SEQ ID NO: 24).

2. Transfection

9020-1RP, 9020-2RP, 9020-6RP, 9020-1RP-1C1, 9020-1RP-1C2, 9020-T1RP, an antibody Rituximab (SEQ ID NO:5 and SEQ ID NO:7) and CD20mAb-SD1(SEQ ID NO:26) in the patent with the application number 201710151979.6 are amplified and extracted by escherichia coli, endotoxin is removed, and the plasmid is prepared by the following steps: Expi-293F cells were transfected at 1 μ g/ml medium. Wherein, the 9020-1RP-1C1, the 9020-1RP-1C2 and the 9020-T1RP double-anti-heavy-chain plasmids are cotransfected with the CD20 antibody light-chain plasmid, and the 9020-2RP, the 9020-6RP double-anti-light-chain plasmids are cotransfected with the CD20 antibody heavy-chain plasmid. The same mutation as 9020-T1RP in the Fc selection of Rituximab was defined as Rituximab-TH as a reference, and the heavy chain gene encodes 451 amino acids (SEQ ID NO: 13). CD20mAb-SD1 (heavy chain SEQ ID NO:26) was also expressed by two plasmid co-transfection.

The transfection reagent used was Expifactamine 293Transfectionkit (Theromfisher, L ot #: A14524) with a cell density of 25 × 10 at transfection⁵cells/ml, 16-18h after transfection, adding expression enhancers Enhancer1 and Enhancer2, and collecting cell supernatant 5 days after transfection.

3. Protein expression purification

Purifying with ProteinA, centrifuging at 4 deg.C at 10000rpm/min for 30min to remove cell debris, balancing 10 column volumes with balancing solution (0.02MPB, 0.15MNaCl, pH7.0), flowing the supernatant through the column at 2ml/min, washing 5 column volumes with the balancing solution, eluting with eluent (0.02MPB, 0.15MNaCl, pH3.0), and dripping the eluent into a collecting tube containing neutralizing solution (1MTris, pH 9.0). The protein eluate was collected, concentrated by ultrafiltration using an ultrafiltration tube (MilliporerUFC 903096)4000G and buffer was replaced with PBS (HyCloneSH30256.01), and stored at-20 ℃ after SDS-PAGE detection. Endotoxin removal, filter sterilization, SDS-PAGE purity determination, as shown in FIG. 2, overall the actual molecular weight close to the theoretical molecular weight. Meanwhile, the bispecific antibody related to the invention does not influence the expression in host cells due to different structures.

4. Protein purity analysis

1mg/ml of bispecific antibody was analyzed for SEC-HP L C purity, with multimer content less than 2% and purity of protein of interest > 96%, as shown in FIG. 3.

Experimental examples bispecific Performance and functional analysis

EXPERIMENTAL EXAMPLE 1 bispecific antibody CD47/CD20 binding assay

1. Binding of diabodies to CD47 was detected by the E L ISA:

hCD47-his (Cat # CD7-H5227, L ot # C56P1-737F1-FA) was coated at 1. mu.g/ml, the coating buffer selected from PBS (HyClone L ot: AC13298279), 100. mu.l/well, coating at room temperature (25 ℃) for 16-18H, washing the plate with TBST for 2 times, blocking with PBS + 3% BSA at 200. mu.l/well, blocking at room temperature (25 ℃) for 16-18H, washing the plate with TBST for 1 time, draining, and drying at 37 ℃ for 2 hours.

Bispecific antibody serial dilutions: 9020-1RP, 9020-2RP, 9020-6RP, 9020-1RP-1C1, 9020-1RP-1C2, 9020-T1RP, CD20mAb-SD1 and the antibody Rituximab were formulated at 330. mu.l of 10. mu.g/ml, with the recombinant protein SIRPaD1, CD002, at 100. mu.g/ml being the first gradient and diluted in 3-fold gradients, for example, the second gradient being 80. mu.l of the first gradient added to 160. mu.l of PBS, and so on for a total of 8 gradient concentrations.

Incubating at 37 ℃ for 1 hour, washing the plate by PBST of an automatic plate washing machine for 3 times, adding 100 mu l of goat anti-human HRP secondary antibody (abcam L ot #: ab98624) diluted at 1: 20000 into each hole, incubating at 37 ℃ for 45 minutes, washing the plate by the automatic plate washing machine for 3 times after the incubation is finished, taking the ELISA plate to the absorbent paper after the last washing is finished, covering the absorbent paper with clean residual liquid, adding 100 mu l of TMB color developing solution into each hole, reacting for 3-5 minutes in a dark place, and adding 50 mu l of 1% H into each hole₂SO₄The reaction was terminated. Setting the light absorption values of reading by an MD (I3X) microplate reader to be 450nm and 630nm, and storing data after automatically reading the values. The results are shown in FIG. 4A. The 9020-1RP, 9020-2RP and 9020-6RP have 10-20 times of reduction in affinity of SIRPaD1 and CD 47; 9020-1RP-1C1 showed a 5-fold decrease in affinity for CD47 relative to CD 002; 9020-1RP-1C2 showed a 10-fold improvement in affinity between 9020-1RP and CD 47.

The results show that the SIRPaD1 can significantly reduce the binding capacity to CD47 no matter the N-terminal of the heavy chain of a CD20 antibody, the N-terminal of the light chain of a CD20 antibody or the C-terminal of a CD20 antibody, so that the potential risk brought by CD47 can be reduced by reducing the affinity of CD47 through the design of a double antibody, and the binding of SIRPaD1 to CD47 in the double antibody can be influenced by the change of L inker.

2. Binding of double antibodies to CD20 was detected by FACS:

the binding activity of 9020-1RP, 9020-2RP, 9020-6RP, 9020-1RP-1C1, 9020-1RP-1C2, 9020-T1RP, the antibody Rituximab, the recombinant protein SIRPaD1, CD002 and a stable cell strain CHO-K1/CD20 expressing human CD20 was determined by a flow cytometer (BDFACSCELESICelestaCellAnalyzer).

The specific implementation is as follows:

after the cells listed were digested separately (suspension cells were not digested), centrifuged at room temperature at 1000rpm for 5mins, supernatant was discarded, washed with PBS, and PBS was resuspended in a flow tube with cell concentration adjusted to 1 × 10⁶cells/ml, PBS volume of 250 μ L per tube, reaction concentration of bispecific antibody 7 gradients, 100 μ g/ml 3-fold gradient dilution, PBS washing 3 times after 1h incubation at room temperature (1500rpm, 5mins), adding 100 μ L secondary antibody Goatanti-humann IgG/Alexa647(Bioss L ot: AE041526) to each tube sample, adding 1m L PBS washing 3 times after 30mins incubation at room temperature in the dark place (1500rpm, 5mins), adding 500 μ L PBS to each tube to resuspend cells, and detecting on-machine the results as shown in FIG. 4B, the binding of 9020-2RP to CD20 is significantly reduced, the binding of other double antibodies to CD20 is slightly reduced compared with Rituximab, indicating that the design of double antibodies not only changes the binding to CD47 but also affects the binding to CD20, and the preferred structure 20-T1RP of the present invention ensures the binding to CD20 while reducing the binding to CD 47.

Experimental example 2 expression level of CD47/CD20 on cell surface

In the target specific detection, reasonable cell strains can be selected as test objects. We performed cell surface CD47 and CD20 expression level detection on multiple tumor cell lines.

The CD20/CD47 expression levels of Raji, Daudi, Ramous, H L60, MDA-MB231, SHP-77 and other hematological and solid tumor cell lines and RBC were determined by flow cytometry (BDFACSCELESTaCellAnalyzer). The reagents and instruments used in this experimental example are shown in Table 1.

TABLE 1 list of reagents and instruments used in Experimental example 2 of the present invention

The specific implementation is as follows:

1. the cells were observed under a microscope, rounded and clear, and collected under normal conditions, counted, and centrifuged at 1500rpm for 5 min.

2. The supernatant was discarded, and after each cell was resuspended in PBS (Hyclone, SH30256.01), the cells were added to a 96-well plate at 25 × 10 per well⁴Cells, 50 μ L per well.

3. At the same time, 10. mu.g/m L and 50. mu. L of corresponding antibody (Rituximab, H5F9-G4, HIgG) was added to each well, and the wells were incubated for 30min at room temperature in the absence of light.

4. After completion of incubation, the cells were washed 3 times by centrifugation at 1500rpm for 5min, and Alexa Fluor 647 AffinipurGoat Anti-Human IgG + IgM (H + L) (Jackson, 109605044) was added.

5. After the incubation was completed, the supernatant was discarded after 3 washes by centrifugation at 1500rpm for 5min, 100. mu. L PBS was added to each well, and the data was analyzed by flow cytometry (Beckman, cytoflex) and plotted using GraphPad Prism.

The detection results of CD20/CD47 expression levels of SHP-77, CHO-K1-Cyno CD47, Ramous, H L, Raji, Daudi, CCRF-CEM, CHO-K1-humancD47, DU145, A431 and RBC are shown in FIG. 5A, and the detection results of CD20/CD47 expression levels of CHO-K1, PC-3, MDA-MB-231, U87, RBC and Jurkat are shown in FIG. 5B. the detection results show that CD20 is mainly expressed in blood tumor cells of Raji, Daudi, Ramous, etc., and CD47 is highly expressed in a plurality of blood tumor and solid tumor cells, especially in tumor cells of PC-3, MDA-MB-231, Jurkat, Daudi, etc., the expression level is significantly higher than the expression level of CD47 in RBC, and the co-expression of CD 20/Raudi 47 is mainly present in tumor cells of Raji, Radumus, Raurus, etc., and the expression level of CD20 is higher than the expression level of CD47 in RBC, etc.

The results suggest that the design of the CD20/CD47 double antibody is mainly demonstrated experimentally by using blood tumor models such as Raji, Daudi, Ramous and the like. Due to the relative specificity of CD20 expression, the high expression of CD47 in RBC, the targeting of double antibody should be inclined to CD20, balancing the efficacy and safety of CD 47.

EXAMPLE 3 bispecific antibody target binding assay (FACS)

The binding activity of 9020-1RP, 9020-2RP, 9020-6RP, 9020-1RP-1C1, 9020-1RP-1C2 and 9020-T1RP as well as an antibody Rituximab, a recombinant protein SIRPaD1 and CD002 to cancer cell lines expressing human CD20/CD47 tumor cell strains Raji, Daudi, Ramous and the like is measured by using a flow cytometer (BDFACSCCelestaCellAnalyzer).

The specific implementation is as follows:

after the cells listed were digested separately (suspension cells were not digested), centrifuged at room temperature at 1000rpm for 5mins, supernatant was discarded, washed with PBS, and PBS was resuspended in a flow tube with cell concentration adjusted to 1 × 10⁶cells/ml, PBS volume per tube 250 u L, reaction concentration of bispecific antibody 7 gradient, 100 u g/ml 3 times gradient dilution, PBS washing 3 times after 1h incubation at room temperature (1500rpm, 5mins), adding 100 u L secondary antibody Goatanti-humann IgG/Alexa647(Bioss L ot: AE041526) to each tube sample, adding 1m L PBS washing 3 times after 30mins incubation at room temperature in the dark place (1500rpm, 5mins), adding 500 u L PBS to each tube to resuspend cells, and detecting on machine.

The binding activity of 9020-1RP, 9020-2RP, 9020-6RP, 9020-1RP-1C1, 9020-1RP-1C2, 9020-T1RP, an antibody Rituximab, a recombinant protein SIRPaD1 and CD002 to a tumor cell line expressing a human CD20/CD47 tumor cell strain Ramous is shown in a figure 6A, the binding activity to a cancer cell line expressing a human CD20/CD47 tumor cell strain Raji is shown in a figure 6B, and the binding activity to a cancer cell line expressing a human CD20/CD47 tumor cell strain Daudi is shown in a figure 6C.

The results show that:

1. in the design of three structures of a double antibody (figure 1), the capacities of 9020-1RP and 9020-6RP targeting CD20/CD47 co-expressed Raji, Daudi, Ramous and other blood tumor cells are obviously stronger than those of 9020-2RP, and the combination of a bispecific antibody and a CD47 targeting result (experimental example 1) can obtain that the N ends of the SIRPaD1 and the CD20 antibody light chain are connected in series, so that the biological activity of the CD20 antibody end is obviously limited.

2. The ability of 9020-1RP-1C1 to target blood tumor cells such as Raji, Daudi, Ramous and the like coexpressed by CD20/CD47 is reduced compared with that of 9020-1RP, while the ability of 9020-1RP-1C1 to target CD47 in experimental example 1 is obviously stronger than that of 9020-1RP, and the result shows that the affinity enhancement of a dual-antibody targeting CD47 end and the structure of 9020-2RP can possibly influence the activity of a dual-antibody targeting CD20 end, namely the reduction of the affinity of the CD47 end in the design of the dual-antibody not only can reduce the potential safety risk, but also can improve the specific targeting ability of the dual-antibody on a CD20/CD47 coexpressed tumor cell line.

3. The binding capacities of 9020-T1RP, 9020-1RP, 9020-6RP, Rituximab and CD20mAb-SD1 to three tumor cells are not obviously different, and 9020-1RP-1C2 is slightly reduced.

EXAMPLE 4 bispecific antibody target binding specificity experiment-I (FACS)

The CD20/CD47 co-expression stable cell line was mixed with the CD20 or CD47 single factor expression stable cell line, and the binding specificity of the bispecific antibody to the CD20/CD47 co-expression cell line was tested. The reagents and instrumentation used in this example are shown in Table 2.

TABLE 2 list of reagents and instruments used in Experimental example 4

The specific implementation is as follows:

1. the CHO-K1/hCD47 or CHO-K1/hCD20 and CHO-K1/hCD20-CD47 cells were observed under a microscope, the cells were collected under normal conditions, counted and centrifuged at 1500rpm for 5 min.

2. The supernatant was discarded, and CHO-K1/hCD47 or CHO-K1/hCD20 cells were resuspended in PBS (Hyclone, SH30256.01), and CFSE (Abcam, Ab113853) was added and the mixture was incubated at 37 ℃ with 5% CO₂Incubate for 15min in the incubator. After the incubation was completed, the cells were centrifuged at 1500rpm for 5min to wash and recounted.

3. CHO-K1/hCD47 or CHO-K1/hCD20 were mixed 1:1 with CHO-K1/hCD20-CD47 cells (2 × 10 per well)⁵cells) 50 mu L per well, and adding corresponding antibodies (9020-1RP, 9020-2RP, 9020-6RP, Rituximab, B6H12, SIRPa, H5F9-G4, HIgG)10 mu G/m L and 50 mu L per well, and incubating for 30min at room temperature in the dark.

Binding of antibody to CHO-K1/hCD47 and CHO-K1/hCD20-CD47 cells As shown in FIG. 7A, 9020-1RP and 9020-6RP specifically target CHO-K1/human CD47/CD20 after mixing of the CD47 single-factor-expressing stable cell line (CHO-K1/human CD47) with the CD20/CD47 co-expressing cell line 1:1(CHO-K1/human CD47/CD 20). 9020-2RP has a significant reduction in binding to CHO-K1/human CD47/CD20, with results similar to SIRPaD 1. The level of the CD47 monoclonal antibody H5F9-G4/B6H12 targeting CHO-K1/human CD47 has no significant difference with the level of the targeting CHO-K1/human CD47/CD20, and the conclusion shows that the improvement of the specificity of the double-antibody targeting CD20/CD47 needs to reduce the affinity of the CD47 end.

Binding of antibody on CHO-K1/hCD20 and CHO-K1/hCD20-CD47 cells As shown in FIG. 7B, when a CD20 single-factor-expressing stable cell line (CHO-K1/human CD20) was mixed with a CD20/CD47 co-expressing cell line 1:1(CHO-K1/human CD47/CD20), 9020-1RP, 9020-6RP, Rituximab had no significant difference in their ability to specifically target CHO-K1/human CD47/CD20, and 9020-2RP, H5F9-G4 and CHO-K1/human CD47/CD20 were relatively weak in binding ability. In addition, the affinity of 9020-1RP, 9020-6RP and Rituximab to CHO-K1/human CD20 is stronger than that of 9020-2RP to CHO-K1/human CD20, and the double-antibody structure of 9020-2RP is proved to limit the biological activity of targeting CD 20.

EXAMPLE 5 bispecific antibody target binding specificity experiment-II (FACS)

CD20/CD47 co-expressed tumor cell line Raji and human Red Blood Cells (RBC) expressed by CD47 single factor are mixed at a ratio of 20:1, and the target specificity of the bispecific antibody on the CD20/CD47 co-expressed tumor cell line is detected.

The specific implementation is as follows:

1. raji cells are observed under a microscope, the cells are collected under a normal state, counted and centrifuged at 1500rpm for 5min, and the cells are round and bright.

2. The supernatant was discarded, Raji cells were resuspended in PBS (Hyclone, SH30256.01), CFSE (Abcam, Ab113853) was added, and the cells were incubated at 37 ℃ in a 5% CO2 incubator for 15 min.

3. After the incubation was completed, the cells were centrifuged at 1500rpm for 5min to wash and recounted.

4. RBC were mixed with Raji cells 20:1 (RBC per well 20 × 10)⁵cells, Raji 1 × 10 per well 10⁵cells) 50 mu L per well, and adding corresponding antibodies (9020-1RP, 9020-T1RP, 9020-1RP-C1, 9020-1RP-C2, CD20mAB-SD1, Rituximab-TH, Rituximab, CD002, SIRPaD1, H5F9-G4, HIgG)10 mu G/m L, 50 mu L per well, and incubating at room temperature in a dark place for 30 min.

5. After completion of incubation, the cells were washed 3 times by centrifugation at 1500rpm for 5min, and Alexa Fluor 647 AffinipurGoat Anti-Human IgG + IgM (H + L) (Jackson, 109605044) was added.

6. After the incubation was completed, the supernatant was discarded after 3 washes by centrifugation at 1500rpm for 5min, 100. mu. L PBS was added to each well, and the data was analyzed by flow cytometry (Beckman, cytoflex) and plotted using GraphPad Prism.

After a CD20/CD47 co-expression cell strain Raji and a CD47 single-factor expression normal human red blood cell RBC are mixed according to a ratio of 20:1, 9020-1RP and 9020-T1RP specifically target the Raji (figure 8A), and 9020-1RP-1C1 and 9020-1RP-1C2 are strongly combined with red blood cells, which shows that the enhancement of the binding capacity of a CD47 end in double antibodies can influence the targeting specificity of the double antibodies; the Raji targeting specificity of 9020-1RP in Raji and RBC mixed cell population is stronger than that of CD20mAb-SD1 (FIG. 8B), and the RBC non-specific binding of 9020-T1RP, 9020-1RP in Raji and RBC mixed cell population is weaker than that of CD20mAb-SD1 (FIG. 8C).

EXAMPLE 6 bispecific antibody erythrocyte binding assay (FACS)

The CD47 mab has the property of binding to Red Blood Cells (RBCs) and for CD47 antibody inhibitors, there is a potential risk of RBC interference in efficacy and tumor off-target, and the efficient design of bispecific antibodies can reduce this risk by reducing the affinity at the CD47 terminus. The safety of the double antibody was assessed by detecting binding of the bispecific antibody to RBC in vitro. The reagents and instrumentation used in this example are shown in Table 3.

TABLE 3 list of reagents and instruments used in Experimental example 6

RBC source: fresh blood was collected from heparin sodium anticoagulation tubes (BD Vacutainer, 367880) on the day, and donors: a company employee.

After the fresh blood was separated by lymphocyte separation medium (Stemcell, 07801), the red blood cells at the bottom were aspirated, and after centrifugation at 1000rpm for 5min for 2 times and at 800rpm for 5min for 1 time, the blood was used for this experiment.

The specific implementation is as follows:

after RBC counting, 2 × 10 per well of 96-well plate⁵Each well was 50 μ L.

2. Simultaneously, each hole is added with corresponding antibody (9020-1RP, 9020-2RP, 9020-6RP, B6H12, 9020-T1RP, 9020-1RP-1C1, 9020-1RP-1C2, CD20mAB-SD1, Rituximab-TH, Rituximab, CD002, SIRPaD1, H5F9-G4 and HIgG)10 mu G/m L, 3 times of gradient dilution, total 8 holes, 50 mu G L and light-shielding incubation for 30min at room temperature.

3. After completion of incubation, the cells were washed 3 times by centrifugation at 1500rpm for 5min, and Alexa Fluor 647 AffinipurGoat Anti-Human IgG + IgM (H + L) (Jackson, 109605044) was added.

4. After the incubation was completed, the supernatant was discarded after 3 washes by centrifugation at 1500rpm for 5min, 100. mu. L PBS was added to each well, and the data was analyzed by flow cytometry (Beckman, cytoflex) and plotted using GraphPad Prism.

As shown in FIG. 9A, either 9020-1RP, 9020-2RP, 9020-6RP did not bind RBC or if bound, when the CD47 partial binding in 9020-1RP was enhanced (9020-1RP-1C1) or L inker changed (9020-1RP-1C2), its bound RBC was significantly enhanced (FIG. 9B). Fc mutant diabody (9020-T1RP) did not bind RBC, weaker than 9020-1RP bound red blood cells.

EXAMPLE 7 bispecific antibody 9020-T1RP binding assay of Fc terminal to receptor (FACS)

Coating E L ISA plate with target proteins RIIIA/CD16a (Catalog: 4325-FC-050) and RIIB/C CD32B/C (Catalog: 1875-CD-050), coating at 1 μ g/ml, standing at 4 deg.C overnight, washing PBST, adding PBST containing 2% BSA, blocking at 37 deg.C for 2h, washing PBST, adding antibodies with different concentrations, reacting at 37 deg.C for 2h, washing PBST, adding goat anti-human IgG-HRP (Catalog: ab98624, abcam), reacting at room temperature for 1h, washing PBST, adding TMB two-components (solution A: cat #: TMB-S-003, solution B: cat #) after washing PBSTTMB-S-003 Chaozhou Yingchuang) method, adding 100 μ l of prepared TMB color developing solution into each well, reacting for 3-5 min in dark place, adding 50 μ l of stop solution 1% H₂SO₄(cat # C1058-500ml solarbio) OD450 was determined. And analyzing the binding capacity of the antibody to be tested to the CD16a and CD32b proteins.

The Fc mutant double antibody or the pre-mutant double antibody can specifically recognize and bind to CD16a and CD32b proteins, and the 9020-T1RP and Rituximab-TH mutant have stronger binding capacity to CD16a protein than the 9020-1RP and Rituximab, and the result is shown in FIG. 10A; 9020-T1RP and the Rituximab-TH mutant bound CD32B/c protein less strongly than the 9020-1RP and Rituximab antibodies, and the results are shown in FIG. 10B.

The increased binding to the ADCC activating receptor CD16a and the decreased binding to the inhibitory receptor CD32b after Fc mutation will lead to increased benefit of the double antibody in patients with CD16-158F homozygote or V/F heterozygote.

EXAMPLE 8 bispecific antibody erythrocyte coagulation assay (Hemagglutination activity)

The hemagglutination activity of 9020-1RP, 9020-2RP, 9020-6RP, 9020-1RP-1C1, 9020-1RP-1C2, 9020-T1RP, SIRPaD1 and CD002 on erythrocytes was measured by using erythrocytes from healthy people, H5F9-G4 is used as a positive control, and 9020-3-991, 9020-3-992 and 9020-3-993 are bispecific antibodies with other structural forms of CD20/CD 47.

The specific implementation is as follows:

collecting whole blood by using anticoagulants such as sodium citrate, putting the whole blood into a 15ml centrifuge tube, supplementing PBS to 15ml, centrifuging at room temperature, centrifuging at 200 × g for 10mins, discarding supernatant, supplementing RBCs to 15m L by PBS, mixing uniformly, centrifuging at room temperature, washing at 1500rpm for 5mins for 3 times, adjusting the concentration of the RBCs to be 2% by using PBS after the last washing (for example, 49ml of PBS is added into 1ml of RBCs, diluting the recombinant protein and the positive antibody according to 2 times of gradient at 200 mu g/ml, totaling 15 concentration gradients, adding 50 mu l of recombinant protein or monoclonal antibody with corresponding concentration into each hole and 50 mu l of RBCs by using a 96-hole round bottom plate, incubating for 2h at room temperature, observing and recording reaction results.

The results (FIG. 11) show that none of 9020-T1RP, 9020-1RP-1C2 caused erythrocyte coagulation, and that H5F9-G4 coagulated blood above the level of 1.25. mu.g. 9020-1RP and 9020-T1RP have higher safety compared with CD47 monoclonal antibody inhibitors such as H5F9-G4 and the like.

EXAMPLE 9 ADCC Activity of bispecific antibodies against tumor cells (FACS)

The ADCC activity of bispecific antibodies against Raji, Ramous, Daudi was evaluated by Jurkat-Fc γ RIIIa-NFAT-L uciferase cells as effector cell assays, wherein 9020-3-991/9020-3-992/9020-3-993 is a diabody with the same structural format as 9020-1RP, and CD47 is partially in the form of ScFv.

TABLE 4 list of reagents and instruments used in Experimental example 9

Name (R)	Brand	Goods number
			White flat-bottom sterile plate	Costar	3917
1640	Gbico	11875-093
			FBS	MRC	CCS30010.02
Bio-glo	promega	G7940
			Enzyme-linked immunosorbent assay (ELISA) instrument	SpectraMax	i3X

The specific implementation is as follows:

1. the target cells were observed under a microscope, and the cells were collected under normal conditions with the cells round and clear, counted, and centrifuged at 1500rpm for 5 min.

2. Discard the supernatant, resuspend the target cells with 1640+ 0.5% FBS, mix well and add a white flat bottom sterile plate, 1 x 10⁴cells/well, 25. mu.l per well.

3. The prepared 4-fold final concentration of the antibody to be detected was added to the plate at 25. mu.l/well, 37 ℃ and 5% CO₂Incubate 30 min.

4. After incubation was complete Jurkat-Fc γ RIIIa-NFAT-L uciferase cells, 6 x 10⁴cells/well, 50. mu.l per well. The volume is less than 100 μ l (control well), and is supplemented to 100 μ l with 1640+ 0.5% FBS medium, mixed well and then 5% CO at 37 deg.C₂Incubate for 6 h.

5. Add 40. mu.l Bio-glo to each well, incubate for 5min in the dark, read chemiluminescence with a microplate reader.

The ADCC activity results show that:

1. 9020-6RP with the strongest ADCC activity and 9020-2RP with the weakest ADCC activity in 9020-1RP, 9020-2RP and 9020-6RP, as shown in figures 12A and 12B. Namely, the ADCC activity is 9020-6RP >9020-1RP >9020-2 RP.

2. The ADCC activity of 9020-T1RP was significantly stronger than Rituximab, Rituximab-TH (Fc mutation) and CD20mAb-SD1 as shown in FIGS. 12C, 12D, 12E. I.e., ADCC activity (9020-T1RP/9020-1RP) > (Rituximab/Rituximab-TH), (9020-T1RP/9020-1RP) > CD20mAb-SD 1.

9020-T1RP showed a strong ADCC activity compared to Rituximab and Rituximab-TH, indicating that the CD47 moiety of the diabody contributes to the enhancement of ADCC activity. In addition, the ADCC activity of 9020-T1RP is also stronger than that of other double antibodies, such as 9020-2RP and CD20mAb-SD1, which suggests that the structure of the double antibodies has a great influence on the ADCC activity.

EXAMPLE 10 ADCC Activity of bispecific antibodies against Normal cells (FACS)

The safety of bispecific antibodies was evaluated by measuring ADCC activity against Red Blood Cells (RBC), Peripheral Blood Mononuclear Cells (PBMC) using Jurkat-Fc γ RIIIa-NFAT-L uciferase cells as effector cells the reagents and instrumentation used in this example are shown in Table 5.

TABLE 5 list of reagents and instruments used in Experimental example 9

The specific implementation is as follows:

3. The prepared 4-fold final concentration of the antibody to be tested was added to the plate at 25. mu.l/well, 37 ℃ and 5% CO₂Incubate 30 min.

As shown in FIG. 13A, the results show that 9020-6RP has ADCC killing activity on normal RBC, and the results of Experimental example 9 show that 9020-6RP has strong ADCC activity on tumor cells, but the strong ADCC effect on RBC reduces the safety of the tumor cells. SIRPaD1 also produced ADCC killing activity against normal RBCs, indicating that CD47 fusion protein inhibitors would pose a significant ADCC safety risk if Fc in the form of IgG1 was used.

9020-T1RP has low ADCC activity on RBC or PBMC, similar to Rituximab in ADCC safety, as shown in FIGS. 13B and 13C.

EXAMPLE 11 CDC Activity of bispecific antibody against tumor cells (FACS)

CDC activity of bispecific antibodies against Raji, Ramous, Daudi was assessed by complement in vitro assay in serum.

The specific implementation is as follows:

1. observing target cells under microscope, collecting target cells under normal state, counting, each antibody, each gradient having 2 duplicate wells, spreading target cells in a row as control wells without antibody, 1.5 × 10⁴cells/well. According to calculationThe desired cells were centrifuged at 1500rpm for 5min and the supernatant was discarded.

2. Resuspending the target cells in 2% FBS +1640 medium (1640: Gbico, REF: 61870-⁴cells/well, 25. mu.l per well.

3. The test antibody was added to the plate at a final concentration of 4 fold diluted in 2% FBS +1640 medium, 25. mu.l per well, at a final concentration of 100. mu.g/ml in the first spot of the test antibody, at a gradient dilution of 10 fold, for a total of 8 spots, and 25. mu.l of 2% FBS +1640 medium was added to the control well. 37 ℃ and 5% CO₂Incubate 30 min.

4. After completion of the incubation, 50. mu.l of 20% Human serum AB (Gemini, Cat: 100-₂Incubate for 4 h. The whole operation process keeps the sterile state.

5. Mu.l of CTG (promega, REF: G7572) was added to each well, incubated for 5 minutes in the absence of light, and then applied to a microplate reader (

i3X) reading chemiluminescence, and analyzing the result.

The results shown in fig. 14A, B, C show that:

1. the 9020-2RP does not generate CDC activity on Raji, Daudi and Ramous tumor cell lines, and the CDC activity of the 9020-1RP and 9020-6RP on tumor cells is stronger than that of CD20mAb-SD1 but weaker than that of Rituximab;

2. the CDC activity of 9020-T1RP was significantly enhanced after Fc mutation of 9020-1RP, comparable to that of Rituximab. The CDC activity of 9020-1RP-1C1 and 9020-1RP-1C2 has no obvious difference with that of 9020-1 RP.

The results show that CDC activity not only needs the participation of Fc, but also needs the binding capacity of target, for example, 9020-2RP has poor targeting capacity, and CDC activity is also poor; furthermore, it is influenced by the structure, such as the structure of the double anti-CD 20mAb-SD1, that two targets are located at both ends of Fc, which also reduces CDC activity on tumor cells.

EXAMPLE 12 CDC Activity of bispecific antibodies against Normal cells (FACS)

The safety of bispecific antibodies was assessed by in vitro detection of CDC activity of the bispecific antibodies against Red Blood Cells (RBCs), Peripheral Blood Mononuclear Cells (PBMCs) by complement in serum.

The specific implementation is as follows:

1. observing target cells under microscope, collecting target cells under normal state, counting, each antibody, each gradient having 2 duplicate wells, spreading target cells in a row as control wells without antibody, 1.5 × 10⁴cells/well. According to the calculation, the required cells are taken out, centrifuged at 1500rpm for 5min, and the supernatant is discarded.

i3X) reading chemiluminescence, and analyzing the result.

As shown in fig. 15A and 15B, 9020-T1RP did not produce CDC activity on PBMC and RBC, which may be associated with low abundance of target expression on the cell surface of PBMC and RBC, and failed to form an efficient polymer structure to produce CDC activity.

EXAMPLE 13 bispecific antibody ADCP activation assay (FACS)

MDM isolation induction of effector cells: extracting 20 persons of venous blood to separate PBMC, separating monocyte, adding GM-CSF/M-CSF to induce differentiation MDM, and identifying differentiated macrophage MDM by CD11b, CD14, CD45, CD163 and CD206 biomarkers after 1 week;

ADCP the target cells H L-60 were removed from the incubator, the cells were collected in a 15ml centrifuge tube, centrifuged, the supernatant discarded, the cells were resuspended in PBS, counted, the target cells were stained with PKH26(SIGMA-A L DRICH), placed at 37 deg.C&5％CO₂Culturing overnight; taking out the target cells from the culture medium the next day, centrifugally collecting the cells, discarding the supernatant, using the complete culture medium for resuspension, and counting; taking target cells, adding a complete culture medium, and adding the cells and a sample to be detected into a corresponding 96-well plate, wherein each cell is 50000 cells/well; incubating for 0.5h at room temperature; taking out effector cells (MDM) from a culture medium, collecting the supernatant, adding PBS for washing, adding Accutase for digesting the MDM, removing the MDM from the wall, adding complete culture medium with the same volume for terminating digestion, transferring the cell suspension into a centrifuge tube, and centrifuging the cell supernatant and the digested cells for 10min according to 300 g; MDM cells were added to a 96-well plate corresponding to the target cells by adding a corresponding volume of MDM cells to the complete medium. Effector cells: target cells were incubated at 37 ℃ for 4h at 1: 1;

adding Accutase, observing whether the adherent cells are digested under a microscope (about 15min), taking out the cells, transferring the cells to another plate, and adding the removed cell suspension into the corresponding hole again after the cells are completely digested; centrifuging, adding detection antibody CD11b, and incubating at 4 deg.C for 15 min; adding PBS to each hole, centrifuging, removing supernatant, and adding PBS for heavy suspension; detecting by a flow cytometer;

data analysis { (PKH26+ & CD11b + cells)/AllPKH26+ cells } × 100% Phagocytosis byMDMS { (PKH26+ & CD11b + cells).

As shown in fig. 16A and 16B, ADCP activation of macrophages by 9020-1RP was similar to CD20mAb-SD1, but ADCP activity of 9020-T1RP was significantly enhanced. And the ADCP activity of 9020-T1RP is stronger than that of SIRPaD 1. In the Daudi cell model, ADCP activity of 9020-T1RP was stronger than that of H5F 9-G4.

Conclusion analysis:

according to the invention, the first functional region SIRPaD1 at the outer end of the human SIRPa membrane and the CD20 antibody are used for constructing the bispecific antibody in different forms, such as 9020-1RP, 9020-2RP and 9020-6RP, and in the three structures, the 9020-1RP is found to have better targeting property and safety. We continued the study on the basis of 9020-1RP, either mutating SIRPaD1 in 9020-1RP to alter the targeting affinity of CD47 (9020-1RP-1C1), or the linker of the bispecific antibody 9020-1RP (9020-1RP-1C2), or mutating the Fc in 9020-1RP to increase CD16a and decrease the affinity of CD32 b.

The research result shows that:

1. 9020-T1RP can simultaneously target CD20/CD47 and has high specificity. In CD20⁺CD47⁺In the experiment of a mixed model of tumor cells and RBC, the 9020-1RP and 9020-T1RP can more specifically target CD20 than monoclonal antibody or other forms of double antibodies⁺CD47⁺A tumor cell.

2. 9020-T1RP has stronger immune activation tumor killing effect. In CD20⁺/CD47⁺Compared with a CD20 monoclonal antibody or other structural forms of double antibodies, the monoclonal antibody can more effectively activate antibody-mediated cell-dependent cytotoxicity (ADCC) and antibody complement-dependent complement killing (CDC) in the tumor cell model. In addition, the bispecific antibody of the invention can activate the phagocytic Activity (ADCP) of macrophages on tumor cells by blocking the binding of CD47 and a ligand SIRPa thereof, and the ADCP activity activated by 9020-T1RP is enhanced compared with an inhibitor of CD47 (a double antibody of CD47 monoclonal antibody or fusion protein or other structural forms).

3. 9020-T1RP has good safety in vitro evaluation. In terms of ADCC/CDC, 9020-T1RP did not have off-target-induced ADCC/CDC side effects on PBMC and RBC relative to CD20 or CD47 mab or other diabodies; in the aspect of blood coagulation, 9020-T1RP has no side effect of blood coagulation.

The bispecific antibody 9020-T1RP in the present invention was compared with the recombinant bifunctional fusion protein (CD20mAb-SD1) of the invention of patent application No. 201710151979.6. The bispecific antibody 9020-T1RP provided by the invention has the effect of resisting tumor cells CD20⁺CD47⁺More specific targeting. 9020-T1The ADCC effect of RP on tumor cells was significantly stronger than that of CD20mAb-SD1, and the CDC effect was also significantly stronger than that of CD20mAb-SD1 on tumor cells.

The structure of CD20mAb-SD1 was Fc located in the middle part of the overall structure, with SIRPaD1 and CD20 antibodies located at both ends of the Fc, potentially limiting the specificity of simultaneous targeting of CD20 and CD47, and not being able to form efficient multimer activation CDC.

The present invention can be further illustrated based on the examples, but is not limited to the examples, and the examples do not limit the scope of the present invention. Various modifications, substitutions and equivalents may occur to those skilled in the art based on the disclosure within the scope of the appended claims.

Sequence listing

<110> times Dada pharmaceutical industries (Suzhou) Co., Ltd

<120> CD20/CD47 bispecific antibody and application

<130>2020

<160>26

<170>SIPOSequenceListing 1.0

<210>1

<211>118

<212>PRT

<213>Artificial Sequence

<400>1

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser

115

<210>2

<211>354

<212>DNA

<213>Artificial Sequence

<400>2

gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60

gctatcctgc attgcaccgt gaccagcctg attcccgtgg gaccaatcca gtggttcaga 120

ggagccggac cagccagaga gctgatctac aaccagaagg agggccactt ccccagagtg 180

acaacagtgt ccgagagcac caagcgggag aacatggact tcagcatcag catcagcaac 240

atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300

accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagc 354

<210>3

<211>118

<212>PRT

<213>Artificial Sequence

<400>3

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser

115

<210>4

<211>354

<212>DNA

<213>Artificial Sequence

<400>4

gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60

gctatcctgc attgcaccgt gaccagcctg tttcccgtgg gaccaatcca gtggttcaga 120

ggagccggac cagccagaga gctgatctac aaccagaggc agggcccttt ccctagagtg 180

acaaccgtgt ccgagaccac caagagggag aacatggact tcagcatcag catcagcgcc 240

atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300

accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagc 354

<210>5

<211>121

<212>PRT

<213>Artificial Sequence

<400>5

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala

115 120

<210>6

<211>363

<212>DNA

<213>Artificial Sequence

<400>6

caggtgcagc tgcagcagcc aggagcagaa ctggtgaagc caggcgccag cgtgaagatg 60

tcttgcaaag ccagcggcta caccttcacc agctacaaca tgcattgggt gaagcagacc 120

ccaggaagag gcctggagtg gatcggtgcc atctaccccg gcaacggcga caccagctac 180

aaccagaagt tcaagggcaa ggccaccctg acagccgata agagcagcag caccgcctac 240

atgcagctgt ctagcctgac cagcgaggat agcgccgtgt actattgcgc caggagcacc 300

tactacggcg gcgattggta cttcaacgtc tggggagccg gaacaacagt gacagtgtcc 360

gca 363

<210>7

<211>106

<212>PRT

<213>Artificial Sequence

<400>7

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>8

<211>318

<212>DNA

<213>Artificial Sequence

<400>8

cagatcgtgc tgtctcagag cccagccatt ctgagcgctt ctccaggcga gaaggtcacc 60

atgacttgca gagccagcag cagcgtgtcc tacatccatt ggttccagca gaagccagga 120

agcagcccta agccttggat ctacgccacc agcaacctgg ctagcggagt gccagtgaga 180

ttcagcggaa gcggaagcgg aaccagctac agcctgacca tcagcagagt ggaggccgaa 240

gacgccgcta catactactg ccagcagtgg accagcaacc ctcctacctt tggcggaggc 300

accaagctgg agatcaag 318

<210>9

<211>330

<212>PRT

<213>Artificial Sequence

<400>9

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>10

<211>990

<212>DNA

<213>Artificial Sequence

<400>10

gccagcacca agggacctag cgtgtttcct ctggcccctt ctagcaagag cacaagcgga 60

ggaacagccg ctctgggctg tctggtgaaa gactactttc ccgagcccgt gaccgtgtct 120

tggaacagcg gagccctgac cagcggagtg cacacatttc cagccgtgct gcagagcagc 180

ggactgtata gcctgagcag cgtggtgacc gtgccttctt cttctctggg cacccagacc 240

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggccgagccc 300

aagtcttgcg acaagaccca cacttgcccc ccttgtccag ctccagaact cctgggagga 360

cctagcgtgt tcctgttccc tcccaagcct aaggacaccc tgatgatcag ccggacccca 420

gaagtgactt gcgtggtggt ggacgtgtcc cacgaagacc ccgaggtcaa gttcaattgg 480

tacgtggacg gagtggaggt gcacaacgct aagaccaagc ccagggagga gcagtacaac 540

agcacctaca gggtggtgtc cgtgctgaca gtgctgcacc aggattggct gaacggcaag 600

gagtacaagt gcaaggtgtc caacaaggcc ctgccagccc ctatcgagaa gaccatcagc 660

aaggccaagg gccagcctag agaacctcag gtgtacaccc tgcccccttc tagagacgag 720

ctgaccaaga accaggtgtc cctgacttgc ctcgtgaagg gcttctaccc cagcgatatc 780

gccgtggagt gggaatctaa cggtcagcca gagaacaact acaagaccac ccccccagtg 840

ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa aagccgctgg 900

cagcagggca acgtgttctc ttgcagcgtg atgcacgagg ccctgcacaa ccactacacc 960

cagaagagcc tgagcctgag cccaggaaag 990

<210>11

<211>330

<212>PRT

<213>Artificial Sequence

<400>11

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Val Gly Gly Pro Ser Val Phe Leu Leu Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>12

<211>990

<212>DNA

<213>Artificial Sequence

<400>12

gccagcacca agggacctag cgtgtttcct ctggcccctt ctagcaagag cacaagcgga 60

ggaacagccg ctctgggctg tctggtgaaa gactactttc ccgagcccgt gaccgtgtct 120

tggaacagcg gagccctgac cagcggagtg cacacatttc cagccgtgct gcagagcagc 180

ggactgtata gcctgagcag cgtggtgacc gtgccttctt cttctctggg cacccagacc 240

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa ggccgagccc 300

aagtcttgcg acaagaccca cacttgcccc ccttgtccag ctccagaact cgtgggagga 360

cctagcgtgt tcctgctgcc tcccaagcct aaggacaccc tgatgatcag ccggacccca 420

gaagtgactt gcgtggtggt ggacgtgtcc cacgaagacc ccgaggtcaa gttcaattgg 480

tacgtggacg gagtggaggt gcacaacgct aagaccaagc cccctgagga gcagtacaac 540

agcaccctga gggtggtgtc cgtgctgaca gtgctgcacc aggattggct gaacggcaag 600

gagtacaagt gcaaggtgtc caacaaggcc ctgccagccc ctatcgagaa gaccatcagc 660

aaggccaagg gccagcctag agaacctcag gtgtacaccc tgcccccttc tagagacgag 720

ctgaccaaga accaggtgtc cctgacttgc ctcgtgaagg gcttctaccc cagcgatatc 780

gccgtggagt gggaatctaa cggtcagcca gagaacaact acaagaccac ccccctggtg 840

ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa aagccgctgg 900

cagcagggca acgtgttctc ttgcagcgtg atgcacgagg ccctgcacaa ccactacacc 960

cagaagagcc tgagcctgag cccaggaaag 990

<210>13

<211>451

<212>PRT

<213>Artificial Sequence

<400>13

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Val Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Leu

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Leu

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>14

<211>584

<212>PRT

<213>Artificial Sequence

<400>14

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu

130 135 140

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

145 150 155 160

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg

165 170 175

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

180 185 190

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

195 200 205

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

210 215 220

Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr

225 230 235 240

Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser

245 250 255

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

260 265 270

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

275 280 285

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

290 295 300

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

305 310 315 320

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

325 330 335

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala

340 345 350

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

355 360 365

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

370 375 380

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

385 390 395 400

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

405 410 415

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

420 425 430

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

435 440 445

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

450 455 460

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

465 470 475 480

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

485 490 495

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

500 505 510

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

515 520 525

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

530 535 540

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

545 550 555 560

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

565 570 575

Ser Leu Ser Leu Ser Pro Gly Lys

580

<210>15

<211>1752

<212>DNA

<213>Artificial Sequence

<400>15

gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60

gctatcctgc attgcaccgt gaccagcctg attcccgtgg gaccaatcca gtggttcaga 120

ggagccggac cagccagaga gctgatctac aaccagaagg agggccactt ccccagagtg 180

acaacagtgt ccgagagcac caagcgggag aacatggact tcagcatcag catcagcaac 240

atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300

accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagcggaggc 360

ggtggaagcg gtggaggcgg atctggcgga ggtggatctc aggtgcagct gcagcagcca 420

ggagcagaac tggtgaagcc aggcgccagc gtgaagatgt cttgcaaagc cagcggctac 480

accttcacca gctacaacat gcattgggtg aagcagaccc caggaagagg cctggagtgg 540

atcggtgcca tctaccccgg caacggcgac accagctaca accagaagtt caagggcaag 600

gccaccctga cagccgataa gagcagcagc accgcctaca tgcagctgtc tagcctgacc 660

agcgaggata gcgccgtgta ctattgcgcc aggagcacct actacggcgg cgattggtac 720

ttcaacgtct ggggagccgg aacaacagtg acagtgtccg cagccagcac caagggacct 780

agcgtgtttc ctctggcccc ttctagcaag agcacaagcg gaggaacagc cgctctgggc 840

tgtctggtga aagactactt tcccgagccc gtgaccgtgt cttggaacag cggagccctg 900

accagcggag tgcacacatt tccagccgtg ctgcagagca gcggactgta tagcctgagc 960

agcgtggtga ccgtgccttc ttcttctctg ggcacccaga cctacatctg caacgtgaac 1020

cacaagcccagcaacaccaa ggtggacaag aaggccgagc ccaagtcttg cgacaagacc 1080

cacacttgcc ccccttgtcc agctccagaa ctcctgggag gacctagcgt gttcctgttc 1140

cctcccaagc ctaaggacac cctgatgatc agccggaccc cagaagtgac ttgcgtggtg 1200

gtggacgtgt cccacgaaga ccccgaggtc aagttcaatt ggtacgtgga cggagtggag 1260

gtgcacaacg ctaagaccaa gcccagggag gagcagtaca acagcaccta cagggtggtg 1320

tccgtgctga cagtgctgca ccaggattgg ctgaacggca aggagtacaa gtgcaaggtg 1380

tccaacaagg ccctgccagc ccctatcgag aagaccatca gcaaggccaa gggccagcct 1440

agagaacctc aggtgtacac cctgccccct tctagagacg agctgaccaa gaaccaggtg 1500

tccctgactt gcctcgtgaa gggcttctac cccagcgata tcgccgtgga gtgggaatct 1560

aacggtcagc cagagaacaa ctacaagacc acccccccag tgctggacag cgacggcagc 1620

ttcttcctgt acagcaagct gaccgtggac aaaagccgct ggcagcaggg caacgtgttc 1680

tcttgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1740

agcccaggaa ag 1752

<210>16

<211>346

<212>PRT

<213>Artificial Sequence

<400>16

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

2025 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu

130 135 140

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

145 150 155 160

Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro

165 170 175

Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val

180 185190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser

195 200 205

Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr

210 215 220

Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

225 230 235 240

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

245 250 255

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

260 265 270

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

275 280 285

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

290 295 300

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

305 310 315 320

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

325 330 335

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

340 345

<210>17

<211>1038

<212>DNA

<213>Artificial Sequence

<400>17

gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60

gctatcctgc attgcaccgt gaccagcctg attcccgtgg gaccaatcca gtggttcaga 120

ggagccggac cagccagaga gctgatctac aaccagaagg agggccactt ccccagagtg 180

acaacagtgt ccgagagcac caagcgggag aacatggact tcagcatcag catcagcaac 240

atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300

accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagcggaggc 360

ggtggaagcg gtggaggcgg atctggcgga ggtggatctc agatcgtgct gtctcagagc 420

ccagccattc tgagcgcttc tccaggcgag aaggtcacca tgacttgcag agccagcagc 480

agcgtgtcct acatccattg gttccagcag aagccaggaa gcagccctaa gccttggatc 540

tacgccacca gcaacctggc tagcggagtg ccagtgagat tcagcggaag cggaagcgga 600

accagctaca gcctgaccat cagcagagtg gaggccgaag acgccgctac atactactgc 660

cagcagtgga ccagcaaccc tcctaccttt ggcggaggca ccaagctgga gatcaagaga 720

accgtggccg ctcctagcgt gttcatcttc cctcccagcg acgagcagct gaagtcagga 780

acagccagcg tcgtgtgtct gctcaacaac ttctacccca gggaggccaa ggtccagtgg 840

aaagtggaca acgccctgca gagcggaaac tctcaggaga gcgtgaccga gcaggacagc 900

aaggacagca cctacagcct gagcagcaca ctgaccctga gcaaggccga ctacgagaag 960

cacaaggtgt acgcttgcga ggtcacacac cagggactgt ctagcccagt gaccaagagc 1020

ttcaaccgag gcgagtgc 1038

<210>18

<211>346

<212>PRT

<213>Artificial Sequence

<400>18

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

210 215 220

Gly Gly Gly Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser

225 230 235 240

Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr

245 250 255

Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro

260 265 270

Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val

275 280 285

Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile

290 295 300

Ser Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val

305 310 315 320

Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly

325 330 335

Thr Glu Leu Ser Val Arg Ala Lys Pro Ser

340 345

<210>19

<211>1038

<212>DNA

<213>Artificial Sequence

<400>19

cagatcgtgc tgtctcagag cccagccatt ctgagcgctt ctccaggcga gaaggtcacc 60

atgacttgca gagccagcag cagcgtgtcc tacatccatt ggttccagca gaagccagga 120

agcagcccta agccttggat ctacgccacc agcaacctgg ctagcggagt gccagtgaga 180

ttcagcggaa gcggaagcgg aaccagctac agcctgacca tcagcagagt ggaggccgaa 240

gacgccgcta catactactg ccagcagtgg accagcaacc ctcctacctt tggcggaggc 300

accaagctgg agatcaagag aaccgtggcc gctcctagcg tgttcatctt ccctcccagc 360

gacgagcagc tgaagtcagg aacagccagc gtcgtgtgtc tgctcaacaa cttctacccc 420

agggaggcca aggtccagtg gaaagtggac aacgccctgc agagcggaaa ctctcaggag 480

agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac actgaccctg 540

agcaaggccg actacgagaa gcacaaggtg tacgcttgcg aggtcacaca ccagggactg 600

tctagcccag tgaccaagag cttcaaccga ggcgagtgcg gaggcggtgg aagcggtgga 660

ggcggatctg gcggaggtgg atctgaagag gagctgcagg tcatccagcc cgacaagtct 720

gtgtccgtgg cagcaggaga aagcgctatc ctgcattgca ccgtgaccag cctgattccc 780

gtgggaccaa tccagtggtt cagaggagcc ggaccagcca gagagctgat ctacaaccag 840

aaggagggcc acttccccag agtgacaaca gtgtccgaga gcaccaagcg ggagaacatg 900

gacttcagca tcagcatcag caacatcaca ccagccgacg ccggcacata ctattgcgtg 960

aagttccgga agggcagccc agataccgag ttcaagagcg gagccggaac agagctgagc 1020

gtgagagcca agcctagc 1038

<210>20

<211>584

<212>PRT

<213>Artificial Sequence

<400>20

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Phe Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu

130 135 140

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

145 150 155 160

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg

165 170 175

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

180 185 190

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

195 200 205

Ser Ser Thr Ala Tyr Met GlnLeu Ser Ser Leu Thr Ser Glu Asp Ser

210 215 220

Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr

225 230 235 240

Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser

245 250 255

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

260 265 270

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

275 280 285

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

290 295 300

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

305 310 315 320

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

325 330 335

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala

340 345 350

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

355 360 365

Pro Glu Leu Leu Gly Gly Pro Ser ValPhe Leu Phe Pro Pro Lys Pro

370 375 380

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

385 390 395 400

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

405 410 415

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

420 425 430

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

435 440 445

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

450 455 460

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

465 470 475 480

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

485 490 495

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

500 505 510

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

515 520 525

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp GlySer Phe Phe Leu Tyr

530 535 540

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

545 550 555 560

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

565 570 575

Ser Leu Ser Leu Ser Pro Gly Lys

580

<210>21

<211>1752

<212>DNA

<213>Artificial Sequence

<400>21

gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60

gctatcctgc attgcaccgt gaccagcctg tttcccgtgg gaccaatcca gtggttcaga 120

ggagccggac cagccagaga gctgatctac aaccagaggc agggcccttt ccctagagtg 180

acaaccgtgt ccgagaccac caagagggag aacatggact tcagcatcag catcagcgcc 240

atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300

accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagcggaggc 360

ggtggaagcg gtggaggcgg atctggcgga ggtggatctc aggtgcagct gcagcagcca 420

ggagcagaac tggtgaagcc aggcgccagc gtgaagatgt cttgcaaagc cagcggctac 480

accttcacca gctacaacat gcattgggtg aagcagaccc caggaagagg cctggagtgg 540

atcggtgcca tctaccccgg caacggcgacaccagctaca accagaagtt caagggcaag 600

gccaccctga cagccgataa gagcagcagc accgcctaca tgcagctgtc tagcctgacc 660

agcgaggata gcgccgtgta ctattgcgcc aggagcacct actacggcgg cgattggtac 720

ttcaacgtct ggggagccgg aacaacagtg acagtgtccg cagccagcac caagggacct 780

agcgtgtttc ctctggcccc ttctagcaag agcacaagcg gaggaacagc cgctctgggc 840

tgtctggtga aagactactt tcccgagccc gtgaccgtgt cttggaacag cggagccctg 900

accagcggag tgcacacatt tccagccgtg ctgcagagca gcggactgta tagcctgagc 960

agcgtggtga ccgtgccttc ttcttctctg ggcacccaga cctacatctg caacgtgaac 1020

cacaagccca gcaacaccaa ggtggacaag aaggccgagc ccaagtcttg cgacaagacc 1080

cacacttgcc ccccttgtcc agctccagaa ctcctgggag gacctagcgt gttcctgttc 1140

cctcccaagc ctaaggacac cctgatgatc agccggaccc cagaagtgac ttgcgtggtg 1200

gtggacgtgt cccacgaaga ccccgaggtc aagttcaatt ggtacgtgga cggagtggag 1260

gtgcacaacg ctaagaccaa gcccagggag gagcagtaca acagcaccta cagggtggtg 1320

tccgtgctga cagtgctgca ccaggattgg ctgaacggca aggagtacaa gtgcaaggtg 1380

tccaacaagg ccctgccagc ccctatcgag aagaccatca gcaaggccaa gggccagcct 1440

agagaacctc aggtgtacac cctgccccct tctagagacg agctgaccaa gaaccaggtg 1500

tccctgactt gcctcgtgaa gggcttctac cccagcgata tcgccgtgga gtgggaatct 1560

aacggtcagc cagagaacaa ctacaagacc acccccccag tgctggacag cgacggcagc 1620

ttcttcctgt acagcaagct gaccgtggac aaaagccgct ggcagcaggg caacgtgttc 1680

tcttgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1740

agcccaggaa ag 1752

<210>22

<211>574

<212>PRT

<213>Artificial Sequence

<400>22

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser Ala Ser Thr Lys Gly Gln Val Gln Leu Gln

115 120 125

Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser

130 135 140

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val

145 150 155 160

Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro

165 170 175

Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr

180 185 190

Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser

195 200 205

Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr

210 215 220

Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val

225 230 235 240

Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

355 360 365

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

370 375 380

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

385 390 395 400

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

405 410 415

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

420 425 430

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

435 440 445

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

450 455 460

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

465 470 475 480

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

485 490 495

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

500 505 510

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

515 520 525

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

530 535 540

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

545 550 555 560

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210>23

<211>1722

<212>DNA

<213>Artificial Sequence

<400>23

gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60

gctatcctgc attgcaccgt gaccagcctg attcccgtgg gaccaatcca gtggttcaga 120

ggagccggac cagccagaga gctgatctac aaccagaagg agggccactt ccccagagtg 180

acaacagtgt ccgagagcac caagcgggag aacatggact tcagcatcag catcagcaac 240

atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300

accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagcgcctct 360

accaagggcc aggtgcagct gcagcagcca ggagcagaac tggtgaagcc aggcgccagc 420

gtgaagatgt cttgcaaagc cagcggctac accttcacca gctacaacat gcattgggtg 480

aagcagaccc caggaagagg cctggagtgg atcggtgcca tctaccccgg caacggcgac 540

accagctaca accagaagtt caagggcaag gccaccctga cagccgataa gagcagcagc 600

accgcctaca tgcagctgtc tagcctgacc agcgaggata gcgccgtgta ctattgcgcc 660

aggagcacct actacggcgg cgattggtac ttcaacgtct ggggagccgg aacaacagtg 720

acagtgtccg cagccagcac caagggacct agcgtgtttc ctctggcccc ttctagcaag 780

agcacaagcg gaggaacagc cgctctgggc tgtctggtga aagactactt tcccgagccc 840

gtgaccgtgt cttggaacag cggagccctg accagcggag tgcacacatt tccagccgtg 900

ctgcagagca gcggactgta tagcctgagc agcgtggtga ccgtgccttc ttcttctctg 960

ggcacccaga cctacatctg caacgtgaac cacaagccca gcaacaccaa ggtggacaag 1020

aaggccgagc ccaagtcttg cgacaagacc cacacttgcc ccccttgtcc agctccagaa 1080

ctcctgggag gacctagcgt gttcctgttc cctcccaagc ctaaggacac cctgatgatc1140

agccggaccc cagaagtgac ttgcgtggtg gtggacgtgt cccacgaaga ccccgaggtc 1200

aagttcaatt ggtacgtgga cggagtggag gtgcacaacg ctaagaccaa gcccagggag 1260

gagcagtaca acagcaccta cagggtggtg tccgtgctga cagtgctgca ccaggattgg 1320

ctgaacggca aggagtacaa gtgcaaggtg tccaacaagg ccctgccagc ccctatcgag 1380

aagaccatca gcaaggccaa gggccagcct agagaacctc aggtgtacac cctgccccct 1440

tctagagacg agctgaccaa gaaccaggtg tccctgactt gcctcgtgaa gggcttctac 1500

cccagcgata tcgccgtgga gtgggaatct aacggtcagc cagagaacaa ctacaagacc 1560

acccccccag tgctggacag cgacggcagc ttcttcctgt acagcaagct gaccgtggac 1620

aaaagccgct ggcagcaggg caacgtgttc tcttgcagcg tgatgcacga ggccctgcac 1680

aaccactaca cccagaagag cctgagcctg agcccaggaa ag 1722

<210>24

<211>584

<212>PRT

<213>Artificial Sequence

<400>24

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu

130 135 140

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

145 150 155 160

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg

165 170 175

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

180 185 190

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

195 200 205

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

210 215 220

Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr

225 230 235 240

Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser

245 250 255

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

260 265 270

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

275 280 285

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

290 295 300

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

305 310 315 320

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

325 330 335

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala

340 345 350

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

355 360 365

Pro Glu Leu Val Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

370 375 380

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

385 390 395 400

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

405 410 415

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

420 425 430

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

435 440 445

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

450 455 460

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

465 470 475 480

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

485 490 495

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

500 505 510

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

515 520 525

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

530 535 540

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

545 550 555 560

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

565 570 575

Ser Leu Ser Leu Ser Pro Gly Lys

580

<210>25

<211>1752

<212>DNA

<213>Artificial Sequence

<400>25

gaagaggagc tgcaggtcat ccagcccgac aagtctgtgt ccgtggcagc aggagaaagc 60

gctatcctgc attgcaccgt gaccagcctg attcccgtgg gaccaatcca gtggttcaga 120

ggagccggac cagccagaga gctgatctac aaccagaagg agggccactt ccccagagtg 180

acaacagtgt ccgagagcac caagcgggag aacatggact tcagcatcag catcagcaac 240

atcacaccag ccgacgccgg cacatactat tgcgtgaagt tccggaaggg cagcccagat 300

accgagttca agagcggagc cggaacagag ctgagcgtga gagccaagcc tagcggaggc 360

ggtggaagcg gtggaggcgg atctggcgga ggtggatctc aggtgcagct gcagcagcca 420

ggagcagaac tggtgaagcc aggcgccagc gtgaagatgt cttgcaaagc cagcggctac 480

accttcacca gctacaacat gcattgggtg aagcagaccc caggaagagg cctggagtgg 540

atcggtgcca tctaccccgg caacggcgac accagctaca accagaagtt caagggcaag 600

gccaccctga cagccgataa gagcagcagc accgcctaca tgcagctgtc tagcctgacc 660

agcgaggata gcgccgtgta ctattgcgcc aggagcacct actacggcgg cgattggtac 720

ttcaacgtct ggggagccgg aacaacagtg acagtgtccg cagccagcac caagggacct 780

agcgtgtttc ctctggcccc ttctagcaag agcacaagcg gaggaacagc cgctctgggc 840

tgtctggtga aagactactt tcccgagccc gtgaccgtgt cttggaacag cggagccctg 900

accagcggag tgcacacatt tccagccgtg ctgcagagca gcggactgta tagcctgagc 960

agcgtggtga ccgtgccttc ttcttctctg ggcacccaga cctacatctg caacgtgaac 1020

cacaagccca gcaacaccaa ggtggacaag aaggccgagc ccaagtcttg cgacaagacc 1080

cacacttgcc ccccttgtcc agctccagaa ctcgtgggag gacctagcgt gttcctgctg 1140

cctcccaagc ctaaggacac cctgatgatc agccggaccc cagaagtgac ttgcgtggtg 1200

gtggacgtgt cccacgaaga ccccgaggtc aagttcaatt ggtacgtgga cggagtggag 1260

gtgcacaacg ctaagaccaa gccccctgag gagcagtaca acagcaccct gagggtggtg 1320

tccgtgctga cagtgctgca ccaggattgg ctgaacggca aggagtacaa gtgcaaggtg 1380

tccaacaagg ccctgccagc ccctatcgag aagaccatca gcaaggccaa gggccagcct 1440

agagaacctc aggtgtacac cctgccccct tctagagacg agctgaccaa gaaccaggtg 1500

tccctgactt gcctcgtgaa gggcttctac cccagcgata tcgccgtgga gtgggaatct 1560

aacggtcagc cagagaacaa ctacaagacc acccccctgg tgctggacag cgacggcagc 1620

ttcttcctgtacagcaagct gaccgtggac aaaagccgct ggcagcaggg caacgtgttc 1680

tcttgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagag cctgagcctg 1740

agcccaggaa ag 1752

<210>26

<211>583

<212>PRT

<213>Artificial Sequence

<400>26

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser

450 455 460

Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu

465 470 475 480

Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg

485 490 495

Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr

500 505 510

Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile

515 520 525

Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe

530 535 540

Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu

545 550 555 560

Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala

565 570 575

Ala Arg Ala Thr Pro Gln His

580

Claims

1. A CD20/CD47 bispecific antibody, which comprises a CD20 antibody of anti-CD 20 and a first structural domain SIRPaD1 of a human SIRPa extracellular segment, wherein the SIRPaD1 is connected to the N terminal of a CD20 antibody heavy chain through a connecting peptide.

2. The CD20/CD47 bispecific antibody of claim 1, wherein the type of said CD20 antibody is of the IgG1 subtype.

3. The CD20/CD47 bispecific antibody of claim 2, wherein the CD20 heavy chain Fc comprises IgG1 wild-type Fc-WT of the sequence set forth in SEQ ID No.9, SEQ ID No.10, or IgG1 combined mutant ADCC-enhanced Fc-MT of the sequence set forth in SEQ ID No.11, SEQ ID No. 12.

4. The CD20/CD47 bispecific antibody of claim 1, wherein the human SIRPa is wild type V2, and the sequence of the SIRPaD1 is shown in SEQ ID NO. 1.

5. A CD20/CD47 bispecific antibody according to claim 1, wherein the linking peptide has the sequence (GGGGS) × 3.

6. The CD20/CD47 bispecific antibody of claim 1, wherein the structure of said CD20/CD47 bispecific antibody is a symmetric homodimer.

7. An amino acid encoding the CD20/CD47 bispecific antibody of any one of claims 1-6.

8. A polynucleotide encoding the amino acid of claim 7.

9. An expression vector comprising the polynucleotide of claim 8.

10. A host cell comprising the expression vector of claim 9.

11. Use of the CD20/CD47 bispecific antibody of any one of claims 1-6 in the preparation of a medicament for the treatment of an overexpression of CD20/CD47, which is a tumor disorder.

12. The use of claim 11, wherein the neoplastic disorder comprises at least one of a hematological neoplastic disorder comprising at least one of acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, mixed leukemia, non-hodgkin's lymphoma.